EP4486441A1

EP4486441A1 - Sila-analogues of vitamin d compounds, method of producing said compounds and uses thereof

Info

Publication number: EP4486441A1
Application number: EP22723183.4A
Authority: EP
Inventors: Rodríguez BORGES; Baltazar Manuel ROMÃO DE CASTRO; Julián LOUREIRO MARTÍNEZ; Ivo Emanuel SAMPAIO DIAS; Román Perez Fernandez; Samuel SEOANE RUZO; Antonio MOURIÑO MOSQUERA
Original assignee: Universidade de Santiago de Compostela; Requimte Rede De Quimica E Tecnologia; Universidade do Porto
Current assignee: Universidade de Santiago de Compostela; Requimte Rede De Quimica E Tecnologia; Universidade do Porto
Priority date: 2022-02-28
Filing date: 2022-03-28
Publication date: 2025-01-08
Also published as: WO2023161688A1; ES2946255B2; PT117825B; PT117825A; ES2946255A1

Abstract

The present application describes the synthesis and important biological applications of novel vitamin D analogues with a carbon-to-silicon exchange in the side chain of the natural hormone and its analogues. In comparison to the natural metabolite 1α,25-dihydroxyvitamin D₃(1,25D) of the vitamin D receptor (VDR), the key biological results of these novel derivatives indicate that: a) these analogues show a similar cell differentiation, b) the proliferation of tumoral cell lines are significantly lower, c) great ability to activate target genes of 1,25D and d) high affinity to the VDR. Furthermore, one of the analogues induces a significant increase in the weight of some animals, which may suggest an increase in bone density.

Description

DESCRIPTION

"SILA-ANALOGUES OF VITAMIN D COMPOUNDS, METHOD OF PRODUCING SAID COMPOUNDS AND USES THEREOF"

Technical field

This application relates to compounds of formula (I), the intermediates, and their synthetic procedures. The present invention further relates to their pharmaceutical uses as therapeutic agents for Vitamin D receptor-related diseases.

Background art

25-Hydroxyvitamin D3, the major metabolite of vitamin D3 undergoes hydroxylation at C-l in the kidneys to generate its biologically active form, la,25-dihydroxyvitamin D3 (1,25D, calcitriol). This natural hormone has a high spectrum of activities, ranging from the participation in mineral homeostasis (calcium and phosphorous metabolism), to numerous antiproliferative, pro-dif ferentiative, and immunomodulatory activities (Vitamin D (D. Feldman, J.W. Pike, J.S. Adams); Two-Volume Set, Elsevier, Academic Press, New York, 2011, Bikie DD. Vitamin D metabolism, mechanism of action, and clinical applications, Chem Biol. 2014, 21, 319-329).

However, due to the calcemic side effects of this hormone at clinical doses, a great number of vitamin D receptor (VDR) ligands have been designed, synthesized, and biologically evaluated to retain or improve the biological activity of 1,25D but with limited or neglected calcemic side effects. Some of these analogues are currently applied for the treatment of different diseases (psoriasis, cancer, or osteoporosis), (J. Chen et al. Eur. J. Med. Chem. 2020,

207, 112738}.

The natural side chain of 1,25D has been the main target of chemical modifications, through the inclusion of rigid units, heteroatoms, or double/ triple bonds. Structure- activity relationship studies of these analogues have shown that a significant dissociation between cell differentiation and calcemic activities can indeed be accomplished, (C. Carlberg et al. J.Med.Chem. 2019, 62, 15, 6854}.

The incorporation of silicon into bioactive compounds is a chemical strategy that has been explored to improve pharmacological and biological performance, {R. Tacke. Curr. Opin. Drug Discov. Devel. 2003, 6, 526}. The silicon derivatives provide increased lipophilicity, solubility, and cell tissue penetration, improving the pharmacokinetic behavior and the agonistic or antagonistic properties. The metabolism of sila-haloperidol represents an example where the incorporation of silicon significantly alters the metabolism compared to haloperidol, avoiding the formation of toxic metabolites, {Tacke et al. Chem. Med. Chem. 2008, 3, 1, 152}.

Another example of silylated drugs with improved activities is the sila-ester indomethacin, which is more potent and less toxic than indomethacin used in the treatment of human pancreatic cancer, {KA Franz et al. J. Med. Chem. 2013, 56, 2, 388}. Summary

The present invention relates to a compound of formula (I), its analogues, or salt thereof:

R¹ and R² and R³ each independently represent a hydrogen atom or hydroxyl group or a branched or non-branched alkyl group having 1 or 3 carbon atoms, or a F, Cl, Br, I or a 0-

(C₁-C₃)alkoxyalkyl group or a (C₁-C₃)alkyl-OH group;

Y represents a hydrogen atom or a (C₁-C₃)alkyl group or an 0- (C₁-C₃)alkoxyalkyl group or 0-(C₁-C₃)0H group;

A is a methylene group or hydrogen atom;

W represents a diyne group -C=C-C=C- or a group wherein X is a (C₂-C₄)alkyl or (C₂-C₄)alkenyl or (C₂- C₄)alkynyl group, or a C₆-aromatic group;

Z represents a straight or branched, substituted or unsubstituted (C₁-C₃)alkyl or 0- (C₁-C₃)alkoxy or a 0- (C₁- C₃)0H group;

R⁴ and R⁵, which may be the same or different, each is independently selected from hydrogen, (C₁-C₁₂)alkyl, (C₆- C₁₀)aryl, and -SiR^aR^bR^c, wherein each R^a, R^b, and R^c is independently selected from (C₁-C₁₂)alkyl, (C₆-C₁₀)aryl, (C₁- C₁₂)alkoxyl, (C₆-C₁₀)aryloxyl and (C₃-C15)heterocycle. In one embodiment in compound of formula (I) R⁴ and R⁵ each represent a hydrogen atom, a methoxymethyl, a methoxyethyl, a trimethylsilylethoxymethyl, a trimethylsilyl, a triethylsilyl, a tert-butyldimethylsilyl, a tri-iso- propylsilyl, a tert-butyldiphenylsilyl , a dimethylphenylsilyl, a dimethylbenzylsilyl, a benzoate, a p-nitrobenzoate, a pyvalate, and an acetate.

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-7a-Methyl-l- [ (R)-5- (trimethylsilyl)pentan-2-yl]octahydro-4H-inden-4- ylidene}ethylidene)-4-methylenecyclohexane-l ,3-diol (P3).

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-7a-Methyl-l- [ (R)-5- (trimethylsilyl)pent-4-yn-2-yl]octahydro-4H-inden-4- ylidene}ethylidene)-4-methylenecyclohexane-l ,3-diol (P4).

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5- (Dimethylsilyl)pent-4-yn-2-yl ]-7a-methyl octahydro-4H- inden-4-ylidene}ethylidene )-4-methylenecyclohexane-l,3-diol (P5).

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(IS,3aS,7aS,E)-7a-Methyl-l- [ (trimethylsilyl)buta-1,3-diyn-l-yl]octahydro-4H-inden-4- ylidene}ethylidene)-4-methylenecyclohexane-l, 3-diol (P4— OH).

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5- (tert- Butyldimethylsilyl)pent-4-yn-2-yl )-7a-methyloctahydro-4H- inden-4-ylidene]ethylidene }-4-methylenecyclohexane-l,3-diol (P2).

In one embodiment in the compound of formula (I) the compound is (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5- (tert- Butyldimethylsilyl)pentan-2-yl ]-7a-methyloctahydro-4H- inden-4-ylidene)ethylidene }-4-methylenecyclohexane-l,3-diol (P1)•

In one embodiment in the ompound of formula (I) the compound is (1R,3S,Z)-5- (2-{(IS,3aS,7aS,E)-7a-Methyl-l-

[(trimethylsilyl)buta-1,3-diyn-l-yl]octahydro-4H-inden-4- ylidene}ethylidene)-4-methylenecyclohexane-l ,3-diol (P6).

The present invention also relates to a pharmaceutical composition comprising the compound of formula (I), its analogues, or salt thereof.

In one embodiment the pharmaceutical composition further comprises an acceptable diluent, carrier, adjuvant and/or excipient.

The present invention also relates to a compound of formula (I), its analogues, or salt thereof, or the pharmaceutical composition for use as a vitamin D receptor activator.

The present invention also relates to a compound of formula (I), its analogues, or salt thereof, or the pharmaceutical composition for use as prophylactic and/or therapeutic agent for vitamin D receptor-related diseases. The present invention also relates to a compound of formula (I), its analogues, or salt thereof, or the pharmaceutical composition for use to increase the weight of animals.

The present invention also relates to a method of producing the compound of formula (I) comprising the steps of:

- Adding a solution of n-butyl lithium or alkyl lithium in an organic solvent with a concentration between 0.5 M and 3.0 M to a solution of phosphine (III) oxide with a concentration between 0.1 M and 1.0 M in an organic solvent that is cooled to a temperature between -40 and -80 °C, and in an inert atmosphere;

- Stirring the mixture for a time between 0.5 h and 4 h;

- Adding a silylated CD bicyclic ketone in a concentration between 0.01 M and 1.0 M in an organic solvent to the mixture of the previous step;

- Colling the mixture to a temperature between -40 and - 80 °C;

- Stirring the mixture for a time between 0.5 h and 4 h; and then allowed to reach a temperature between 20 and 30 °C;

- Adding aqueous NH₄CI in a concentration between 7.0 M and 12 M;

- Extraction with an organic solvent and evaporation of the solvent to obtain the product as a residue;

- Selective deprotection of the hydroxyl groups at C-l and C-3 of the product obtained in the previous step achieved by a desilylation reaction by:

- adding aqueous hydrofluoric acid (48%) to the solution of the residue with a concentration between 0.02 M and 0.08 M in an organic solvent and stirring at a temperature between 20 and 30°C for a time between 1 and 24 hours; or - adding a solution of tetra-n-butylammonium fluoride with a concentration between 0.5 M and 2.0 M in an organic solvent to the dissolved residue with a concentration between 0.02 M and 1.0 M in an organic solvent and allowed to stir at a temperature between 20 and 30°C for a time between 12 and 72 h;

- adding aqueous NaHCO₃ in a concentration between 0.9 M and 1.1 M followed by extraction with an organic solvent and evaporation of solvent.

The invention also relates to a method further comprising the following steps to produce (1R,3S,Z)-5- (2-

{ (lR,3aS,7aR,E)-l-[(R)-5- (Hydroxydimethylsilyl)pent-4-yn-2- yl]-7a-methyloctahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol :

- adding catalytic amounts between 0.01 and 20 moll of dichloro (p-cymene)ruthenium (II) dimer or Rd (OH)2/C or Pd/C to a solution of hydrosilane with a concentration between 0.01 M and 1.0 M in an organic solvent and stirring at a temperature between 20 and 30°C for a time between 5 and 120 minutes;

- Filtering the reaction mixture to remove the catalyst;

- Addition of aqueous HCl in a concentration between 2.0 M and 5.0 M followed by extraction with an organic solvent and evaporation of the solvent.

General description

The present application is directed to compounds of pharmaceutical interest. In particular, it is focused on 1,25D derivatives, the procedures for obtaining them, their intermediates, and their pharmacological and biological applications.

Considering the significance of silicon incorporation as a convenient strategy for enhancing the biological performance of bioactive compounds, the present invention aims at the development of new vitamin D analogues based on the C-to-Si replacement in the side chain, along with several groups at Si atom, such as alkyl, hydrogen, or hydroxyl.

Brief description of drawings

For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

Figure 1. Shows the compound of Formula (I).

