# First Sila-Vitamin D Analogues: Design, Synthesis, Structural Analysis and Biological Activity

Julian Loureiro, Samuel Seoane, Ivo Sampaio-Dias, Carole Peluso-Iltis, Thierry Guiberteau, Beatriz Brito, Carlos Gregorio, Román Pérez-Fernández, Natacha Rochel, Antonio Mouriño, et al.

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## To cite this version:

Julian Loureiro, Samuel Seoane, Ivo Sampaio-Dias, Carole Peluso-Iltis, Thierry Guiberteau, et al. First Sila-Vitamin D Analogues: Design, Synthesis, Structural Analysis and Biological Activity. Journal of Medicinal Chemistry, 2024, 67 (23), pp.21505-21519. 10.1021/acs.jmedchem.4c02404. hal-04796996

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First Sila-Vitamin D Analogues: Design, Synthesis, Structural
Analysis and Biological Activity

Julian Loureiro,ª Samuel Seoane,ᵇ Ivo E. Sampaio-Dias,ª Carole Peluso-Iltis,ᶜ Thierry

Guiberteau,ᶜ Beatriz Brito,ᵈ Carlos Gregorio,ᵈ Román Pérez-Fernández,ᵇ\* Natacha

Rochel,ᶜ\* Antonio Mouriño,ᵈ\* José E. Rodríguez-Borgesª\*

ªLAQV/REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences,
University of Porto, Porto 4169-007, Portugal

ᵇDepartment of Physiology, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Santiago de Compostela 15706, Spain

ᶜInstitut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 67400 Illkirch, France; Institut National de La Santé et de La Recherche Médicale (INSERM), U1258, 67400 Illkirch, France; Centre National de Recherche Scientifique (CNRS), UMR7104, 67400 Illkirch, France; Université de Strasbourg, 67400 Illkirch, France

ᵈDepartment of Organic Chemistry, Ignacio Ribas Research Laboratory, University of
Santiago de Compostela, Santiago de Compostela 15782, Spain

ᵉLaboratoire ICube - Université de Strasbourg, CNRS UMR 7357, 67000 Strasbourg,
France

Corresponding authors:

* E-mail: roman.perez.fernandez@usc.es

* E-mail: rochel@igbmc.fr

* E-mail: antonio.mourino@usc.es

* E-mail: jrborges@fc.up.pt
The incorporation of silicon bioisosteres into pharmacological structures has been used as a strategy to improve the therapeutic potential of drugs. However, no secosteroidal silicon-containing VDR ligands have been developed. Here we report the design, synthesis, and biological activity of six analogues of the natural hormone 1,25-dihydroxyvitamin D (1,25D₃), which incorporate a silicon atom as a side chain-C25 isostere. The analogues were synthesized by the Wittig-Horner approach starting from Inhoffen-Lythgoe diol. The crystal structures of the complexes formed by the sila-analogues with the ligand binding domain of VDR revealed additional interactions of the sila-containing side chains that stabilize the VDR active conformation. These sila-analogues show similar VDR binding and transcriptional activity in comparison with the natural hormone 1,25D₃, but with significantly less hypercalcemic activity. The new analogues, when combined with chemotherapy, significantly decrease cell proliferation.

KEYWORDS: noncalcemic sila-vitamin D analogs; calcemia; antiproliferation; design; synthesis; crystal structure.
INTRODUCTION

Solar radiation of 7-dehydrocholesterol (1) in the skin produces the secosteroid previtamin D₃ (2), which can be converted to other photoproducts such as tachysterol or lumisterols, or to vitamin D₃ by thermal isomerization.[1] Vitamin D₃ and previtamin D₃-photoproducts can further be hydroxylated by cytochrome P450 enzymes to produce biologically active hydroxylated derivatives.[2,3] The best-known and most active vitamin D₃-hydroxylated metabolite is 1α,25-dihydroxyvitamin D₃ (4, calcitriol, 1,25(OH)₂D₃, 1,25D₃), which is enzymatically produced in almost every tissue in the body (Figure 1).[4,5] This secosteroid hormone, the natural ligand of the vitamin D receptor (VDR) exhibits a wide range of biological activities including regulation of calcium and bone metabolism, cell proliferation-differentiation, gene transcription, and the immune system.[6-8] In addition, vitamin D₃ metabolites have been shown to act on alternative nuclear receptors.[2] Low levels of 1,25D₃ have been associated with cancer, autoimmune diseases, cardiovascular and kidney dysfunctions, bone and skin diseases, and more recently with brain diseases.[9-11] A large number of biologically active secosteroidal VDR ligands have been developed, some of which found clinical applications although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages cause toxicity.[12-14] Recent synthetic efforts have led to the discovery of new promising highly active and non-calcemic seco-steroidal (5-7),[15-17] steroidal (8 and 9),[18] and non-steroidal (10)[19] VDR ligands (Figure 2). Surprisingly and despite the large number of known vitamin D analogues synthesized,[20] no seco-steroidal VDR ligands incorporating silicon atoms as carbon isosteres or other silicon-containing moieties have been explored.
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<figure><img src="image_6.png" /><figcaption>**Figure 1.** Structures of 7-Dehydrocholesterol, Previtamin D₃, Vitamin D₃, and 1α,25-Dihydroxyvitamin D₃.</figcaption></figure>

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<figure><img src="image_11.png" /><figcaption>**Figure 2.** Active non-calcemic VDR ligands.</figcaption></figure>

Silicon (Si) is a nonmetallic element located just below the carbon atom (C) in the periodic table. Like carbon, the Si atom can form covalent bonds with other atoms usually C, H, and/or O, but not π bonds. The electropositivity of the Si atom, which inverts the polarity of the Si-H bond, and its greater size in comparison with C, provide Si-derivatives with improved lipophilicity that favor cell penetration, accumulation in tumors, pharmacokinetics, and agonistic or antagonistic properties. The incorporation of
a silicon group into a known scaffold or replacement of a carbon by silicon (carbon bioisostere) has been used to modify the known biological activity of a molecule for medicinal applications.[21-26] For example, in comparison with the corresponding carbon compound, karenitecin (11) (Figure 3), an analog of camptothecin, increases tissue penetration in tumors,[27] tamibarotene (12) bearing two silicon atoms is 10-fold more active,[28] retinoid 13, a sila-retinobenzoic acid analog bearing a *t*-butyldimethylsilyl (TBS) moiety shows higher activity towards RARγ,[29] silinezolid (14) increases brain penetration,[30] and sila-ibuprofen 15 shows higher potency than ibuprofen,[31] biphenyl ligands of type 16 behave like androgen receptor (AR) antagonists and weak vitamin D receptor (VDR) agonists.[32] However, no Si-containing drugs have been approved yet. The biological activity of the silicon derivatives shown above (Figure 3) prompted us to initiate a program directed toward the development of Si-containing vitamin D analogues as new ligands of the vitamin D receptor. Here we describe the design, synthesis, structural studies, and evaluation of the first sila-vitamin D₃ analogues **Sila-W** (Sila-1-6, Figure 4), which contain a Si atom replacing the side chain-C25 of 1,25D₃.

<figure><img src="image_12.png" /><figcaption>**Figure 3.** Selected biologically active silicon-containing derivatives.</figcaption></figure>

The choice of the **Sila-W** analogues was based on the side-chains of the natural hormone 1,25D₃ and the highly active and non-calcemic 1,25D₃-analogues 1α,25-
dihydroxy-16-ene-23-yne-vitamin D₃,[³³,³⁴] 1α,25-dihydroxy-19-nor-14-epi-23-yne-vitamin D₃,[³⁵] 1α,25-dihydroxy-21-nor-20(22),23-diyne-vitamin D₃,[³⁶] and 1α,25-dihydroxy-17α-methyl-21-nor-20(22),23-diyne-vitamin D₃.[³⁷] The presence of a double or triple bond in the side chain of 1,25D₃ analogues has been associated with the enzymatic blockade of hydroxylation at carbons adjacent to the unsaturation.[³⁸,³⁹] We also considered the possibility of enzymatic hydroxylation of analogue **Sila-5** at Si25 or analogues **Sila-1-4,6** at the respective adjacent carbons to Si25 to give rise to hydroxylated metabolites with beneficial pharmacodynamic or pharmacokinetic properties. Analogues that lack the 25-hydroxyl group[⁴⁰] or contain short nonhydroxylated side chains[⁴¹] have been shown to retain activity however lower in comparison with the natural hormone 1,25D₃.