Figure 2. Representative structures of sila-vitamin D derivatives.

Figure 3. The general method for the preparation of compounds of formula (I).

Figure 4. Examples for obtaining of compounds represented by general formula (I).

Figure 5. Examples for obtaining of representative diyne compounds of formula (I). Figure 6. Cell differentiation assay in HaCaT cells (human keratinocyte cell line). HaCaT cells were seeded in 60 well plates. After 24 h were treated with ethanol (controls) and with 1,25D, and the sila-derivatives (P1, P2, P3, P4, P4- OH, P5, and P6) at 10^-7M for 48 h. Differentiation of HaCaT cells was evaluated by phase-contrast micrographs.

Figure 7. Proliferation assay in HaCaT cells (normal human keratinocyte cell line). Cells were seeded in 24-well plates and 24 h after were treated with ethanol (controls) and 1,25D and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10^-7 M for 48 h, and then incubated with the MTT reagent for 1 h. Absorbance was determined at 570 nm. The values were plotted as mean ± SD. Proliferation was considered as 100% in cells treated with vehicle (ethanol, control cells).

Figure 8. Proliferation assay in the human breast adenocarcinoma MCF-7 cell line. MCF-7 cells were seeded in 24-well plates and 24 h after were treated with ethanol (controls) and 1,25D and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10^-7 M for 48 h, and then incubated with the MTT reagent for 1 h. Absorbance was determined at 570 nm. The values were plotted as mean ± SD. Proliferation was considered as 100% in cells treated with vehicle (ethanol, control cells).

Figure 9. Proliferation assay in the human ovary adenocarcinoma SKOV-3 cells. Cells were seeded in 24-well plates and 24 h after were treated with ethanol (controls) and 1,25D and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10^-7 M for 48 h, and then incubated with the MTT reagent for 1 h. Absorbance was determined at 570 nm. The values were plotted as mean ± SD. Proliferation was considered as 100% in cells treated with vehicle (ethanol, control cells).

Figure 10. Proliferation assay in the human prostate adenocarcinoma PC-3 cell line. Cells were seeded in 24-well plates and 24 h after were treated with ethanol (controls) and 1,25D and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10^-7 M for 48 h, and then incubated with the MTT reagent for 1 h. Absorbance was determined at 570 nm. The values were plotted as mean ± SD. Proliferation was considered as 100% in cells treated with vehicle (ethanol, control cells).

Figure 11. Regulation of 1,25D target genes in HaCaT cells. Cells were seeded in 24-well plates, and 24 h later were treated either with ethanol (controls), 1,25D, and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10- ⁷ M for 48 h. CYP24A1 mRNA levels (24-hydroxylase, a target gene of 1,25D) were quantified by a real time-PCR.

Figure 12. VDR binding assay. The 1,25D and the sila- derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) (from IO^-11 to 10^-6 M) were incubated with VDR in a competitive binding assay. The IC50 value (calculated from values of the dose- response curves, corresponding to 50% of the inhibition of each compound) is shown.

Figure 13. Calcium levels in vivo. CD-I mice (6-8 weeks old, n=5 per group) were injected intraperitoneally (ip) with 0.5 μg/kg weight every other day for 21 days with 1,25D and the sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6), and with sesame oil (controls). Calcium levels were determined by QuantiChomCalciumAssay Kit. Values are expressed in mg/dl.

Figure 14. Body weight of mice treated as described in Figure 10. Values are expressed as grams.

Figure 15. Reaction scheme to obtain compound 2a.

Figure 16. Reaction scheme to obtain compound 3a.

Figure 17. Reaction scheme to obtain compound 4a.

Figure 18. Reaction scheme to obtain compound 5a.

Figure 19. Reaction scheme to obtain compound 6a.

Figure 20. Reaction scheme to obtain compound P3.

Figure 21. Reaction scheme to obtain compound P4.

Figure 22. Reaction scheme to obtain compound 8.

Figure 23. Reaction scheme to obtain compound P5.

Figure 24. Reaction scheme to obtain compound P4-OH.

Figure 25. Reaction scheme to obtain compound 2b.

Figure 26. Reaction scheme to obtain compound 3b.

Figure 27. Reaction scheme to obtain compound 4b.

Figure 28. Reaction scheme to obtain compound P2. Figure 29. Reaction scheme to obtain compound 5b.

Figure 30. Reaction scheme to obtain compound 6b.

Figure 31. Reaction scheme to obtain compound P1.

Figure 32. Reaction scheme to obtain compound P6.

Figure 33. Reaction scheme to obtain compound 10.

Figure 34. Reaction scheme to obtain compound 11.

Description of embodiments

Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

The present application is directed to compounds of pharmaceutical interest. In particular, it is focused on 1,25D derivatives, the procedures for obtaining them, their intermediates, and their pharmacological and biological applications. Bioactive sila-derivatives of vitamin D were never described in the literature.

The present invention is related to compounds of formula (I) represented in Figure 1, with a silicon atom at the side chain as well as several other substitutions in this atom.

R¹ and R² and R³ each independently represent a hydrogen atom or hydroxyl group or a branched or non-branched alkyl group having 1 or 3 carbon atoms, or a F, Cl, Br, I or a 0- (C₁-C₃)alkoxyalkyl group or a (C₁-C₃)alkyl-OH group;

A is a methylene group or hydrogen atom;

Z represents a straight or branched, substituted or unsubstituted (C₁-C₃)alkyl or 0- (C₁-C₃)alkoxy or a 0- (C₁-C₃)0H group;

R⁴ and R⁵, which may be the same or different, each is independently selected from hydrogen, (C₁-C₁₂)alkyl, (C₆- C₁₀)aryl, and -SiR^aR^bR^c, wherein each R^a, R^b, and R^c is independently selected from (C₁-C₁₂)alkyl, (C₆-C₁₀)aryl, (C₁- C₁₂)alkoxyl, (C₆-C₁₀)aryloxyl and (C₃-C₁₅)heterocycle. Preferably R⁴ and R⁵ each represent a hydrogen atom, a methoxymethyl, a methoxyethyl, a trimethylsilylethoxymethyl, a trimethylsilyl, a triethylsilyl, a tert-butyldimethylsilyl, a tri-iso- propylsilyl, a tert-butyldiphenylsilyl , a dimethylphenylsilyl, a dimethylbenzylsilyl, a benzoate, a p-nitrobenzoate, a pyvalate, and an acetate.

These novel vitamin D analogues show interesting biological profiles with diminished or non-calcemic side effects that can find application as a treatment for several VDR-related pathologies .

Compounds of formula (I) have a silicon atom attached to a C-position of different side chains attached to the CD bicyclic moiety. These analogues display: a) similar cell differentiation in comparison with 1,25D, b) slower proliferation rates of tumoral cells, c) the ability to activate target genes of 1,25D, and d) high affinity for the VDR. Furthermore, one of the analogues induces a significant increase in the body weight of the animals (mice), which may suggest an increase in bone density.

These unprecedented analogues may provide the Pharmaceutical Industry and society with a new generation of more efficient drugs in terms of antitumor action and for the treatment of other diseases related to VDR receptors (psoriasis, Alzheimer's, osteoporosis, diabetes, obesity, hypertension, low immunity, rickets) with low or negligible toxic calcemic side effects.

The present application also relates to a pharmaceutical composition comprising the compound of formula (I), its analogue, or salt thereof. The pharmaceutical composition further comprises an acceptable diluent, carrier, adjuvant and/or excipient.

The compound of formula (I) or the pharmaceutical composition comprising the compound of formula (I) are used as a vitamin D receptor activator.

The compound of formula (I) or the pharmaceutical composition comprising the compound of formula (I) are used as prophylactic and/or therapeutic agent for vitamin D receptor-related diseases.

The compound of formula (I) or the pharmaceutical composition comprising the compound of formula (I) are used to increase the weight of animals.

Synthesis of compounds

Methods and results

It has been synthesized several 1,25D-silicon derivatives of general formula (I) represented in Figure 2. The present application furnishes a synthetic method for different compounds of the general formula (I) but is not limited to the examples presented in Figure 2.

The compounds represented by formula (I) may be used as drugs or prodrugs. After being administered to a living body, the prodrug is a pharmaceutically active compound by the action of an enzyme, metabolic hydrolysis, and other routes.

The preparation of compounds with formula (I) was achieved by using a general method that includes the Wittig-Horner type reaction between a silylated CD bicyclic ketone of Windaus-Grundmann type (II) with the A-ring allylic phosphine oxide (III) followed by selective deprotection of the hydroxyl groups at C-l and C-3 positions (Figure 3).

The method to produce the compound of formula (I) comprises the following steps:

- Adding a solution of n-butyl lithium or alkyl lithium in an organic solvent (for example selected from: hexane, heptane, tetrahydrofuran (THF), tert-butyl methyl ether (TBME), Et2O) with a concentration between 0.5 M and 3.0 M to a solution of phosphine (III) oxide with a concentration between 0.1 M and 1.0

M in an organic solvent (for example selected from THF, hexane, heptane, TBME, Et2O)that is cooled to a temperature between -40 and -80 °C, and in an inert atmosphere (Ar or N2);

- Stirring the mixture for a time between 0.5 h and 4 h;

- Adding a silylated CD bicyclic ketone (for example, 4a, 4b, 6a, 6b and 9) in a concentration between 0.01 M and 1.0 M in an organic solvent (for example selected from: THF, hexane, heptane, TBME, Et2O) to the mixture of the previous step;

- Colling the mixture to a temperature between -40 and - 80 °C;

- Stirring the mixture for a time between 0.5 h and 4 h; and then allowed to reach a temperature between 20 °C and 30 °C;

- Adding aqueous NH4CI in a concentration between 7.0 M and 12 M;

- Extraction with an organic solvent (for example, Et2O, TBME, EtOAc, Hexanes) and evaporation of the solvent to obtain the product as a residue; - Selective deprotection of the hydroxyl groups at C-l and C-3 of the product obtained in the previous step achieved by a desilylation reaction by:

- adding aqueous hydrofluoric acid (HF) (48%) to the solution of the residue with a concentration between 0.02 M and 0.08 M in an organic solvent (for example selected from: CH2CI2, CHCI3, 1,2- dichloroethane, acetonitrile, acetonitrile (MeCN)) and stirring at a temperature between 20 and 30°C for a time between 1 and 24 hours; or

- adding a solution of tetra-n-butylammonium fluoride (TBAF) with a concentration between 0.5 M and 2.0 M in an organic solvent (for example selected from: THF, TBME, Et₂O, MeCN, DMSO) to the dissolved residue with a concentration between 0.02 M and 1.0 M in an organic solvent (for example selected from: THF, hexane, heptane, TBME, Et2O) and allowed to stir at a temperature between 20 and 30°C for a time between 12 and 72 h;

- adding aqueous NaHCO₃ in a concentration between 0.9 M and 1.1 M followed by extraction with an organic solvent (for example selected from: Et2O, TBME, EtOAc, Hexanes, CH2CI2, CHCI3, 1,2-dichloroethane) and evaporation of solvents.

The product is purified by chromatography and obtained in high yields (60-80%).

More specifically, silanol (P4-OH) is prepared from corresponding Hydrosilane (P5) by oxidation adding catalytic amounts (between 0.01 and 20 mol%)of dichloro (p- cymene)ruthenium (II) dimer or Pd(OH)2/C or Pd/C to a solution of hydrosilane with a concentration between 0.01 M and 1.0 M in an organic solvent (for example selected from: water, MeCN, THF, TBME, Et2O, DMSO) and stirring at a temperature between 20 and 30°C for a time between 5 and 120 minutes. The reaction mixture was filtered to remove the catalyst. Addition of aqueous HCl in a concentration between 2.0 M and 5.0 M followed by extraction with an organic solvent (for example selected from: EtOAc, Et2O, TBME, Hexanes, CH₂CI₂, CHCI₃, 1,2-dichloroethane), evaporation of the solvent. Chromatographic purification provides the silanol in high yields (80-87%).