<figure><img src="image_13.png" /><figcaption>**Figure 4.** Structure of target sila-vitamin D₃ analogues **Sila-W**.</figcaption></figure>

## RESULTS AND DISCUSSION

**Docking Studies.** To obtain information about the affinity of the new **Sila-W** analogues within the binding site of the hVDR, docking studies were carried out with the GOLD program[⁴²] on the *h*VDR LBD crystal structure (PDB 1DB1),[⁴³] which lacks the flexible insertion domain in the ligand-binding domain (LBD) and has been successfully crystallized in complex with several 1,25D₃ analogs.[⁴⁴] The docking studies employing the *h*VDR LBD have proven useful in designing active 1,25D₃ analogs, including VDR super agonists.[⁴⁴,⁴⁵]

The binding affinities of sila-analogues (**Sila-1-6**) to *h*VDR LBD were compared to the natural hormone 1,25D₃ and their **25C** counterparts. The molecular docking results are shown in Figure S1 and the relative affinities are listed in Table 1. In this series, all sila-analogues (**Sila-1-6**) bind efficiently to the *h*VDR LBD relative to 1,25D₃ (100%): **Sila-
1 (95%), Sila-2 (98%), Sila-3 (95%), Sila-4 (98%), Sila-5 (94%), and Sila-6 (90%). While Sila-1 and Sila-2 display an alkyl chain similar to that of the natural hormone, Sila-3-6 exhibit more rigid side chains by introduction of one (Sila-3-5) or two (Sila-6) triple bonds. In respect with Si functionalization, Sila-1, Sila-3, and Sila-6 exhibit a trimethylsilyl group (TMS), Sila-2 and Sila-4 display a *tert*-butyldimethylsilyl group (TBS), and Sila-5 a dimethylsilane. Notably, the introduction of the silicon at the side chain of 1,25D₃ sila-based counterparts (Sila-1-6) does not significantly alter the overall shape, with all sila-analogues adopting the typical elongated conformation of the natural hormone 1,25D₃ in the binding pocket (Figure S1). The absence of polar groups at the side chain of Sila-1-6 results in increased hydrophobicity of these analogues in comparison with natural hormone, thus affecting the hydrogen bonding network of VDR. The hydrogen-bonding interactions between the C25-OH group of the 1,25D₃ and the amino acid residues His397 (2.6 Å) and His305 (2.9 Å) are replaced in the new analogues by longer van der Walls interactions except for Sila-2 and 25C-2, which show shorter distances of 2.7 Å and 2.6 Å, respectively, in respect with His305 (Table 2). Sila-1 shows a longer distance to Phe422 (4.8 Å) in comparison with that of 25C-1 (3.4 Å) indicating a remarkable isostere effect. The distances to the critical amino acid residues to activate transcription Val-418 and Phe-422 on H12 are similar to those of 1,25D₃ (Table 2).

Despite the different alkyl chains and silyl groups present in Sila-1-6, the molecular docking experiments demonstrate that these chemical modifications does not interfere significantly with the anchoring of the A-ring and CD bicyclic scaffold (Figure S1). The A-ring-hydroxyl groups of all new analogues bind by hydrogen bonding to the same amino acid residues as the native 1,25D₃: C1-OH with Ser-237 and Arg-274; C3-OH Tyr-143 and Ser-278.

<table><caption>**Table 1.** Gold relative affinities of target Sila-1-6 ligands for the VDR LBD in comparison with that of 1,25D₃ (100%).</caption><thead><tr><th>Ligand</th><th>1,25D<sub>3</sub></th><th>Sila-1</th><th>Sila-2</th><th>Sila-3</th><th>Sila-4</th><th>Sila-5</th><th>Sila-6</th></tr></thead><tbody><tr><td>Soln 1<sup>a</sup></td><td>109.53</td><td>102.67</td><td><strong>106.68</strong></td><td>103.14</td><td><strong>106.62</strong></td><td>101.88</td><td>97.50</td></tr><tr><td>Soln 2<sup>a</sup></td><td>108.45</td><td>103.54</td><td>105.69</td><td>103.06</td><td>106.61</td><td>101.85</td><td>97.68</td></tr><tr><td>Soln 3<sup>a</sup></td><td>108.42</td><td><strong>103.76</strong></td><td>105.85</td><td><strong>103.15</strong></td><td>106.22</td><td><strong>101.92</strong></td><td><strong>97.72</strong></td></tr><tr><td>Average score<sup>b</sup></td><td>108.80</td><td>103.32</td><td>106.07</td><td>103.12</td><td>106.48</td><td>101.88</td><td>97.63</td></tr><tr><td>(%)<sup>c</sup></td><td>100.0</td><td>95.0</td><td>97.5</td><td>94.8</td><td>97.9</td><td>93.6</td><td>89.7</td></tr></tbody></table>
ᵃFitness score associated with the three best solutions for each ligand. ᵇMean score obtained for each ligand. ᶜRelative affinity to VDR-1,25D docking. 1,25D score was normalized to 100%.

<table><caption>**Table 2.** Selected shortest distances (Å) from the docked **Sila-1-6** analogs, the 25C-counterparts **25C-1-6** (parenthesis), and the natural hormone 1,25D₃ to *h*VDR LBD.</caption><thead><tr><th rowspan="2">Residue</th><th colspan="7">Distance / Å</th></tr><tr><th>Sila-1</th><th>Sila-2</th><th>Sila-3</th><th>Sila-4</th><th>Sila-5</th><th>Sila-6</th><th>1,25D<sub>3</sub></th></tr></thead><tbody><tr><td>Tyr-143</td><td>2.7 (2.6)</td><td>2.7 (2.7)</td><td>2.6 (2.6)</td><td>2.5 (2.6)</td><td>2.7 (2.6)</td><td>2.6 (2.6)</td><td>2.6</td></tr><tr><td>Ser-237</td><td>3.1 (2.9)</td><td>3.0 (3.1)</td><td>3.1 (3.0)</td><td>3.0 (3.0)</td><td>3.1 (3.0)</td><td>3.1 (3.1)</td><td>3.0</td></tr><tr><td>Arg-274</td><td>3.1 (3.0)</td><td>3.1 (3.0)</td><td>3.1 (3.1)</td><td>3.0 (3.0)</td><td>3.0 (3.1)</td><td>3.0 (3.0)</td><td>3.1</td></tr><tr><td>Ser-278</td><td>3.0 (2.9)</td><td>3.0 (2.9)</td><td>3.0 (2.9)</td><td>3.0 (2.9)</td><td>3.0 (3.0)</td><td>3.0 (3.0)</td><td>3.0</td></tr><tr><td>His-305</td><td>3.4 (3.5)</td><td>2.7 (2.6)</td><td>3.5 (3.6)</td><td>3.0 (3.3)</td><td>3.7 (4.0)</td><td>3.3 (3.3)</td><td>2.9</td></tr><tr><td>His-397</td><td>3.6 (3.5)</td><td>3.3 (3.4)</td><td>3.8 (3.6)</td><td>3.3 (2.8)</td><td>3.4 (3.4)</td><td>3.2 (3.5)</td><td>2.6</td></tr><tr><td>Val-418</td><td>4.3 (4.0)</td><td>4.2 (3.7)</td><td>3.6 (4.3)</td><td>3.2 (3.5)</td><td>4.4 (4.5)</td><td>4.0 (4.3)</td><td>4.5</td></tr><tr><td>Phe-422</td><td>4.8 (3.4)</td><td>3.5 (3.2)</td><td>3.4 (3.4)</td><td>3.5 (3.4)</td><td>4.2 (3.5)</td><td>3.5 (3.5)</td><td>3.8</td></tr></tbody></table>

**Retrosynthetic analysis of Sila-W (Sila-1-6).** The synthetic plan for the target compounds **Sila-1-6** is outlined in Scheme 1. The commercial availability of phosphine oxide **18** led us to choose the Wittig-Horner coupling approach[46] to generate the triene system of vitamin D protected intermediates **17**, **22**, and **25** utilizing the corresponding CD-side chain ketones **19**, **23**, and the known ketone **26**, which in turn would be prepared from the Inhoffen-Lythoe diol (**21**) [47,48] through the key alkynyl silanes **20**, and the known alkynyl iodide **27**.[37,49] **Sila-5** would arise from alkyne **24** by reaction of the corresponding alkynyllithium derivative with HSi(Cl)Me₂.