All compounds of formula (I) and some precursors were obtained using the same methodology. All of them include silicon atoms attached to the terminus of the side chain carbon. The alkyne-side chain moiety intermediates were used to prepare the saturated derivatives.

Methods for producing representative compounds encompassed by general formula (I) are described in detail by the following examples, but the invention is not limited to the given examples.

The method for the preparation of compounds of formula (I) with the natural side chain of the hormone or with an alkyne moiety between the C₂₃ and C₂₄ atoms is described in Figure 4 starting from compound 1 as per K. Nagasawa et al. Molecules 2003, 8, 6, 488-499. Examples are P1 to P6.

The method for the preparation of compounds of formula (I) possessing a diyne side chain moiety is described in Figure 5. The synthesis of these compounds (exemplified for compounds P6 and P7) starts from ketone 9, which was synthesized following known procedures, (R. Sigueiro et al.

Org. Lett. 2018, 20, 9, 2641-2644}.

The compounds of the present invention are novel lα,25(OH)2D3 derivatives displaying high biological activity in comparison with the natural hormone la,25 (OH)2D3. They are useful as pharmaceuticals displaying the inhibition of the cellular proliferation in a panel of different cell lines (HaCat, MCF-7, SKOV-3, and PC-3 cell lines) with similar values as the natural hormone la,25 (OH)2D3. All compounds activate the expression of the CYP24A enzyme, with higher values for P2 and P3, while compounds P1 to P6 show lower calcemic side effects than the parent hormone, except for P4-OH which have a similar calcemic effect to the natural hormone. P1 and P2 analogues display no calcemic effects.

Biological activity Methods and results Cell culture

The cell lines of human breast (MCF-7), prostate (PC3), and ovarian (SKOV-3) adenocarcinoma were obtained from the European Collection of Cell Cultures. The normal human keratinocyte cell line (HaCaT) keratinocyte was obtained from Dr. Miguel Quintanilla (Department of Cancer Biology, TIB Alberto Sols, CSIC, Madrid). The cells were cultured in 90-mm plates in essential culture medium DMEM, supplemented with 10% bovine fetal serum free of steroid hormones, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM non- essential amino acids (L-glutamine) at 37°C in a humid atmosphere O2/CO2 (95/5%).

Compounds 1,25D and sila-derivatives (P1, P2, P3, P4, P4-0H, P5, and P6) were resuspended in absolute ethanol at a concentration of 10^-4 M, and from this solution, the concentrations used for each experiment were obtained by dilution of the stock solution (from 10^-5 to 10^-11M).

Cell differentiation

Cell differentiation assay was carried out in HaCaT cells. HaCaT cells were seeded in 60 well plates and treated with 1,25D and sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) at 10^-7 M for 48 h. Differentiation of HaCaT cells was confirmed by phase-contrast micrographs. The results obtained indicated that cells treated with the sila- derivatives exhibit a similar differentiation phenotype as the ones treated with 1,25D (Figure 6).

Inhibition of cell proliferation

Cell proliferation/toxicity in cell lines was assessed by the MTT assay, an indirect method for determining living cells, wherein the yellow water-soluble MTT is reduced in mitochondria, thus generating a purple insoluble product (formazan). After seeding 5xl0⁴ cells per well in 24-well plates for 24 h, sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) and 1,25D were administered at a concentration of 10^-7 M and incubated for 48 h. MTT was added (500 mg/ml) for 1 h. The culture medium was removed and 500 pL of DMSO was added to each well to solubilize the formazan incorporated by the cells. Finally, the absorbance was measured in each well in an automatic plate reader at a wavelength of 590 nm.

The results obtained indicated that cell proliferation in different cell lines after treatment either with 10^-7 M 1,25D or sila-derivatives analogues is significantly lower in all cell lines analyzed with respect to the control (untreated) group (100% in control cells) (Figures 7-11; Table 1).

Specifically, proliferation of the HaCaT cell line after treatment with 1,25D at 10^-7 M was 85% (100% in control cells), and with 10^-7 M of sila-derivatives: P1: 80%; P2: 65%; P3: 72%; P4: 82%; P4-OH: 72%, P5: 72%; and P6: 76% (Figure 7; Table 1). Proliferation of the MCF-7 cells after treatment with 1,25D at 10^-7 M was 80% (100% in control cells), and with 10^-7 M of sila-derivatives P1: 71%; P2: 68%; P3: 67%; P4: 76%; P4-OH: 66%, P5: 71%; and P6: 69% (Figure 8; Table 1). Proliferation of the SKOV-3 cells after treatment with 1,25D at 10^-7 M was 88% (100% in control cells), and with 10^-7 M of sila-derivatives P1: 82%; P2: 81%; P3: 78%; P4: 84%; P4-OH: 72%, P5: 78%; and P6: 78% (Figure 9; Table 1). Proliferation of the PC-3 cells after treatment with 1,25D at 10^-7 M was 84% (100% in control cells), and with 10^-7 M of sila-derivatives P1: 59%; P2: 62%; P3: 60%; P4: 77%; P4-OH: 67%, P5: 64%; and P6: 65% (Figure 10; Table 1).

Table 1. Cellular proliferation (MTT) analysis.

Transactivation assays

To assess the effect of the administration of 1,25D and sila-derivatives on 1,25D target genes, the vitamin D 24- hydroxylase (CYP24A1) mRNA levels were quantified using a real-time PCR after treatment of HaCaT cells with 1,25D and P1, P2, P3, P4, P4-OH, P5, and P6 at 10^-7 M for 48 h. The results indicated that the compounds P2 and P3 are those with greater ability to activate CYP24A1 (398 and 309-fold, respectively) relative to the natural hormone 1,25D (335-fold, compared to control). The other compounds have the following transactivation capacity values (relative to control): P1: 136, P4: 110, P4-OH: 166, P5: 214, and P6: 240 (Figure 11; Table 2). Table 2. CYP24A1 mRNA regulation by 1,25D and sila- derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) in HaCaT cells.

The binding affinity of 1,25D and sila-derivatives (P1, P2, P3, P4, P4-0H, P5, and P6) to the VDR

The affinity of the sila-derivatives for the vitamin D receptor was determined by competitive displacement assay (Polarscreen Vitamin D receptor competitor assay, Red, Invitrogen). Polarized fluorescence was determined in 384- well black plates for 200 ms/well using a Mithras LB 940 device. The sila-derivatives and 1,25D (as control) were assessed at a range of concentrations from IO^-11 to 10^-6 M, and the half maximal inhibitory concentration (IC₅₀) for 1,25D and each sila-derivatives (P1, P2, P3, P4, P4-OH, P5, and P6) was calculated. The activity of each sila- derivatives was also expressed as a percentage relative to the activity of the natural hormone (1,25D), which was normalized at 100%. The results indicated that the sila- derivatives P1 and P6 have the higher affinity to VDR

(IC₅₀: 3.96 x 10^-9 M: 70%; and 4.14 x 10^-9 M: 67% respectively), with respect to the 1,25D hormone (IC₅₀:

2.78 x 10^-9 M: 100% binding). The other compounds have the following affinity percentage: P2: 46%, P3: 31%; P4: 28%;

P4-OH: 44%, and P5: 55% (Figure 12; Table 3).

Table 3. VDR binding assays of 1,25D and the sila- derivatives .

Calcemic effects and body weight in mice

Calcium mobilization was determined in 45 male CD-1 mice (6-8 weeks old). Nine groups of 5 mice each were used. Mice were intraperitoneally (ip) injected with 1,25D or sila- derivatives at a dose of 0.5 μg/kg body weight dissolved in sesame oil (vehicle) every other day for 21 days. The control group was treated only with vehicle (sesame oil). Calcium levels in blood were determined by QuantiChomCalciumAssay Kit (BioAssaySystems, Hayward, CA, USA). The results indicated that treatment with 1,25D induced a significant increase in calcium plasma levels (13.7 ± 2.0 mg/dl, 143%) compared to controls (9.6 ± 1.7 mg/dl, 100%) (Figure 13 and Table 4). The calcium levels in mice treated with sila-derivatives (expressed in percentage with respect to control mice, 100%) were: P1: 106%; P2: 104%; P3: 121%; P4: 113%; P4-OH: 140%; P5: 122%, and P6:

112% (Figure 13); Table 4). Table 4. Calcium levels.

Body weight

Body weight of 1,25D and sila-derivatives-treated animals and controls was also quantified. Treatment with 1,25D for 21 days produced a significant decrease in the body weight of mice (34.3 ± 5.4 g) as compared with the control group (40.1 ± 3.9 g). The sila-derivatives P1, P2, P3, P4, P5, and P6 treatment does not induce significant body weight changes with respect to controls. However, the group of mice treated with P4-OH significantly increased their body weight (49.2 ± 4.7) with respect to both the control group and the group treated with 1,25D (Figure 14; Table 5).

Table 5. Body weight in controls and 1,25D- and sila- derivatives-treated mice.

Statistical analysis

Each experiment was performed at least three times. Values are expressed as meanlSD. Means were compared using 2- tailed Student's t-test or 1-way ANOVA, with the Tukey- Kramer multiple comparison test for post-hoc comparisons.

Synthesis of compounds Butyldimethyl {[(1R,3aR,4S,7aR)-7a-methyl-1-[(R)-5- (trimethylsilyl)pent-4-yn-2-yl]octahydro-lH-inden-4- yl]oxy}silane (2a) (Figure 15)

A solution of n-butyl lithium in hexanes (2.5 M, 3.65 mL, 9.12 mmol, 4.0 equiv.) was added dropwise to a 0 °C cooled solution of TMSC=CH (1.4 mL, 9.8 mmol, 4.3 equiv) in dioxane (10 mL). The mixture was stirred for 1 h. Then a solution of 1 (1.1 g, 2.28 mmol, 1 equiv) in dioxane (7 mL) was added. The mixture was heated at 100 °C under stirring for 48 h. The reaction mixture was allowed to reach room temperature. The reaction was quenched by the addition of drops of water. The mixture was extracted with TBME (3 x 20 mL). The combined organic layers were dried (anhydrous Na2SO₄), filtered, and concentrated in vacuo. The residue was flash chromatographed (SiO₂ 1 x 8 cm, hexanes) to give 2a [740 mg, 80%, Rf = 0.58 (hexanes), colorless oil, [«]D + 122.4 (cl.48, CHCl₃)].

¹H NMR (400 MHz, CDCI₃) 5 4.00 (d, J = 2.5 Hz, 1H, H-8), 2.28 (dd, J = 16.8, 3.5 Hz, 1H), 2.02 - 1.92 (m, 2H), 1.85 - 1.75 (m, 2H), 1.68 (dd, J = 14.5, 3.1 Hz, 1H), 1.61 - 1.52 (m, 2H), 1.41 - 1.33 (m, 3H), 1.28 - 1.22 (m, 2H), 1.18 - 1.10 (m, 2H), 1.05 (d, J = 6.6 Hz, 3H, Me-21), 0.92 (s, 3H, Me-18), 0.89 (s, 9H, tBu-Si), 0.15 (s, 9H, 3xMe- Si), 0.01 (s, 3H, Me-Si), -0.00 (s, 3H, Me-Si).

¹³C NMR (101 MHz, CDCI3) 5 106.9 (C, C-24), 85.5 (C, C-23),

69.6 (CH, C-8), 56.2 (CH), 53.2 (CH), 42.3 (C, C-13), 40.7 (CH₂), 35.4 (CH), 34.6 (CH₂), 27.3 (CH₂), 27.1 (CH₂), 26.0 (CH₃, tBu-Si), 23.2 (CH₂), 19.0 (CH₃, Me-21), 18.2 (C, C- Si), 17.8 (CH2), 14.0 (CH₂, Me-18), 0.4 (CH₃, 3xMe-Si), -

4.6 (CH₃,Me-Si), -5.0 (CH₃,Me-Si).

(1R,3aR,4S,7aR)-7a-methyl-l- ((R)-5- (trimethylsilyl)pent-4- yn-2-yl)octahydro-lH-inden-4-ol (3a) (Figure 16)

HF (48% in water, 5 drops) was added to a solution of 2a (105 mg, 0.26 mmol, 1 equiv) in CH₂Cl₂/MeCN (2/1, 5 mL). The mixture was stirred at room temperature for 4 h and added to a saturated solution of NaHCO₃ (20 mL). The mixture was stirred for 30 min and then extracted with CH₂Cl₂ (4 x 10 mL). The combined organic layers were dried (anhydrous Na₂SO₄), filtered, and concentrated in vacuo. The residue was flash chromatographed (SiO₂ 1 x 8 cm, 10% EtOAc/hexanes) to give 3a [77 mg, 89%, Rf = 0.24 (20% EtOAc/hexanes), colorless oil, [«]D +126.0 (c1.0, CHCI3)].

¹H NMR (400 MHz, CDCI3) 5 4.08 (dd, J = 5.4, 2.6 Hz, 1H, H- 8), 2.29 (dd, J = 16.8, 3.5 Hz, 1H), 2.04 - 1.96 (m, 2H), 1.83 (tdt, J = 11.3, 9.2, 5.6 Hz, 3H), 1.59 - 1.44 (m, 6H), 1.37 - 1.27 (m, 2H), 1.21 - 1.13 (m, 2H), 1.06 (d, J = 6.6 Hz, 3H, Me-21), 0.94 (s, 3H, Me-18), 0.15 (s, 9H, 3xMe-Si).

¹³C NMR (101 MHz, CDCl₃) 5 106.7 (C, C-23), 85.7 (C, C-24), 69.5 (CH, C-8), 55.9 (CH), 52.7 (CH), 42.0 (C, C-13), 40.4 (CH₂), 35.4 (CH), 33.7 (CH₂), 27.1 (CH₂), 27.0 (CH₂), 22.7 (CH₂), 19.0 (CH₃, Me-21), 17.5 (CH₂), 13.8 (CH₃, Me-18), 0.4 (CH₃, 3xMe-Si).

(1R,3aR,7aR)-7a-Methyl-l- [(R)-5- (trimethylsilyl)pent-4-yn- 2-yl]octahydro-4H-inden-4-one (4a) (Figure 17)

Dess-Martin periodinane (DMP, 107 mg, 0.25 mmol, 1.1 equiv) was added to a solution of 3a (77 mg, 0.23 mmol, 1 equiv) in CH₂Cl₂. The mixture was stirred at room temperature for 30 min. The reaction mixture was filtered through pad of celite. The residue after concentration was flash chromatographed (SiO₂, 1 x 6 cm, 5% EtOAc/hexanes) to give 4a [63 mg, 95%, Rf = 0.45 (10% EtOAc/hexanes), yellow oil, [α]_D +48.8 (cl.03, CHCl₃)].

¹H NMR (400 MHz, CDCl₃) 5 2.46 (dd, J = 11.6, 7.5 Hz, 1H), 2.33 - 2.20 (m, 3H), 2.06 (dddd, J = 12.9, 9.3, 4.3, 2.3 Hz, 3H), 1.95 - 1.85 (m, 2H), 1.77 - 1.70 (m, 1H), 1.63 - 1.50 (m, 4H), 1.30 (ddd, J = 7.3, 6.0, 2.2 Hz, 1H), 1.10 (d, J = 6.2 Hz, 3H, Me-21), 0.64 (s, 3H, Me-18), 0.15 (s, 9H, 3xMe-Si).

¹³C NMR (101 MHz, CDCl₃) 5 211.9 (C, C-8), 106.0 (C, C-23), 86.1 (C, C-24), 62.0 (CH), 55.8 (CH), 49.9 (C, C-13), 41.0 (CH₂), 39.0 (CH₂), 35.4 (CH), 27.5 (CH₂), 27.0 (CH₂), 24.1 (CH₂), 19.2 (CH₂), 19.1 (CH₃, Me-21), 12.7 (CH₃, Me-18), 0.3

(CH₃, 3xMe-Si). (1R,3aR,AS,7aR)-7a-Methyl-l-[(R)-5- (trimethylsilyl)pentan- 2-yl]octahydro-lH-inden-4-ol (5a) (Figure 18)

Pd/C (10%, 2 mg) was added to a solution of 3a (20 mg, 0.068 mmol, 1 equiv) in EtOAc (5 mL). The mixture was stirred under an H2 atmosphere (balloon pressure) at room temperature for 16h. The reaction mixture was filtered through a pad of celite. The residue afer concentration was flash chromatographed (SiO₂, 1x6 cm, 5% EtOAc/hexanes) to give 5a [19 mg, 0.06 mmol, 93%, Rf = 0.25 (20%

EtOAc/hexanes), colorless oil], [«]D +119.5 (cl.0, CHCI3).

¹H NMR (400 MHz, CDCI3) 5 4.07 (dd, J = 5.4, 2.6 Hz, 1H, H- 8), 2.00 (dd, J = 10.1, 3.0 Hz, 1H), 1.85 - 1.78 (m, 3H), 1.52 - 1.40 (m, 5H), 1.29 - 1.02 (m, 8H), 0.93 (s, 3H, Me- 18), 0.88 (d, J = 6.6 Hz, 3H, Me-21), 0.51 - 0.34 (m, 2H, H-24), -0.03 (s, 9H, 3xMe-Si).

¹³C NMR (101 MHz, CDCI3) 5 213.7 (C, C-8), 63.6 (CH), 58.4 (CH), 51.5 (C, C-13), 42.6 (CH₂), 41.4 (CH₂), 40.6 (CH₂), 37.0 (CH), 29.1 (CH₂), 25.7 (CH₂), 22.1 (CH₂), 20.7 (CH₂), 20.2 (CH₃, Me-21), 18.6 (CH₂), 14.1 (CH₃, Me-18), 0.00 (CH₂, 3xMe-Si).

(1R,3aR,7aR)-7a-Methyl-l-[(R)-5- (trimethylsilyl)pentan-2- yl]octahydro-4H-inden-4-one (6a) (Figure 19)

DMP (42 mg, 0.10 mmol, 1.1 equiv) was added to a solution of 5a (26 mg, 0.09 mmol, 1 equiv) in CH₂Cl₂ (5 mL). The mixture was stirred at room temperature for 30 min. The reaction mixture was filtered through celite. The residue after concentration was Flash chromatographyed (SiO₂, 1 x 6 cm, 5% EtOAc/hexanes) to give 6a [25 mg, 98%, Rf = 0.25 (10% EtOAc/hexanes), yellow oil, [a]_D +48.8 (cl.03,

CHCI3)].

¹H NMR (400 MHz, CDCl₃) 5 2.43 (dd, J = 11.6, 7.5 Hz, 1H),

2.29 - 2.19 (m, 2H), 2.14 - 2.09 (m, 1H), 2.02 - 1.96 (m,

1H), 1.95 - 1.85 (m, 2H), 1.71 (ddd, J = 11.6, 6.3, 1.6 Hz,

1H), 1.61 - 1.49 (m, 2H), 1.40 - 1.26 (m, 5H), 0.93 (d, J =

6.1 Hz, 3H, Me-21), 0.63 (s, 3H, Me-18), -0.04 (s, 9H,

3xMe-Si) .

¹³C NMR (101 MHz, CDCI3) 5 213.7 (C, C-8), 63.6 (CH), 58.4 (CH), 51.5 (C, C-13), 42.6 (CH₂), 41.4 (CH₂), 40.6 (CH₂), 37.0 (CH), 29.1 (CH₂), 25.7 (CH₂), 22.1 (CH₂), 20.7 (CH₂), 20.2 (CH₃, Me-21), 18.6 (CH₂, C-22), 14.1 (CH₃, Me-18), 0.0 (CH₃, 3xMe-Si).

(1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-7a-Methyl-l-[(R)-5- (trimethylsilyl)pentan-2-yl]octahydro-4A-inden-4 ylidene}ethylidene )-4-methylenecyclohexane-l,3-diol (P3) (Figure 20)

A solution of n-butyl lithium in hexanes (1 M, 0.14 mL, 0.19 mmol, 3 equiv) was added to a -80 °C cooled solution of phosphine oxide 7 (118 mg, 0.2 mmol, 3.1 equiv) in THE (1.5 mL). The mixture was stirred for 1.5 h. Ketone 6a (19 mg, 0.064 mmol, 1 equiv) in THE (1.5 mL) was added via cannula. The mixture was stirred for 1.5 h and then allowed to reach room temperature. A saturated solution of NH4CI (10 mL) was added. The mixture was extracted with EtOAc (3 x 10 mL). The organic layer was dried, filtered, and concentrated in vacuo. A mixture of CH2CI2 and MeCN (1:1, 5 mL) and HF (48% aq, 10 drops) were successively added. The mixture was stirred for 2 h at room temperature and then poured onto a NaHCOs saturated solution. The mixture was stirred for 30 min. The combined organic layers were extracted with CH2CI2 (4 x 10 mL), dried and concentrated in vacuo. The residue was purified by HPLC (Shimadzu preparative liquid chromatograph model LC-8A equipped with a TSP UV-1 absorbance detector, using HPLC Phenomenex Luna column (SiO2, 5 μm, 0 250 mmx21.2 mm, 10% iPrOH/hexanes)) to give P3 [17.5 mg, 63%, white solid, Rf = 0.3 (75%

EtOAc/hexanes), [«]D +31.2 (c 0.87, CHCI3)].

¹H NMR (400 MHz, CDCI3) 5 6.38 (d, J = 11.2 Hz, 1H, =CH), 6.02 (d, J = 11.3 Hz, 1H, =CH), 5.33 (s, 1H, H-19), 5.00 (s, 1H, H-19), 4.43 (dd, J = 7.6, 4.3 Hz, 1H, H-l), 4.25 -

4.20 (m, 1H, H-3), 2.85 - 2.79 (m, 1H), 2.60 (dd, J = 13.4, 3.0 Hz, 1H), 2.31 (dd, J = 13.4, 6.5 Hz, 1H), 2.04 - 1.85

(m, 6H), 1.72 - 1.61 (m, 5H), 1.55 - 1.39 (m, 6H), 1.12 -

0.99 (m, 2H), 0.91 (d, J = 6.4 Hz, 3H, Me-21), 0.54 (s, 3H, Me-18), 0.50 - 0.35 (m, 2H, H-24), -0.03 (s, 9H, 3xMe-Si).