**Scheme 1.** Retrosynthetic analysis of sila-vitamin D analogs (Sila-1-6).
<figure><img src="image_14.png" /></figure>

**Synthesis.** The synthesis of our target sila-vitamin D analogues is outlined in Scheme 2. The synthesis of **Sila-1** and **Sila-2** bearing a silicon atom replacing the C25 of vitamin D₃ side chain started from Inhoffen-Lythgoe diol (21)[48,49] which was converted in 92% yield through alcohol 28 to protected tosylate 29 as previously described.[50,51] Tosylate displacement with trimethylsilyl and *tert*-butyldimethylsilyl lithium acetylides in dioxane provided the respective silylalkynes 20a (80%) and 20b (87%), which were deprotected with aqueous hydrogen fluoride in CH₃CN/CH₂Cl₂ to give the corresponding alcohols 30a (89%) and 30b (98%). The next task was to reduce the side chain-triple bonds to the desired saturated side-chains. Individual hydrogenation of 30a and 30b in the presence of catalytic Pd/C in EtOAc provided the respective saturated silanes 31a (93%) and 31b (99%), which were converted to the desired ketones 19a (98%) and 19b (98%) by Dess-Martin periodinane oxidation in CH₂Cl₂. Wittig-Horner coupling of ketones 19a and 19b with the lithium ylide of phosphine oxide 18 furnished the corresponding protected sila-vitamins D compounds 17a and 17b, which were deprotected with aqueous hydrogen fluoride in CH₃CN/CH₂Cl₂ to provide the
respective desired sila-vitamin D analogues **Sila-1** (63%, two steps; 37% from **5**, 8 steps) and **Sila-2** (65%, two steps, 49% from **21**, 8 steps).

<figure><img src="image_15.png" /><figcaption>**Scheme 2.** Synthesis of sila-vitamin D analogues **Sila-1-6** TMS = SiMe₃, TBS = Sit-BuMe₂</figcaption></figure>

The synthesis of **Sila-3** and **Sila-4** started from alcohols **30a** and **30b**, whose Dess-Martin periodinane oxidations in CH₂Cl₂ provided the corresponding alkynylsilyl ketones **23a** (95%) and **23b** (98%). Wittig-Horner coupling of these ketones with **18-Li** as above provided the protected analogs **22a** and **22b**, which were individually deprotected with aqueous hydrogen fluoride in CH₃CN/CH₂Cl₂ to afford the desired vitamin D analogs **Sila-3** (67%, two steps; 43%, 7 steps from **21**) and **Sila-4** (62%, two steps; 47%, 7 steps from **21**).

Desilylation of **22a** with tetrabutylammonium fluoride in THF provided alkynyl diol **24** in 86% yield, whose metalation with an excess of *n*-butyllithium in THF and subsequent
reaction of the resulting organolithium derivative with chlorodimethylsilane afforded the desired **Sila-5** vitamin D analogue in 70% yield (60%, 2 steps). **Sila-6** was synthesized from known ketone **26**[37,49] in a similar way. Wittig-Horner coupling between the lithium ylide of phosphine oxide **18** with ketone **26** provided protected compound **25**, which was *O*-desilylated with aq HF to give the desired diynic analogue **Sila-6** (78%, two steps; 19% from **21**, 12 steps).

**Biological Assays.** A competitive binding assay was used to quantify the binding of the **Sila-W** analogues for the VDR. The **Sila-W** compounds, including 1,25D₃, bind to VDR with a similar affinity (10⁻⁹ M) except **Sila-1** and **Sila-3** analogues, whose binding affinities are around ten times lower (Figure 5A). Interestingly, the biological activity of the previously reported carbon congener of **Sila-1**, compound **25C-1**, was 80% of that of the native hormone 1,25D₃.[52] Evaluation of the transcriptional activities of the sila-analogs on the well-known vitamin D target gene *CYP24A1* (also known as 24-hydroxylase, responsible for 1,25D₃ catabolism) was performed by qPCR. Our data indicate a significant (P<0.001) increase of *CYP24A1* mRNA expression in the human breast adenocarcinoma MCF-7 cell line (luminal A subtype -positive in estrogen and progesterone receptors-) after administration of 1,25D₃ or **Sila-1** and **Sila-4** analogues as compared to control. In addition, the **Sila-2,3,5,6** analogues induced a significant (P<0.001) transcriptional activation of this gene compared to control cells (Figure 5B), however lower than 1,25D₃. To confirm the regulation of other vitamin D target genes by the **Sila-W** analogues, the e-cadherin mRNA and protein expression, as well as the cyclin D1, and p27 protein expression were also evaluated in MCF-7 cells treated 10⁻⁷ M with 1,25D₃, **Sila-W** analogues or vehicle. Except **Sila-2** and **Sila-6**, the sila-analogues induced e-cadherin mRNA similarly to 1,25D₃ (Figure 5C). This effect was also observed when the human breast adenocarcinoma MDA-MB-231 cell line (triple-negative subtype) was treated (Supporting Information Figure S2B). E-cadherin protein expression also rises, as shown by Western blot (Figure 5D) and immunofluorescence (Figure 5E). Cyclin D1 and p27 protein expression indicate a similar increase when treated with 1,25D₃ and **Sila-W** analogues (Figure 5D).

As previously commented, hypercalcemia is one of the main problems for the pharmacological use of 1,25D₃ in the treatment of several diseases, such as cancer.[12,14,53] [20,22,44] Thus, our next step was to evaluate the effect of the **Sila-W** analogues on calcium levels *in vivo*. Normal mice were treated with **Sila-W** analogues,
1,25D₃, or sesame oil every other day for 21 days, and then calcium levels and body weight were determined. 1,25D₃ administration significantly (P<0.05) raised calcium in sera, but none of the analogs significantly increased calcium, as compared to sesame oil-treated mice (Figure 5F). In addition, while the administration of 1,25D₃ produced a significant decrease (P<0.05) in body weight compared to controls, mice treated with the sila-analogs did not modify body weight (Supporting Information Figure S2A).

<figure><img src="image_16.png" /></figure>

**Figure 5.** Biological properties of the vitamin D analogs. (A) Competitive binding of 1,25D₃ (as control) and Sila-W analogues to the human vitamin D receptor (VDR). Dose response curves (10⁻¹¹ to 10⁻⁶ M) were used to calculate the IC₅₀ values. (B) CYP24A1 mRNA levels were
evaluated by real-time PCR in MCF-7 cells treated for 48 h with 1,25D and the **Sila-W** compounds at 10⁻⁷ M (ethanol was used as control, C). (C) *E-cadherin* mRNA levels were evaluated by real-time PCR in MCF-7 cells treated with ethanol (as control), 1,25D₃, and **Sila-W** analogues (10⁻⁷ M) for 48 h. (D) Western blot of e-cadherin, cyclin D1, p-27, and GAPDH (used as loading control) in MCF-7 cells treated as in D. (E) Representative images of e-cadherin (in green color) after fluorescence microscopy in MCF-7 cells treated as in D. DAPI (in blue color) was used as nuclei marker. Scale bar: 20 µm. (F) Serum calcium levels in mice treated every other day for 21 days with sesame oil (as control) and 0.5 µg/kg of 1,25D₃ and **Sila-W** compounds. Data are expressed as mean ± SD. *P*<0.05, * *P*<0.01, and ** **P**<0.001.