¹³C NMR (101 MHz, CDCI3) 5 147.8 (C, C-10), 143.5 (C C-5)

132.9 (C, C-8), 125.2 (CH, C-6) 117.1 (CH C-7) 111.9

(CH₂ C-19), 71.0 (CH C-l), 67.0 (CH C-3) 56.9 (CH)

56.5 (CH) 46.1 (C, C-13) 45.4 (CH₂) 43.0 (CH₂) 40.7

(CH₂) 40.2 (CH₂), 36.1 (CH) 29.3 (CH₂) 27.8 (CH₂) 23.8

(CH₂) 22.4 (CH₂), 20.7 (CH₂) 18.9 (CH₃, Me-21), 17.2 (CH₂

C-24) 12.2 (CH₂, Me-18), -1.41 (CH₃, 3xMe-Si).

(1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-7a-Methyl-l-[(R)-5-

(trimethylsilyl)pent-4-yn-2-yl]octahydro-4H-inden-4- ylidene}ethylidene )-4-methylenecyclohexane-l,3-diol_ (P4)

(Figure 21)

A solution of n-butyl lithium in hexanes (IM, 0.165 mL, 0.23 mmol, 2.9 equiv) was added to a -80 °C cooled solution of phosphine oxide 7 (140 mg, 0.24 mmol, 3 equiv) in THE (1.5 mL). The mixture was stirred for 1.5 h. Ketone 4a (23 mg, 0.08 mmol, 1 equiv) in THF (1.5 mL) was added via cannula. The mixture was stirred for 1.5 h. The resulting mixture allowed to reach room temperature and treated with a saturated solution of NH4CI (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. A mixture of CH2CI2 and MeCN (1:1, 5 mL) and aq HF (48%, 10 drops) were successively added. The mixture was stirred at room temperature for 2 h and the poured onto sat NaHCOs (10 mL). The mixture was stirred for 30 min and then extracted with CH2CI2 (4 x 10 mL). The combined organic extracts were dried, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, 1 x 7 cm, 70% EtOAc/hexanes) to give P4 [22 mg, 67% (2 steps), Rf = 0.3 (75% EtOAc/hexanes), white solid, [«]D +48.8 (cl.03,

CHCI3)].

1H NMR (400 MHz, CDCI3) 5 6.35 (d, J = 11.2 Hz, 1H, =CH), 6.00 (d, J = 11.3 Hz, 1H, =CH), 5.31 (s, 1H, H-19), 4.98 (s, 1H, H-19), 4.40 (dd, J = 7.6, 4.3 Hz, 1H, H-l), 4.23 - 4.17 (m, 1H, H-3), 2.81 (dd, J = 12.1, 3.9 Hz, 1H), 2.57 (dd, J = 13.4, 3.2 Hz, 1H), 2.32 - 2.26 (m, 2H), 2.09 - 1.96 (m, 6H), 1.89 (m, 2H), 1.68 (m, 2H), 1.59 - 1.45 (m, 4H), 1.37 - 1.23 (m, 3H), 1.07 (d, J = 6.5 Hz, 3H, Me-21), 0.54 (s, 3H, Me-18), 0.14 (s, 9H, 3xMe-Si).

13C NMR (101 MHz, CDCI3) 5 147.7 (C, =C), 142.9 (C, =C), 133.3 (C, =C), 124.9 (CH), 117.3 (CH), 112.0 (CH₂, C-19), 106.7 (C, =C), 85.7 (C, =C), 70.9 (CH, C-l), 66.9 (CH, C-

3), 56.4 (CH), 55.8 (CH), 45.9 (C, C-13), 45.3 (CH₂), 42.9 (CH₂), 40.4 (CH₂), 36.1 (CH), 29.1 (CH₂), 27.6 (CH₂), 27.1 (CH₂), 23.6 (CH₂), 22.4 (CH₂), 19.2 (CH₃, Me-21), 12.2 (CH₃,

Me-18), 0.3 (3XCH₃, Me-Si).

(1R,3S,Z)-5-{2-[(1R,3aS,7aR,E)-7a-Methyl-l- ((R)-pent-4-yn- 2-yl)octahydro-4H-inden-4-ylidene ]ethylidene}-4- methylenecyclohexane-1 ,3-diol (8) (Figure 22)

A solution of n-butyl lithium in hexanes (IM, 0.250 mL, 0.348 mmol, 2.9 equiv) was added to a -80 °C cooled solution of phosphine oxide 7 (212 mg, 0.36 mmol, 3 equiv) in THE (1.5 mL). The mixture was stirred for 1.5 h. Ketone 4a (40 mg, 0.12 mmol, 1 equiv) in THE (1.5 mL) was added via cannula. The mixture was stirred for 1.5 h. The resulting mixture allowed to reach room temperature and treated with a saturated solution of NH4CI (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. The residue was dissolved in dry THE (10 mL) and a solution of TBAF (IM in THE) (0.48 mL, 0.48 mmol, 4 equiv) was added and stirred at room temperature for 16 h. The resulting mixture was treated with an aqueous saturated solution of NH4CI and extracted with EtOAc (3 x 10 mL). The combined organic extracts were dried, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂, 1 x 7 cm, 70% EtOAc/hexanes) to give 8 [36 mg, 86 % (2 steps), Rf = 0.25 (70% EtOAc/hexanes), white foam, [«]D + 25.6 (cl.05, CHCl₃)].

!H NMR (400 MHz, CDCl₃) 5 6.37 (d, J = 11.2 Hz, 1H, =CH), 6.02 (d, J = 11.3 Hz, 1H, =CH), 5.37 - 5.30 (m, 1H, H-19), 5.00 (s, 1H, H-19), 4.43 (dd, J = 7.8, 4.4 Hz, 1H, H-l), 4.26 - 4.19 (m, 1H, H-3), 2.83 (dd, J = 12.1, 4.2 Hz, 1H), 2.60 (dd, J = 13.4, 3.4 Hz, 1H), 2.34 - 2.23 (m, 2H), 2.09 - 1.97 (m, 4H), 1.95 (t, J = 2.6 Hz, 1H, H-24), 1.89 (ddd, J = 13.2, 8.8, 5.7 Hz, 2H), 1.68 - 1.24 (m, 12H), 1.10 (d, J = 6.6 Hz, 3H, Me-21), 0.56 (s, 3H, Me-18).

¹³C NMR (101 MHz, CDCl₃) 5 147.6 (C, C-10), 142.9 (C, C-5), 133.0 (C, C-8), 124.9 (CH, C-7), 117.1 (CH, C-6), 111.8

(CH₂, C-19), 83.3, (C, C-23), 70.8 (CH, C-l), 69.2 (CH, C-

24), 66.8 (CH, C-3), 56.2 (CH), 55.3 (CH), 45.8 (C, C-13),

45.3 (CH₂), 42.8 (CH₂), 40.2 (CH₂), 35.6 (CH), 29.0 (CH₂),

27.4 (CH₂), 25.6 (CH₂), 23.5 (CH₂), 22.2 (CH₂), 19.1 (CH₃,

Me-21), 12.1 (CH₃, Me-18).

(1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5-

(Dimethylsilyl)pent-4-yn-2-yl]-7a-methyl_ octahydro-4H- inden-4-ylidene }ethylidene)-4-methylenecyclohexane-l,3-diol (P5) (Figure 23)

A solution of n-butyl lithium in hexanes (0.195 mL, 0.293 mmol, 4 equiv) was added to a 0 °C cooled solution of diol 8 (26 mg, 0.073 mmol, 1 equiv) in THF (5 mL). The mixture was stirred for 30 min. ClSiHMe₂ (0.045 mL, 0.401 mmol, 5.5 equiv) was added dropwise. The mixture was allowed to reach room temperature for 1 h. The reaction was quenched by the addition of a solution of HCl (10%, 10 mL). The mixture was extracted with EtOAc (3 x 10 mL). The combined organic layers were dried, filtered, and concentrated. After purification by flash chromatography (SiO2, 1 x 7 cm, 70% EtOAc/hexanes) to give P5 [21 mg, 70%, Rf = 0.29 (70% EtOAc/hexanes), white foam], [«]D +37.8 (c0.78, CHCl₃)].

¹H NMR (400 MHz, CDCl₃) 5 6.37 (d, J = 11.2 Hz, 1H, =CH), 6.01 (d, J = 11.3 Hz, 1H, =CH), 5.32 (s, 1H, H-19), 5.00 (s, 1H, H-19), 4.43 (dd, J = 7.7, 4.3 Hz, 1H, H-3), 4.22 (dt, J = 10.2, 3.4 Hz, 1H, H-l), 4.15 - 4.08 (m, 1H, H-Si), 2.83 (dd, J = 12.1, 4.0 Hz, 1H), 2.59 (dd, J = 13.4, 3.3 Hz, 1H), 2.31 (dd, J = 12.4, 5.4 Hz, 2H), 2.06 - 1.97 (m,

4H), 1.90 (ddd, J = 8.8, 6.6, 3.2 Hz, 2H), 1.68 (d, J =

10.3 Hz, 3H), 1.52 - 1.47 (m, 2H), 1.38 (dt, J = 7.7, 2.5

Hz, 2H), 1.28 (ddd, J = 19.3, 11.3, 6.0 Hz, 4H), 1.09 (d, J

= 6.6 Hz, 3H, Me-18), 0.55 (s, 3H, Me-18), 0.22 (d, J =

3.7 Hz, 6H, 2xMe-Si).

¹³C NMR (101 MHz, CDCl₃) 5 147.8 (C, C-10), 143.0 (C, C-5), 133.2 (C, C-8), 125.1 (CH, C-6), 117.3 (CH, C-7), 112.0

(CH₂, C-19), 108.3 (C, C-23), 82.7 (C, C-24), 71.0 (CH, C- 1), 67.0 (CH, C-3), 56.4 (CH), 55.8 (CH), 46.0 (CH₂), 45.4 (CH₂), 43.0 (CH₂), 40.4 (CH₂), 36.1 (CH), 29.2 (CH₂), 27.6 (CH₂), 27.2 (CH₂), 23.7 (CH₂), 22.4 (CH₂), 19.3 (CH₃, Me-21), 12.2, (CH₃, Me-18) -2.5 (CH₃, 2xMe-Si).

(Hydroxydimethylsilyl)pent-4-yn-2-yl]-7a-methyloctahydro- 4H-inden-4-ylidene }ethylidene)-4-methylenecyclohexane-l,3- diol (P4-OH) (Figure 24)

Dichloro (p-cymene)ruthenium (II) dimer (3 mg, cat) was added to a solution of hydrosilane P5 (30 mg, 0.07 mmol, 1 equiv) in acetonitrile (5 mL). The mixture was and stirred at room temperature for 5 min. Water (0.5 mL) was added. The mixture was stirred for 10 min. The reaction mixture was filtered to remove the catalyst. Aq HCl (10%, 10 mL) was added. The aqueous layer was extracted with EtOAc (3 x 10 mL). The combined organic layers were dried (anhydrous Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂ 1 x 8 cm, 70% EtOAc/hexanes) to give P4-OH [25 mg, 0.06 mmol, 82%, Rf = 0.34 (70% EtOAc/hexanes), white foam, [«]D + 30.9 (cl.02, CHCl₃)]. ¹H NMR (400 MHz, CDCI3) 5 6.37 (d, J = 11.2 Hz, 1H, =CH), 6.01 (d, J = 11.3 Hz, 1H, =CH), 5.32 (s, 1H, H-19), 4.99 (s, 1H, H-19), 4.43 (dd, J = 7.7, 4.3 Hz, 1H, H-l), 4.22 (dt, J = 10.2, 3.4 Hz, 1H, H-3), 2.82 (dd, J = 12.2, 4.0 Hz, 1H), 2.59 (dd, J = 13.4, 3.4 Hz, 1H), 2.34 - 2.28 (m, 2H), 2.02 - 1.85 (m, 6H), 1.81 (br s, 3H, 3xOH), 1.70 - 1.35 (m, 9H), 1.09 (d, J = 6.6 Hz, 3H, Me-21), 0.55 (s, 3H, Me-18), 0.28 (s, 6H, 2xMe-Si).