Our data suggest that the **Sila-W** analogues could be useful for possible treatment of
cancer, either alone or combined with conventional chemotherapy. Thus, we first study
the effect of our compounds on cell proliferation, one of the key steps in cancer
progression. Four different human cell lines, the normal human keratinocytes HaCaT
cells with P53 mutation, the human breast adenocarcinoma MCF-7 cells, the human
ovary adenocarcinoma SKOV-3 cells, and the human prostate adenocarcinoma PC-3
cells, were treated with 1,25D₃ or the **Sila-W** -analogues for 48 h (Supporting
Information Figure S3A-D). As compared to control vehicle-treated cells, both 1,25D₃
and all sila-analogues significantly decreased cell proliferation in all cell lines.
Numerous studies have demonstrated that vitamin D analogues decrease proliferation,
induce cell differentiation, and could be useful either alone or combined with
conventional chemotherapy in breast cancer (BC).[12] Therefore, we evaluate the effect
of the **Sila-W** analogues in triple-negative breast cancer (TNBC) cells. TNBC is a
metastatic subtype of BC with the worst prognostic, being conventional chemotherapy
the most useful treatment.[14] We used the TNBC cell line MDA-MB-231, to test the
effect of **Sila-W** analogues on cell proliferation. Except for **Sila-5**, all compounds
significantly (P<0.05) reduced cell proliferation similarly to 1,25D₃ effects (Figure 6A).
Similar effects were observed in spheroids of MDA-MB-231 cells treated as the 2D
cultures (Figure 6B-C). In addition, MDA-MB-231 cells co-treated with the **Sila-3-6**
analogues and paclitaxel (PT, 2 nM), one of the most used chemotherapies for TNBC
treatment in patients, exhibit a significant (P<0.01) decrease in cell proliferation as
compared with cells treated with PT alone (Figure 6B-C), whereas no significant effect
was observed for cells co-treated with PT and 1,25D₃, **Sila-1** or **Sila-2**.
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /><figcaption>**Figure 6.** Effects of the **Sila-1-6** analogues in breast cancer cell proliferation. (A) MDA-MB-231 cells seeded on 24-well plates were treated with ethanol (control, C, 100%) and with paclitaxel (PT, 2 nM), and with $10^{-7}$ M of $1,25D_3$, and the sila-analogues alone or combined with paclitaxel (+PT) for two days, and cell proliferation was measured. (B-C) MDA-MB-231 cells were grown in three-dimensional (3D) cultures for 6 days and then treated on days 6 and 8 with ethanol (control, C), paclitaxel (2 nM, PT), and with $1,25D_3$ ($10^{-7}$ M), and the sila-analogs ($10^{-7}$ M) alone or with paclitaxel (+PT). On day 10, spheres were stained, photographed, and fluorescence quantified (represented as relative units, RU). Scale bar: 200 µm. Values are plotted as mean ± SD. ns= not significant, * = P < 0.05, ** = P < 0.01.</figcaption></figure>
Taken together, our results indicate that the sila-analogs increase transcriptional activities and exert antiproliferative effects similarly to the natural hormone 1,25D₃, but without inducing hypercalcemic effects, at least at the doses employed in this study. These results suggest that the sila-analogs could be usefully combined with conventional chemotherapy in certain pathologies such as triple-negative breast cancer.

**Crystal Structures of VDR LBD complexes.** Except for **Sila-2** compound, the sila-derivatives **Sila-1**, **Sila-3**, **Sila-4**, **Sila-5**, and **Sila-6** were crystallized in complex with the zebrafish (z) VDR LBD and NCOA2 peptide encompassing the second nuclear receptor LXXLL-interacting motif. We used zVDR LBD as a surrogate of the human receptor as we have previously demonstrated by x-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry that the binding mode of the natural ligand is identical in zVDR LBD and hVDR LBD as well as in the full-length hVDR.[54,55] The crystal structures of the complexes were refined with a resolution ranging from 1.9 Å to 2.2 Å. The sila-analogues are accommodated in the VDR ligand binding pocket (LBP) and bind to VDR in a similar way as 1,25D₃ (Figure 7). The overall structures are highly homologous to the VDR-1,25D₃ structure with a root mean square deviations of 0.2-0.3 Å over ~234-239 residues when comparing the Cα atoms. Differences with the docking data were observed due to protein-ligand mutual adaptations. Distances between ligands and the two Histidines were in agreement with the docking data only for **Sila-3** and **Sila-4** and those with C-terminal residues Val and Phe only for **Sila-5**.

<figure><img src="image_20.png" /><figcaption>**Figure 7.** X-ray crystal structures of zVDR LBD in complex with sila-analogues. (A) Superposition of sila-analogues with 1,25D₃ (grey, PDB 2HC4) in VDR pocket. **Sila-1** (green, PDB 9GY8), **Sila-3** (orange, PDB 9GYJ), **Sila-4** (blue, PDB 9GYC), **Sila-5** (pink, PDB</figcaption></figure>
9GYA), Sila-6 (cyan, PDB 9GYK). (B-F) Hydrogen bonds formed by 1,25D₃ (grey) and Sila-1 (B), Sila-3 (C), Sila-4 (D), Sila-5 (E) and Sila-6 (F).

The A-, seco-B and C/D- rings of the five compounds exhibit similar conformations and form similar interactions with VDR LBD than those observed with 1,25D₃ (Figure 7A). In particular, the C1-OH and C3-OH groups formed similar hydrogen bonds with the zVDR anchoring residues (Figure 7B-E). As expected, differences are observed around the side chains. The distances between the C1-OH and Si25 are: 11.7 Å, 11.7 Å, 12.6, 11.7 Å and 12.3 Å for Sila-1, Sila-3, Sila-4, Sila-5, and Sila-6 in comparison to 12.3 Å for 1,25D₃. Due to the absence of the C25-OH group, hydrogen bonds with zHis333 and zHis423 are not formed but are replaced by Van der Waals interactions. Within the series, Sila-1 followed by Sila-3 are forming weaker interactions with the anchoring Histidines (Supporting Information Table S1), which may explain the observed lower binding affinity for VDR.

In the Sila-1 complex, the Si atom is shifted 0.9 Å towards H3 and H12 in comparison to the carbon counterpart of 1,25D₃. Consequently, Sila-1 geminal methyl groups form stronger interactions, notably with Leu258 and Ala259 in H3 and with Val444 and Phe448 in H12 in comparison to 1,25D₃ (Figure 8A and Supporting Information Table S1).

Sila-3-5 contain a 23yne group in their side chains. Whereas Sila-5 side chain is almost similarly positioned as its C25-OH counterpart compound, TX522,[56] Si25 of Sila-3 is shifted by 0.8 Å (Supporting Information Figure S4B). Sila-3 forms weaker interactions with C-terminal Val444 and Phe448 in comparison to 1,25D₃, but forms stronger interactions with Ala331, Ala259 and Ile296 (Figure 8B and Supporting Information Table S1). Sila-5 which lacks one methyl group of Sila-3, forms as well weaker interactions with C-terminal residues but interacts more strongly with Ile296 (Figure 8C and Supporting Information Table S1).

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>
**Figure 8.** Interactions of Sila analogues with zVDR LBD around the side chains of the ligand. (A-C) Interactions of **Sila-1** (PDB 9GY8) (A), **Sila-3** (PDB 9GYJ) (B) and **Sila-5** (PDB 9GYA) (C) with VDR residues within a 4 Å distance. Residues that interact more strongly than 1,25D₃ are underlined.

The additional *t*-butyl group in **Sila-4** requires some side-chain conformational changes, notably for His333, His397 and Tyr427 (Figure 9A). Of note, **Sila-4** stabilizes the most the VDR active conformation with increased Van der Waals interactions with His333, His423, Tyr427, Leu440 and Phe448 (Figure 9A and Supporting Information Table S1). The most rigid side chain of **Sila-6** bearing a diyne group is similarly positioned as its C25 carbon counteract compound, Xe[57] (Supporting Information Figure S4C). **Sila-6** forms weaker interactions with Val444 and Phe448 but interacts more strongly with Ala259, Ala331, Tyr427 and Leu430 (Figure 9B and Supporting Information Table S1).

<figure><img src="image_24.png" /></figure>

**Figure 9.** Structural comparisons between zVDR LBD in complex with 1,25D₃ (grey) and **Sila-4** (PDB 9GYC) (A) or **Sila-6** (PDB 9GYK) (B) around the side chain of the ligands. VDR residues that interact with the ligands within a 4 Å distance are labeled and those interacting more strongly with Sila analogues compared to 1,25D₃ are underlined.

The sila-analogues lack the C25-OH but in contrast to analogues with nonhydroxylated[52] and short side chains,[58] that were shown to be at least 10 times less active, they efficiently stabilize the VDR agonist conformation due to increased interactions of the Si-containing side chains

## CONCLUSIONS

C/Si switch is an attractive strategy for the design of new nuclear receptor ligands. We demonstrate here that secosteroidal VDR agonists can be designed by this strategy. The
new **Sila-W** analogues of the hormone 1,25D₃, whose side chains lack the C25OH group and contain a Si25 atom replacing the C25, were efficiently synthesized by the Wittig-Horner approach starting from the Inhoffen-Lythgoe diol. In agreement with the docking data, the binding assay indicated that the compounds bind to VDR with a similar affinity to the natural hormone 1,25D₃. Structural analysis indicated that the sila-analogues formed different interactions with VDR stabilizing the active conformation.
**Sila-1-6** vitamin D analogues bind to the VDR and induce significantly less hypercalcemic activity. Interestingly, the new analogs, when combined with chemotherapy, significantly decrease cell proliferation. Further studies on sila-analogs of 1,25D₃ are underway.

## EXPERIMENTAL SECTION

**Molecular docking procedure.** Molecular docking studies of the new VDR ligands were carried out using the GOLD program (version 2022.3.0) with a modified crystal structure (addition of hydrogens and correction of His tautomers) of the complex between 1,25D-hVDR(LBD) (PBD 1DB1).[43] Energy minimization was not performed on the protein. The target ligands were built with the editing tool provided by the Chem3D software. The receptor and the ligands were used as MOL2 files. The ligand binding pocket of the mutant LBD was defined as the binding site with the automatic active-site detection on, and the radius was set to 10 Å. The conformations for each ligand in the active site of the protein were sampled using a genetic algorithm (GA). The ligands were docked in 25 independent genetic algorithm (GA) runs, for each of which a maximum of 125000 GA operations were performed on a single population of 100 individuals. Operator weights for crossover, mutation, and migration in the entry box were used as default parameters (95, 95, and 10, respectively). The “flip ring corners” flag was switched off, while all the other flags were on. The different poses were ranked according to the force-field-based CHEMLP scoring function and GoldScore was used as a re-scoring function. The best three solutions for each ligand were obtained and compared with the solutions for 1,25D₃. [43]

**CHEMISTRY-General.** All reactions involving oxygen- or moisture-sensitive compounds were carried out under a dry Ar atmosphere using oven-dried or flame-dried glassware and standard syringe/septa techniques, unless when indicated. Liquid reagents
or solutions of reagents were added by syringe or cannula techniques. Reaction
temperatures refer to external bath temperatures. Acetone-dry ice baths were used for
reactions at low temperatures. Alternatively, methanol or acetone baths were cooled
with a CRYOCOOL immersion cooler, and provided with a temperature regulator.
Organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated using a
rotary evaporator at aspirator pressure (20-30 mmHg). Reagents were obtained from
Aldrich Chemical (www sigma-aldrich.com) or Acros Organics (www acros.com) and
used without further purification. All dry solvents were distilled under Ar immediately
before use. Tetrahydrofuran (THF) Na/benzophenone. CH₂Cl₂ was distilled from P₂O₅.
Piperidine was distilled from CaH₂. *tert*-Butyl methyl ether (TBME), EtOAc, hexanes,
and CH₂Cl₂ for extractions were used as received. CuI was purified by suspension in
boiling water followed by filtration, washing with EtOH (96%), and drying at 100 °C
under vacuum over P₂O₅. Solutions of *n*-BuLi in hexanes, and *n*-HexLi in pentane were
titrated with *N*-benzylbenzamide before use. Reactions were monitored by thin-layer
chromatography (TLC) using aluminum-backed MERCK 60 silica gel plates (0.2 mm
thickness). The chromatograms were visualized first with ultraviolet light (254 nm) and
then by immersion in solutions of ceric ammonium molybdate or *p*-anisaldehyde
followed by heating with a heater gun. Flash column chromatography was performed
with Merck silica gel 60 (230-400 mesh). All NMR spectra were measured with
solutions in CDCl₃ in a Bruker DPX-250 (250 MHz) or Varian Inova 400 (400 MHz)
spectrometers. Chemical shifts are reported on the *δ* scale (ppm) downfield from
tetramethylsilane (*δ* = 0.0 ppm) using the residual solvent signal at *δ* = 7.26 ppm (¹H, s,
CDCl₃) or *δ* = 77.0 ppm (¹³C, t, CDCl₃). Coupling constants (*J*) are reported in Hz.
Distortionless Enhancement by Polarization Transfer (DEPT-135) was used to assign
carbon types. NMR signals have been assigned using steroidal numbering. High-
resolution mass spectra (HRMS) were performed in a Micromass Instruments Autospec,
a Thermo Finnigan MAT95XP, and an Applied Biosystems QSTAR Elite spectrometer.
UV spectra were registered in a Hewlett Packard 8452A spectrometer. Optical rotations
were measured on a Jasco DIP-370 polarimeter in a 1 dm cell. [α] and *c* are given in
deg cm³ g⁻¹ dm⁻¹ and g cm⁻³ respectively. The purities of the Sila-vitamin D analogs
were determined by HPLC [Shimadzu preparative liquid chromatograph model LC-8A
equipped with a TSP UV-1 absorbance detector, using a Phenomenex Luna column [5u
Silica(2) 100A, 250 x 21.2 mm], normal phase; and isocratic mode. Their purities of
sila-compounds were ≥ 99%.

Synthesis.

tert-Butyldimethyl{[(1R,3aR,4S,7aR)-7a-methyl-1-[(R)-5-(trimethylsilyl)pent-4-yn-2-yl]octahydro-1H-inden-4-yl]oxy}silane (20a). A solution of *n*-butyllithium 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. A solution of 29[51] (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 and then 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, filtered, and concentrated in vacuo. The residue was flash chromatographed (SiO₂ 1 x 8 cm, hexanes) to give 20a [740 mg, 80%, Rf = 0.58 (hexanes), colorless oil, [α]D +122.4 (c1.48, CHCl₃)]. ¹H NMR (400 MHz, CDCl₃) δ 4.00 (d, *J* = 2.5 Hz, 1H, H-8), 2.25 (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, *t*-Bu-Si), 0.15 (s, 9H, 3 x Me-Si), 0.01 (s, 3H, Me-Si), -0.00 (s, 3H, Me-Si). ¹³C NMR (101 MHz, CDCl₃) δ 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, *t*-Bu-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, 3 x Me-Si), -4.6 (CH₃,Me-Si), -5.0 (CH₃,Me-Si). HRMS ([ESI]⁺, m/z) [M+H]⁺ calc. for C₂₄H₄₇OSi₂ 407.3167; found. 407.3164.

(1R,3aR,4S,7aR)-7a-methyl-1-((R)-5-(trimethylsilyl)pent-4-yn-2-yl)octahydro-1H-
inden-4-ol (30a). An aqueous solution of HF (48%, 5 drops) was added to a solution of
20a (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 saturated 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, filtered, and concentrated in vacuo. The residue
was flash chromatographed (SiO₂ 1 x 8 cm, 10% EtOAc/hexanes) to give 30a [77 mg,
89%, Rf = 0.24 (20% EtOAc/hexanes), colorless oil, [α]D +126.0 (c1.0, CHCl₃)]. ¹H
NMR (400 MHz, CDCl₃) δ 4.08 (dd, J = 5.4, 2.6 Hz, 1H, H-8), 2.29 (dd, J = 16.8, 3.5
Hz, 1H), 2.01 (dd, *J* = 5.4, 2.6 Hz, 2H), 1.90 – 1.76 (m, 3H), 1.59 – 1.44 (m, 6H), 1.38 – 1.32 (m, 1H), 1.31 – 1.23 (m, 1H), 1.21 – 1.11 (m, 2H), 1.06 (d, *J* = 6.6 Hz, 3H, Me-21), 0.94 (s, 3H, Me-18), 0.15 (s, 9H, 3 x Me-Si). <b>¹³C NMR</b> (101 MHz, CDCl₃) d 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). <b>HRMS</b> ([ESI]⁺, m/z) [M+H]⁺ calc. for C₁₈H₃₃OSi 293.2302; found. 293.2305.