¹³C NMR (101 MHz, CDCI3) 5 147.8 (C, =C), 143.0 (C, =C), 133.2 (C, =C), 125.1 (CH, =C), 117.3 (CH, =C), 112.0 (CH₂, C-19), 106.8 (C, =C), 84.9 (C, =C), 71.0 (CH, C-l), 67.0 (CH, C-3), 56.4 (CH), 55.8 (CH), 46.0 (C, C-13), 45.4

(CH₂), 43.0 (CH₂), 40.4 (CH₂), 36.0 (CH), 29.2 (CH₂), 27.6 (CH₂), 27.0 (CH₂), 23.6 (CH₂), 22.4 (CH₂), 19.3 (CH₃, Me- 21), 12.2 (CH₃, Me-18), 1.9 (CH₃, 2xMe-Si). Butyl ((R)-4-{(1R,3aR,4S,7aR)-4-[(tert- butyldimethylsilyl )oxy]-7a-methyloctahydro-lH-inden-l- yl}pent-l-yn-l-yl )dimethylsilane (2b) (Figure 25)

A solution of n-butyl lithium in hexanes (1.4 M, 0.93 mL, 1.30 mmol, 4.8 equiv) was added dropwise to a 0 °C cooled solution of TBSC=CH (0.252 mL, 1.35 mmol, 5.0 equiv) in toluene (10 mL). The mixtures solution was stirred for 1 h. A solution of 1 (130 mg, 0.027 mmol, 1 equiv) in toluene (5 mL) was added via cannula. The mixture was stirred at 100 °C for 24 h. The reaction mixture was allowed to reach room temperature. The reaction was quenched by the addition of drops of water. The mixture was extracted with hexanes. The combined organic layers were dried (anhydrous Na2SO₄), filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂ 1 x 8 cm, hexanes) to give 2b [105 mg, 0.23 mmol, 87%, Rf 0.64 (hexanes), colorless oil, [«]D + 30.9 (cl.02, CHCI3)].

¹H NMR (400 MHz, CDCI3) 5 4.00 (d, J = 2.5 Hz, 1H, H-8), 2.26 (dd, J = 16.8, 3.5 Hz, 1H), 2.07 (dd, J = 16.8, 7.5 Hz, 1H), 1.94 (dt, J = 5.0, 2.7 Hz, 1H), 1.86 - 1.76 (m, 2H), 1.70 - 1.65 (m, 1H), 1.57 (ddd, J = 11.1, 5.9, 2.8 Hz,

2H), 1.38 (ddd, J = 12.2, 7.4, 4.3 Hz, 3H), 1.28 - 1.18 (m,

4H), 1.06 (d, J = 6.6 Hz, 3H, Me-21), 0.94 (s, 9H, tBu-Si), 0.92 (s, 3H, Me-18), 0.89 (s, 9H, tBuSi), 0.08 (s, 6H,

2xMe-Si), 0.01 (s, 3H, Me-Si), -0.00 (s, 3H, Me-Si).

¹³C NMR (101 MHz, CDCI3) 5 107.2 (C, C-23), 83.6 (C, C-24), 69.5 (CH, C-8), 55.8 (CH), 53.2 (CH), 42.2 (C, C-13), 40.7 (CH₂), 35.3 (CH), 34.6 (CH₂), 27.3 (CH₂), 27.1 (CH₂), 26.3

(CH₃ tBu-Si), 26.0 (CH₃ tBu-Si), 23.2 (CH₂), 19.1 (CH₃, Me- 21), 18.2 (C, tBu-Si) , 17.8 (CH₂), 16.8 (C, tBu-Si), 14.0 (CH₃, Me-18), -4.23 (CH₃, 2xMe-Si), -4.6 (CH₃, Me-Si), -5.0 (CH₃, Me-Si).

(1R,3aR,4S,7aR)-1-[(R)-5- Butyldimethyl silyl)pent-4- yn-2-yl]-7a-methyloctahydro-lH-inden-4-ol (3b) (Figure 26) HF (48% in water, 5 drops) was added to a solution of 2b (110 mg, 0.245 mmol, 1 equiv) in CH₂Cl₂/MeCN (2/1, 5 mL). The mixture was at room temperature for 4h at room temperature. The reaction mixture was poured onto sat NaHCOs and stirred for 30 min. The organic layer was extracted with CH₂Cl₂ (4 x 10 mL). The combined organic layers were dried (anhydrous Na₂SO₄), filtered, and concentrated in vacuo. Purification by flash chromatography (SiO₂ 1 x 6 cm, 15% EtOAc/hexanes) gave 3b [80 mg, 0.24 mmol, 98%, Rf = 0.24 (20% EtOAc/hexanes), colorless oil, [a]_D -64.9 (cO.92, CHCI3)]. ¹H NMR (400 MHz, CDCI3) 5 4.08 (d, J = 2.6 Hz, 1H, H-8), 2.27 (dd, J = 16.8, 3.6 Hz, 1H), 2.07 (dd, J = 15.1, 7.7 Hz, 1H), 2.01 - 1.96 (m, 1H), 1.84 (ddt, J = 14.4, 11.0, 5.2 Hz, 3H), 1.62 - 1.54 (m, 2H), 1.49 - 1.40 (m, 4H), 1.30 - 1.23 (m, 4H), 1.07 (d, J = 6.6 Hz, 3H, Me-21), 0.94 (s, 3H, Me-18), 0.93 (s, 9H, tBu-Si), 0.08 (s, 6H, 2x Me-Si).

¹³C NMR (101 MHz, CDCI3) 5 106.8 (C, C-24), 83.7 (C, C-23), 69.3 (CH, C-8), 55.5 (CH), 52.6 (CH), 41.8 (C, C-13), 40.2 (CH₂), 35.0 (CH), 33.5 (CH₂), 27.0 (CH₂), 26.9 (CH₂), 26.1 (CH₃, tBu-Si), 22.5 (CH₂), 18.9 (CH₃, Me-21), 17.4 (CH₂), 16.6 (CH, tBu-Si), 13.7 (CH₃, Me-18), -4.4 (CH₃, 2xMe-Si).

(1R,3aR,7aR)-1-[(R)-5- Butyldimethyl silyl)pent-4-yn-2- yl]-7a-methyloctahydro-4A-inden-4-one (4b) (Figure 27)

DBM (50 mg, 0.12 mmol, 1.1 equiv) was added to a solution of 3b (36 mg, 0.11 mmol, 1 equiv) in CH2CI2 (5 mL). The mixture was stirred for 30 min at room temperature. The reaction mixture was filtered through pad of celite. The residue after concentration was flash chromatographed (SiO2, 1 x 6 cm, 5% EtOAc-Hexanes) to give 4b [35 mg, 0.11 mmol, 98%, Rf = 0.73 (20% EtOAc-Hexanes), yellow oil, [«]D +25.1 (cl.4, CHCI3)].

¹H NMR (400 MHz, CDCI3) 5 2.44 (dd, J = 11.6, 7.5 Hz, 1H), 2.26 (ddd, J = 11.1, 9.2, 4.2 Hz, 2H), 2.16 - 1.87 (m, 5H),

1.74 (ddd, J = 17.5, 9.4, 5.6 Hz, 1H), 1.64 - 1.50 (m, 4H),

1.35 - 1.23 [(m, 2H, H-20), d, J = 6.1 Hz, 3H, Me-21)],

0.93 (s, 9H, tBu-Si), 0.64 (s, 3H, Me-18), 0.08 (s, 6H,

2xMe-Si). ¹³C NMR (101 MHz, CDCI3) 5 211.9 (C, C-8), 106.2 (C, C-24),

84.2 (C, C-23), 62.1 (CH), 55.5 (CH), 49.9 (C, C-13), 41.0

(CH₂), 39.0 (CH₂), 35.2 (CH), 27.4 (CH₂), 27.1 (CH₂), 26.2 (CH₃, tBu-Si), 24.1 (CH₂), 19.2 (CH₃, Me-21), 19.2 (CH₂, C-

22), 16.7 (C, C-tBu-Si), 12.7 (CH₃, Me-18), -4.28 (CH₃,

2xMe-Si) .

(1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5- (tert- Butyldimethylsilyl )pent-4-yn-2-yl)-7a-methyloctahydro-4H- inden-4-ylidene]ethylidene }-4-methylenecyclohexane-l,3-diol (P2) (Figure 28)

A solution of n-butyl lithium in hexanes (IM, 0.081 mL, 0.13 mmol, 2.0 equiv) was added to a -80 °C cooled solution of phosphine oxide 7 (76 mg, 0.13 mmol, 2.1 equiv) in THF (1.5 mL). The mixture was stirred for 1.5 h. Ketone 4b (22 mg, 0.065 mmol, 1 equiv) in THF (1.5 mL) was added via cannula. The mixture was stirred for 1.5 h and then allowed to reach room temperature. Sat NH4CI (10 mL) was added. The mixture was extracted with EtOAc (3 x 10 mL). The organic layer was dried, filtered, and concentrated in vacuo. A mixture of CH₂Cl₂ and MeCN (1:2, 5 mL) and HF (48% aq, 5 drops) were successively added. The mixture was stirred for 4 h at room temperature and then poured onto a NaHCO₃ saturated solution. The mixture was stirred for 30 min. The combined organic layers were extracted with EtOAc (3 x 10 mL), dried and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂, 1 x 7 cm, 70% EtOAc/Hexanes) to give P2 [19.0 mg, 62% (2 steps), white foam, Rf = 0.3 (70% EtOAc-Hexanes), [«]D +175.6 (c0.85

CHCl₃)].

¹H NMR (400 MHz, CDCl₃) 5 6.37 (d, J = 11.2 Hz, 1H, =CH),

6.01 (d, J 11.2 Hz, 1H, =CH), 5.32 (s, 1H, H-19), 5.00 (s, 1H, H-19), 4.43 (dd, J = 7.5, 4.3 Hz, 1H, H-l), 4.22 (dt, J = 10.1, 3.4 Hz, 1H, H-3), 2.82 (dd, J = 12.0, 3.9 Hz, 1H), 2.60 (dd, J = 13.3, 3.3 Hz, 1H), 2.33 - 2.25 (m, 2H), 2.13 - 2.07 (m, 1H), 2.02 - 1.87 (m, 5H), 1.72 - 1.47 (m, 11H), 1.09 (d, J = 6.5 Hz, 3H, Me-21), 0.94 (s, 9H, tBu-Si), 0.55 (s, 3H, Me-18), 0.08 (s, 6H, 2xMe-Si).

¹³C NMR (101 MHz, CDCl₃) 5 147.8 (C, C-8), 143.1 (C, C-5), 133.1 (C, C-10), 125.1 (CH, C-7), 117.3 (CH, C-6), 112.0 (CH₂, C-19), 106.9 (C, C-23), 83.9 (C, C-24), 71.0 (CH, C- 1), 67.0 (CH, C-3), 56.4 (CH), 55.6 (CH), 45.9 (CH₂), 45.4 (C, C-13), 43.0 (CH₂), 40.5 (CH₂), 36.0 (CH), 29.2 (CH₂), 27.6 (CH₂), 27.2 (CH₂), 26.3 (CH₃, tBu-Si), 23.7 (CH₂), 22.4 (CH₂), 19.3 (CH₃, Me-21), 16.8 (C, C-tBu-Si), 12.3 (CH₃, Me- 18), -4.25 (CH₃, 2xMe-Si).

(1R,3aR,4S,7aR)-1-[(R)-5- butyldimethyl silyl)pentan-2- yl]-7a-methyloctahydro-lH-inden-4-ol (5b) (Figure 29)

A catalytic amount of Pd/C (10%, 2 mg) was added to a solution of 3b (80 mg, 0.24 mmol, 1 equiv) in EtOAc (5 mL). The mixture was stirred under an H₂ atmosphere (balloon pressure) at room temperature for 16 h. The mixture was filtered through a pad of celite. The residue after concentration was purified by flash chromatography (SiO₂, 1 x 6 cm, 5% EtOAc/hexanes) 5b to give [81 mg, 0.24 mmol, 99%, [a]_D +99.9 (cl.00 CHCl3), Rf = 0.48 (15%

EtOAc/hexanes), colorless oil].

¹H NMR (400 MHz, CDCl₃) 5 4.07 (d, J = 2.6 Hz, 1H, H-8), 2.02 - 1.97 (m, 1H), 1.88 - 1.78 (m, 3H), 1.62 (s, 1H), 1.48 - 1.28 (m, 10H), 1.20 - 0.99 (m, 5H), 0.93 (s, 3H, Me- 18), 0.88 (d, J = 6.6 Hz, 3H, Me-21), 0.86 (s, 9H, tBu-Si), -0.08 (s, 6H, 2xMe-Si). ¹³C NMR (101 MHz, CDCI3) 5 69.5 (CH, C-8), 56.8 (CH), 52.6 (CH), 41.9 (C, C-13), 40.4 (CH₂), 40.3 (CH₂), 35.2 (CH₃), 33.6 (CH₂), 27.2 (CH₂), 26.6 (CH₃, tBu-Si), 22.5 (CH₂), 20.9 (CH₂), 18.5 (CH₃, Me-18), 17.4 (CH₂), 16.5 (C, C-tBu-Si), 13.5 (CH₃, Me-21), 12.9 (CH₂, C-24), -6.2 (CH₃, Me-Si), -6.3 (CH₃, Me-Si).

(1R,3aR,7aR)-1- ((R)-5- Butyldimethyl silyl)pentan-2- yl)-7a-methyloctahydro-4H-inden-4-one (6b) (Figure 30) DBM (51 mg, 0.12 mmol, 1.1 equiv) was added to a solution of 5b (37 mg, 0.11 mmol, 1 equiv) in CH₂Cl₂ (5 mL). The mixture was stirred for 30 min at room temperature. The reaction mixture was filtered through pad of celite. The residue after concentration was flash chromatographed (SiO₂, 1 x 6 cm, 5% EtOAc-Hexanes) to give 6b [36 mg, 0.11 mmol, 98%, Rf = 0.60 (15% EtOAc/Hexanes), yellow oil, [«]D +55.1 (c0.9, CHCl₃)].

¹H NMR (400 MHz, CDCl₃) 5 2.44 (dd, J = 11.6, 7.5 Hz, 1H), 2.32 - 2.19 (m, 2H), 2.15 - 2.09 (m, 1H), 2.03 - 1.97 (m, 1H), 1.94 - 1.85 (m, 2H), 1.77 - 1.69 (m, 1H), 1.65 - 1.48

(m, 3H), 0.93 (d, J = 6.0 Hz, 3H, Me-21), 0.86 (s, 9H, tBu-

Si), 0.63 (s, 3H, Me-18), 0.52 - 0.35 (m, 2H, H-24), -0.08

(s, 6H, 2xMe-Si).

¹³C NMR (101 MHz, CDCl₃) 5 212.2 (C, C-8), 62.0 (CH), 56.9 (CH), 50.0 (C, C-13), 41.0 (CH₂), 40.3 (CH₂), 39.0 (CH₂), 35.4 (CH), 27.6 (CH₂), 26.6 (CH₃, tBu-Si), 24.1 (CH₂), 20.9 (CH₂), 19.1 (CH₂), 18.7 (CH₃, Me-21), 16.5 (C, tBu-Si), 12.9 (CH₂, C-24), 12.5 (CH₃, Me-18), -6.2 (CH₂, Me-Si), -6.2

(CH₂, Me-Si). (1R,3S,Z)-5- (2-{(1R,3aS,7aR,E)-1-[(R)-5- (tert- Butyldimethylsilyl )pentan-2-yl]-7a-methyloctahydro-4H- inden-4-ylidene)ethylidene }-4-methylenecyclohexane-l,3-diol (P1) (Figure 31)

A solution of n-butyl lithium in hexanes (84 pL, 0.1 mmol, 2.9 equiv) was added to a -78 °C cooled solution of phosphine oxide 7 (59 mg, 0.1 mmol, 3 equiv) in THF (1.5 mL). The mixture was stirred for 1.5 h. A solution of ketone 6b (12 mg, 0.035 mmol, 1 equiv) in THF (1.5 mL) was added via cannula. The mixture was stirred for 1.5 h and then allowed to reach room temperature. The resulting mixture was treated with a saturated solution of NH4CI (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. A mixture of CH2CI2 and MeCN (5 mL, 1:1) and aq HF (48%, 10 drops) were successively added. The mixture was stirred at room temperature for 2 h and then poured onto sat NaHCOs. The mixture was stirred for 30 min. The combined organic layers were extracted with CH2CI2 (4 x 10 mL), dried, and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, 1x7 cm, 70% EtOAc/hexanes) and HPLC (Shimadzu preparative liquid chromatograph model LC-8A equipped with a TSP UV-1 absorbance detector, using HPLC Phenomenex Luna column (SiO2, 5 pm, 0 250 mmx21.2 mm, 10% iPrOH/hexanes) to give P1 [11 mg, 65% (2 steps), Rf = 0.3 (70% EtOAc/hexanes), white foam, [«]D +175.6 (c0.85 CHCI3)].

!H NMR (400 MHz, CDCI3) 5 6.38 (d, J = 11.2 Hz, 1H, =CH), 6.02 (d, J = 11.2 Hz, 1H, =CH), 5.33 (s, 1H, H-19), 5.00

(s, 1H, H-19), 4.43 (dd, J = 7.6, 4.3 Hz, 1H, H-l), 4.23 (dd, J = 6.7, 3.4 Hz, 1H, H-3), 2.85 - 2.78 (m, 1H), 2.63 -

2.55 (m, 1H), 2.31 (dd, J = 13.3, 6.6 Hz, 1H), 2.06 - 1.86 (m, 5H), 1.69 - 1.55 (m, 5H), 1.40 - 1.25 (m, 10H), 0.91 (d, J = 6.4 Hz, 3H, Me-21), 0.86 (s, 9H, tBu-Si), 0.55 (s, 3H, Me-18), 0.51 - 0.36 (m, 2H, H-24), -0.08 (s, 6H, 2xMe- Si).

¹³C NMR (101 MHz, CDCI3) 5 147.8 (C, C-10), 143.5 (C, C-5), 132.9 (C, C-8), 125.2 (CH, C-6), 117.1 (CH, C-7), 111.9

((CH₂, C-19), 71.0 (CH, C-l), 67.0 (CH, C-3), 56.9 (CH), 56.5 (CH), 46.1 (C, C-13) , 45.4 (CH₂), 43.0 (CH₂), 40.7

(CH₂), 40.6 (CH₂), 36.2 (CH), 29.3 (CH₂), 27.8 (CH₂), 26.8

(CH₃,tBu-Si), 23.8 (CH₂), 22.4 (CH₂), 21.1 (CH₂), 19.0 (CH₃,

Me-21), 16.7 (C, tBu-Si), 13.0 (CH₂), 12.2 (CH₃, Me-18), - 6.0 (CH₂, C-24), -6.1 (CH₂, 2xMe-Si).

(1.R,3S,Z)-5- (2-{(IS,3aS,7aS,Z)-7a-Methyl-l-

[(trimethylsilyl)buta-1,3-diyn-l-yl]octahydro-4H-inden-4- ylidene}ethylidene )-4-methylenecyclohexane-l,3-diol (P6) (Figure 32)

A solution of n-butyl lithium in hexanes (IM, 0.66 mL, 0.86 mmol, 2.0 equiv) was added to a -78 °C cooled solution of phosphine oxide 7 (513 mg, 0.88 mmol, 2.1 equiv) in THF (1.5 mL). The mixture was stirred for 1.5 h. Ketone 9 (120 mg, 0.44 mmol, 1.0 equiv) in THF (0.5 mL) was added via cannula. The mixture was stirred for 1.5 h. The resulting mixture allowed to reach room temperature and treated with sat NH4Cl (10 mL). The mixture was extracted with EtOAc (4 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. A mixture of CH2CI2 and MeCN (5 mL, 1:2) and 0.5 mL of aq HF (48%) were successively added. The mixture was stirred at room temperature for 2 h and then poured onto sat NaHCO₃ (20 x mL). The mixture was extracted with CH2CI2 (3 x 10 mL). The combined organic extracts were dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂, 1 x 6 cm, 50% EtOAc/hexanes) to give P6 [140 mg, 78% (2 steps), Rf = 0.89 (20% EtOAc/hexanes), colorless oil, [«]D +1.5 (c 1.05 CHCl₃].

¹H NMR (400 MHz, CDCI3) 5 6.35 (d, J = 11.2 Hz, 1H, H-7), 6.01 (d, J = 11.2 Hz, 1H, H-6), 5.32 (s,, 1H, H-19), 4.98 (s, 1H, H-19), 4.43 (dd, J = 7.6, 4.3 Hz, 1H, H-l), 4.25 - 4.21 (m, 1H, H-3), 2.87 - 2.82 (m, 1H), 2.59 (dd, J = 13.3, 3.2 Hz, 1H), 2.42 (t, J = 9.3 Hz, 1H), 2.34 - 2.29 (m, 1H), 2.05 - 1.98 (m, 2H), 1.95 - 1.88 (m, 3H), 1.76 - 1.58 (m, 7H), 0.67 (s, 3H, Me-18), 0.18 (s, 9H, 3xMe-Si).

¹³C NMR (101 MHz, CDCI3) 5 147.7 (C, C-8), 141.9 C, C-5), 134.0 (C, C-10), 124.7 (CH, C-7), 117.7 (CH, C-6), 112.0

(CH₂, C-19), 88.6 (C, C=), 83.8 (C, C=), 81.4 (C, C=), 70.9

(CH, C-l), 67.4 (C, C=), 67.0 (CH, C-3), 54.7 (CH), 48.0 (C, C-13), 45.4 (CH₂), 43.0 (CH₂), 42.9 (CH), 38.1 (CH₂),

29.8 (CH₂), 28.8 (CH₂), 28.2 (CH₂), 23.2 (CH₂), 22.8 (CH₂),

14.0 (CH₃, Me-18), -0.17 (CH₃, 3xMe-Si).

(1R,3S,Z)-5- (2- ((IS,3aS,7aS,E)-1- (Buta-1,3-diyn-l-yl)-7a- methyloctahydro-4H-inden-4-ylidene )ethylidene)-4- methylenecyclohexane-1 ,3-diol (10) (Figure 33)

A solution of n-butyl lithium in hexanes (1 M, 133 pL, 0.186 mmol, 2.0 equiv) was added to a -78 °C cooled solution of phosphine oxide 7 (113.8 mg, 0.195 mmol, 2.1 equiv) in THE (1.5 mL). The mixture was stirred for 1.5 h. Ketone 9 (25 mg, 0.093 mmol, 1.0 equiv) in THE (0.5 mL) was added via cannula. The mixture was stirred for 1.5 h. The resulting mixture allowed to reach room temperature and treated with an sat NH4CI (10 mL) and extracted with EtOAc (4 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. The residue was dissolved in dry THF (5 mL) and a solution of TBAF (IM in THF) (0.465 mL, 0.465 mmol, 5 equiv) was added and stirred at room temperature for 24 h. The resulting mixture was treated with an aqueous saturated solution of NaHCOs (20 mL) and extracted with CH2CI2 (3 x 20 mL). The combined organic extracts were dried, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, 1 x 6 cm, 50% EtOAc/hexanes) to give 10 [26 mg, 83% (2 steps), Rf = 0.25 (70% EtOAc/hexanes), colorless oil, [a]_D -18.2° (cl.3, CHCI3).