(1R,3aR,4S,7aR)-7a-Methyl-1-[(R)-5-(trimethylsilyl)pentan-2-yl]octahydro-1H-inden-4-ol (31a). Pd/C (10%, 2 mg) was added to a solution of 30a (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 16 h. The mixture was filtered through a pad of celite and concentrated. The residue was flash chromatographed (SiO₂, 1 x 6 cm, 5% EtOAc/hexanes) to give 31a [19 mg, 0.06 mmol, 93%, Rf = 0.25 (20% EtOAc/hexanes), colorless oil], [α]D +119.5 (c1.00, CHCl₃). <b>¹H NMR</b> (400 MHz, CDCl₃) δ 4.07 (dd, *J* = 5.4, 2.6 Hz, 1H, H-8), 2.00 (d, *J* = 12.8 Hz, 1H), 1.90 – 1.75 (m, 3H), 1.57 – 1.22 (m, 12H), 1.20 – 0.99 (m, 4H), 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, 3 x Me-Si). <b>¹³C NMR</b> (101 MHz, CDCl₃) δ 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, 3 x Me-Si). <b>HRMS</b> ([ESI]⁺, m/z) [M+H]⁺ calc'd for C₁₈H₃₇OSi, 297.2615; found. 297.2617.

(1R,3aR,7aR)-7a-Methyl-1-[(R)-5-(trimethylsilyl)pentan-2-yl]octahydro-4H-inden-4-one (19a). DMP (42 mg, 0.10 mmol, 1.1 equiv) was added to a solution of 31a (26 mg, 0.09 mmol, 1 equiv) in CH₂Cl₂ (5 mL). The mixture was stirred at room temperature for 30 min and then filtered through celite, and concentrated. The residue was flash chromatographed (SiO₂, 1 x 6 cm, 5% EtOAc/hexanes) to give 19a [25 mg, 98%, Rf = 0.25 (10% EtOAc/hexanes), yellow oil, [α]D +48.8 (c1.03, CHCl₃)]. <b>¹H NMR</b> (400 MHz, CDCl₃) δ 2.44 (dd, *J* = 11.7, 7.4 Hz, 1H), 2.30 – 2.17 (m, 2H), 2.14 – 2.09 (m, 1H), 2.04 – 1.96 (m, 1H), 1.95 – 1.84 (m, 2H), 1.72 (dtd, *J* = 13.3, 11.6, 6.3 Hz, 1H), 1.62 – 1.47 (m, 2H), 1.40 – 1.26 (m, 5H), 0.93 (d, *J* = 6.1 Hz, 3H, Me-21), 0.91 – 0.83 (m, 2H), 0.64 (s, 3H, Me-18), 0.54 – 0.64 (m, 2H). -0.03 (s, 9H, 3 x Me-Si). <b>¹³C NMR</b> (101 MHz, CDCl₃) δ 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).
(CH₂), 20.7 (CH₂), 20.2 (CH₃, Me-21), 18.6 (CH₂, C-22), 14.1 (CH₃, Me-18), 0.0 (CH₃, 3 x Me-Si). **HRMS** ([ESI]⁺, m/z) [M+H]⁺ calcd for C₁₈H₃₅OSi, 295.2459; found. 295.2461.

**25,26,27-Trisnor-24-trimethylsilyl-1α-hydroxy-vitamin D₃ (Sila-1).** 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 **18** (118 mg, 0.2 mmol, 3.1 equiv) in THF (1.5 mL). The mixture was stirred for 1.5 h. Ketone **19a** (19 mg, 0.064 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. A saturated solution of NH₄Cl (10 mL) was added and the resulting mixture was extracted with EtOAc (3 x 10 mL). The combined organic layer was dried, filtered, and concentrated in vacuo. A mixture of CH₂Cl₂ and MeCN (1:1, 5 mL), and HF (aq 48%, 10 drops) were successfully added. The mixture was stirred at room temperature for 2 h and then poured into saturated NaHCO₃. The mixture was stirred for 30 min. The combined organic layers were extracted with CH₂Cl₂ (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, Ø 250 mm x 21.2 mm, 10% *i*-PrOH / hexanes) to give **Sila-1** [17.5 mg, 63%, Rf = 0.3 (75% EtOAc/hexanes), white solid]. [α]D +31.2 (c0.87, CHCl₃)]. **¹H NMR** (400 MHz, CDCl₃) d 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-1), 4.23 (dt, *J* = 6.6, 3.2 Hz, 1H, H-3), 2.82 (dd, *J* = 12.0, 4.2 Hz, 1H), 2.60 (dd, *J* = 13.4, 3.8 Hz, 1H), 2.31 (dd, *J* = 13.4, 6.6 Hz, 1H), 2.04 – 1.84 (m, 4H), 1.82 – 1.61 (m, 4H), 1.55 – 1.43 (m, 3H), 1.41 – 1.33 (m, 3H), 1.31 – 1.23 (m, 4H), 1.232H), 0.54 (s, 3H, Me-18), 0.50 – 0.35 (m, 2H, H-24), -0.03 (s, 9H, 3 x Me-Si). **¹³C NMR** (101 MHz, CDCl₃) d 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-1), 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, 3 x Me-Si). **IR** (film, cm⁻¹): 3394 (νOH), 2974, 2925, 2873, 2854, 1653, 1636, 1624, 790). **UV** (96% EtOH): λmax = 265 nm (ε = 21095), λmin = 230 nm]. **HRMS** ([ESI]⁺, m/z) [M+Na]⁺ calcd for C₂₇H₄₆O₂SiNa, 453.3165; found. 453.3163.
(1R,3aR,7aR)-7a-Methyl-1-[(R)-5-(trimethylsilyl)pent-4-yn-2-yl]octahydro-4H-
inden-4-one (23a). Dess-Martin periodinane (DMP, 107 mg, 0.25 mmol, 1.1 equiv) was
added to a solution of 30a (77 mg, 0.23 mmol, 1 equiv) in CH2Cl2. The mixture was
stirred at room temperature for 30 min and then filtered through a pad of celite and
concentrated. The residue was flash chromatographed (SiO2, 1 x 6 cm, 5% EtOAc
/hexanes) to give 23a [63 mg, 95%, Rf = 0.45 (10% EtOAc/hexanes), yellow oil, [α]D
+48.8 (c1.03, CHCl3)]. 1H NMR (400 MHz, CDCl3) δ 2.46 (dd, J = 11.6, 7.5 Hz, 1H),
2.33 – 2.20 (m, 3H), 2.14 – 1.97 (m, 3H), 1.95 – 1.84 (m, 2H), 1.79 – 1.69 (m, 1H), 1.63
– 1.49 (m, 4H), 1.34 – 1.25 (m, 1H), 1.10 (d, J = 6.1 Hz, 3H, Me-21), 0.64 (s, 3H, Me-
18), 0.15 (s, 9H, 3 x Me-Si). 13C NMR (101 MHz, CDCl3) δ 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, 3 x Me-Si). HRMS ([ESI]+, m/z) [M+H]⁺ calcd for
C18H31OSi2, 291.2146; found 291.2144.

**25,26,27-Trinor-24-trimethylsilyl-23-yne-1α-hydroxy-vitamin D₃ (Sila-3.**
**Procedure as for Sila-1.** 67%, Rf = 0.3 (75% EtOAc/hexanes), white solid, [α]D +48.8 (c1.03, CHCl3)]. 1H NMR (400 MHz, CDCl3) δ 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-1), 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), 2.06 – 1.95 (m, 6H), 1.93 – 1.82 (m, 2H), 1.72 – 1.63 (m, 2H), 1.60 – 1.43 (m, 4H), 1.40 – 1.21 (m, 3H), 1.07 (d, J = 6.5 Hz, 3H, Me-21), 0.54 (s, 3H, Me-18), 0.14 (s, 9H, 3 x Me-Si). 13C NMR (101 MHz, CDCl3) δ 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-1), 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). IR (film, cm⁻¹): 3396 (νOH), 2973, 2922, 2871, 2855, 2182, 1652, 1634, 1626, 793. UV (96% EtOH): λmax = 265 nm (ε = 16953), λmin = 230 nm]. HRMS ([ESI]⁺, m/z) [M+Na]⁺ calcd for C27H42O2SiNa, 449.2852; found. 449.2849.

**25,26,27-Trinor-23(24)-yne-1α-hydroxy-vitamin D₃ (24).** A solution of *n*-butyllithium in hexanes (1 M, 0.250 mL, 0.348 mmol, 2.9 equiv) was added to a -80 °C
cooled solution of phosphine oxide **18** (212 mg, 0.36 mmol, 3 equiv) in THF (1.5 mL). The mixture was stirred for 1.5 h. Ketone **23a** (40 mg, 0.12 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 mixture was treated with saturated NH₄Cl (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried, filtered, and concentrated in vacuo. The residue containing **22a** was immediately dissolved in dry THF (10 mL). A solution of TBAF in THF (1 M, 0.48 mL, 0.48 mmol, 4 equiv) was added. The mixture was stirred at room temperature for 16 h and then treated with saturated NH₄Cl. The resulting mixture was 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 **24** [36 mg, 86 % (2 steps), Rf = 0.25 (70% EtOAc/hexanes), white foam, [α]D₅ + 25.6 (c1.05, CHCl₃)]. **¹H NMR** (400 MHz, CDCl₃) δ 6.37 (d, *J* = 11.2 Hz, 1H, =CH), 6.02 (d, *J* = 11.3 Hz, 1H, =CH), 5.32 (s, 1H, H-19), 5.00 (s, 1H, H-19), 4.41 (dd, *J* = 7.6, 4.3 Hz, 1H, H-1), 4.23 (tt, *J* = 6.8, 3.7 Hz, 1H, H-3), 2.83 (dd, *J* = 12.1, 3.9 Hz, 1H), 2.59 (dd, *J* = 13.4, 3.2 Hz, 1H), 2.31 (dd, *J* = 13.9, 7.1 Hz, 1H), 2.25 (dt, *J* = 16.5, 3.1 Hz, 1H), 2.09 – 1.96 (m, 4H), 1.95 (t, *J* = 2.6 Hz, 1H), 1.93 – 1.83 (m, 2H), 1.74 – 1.23 (m, 11H), 1.10 (d, *J* = 6.5 Hz, 3H, Me-21), 0.56 (s, 3H, Me-18). **¹³C NMR** (101 MHz, CDCl₃) δ 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-1), 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). **HRMS** ([ESI]⁺, m/z) [M+H]⁺ calcd for C₂₄H₃₅O₂, 355.2639; found. 355.2641.

**25,26,27-Trisnor-23(24)-yne-24-dimethylsilyl-1α-hydroxy-vitamin D₃ (Sila-5).** A solution of *n*-butyllithium in hexanes (0.195 mL, 0.293 mmol, 4 equiv) was added to a 0 °C cooled solution of diol **24** (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 an aqueous 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. The residue was flash chromatographed (SiO₂, 1 x 7 cm, 70% EtOAc/hexanes) to give **Sila-5** [21 mg, 70%, Rf = 0.29 (70% EtOAc/hexanes),
white foam], [α]<sub>D</sub> +37.8 (c0.78, CHCl<sub>3</sub>]. ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ 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), 4.46 – 4.40 (m, 1H, H-3), 4.23 (tt, J = 6.8, 3.6 Hz, 1H, H-1), 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), 2.35 – 2.22 (m, 2H), 2.09 – 1.83 (m, 6H), 1.63 – 1.23 (m, 11H), 1.09 (dd, J = 6.5, 3.5 Hz, 3H, Me-18), 0.55 (s, 3H, Me-18), 0.22 (d, J = 3.7 Hz, 6H, 2 x Me-Si). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ 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 (CH<sub>2</sub>, 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 (CH<sub>2</sub>), 45.4 (CH<sub>2</sub>), 43.0 (CH<sub>2</sub>), 40.4 (CH<sub>2</sub>), 36.1 (CH), 29.2 (CH<sub>2</sub>), 27.6 (CH<sub>2</sub>), 27.2(CH<sub>2</sub>), 23.7 (CH<sub>2</sub>), 22.4 (CH<sub>2</sub>), 19.3 (CH<sub>3</sub>, Me-21), 12.2, (CH<sub>3</sub>, Me-18) -2.5 (CH<sub>3</sub>, 2 x Me-Si). IR (film, cm<sup>-1</sup>): 3393 (n<sub>OH</sub>), 2972, 2923, 2870, 2857, 2182, 1650, 1633, 1624, 792, 650). UV (96% EtOH): λ<sub>max</sub> = 265 nm (ε = 18025), λ<sub>min</sub> = 230 nm]. HRMS ([ESI]<sup>+</sup>, m/z) [M+Na]<sup>+</sup> calcd for C<sub>26</sub>H<sub>40</sub>O<sub>2</sub>SiNa, 435.2696; found. 435.2694.

tert-Butyl((R)-4-{(1R,3aR,4S,7aR)-4-[(tert-butyldimethylsilyl)oxy]-7a-methyloctahydro-1H-inden-1-yl}pent-1-yn-1-yl) dimethylsilane (20b). Procedure as for 20a. 20b: 87%, Rf = 0.64 (hexanes), colorless oil, [α]<sub>D</sub> + 30.9 (c1.02, CHCl<sub>3</sub>)]. ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ 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), 1.84 – 1.75 (m, 2H), 1.71 – 1.63 (m, 1H), 1.62 – 1.51 (m, 2H), 1.41 – 1.31 (m, 3H), 1.28 – 1.19 (m, 3H), 1.18 – 1.08 (m, 1H), 1.06 (d, J = 6.6 Hz, 3H, Me-21), 0.93 (s, 9H, *t*-Bu-Si), 0.92 (s, 3H, Me-18), 0.89 (s, 9H, *t*-BuSi), 0.08 (s, 6H, 2 x Me-Si), 0.01 (s, 3H, Me-Si), -0.00 (s, 3H, Me-Si). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ 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 (CH<sub>2</sub>), 35.3 (CH), 34.6 (CH<sub>2</sub>), 27.3 (CH<sub>2</sub>), 27.1 (CH<sub>2</sub>), 26.3 (CH<sub>3</sub> *t*-Bu-Si), 26.0 (CH<sub>3</sub> *t*-Bu-Si), 23.2 (CH<sub>2</sub>), 19.1 (CH<sub>3</sub>, Me-21), 18.2 (C, *t*-Bu-Si), 17.8 (CH<sub>2</sub>), 16.8 (C, *t*-Bu-Si), 14.0 (CH<sub>3</sub>, Me-18), -4.23 (CH<sub>3</sub>, 2 x Me-Si), -4.6 (CH<sub>3</sub>, Me-Si), -5.0 (CH<sub>3</sub>, Me-Si). HRMS ([ESI]<sup>+</sup>, m/z) [M+H]<sup>+</sup> calcd for C<sub>27</sub>H<sub>53</sub>OSi<sub>2</sub>, 449.3637; found 449.3639.

(1R,3aR,4S,7aR)-1-[(R)-5-(tert-Butyldimethylsilyl)pent-4-yn-2-yl]-7a-methyloctahydro-1H-inden-4-ol (30b). Procedure as for 30a. 30b: 98%, Rf = 0.24 (20% EtOAc/hexanes), colorless oil, [α]<sub>D</sub> -64.9 (c0.92, CHCl<sub>3</sub>)]. ¹H NMR (400 MHz,
CDCl₃) δ 4.08 (d, *J* = 2.6 Hz, 1H, H-8), 2.26 (dd, *J* = 16.8, 3.6 Hz, 1H), 2.08 (dd, *J* = 15.1, 7.7 Hz, 1H), 2.01 – 1.95 (m, 1H), 1.91 – 1.75 (m, 2H), 1.62 – 1.52 (m, 2H), 1.51 – 1.40 (m, 3H), 1.37 – 1.23 (m, 4H), 1.14 (td, *J* = 13.0, 3.5 Hz, 1H), 1H), 1.06 (d, *J* = 6.6 Hz, 3H, Me-21), 0.94 (s, 3H, Me-18), 0.93 (s, 9H, *t*-Bu-Si), 0.08 (s, 6H, 2 x Me-Si). <strong>¹³C NMR</strong> (101 MHz, CDCl₃) δ 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, *t*-Bu-Si), 22.5 (CH₂), 18.9 (CH₃, Me-21), 17.4 (CH₂), 16.6 (CH, *t*-Bu-Si), 13.7 (CH₃, Me-18), -4.4 (CH₃, 2 x Me-Si). <strong>HRMS</strong> ([ESI]⁺, m/z) [M+H]⁺ calcd for C₂₁H₃₉OSi, 335.2772; found. 335.2770.