¹H NMR (400 MHz, CDCI3) 5 6.35 (d, J = 11.2 Hz, 1H, H-6), 6.01 (d, J = 11.2 Hz, 1H, H-7), 5.32 (s, 1H, H-19), 4.98 (s, 1H, H-19), 4.43 (dd, J = 7.8, 4.3 Hz, 1H, H-l), 4.26 - 4.20 (m, 1H, H-3), 2.88 - 2.82 (m, 1H), 2.59 (dd, J = 13.4, 3.3 Hz, 1H), 2.40 (t, J = 9.3 Hz, 1H), 2.33 - 2.28 (m, 1H), 2.10 - 2.03 (m, 2H), 1.95 - 1.86 (m, 4H), 1.74 - 1.57 (m, 5H), 1.29 - 1.20 (m, 4H), 0.67 (s, 3H, Me-18).

¹³C NMR (101 MHz, CDCI3) 5 147.7 (C, C-10), 141.8 (C, C-8), 134.0 (C, C-5), 124.7 (CH, C-6), 117.8 (CH, C-7), 112.0 (CH₂, C-19), 79.8 (C, C=), 70.9 (CH, C-l), 68.7 (C, C=), 67.0 (CH, C-3), 66.6 (C, C=), 65.3 (CH, C-24), 54.7 (CH), 47.9 (C, C-13), 45.3 (CH₂), 43.0 (CH₂), 42.7 (CH), 38.1 (CH₂), 28.8 (CH₂), 28.1 (CH₂), 23.2 (CH₂), 22.8 (CH₂), 14.0 (CH₃, Me-18).

(1R,3S,Z)-5- (2-{(IS,3aS,7aS,E)-1-[(Dimethylsilyl)buta-1,3- diyn-l-yl]-7a-methyloctahydro-4H-inden-4- ylidene}ethylidene)-4-methylenecyclohexane-l ,3-diol_ (11)

(Figure 34) A solution of n-butyl lithium (212 pL, 0.297 mmol, 4 equiv) in hexanes was added dropwise to a -78 °C cooled solution of alkyne 10 (25 mg, 0.074 mmol, 1 equiv) in THF (5 mL). The mixture was stirred for 30 min. ClSiHMe2 (35 pL, 0.312 mmol, 4.2 equiv) was added. The mixture allowed to reach room temperature under stirring. Water (20 mL) was added. The mixture was extracted with TBME (3 x 20 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂, 1 x 6 cm, 50% EtOAc/hexanes) to give 11 [27 mg, 92%, Rf = 0.25 (70% EtOAc/hexanes), white solid, [a]_D +21.0 (cl.1, CHCl₃)].

¹H NMR (400 MHz, CDCI3) 5 6.35 (d, J = 11.2 Hz, 1H, H-7), 6.01 (d, J = 11.2 Hz, 1H, H-6), 5.34 - 5.31 (m, 1H, H-19), 4.98 (s, 1H, H-19), 4.43 (dd, J = 7.8, 4.3 Hz, 1H, H-l), 4.25 - 4.21 (m, 1H, H-3), 4.14 (dp, J = 7.5, 3.7 Hz, 1H, H- Si), 2.87 - 2.81 (m, 1H), 2.59 (dd, J = 13.3, 3.2 Hz, 1H), 2.42 (t, J = 9.3 Hz, 1H), 2.33 - 2.28 (m, 1H), 2.06 - 1.99 (m, 2H), 1.95 - 1.88 (m, 3H), 1.79 - 1.57 (m, 9H), 0.67 (s, 3H, Me-18), 0.24 (d, J = 3.8 Hz, 6H, 2xMe-Si).

¹³C NMR (101 MHz, CDCI3) 5 147.6 (C, C-8), 141.7 (C, C-5), 133.9 (C, C-10), 124.6 (CH, C-7), 117.6 (CH, C-6), 111.9 (CH₂, C-19), 89.8 (C, C-20), 81.6 (C, C-22), 80.6 (C, C- 23), 70.8 (CH, C-l), 67.2 (C, C-24), 66.8 (CH, C-3), 54.5

(CH), 47.8 (CH), 45.2 (CH₂), 42.8 (CH₂), 42.7 (CH), 38.0

(CH₂), 28.7 (CH₂), 28.1 (CH₂), 23.1 (CH₂), 22.7 (CH₂), 13.8

(CH₃, Me-18), -3.3 (CH₃, 2xMe-Si).

The project related to this invention: PTDC/BIA-

MIB/29059/2017 - "Synthesis and Biological Evaluation of

Sila-Secosteroids" This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

Claims

A compound of formula (I), its analogues, or salt thereof :

R¹ and R² and R³ each independently represent a hydrogen atom or hydroxyl group or a branched or non-branched alkyl group having 1 or 3 carbon atoms, or a F, Cl, Br, I or a O-

(C₁-C₃)alkoxyalkyl group or a (C₁-C₃)alkyl-OH group;

Y represents a hydrogen atom or a (C₁-C₃)alkyl group or an

0- (C₁-C₃)alkoxyalkyl group or 0-(C₁-C₃)0H group;

A is a methylene group or hydrogen atom;

R⁴ and R⁵, which may be the same or different, each is independently selected from hydrogen, (C₁-C₁₂)alkyl, (C₆- C₁₀)aryl, and -SiR^aR^bR^c, wherein each R^a, R^b, and R^c is independently selected from (C₁-C₁₂)alkyl, (C₆-C₁₀)aryl, (C₁-C₁₂)alkoxyl, (C₆-C₁₀)aryloxyl and (C₃-C₁₅)heterocycle.

2. Compound of formula (I) according to the previous claim, wherein R⁴ and R⁵ each represent a hydrogen atom, a methoxymethyl, a methoxyethyl, a trimethylsilylethoxymethyl, a trimethylsilyl, a triethylsilyl, a tert-butyldimethylsilyl, a tri-iso- propylsilyl, a tert-butyldiphenylsilyl , a dimethylphenylsilyl, a dimethylbenzylsilyl, a benzoate, a p-nitrobenzoate, a pyvalate, and an acetate.

3. Compound of formula (I) according to any of the previous claims, wherein the compound is (1R,3S,Z)-5- (2- { (lR,3aS,7aR,E)-7a-Methyl-l-[(R)-5- (trimethylsilyl)pentan- 2-yl]octahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol (P3).

4. Compound of formula (I) according to any of the claims

1 to 2, wherein the compound is (1R,3S,Z)-5- (2-

{ (lR,3aS,7aR,E)-7a-Methyl-l-[(R)-5- (trimethylsilyl)pent-4- yn-2-yl]octahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol (P4).

5. Compound of formula (I) according to any of the claims

1 to 2, wherein the compound is (1R,3S,Z)-5- (2- { (lR,3aS,7aR,E)-l-[(R)-5- (Dimethylsilyl)pent-4-yn-2-yl]-7a- methyl octahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol (P5).

6. Compound of formula (I) according to any of the claims 1 to 2, wherein the compound is (1R,3S,Z)-5- (2- { (!S,3aS,7aS,E)-7a-Methyl-l-[(trimethylsilyl)buta-1,3-diyn- 1-yl]octahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol (P4-OH).

7. Compound of formula (I) according to any of the claims

1 to 2, wherein the compound is (1R,3S,Z)-5- (2-

{ (lR,3aS,7aR,E)-l-[(R)-5- (tert-Butyldimethylsilyl)pent-4- yn-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene] ethylidene}- 4-methylenecyclohexane-l ,3-diol (P2).

8. Compound of formula (I) according to any of the claims

1 to 2, wherein the compound is (1R,3S,Z)-5- (2-

{ (lR,3aS,7aR,E)-l-[(R)-5- (tert-Butyldimethylsilyl)pentan-2- yl]-7a-methyloctahydro-4H-inden-4-ylidene) ethylidene}-4- methylenecyclohexane-1,3-diol (P1).

9. Compound of formula (I) according to any of the claims

1 to 2, wherein the compound is (1R,3S,Z)-5- (2-

{ (lS,3aS,7aS,E)-7a-Methyl-l-[(trimethylsilyl)buta-1,3-diyn- 1-yl]octahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol (P6).

10. A pharmaceutical composition comprising the compound of formula (I), its analogues, or salt thereof disclosed in any of the claims 1 to 9.

11. Pharmaceutical composition according to the previous claim, wherein the composition further comprises an acceptable diluent, carrier, adjuvant and/or excipient.

12. Compound of formula (I), its analogues, or salt thereof described in any of the claims 1 to 9, or the pharmaceutical composition described in any of the claims 10 to 11, for use as a vitamin D receptor activator.

13. Compound of formula (I), its analogues, or salt thereof described in any of the claims 1 to 9, or the pharmaceutical composition described in any of the claims 10 to 11, for use as prophylactic and/or therapeutic agent for vitamin D receptor-related diseases.

14. Compound of formula (I), its analogues, or salt thereof described in any of the claims 1 to 9, or the pharmaceutical composition described in any of the claims 10 to 11, for use to increase the weight of animals.

15. Method of producing the compound of formula (I) disclosed in any of the claims 1 to 9, comprising the steps of:

- Stirring the mixture for a time between 0.5 h and 4 h;

- Colling the mixture to a temperature between -40 and - 80 °C;

Adding aqueous NH4CI in a concentration between 7.0 M and 12 M; - Extraction with an organic solvent and evaporation of the solvent to obtain the product as a residue;

- Selective deprotection of the hydroxyl groups at C-1 and C-3 of the product obtained in the previous step achieved by a desilylation reaction by:

- adding aqueous hydrofluoric acid (48%) to the solution of the residue with a concentration between 0.02 M and 0.08 M in an organic solvent and stirring at a temperature between 20 and 30°C for a time between 1 and 24 hours; or

- adding a solution of tetra-n-butylammonium fluoride with a concentration between 0.5 M and 2.0 M in an organic solvent to the dissolved residue with a concentration between 0.02 M and 1.0 M in an organic solvent and allowed to stir at a temperature between 20 and 30°C for a time between 12 and 72 h;

16. Method according to the previous claim, further comprising the following steps to produce (1R,3S,Z)-5- (2- { (lR,3aS,7aR,E)-l-[(R)-5- (Hydroxydimethylsilyl)pent-4-yn-2- yl]-7a-methyloctahydro-4H-inden-4-ylidene }ethylidene)-4- methylenecyclohexane-1,3-diol :

- adding catalytic amounts between 0.01 and 20 mol% of dichloro (p-cymene)ruthenium (II) dimer or Rd (OH)2/C or Pd/C to a solution of hydrosilane with a concentration between 0.01 M and 1.0 M in an organic solvent and stirring at a temperature between 20 and 30°C for a time between 5 and 120 minutes;

- Filtering the reaction mixture to remove the catalyst; - Addition of aqueous HCl in a concentration between 2.0 M and 5.0 M followed by extraction with an organic solvent and evaporation of the solvent.