(1R,3aR,7aR)-1-[(R)-5-(tert-Butyldimethylsilyl)pent-4-yn-2-yl]-7a-methyloctahydro-4H-inden-4-one (23b). Procedure as for 23a. 23b: 98%, Rf = 0.73 (20% EtOAc/hexanes), yellow oil, [α]D +25.1 (c1.40, CHCl3)]. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 2.44 (dd, *J* = 11.6, 7.5 Hz, 1H), 2.31 – 2.07 (m, 4H), 2.01 (dddt, *J* = 14.3, 7.3, 4.9, 2.4 Hz, 1H), 1.96 – 1.83 (m, 2H), 1.74 (dttd, *J* = 13.3, 11.8, 6.2 Hz, 1H), 1.66 – 1.48 (m, 4H), 1.38 – 1.21 (m, 2H), 1.42 – 1.21 (m, 2H, H-22); 1.11 (d, *J* = 6.0 Hz, 3H, Me-21), 0.93 (s, 9H, *t*-Bu-Si), 0.64 (s, 3H, Me-18), 0.08 (s, 6H, 2 x Me-Si). <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ 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 (CH<sub>2</sub>), 39.0 (CH<sub>2</sub>), 35.2 (CH), 27.4 (CH<sub>2</sub>), 27.1 (CH<sub>2</sub>), 26.2 (CH<sub>3</sub>, *t*-Bu-Si), 24.1 (CH<sub>2</sub>), 19.2 (CH<sub>3</sub>, Me-21), 19.2(CH<sub>2</sub>, C-22), 16.7 (C, C-*t*-Bu-Si), 12.7 (CH<sub>3</sub>, Me-18), -4.28 (CH<sub>3</sub>, 2 x Me-Si). <strong>HRMS</strong> ([ESI]⁺, m/z) [M+H]⁺ calcd for C₂₁H₃₇OSi, 333.2615; found. 333.2618.

25,26,27-Trisnor-23(24)-yne-24-dimethylsilyl-1α-hydroxy-vitamin D₃ (Sila-4).

**Procedures as for Sila-1. Sila-4:** 62% (two steps), Rf = 0.3 (70% EtOAc/hexanes), white foam]. [α]D +175.6 (c0.85, CHCl3)]. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 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-1), 4.23 (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, *t*-Bu-Si), 0.55 (s, 3H, Me-18), 0.08 (s, 6H, 2 x Me-Si). <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ 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, *t*-Bu-Si), 23.7 (CH₂), 22.4 (CH₂), 19.3 (CH₃, Me-21), 16.8 (C, C-*t*-Bu-Si), 12.3 (CH₃, Me-18), -4.25 (CH₃, 2 x Me-Si). **IR** (film, cm⁻¹): 3393 (n<sub>OH</sub>), 2971, 2921, 2870, 2854, 2183, 1653, 1633, 1625, 792. **HRMS** ([ESI]<sup>+</sup>, m/z) [M+Na]<sup>+</sup> calcd for C₃₀H₄₈O₂SiNa, 491.3322; found. 491.3320. **UV** (96% EtOH): λ<sub>max</sub> = 265 nm (ε = 18010), λ<sub>min</sub> = 230 nm].

(1R,3aR,4S,7aR)-1-[(R)-5-(tert-Butyldimethylsilyl)pentan-2-yl]-7a-methyloctahydro-1H-inden-4-ol (31b). Procedure as for 31a. 31b: 99%, Rf = 0.48 (15% EtOAc / hexanes), colorless oil. [α]<sub>D</sub> +99.9 (c1.00, CHCl<sub>3</sub>). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ 4.07 (d, *J* = 2.6 Hz, 1H, H-8), 2.02 – 1.97 (m, 1H), 1.88 – 1.78 (m, 2H), 1.57 – 1.28 (m, 11H), 1.25 (br s, 1H), 1.18 – 1.00 (m, 3H), 0.93 (s, 3H, Me-18), 0.88 (d, *J* = 6.6 Hz, 3H, Me-21), 0.86 (s, 9H, *t*-Bu-Si), 0.54 – 0.35 (m, 2H, H-24) -0.08 (s, 6H, 2 x Me-Si). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ 69.5 (CH, C-8), 56.8 (CH), 52.6 (CH), 41.9 (C, C-13), 40.4 (CH<sub>2</sub>), 40.3 (CH<sub>2</sub>), 35.2 (CH<sub>3</sub>), 33.6 (CH<sub>2</sub>), 27.2 (CH<sub>2</sub>), 26.6 (CH<sub>3</sub>, *t*-Bu-Si), 22.5 (CH<sub>2</sub>), 20.9 (CH<sub>2</sub>), 18.5 (CH<sub>3</sub>, Me-18), 17.4 (CH<sub>2</sub>), 16.5 (C, C-*t*-Bu-Si), 13.5 (CH<sub>3</sub>, Me-21), 12.9 (CH<sub>2</sub>, C-24), -6.2 (CH<sub>3</sub>, Me-Si), -6.3 (CH<sub>3</sub>, Me-Si). **HRMS** ([ESI]<sup>+</sup>, m/z) [M+H]<sup>+</sup> calcd for C<sub>21</sub>H<sub>43</sub>OSi, 339.3085; found. 339.3083.

(1R,3aR,7aR)-1-((R)-5-(tert-Butyldimethylsilyl)pentan-2-yl)-7a-methyloctahydro-4H-inden-4-one (19b). Procedure as for 19a. 19b: 98%, Rf = 0.60 (15% EtOAc/hexanes), yellow oil). [α]<sub>D</sub> +55.1 (c0.9, CHCl<sub>3</sub>). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ 2.44 (dd, *J* = 11.6, 7.5 Hz, 1H), 2.29 – 2.16 (m, 2H), 2.14 – 2.07 (m, 1H), 2.03 – 1.94 (m, 1H), 1.93 – 1.82 (m, 2H), 1.71 (dtd, *J* = 13.2, 11.5, 6.3 Hz, 1H), 1.62 – 1.44 (m, 2H), 1.43 – 1.23 (m, 5H), 1.21 – 1.00 (m, 2H), 0.92 (d, *J* = 6.0 Hz, 3H, Me-21), 0.84 (s, 9H, *t*-Bu-Si), 0.62 (s, 3H, Me-18), 0.53 – 0.34 (m, 2H, H-24), -0.09 (s, 6H, 2 x Me-Si). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ 212.2 (C, C-8), 62.0 (CH), 56.9 (CH), 50.0 (C, C-13), 41.0 (CH<sub>2</sub>), 40.3 (CH<sub>2</sub>), 39.0 (CH<sub>2</sub>), 35.4 (CH), 27.6 (CH<sub>2</sub>), 26.6 (CH<sub>3</sub>, *t*-Bu-Si), 24.1 (CH<sub>2</sub>), 20.9 (CH<sub>2</sub>), 19.1 (CH<sub>2</sub>), 18.7 (CH<sub>3</sub>, Me-21), 16.5 (C, *t*-Bu-Si), 12.9 (CH<sub>2</sub>, C-24), 12.5 (CH<sub>3</sub>, Me-18), -6.2 (CH<sub>2</sub>, Me-Si), -6.2 (CH<sub>2</sub>, Me-Si). **HRMS** ([ESI]<sup>+</sup>, m/z) [M+H]<sup>+</sup> calcd for C<sub>21</sub>H<sub>41</sub>OSi, 337.2928; found. 337.2925.
**25,26,27-Trisnor-24-tert-butyldimethylsilyl-1α-hydroxy-vitamin D₃ (Sila-2).**

**Procedures as for Sila-1. Sila-2:** 65% (two steps), Rf = 0.3 (70% EtOAc / hexanes), white foam]. [α]D +175.6 (c0.85, CHCl3)]. ¹H NMR (400 MHz, CDCl3) δ 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-1), 4.23 (tt, J = 6.5, 3.6 Hz, 1H, H-3), 2.85 – 2.78 (m, 1H), 2.60 (dd, J = 6.5, 3.7 Hz, 1H), 2.31 (dd, J = 13.3, 6.6 Hz, 1H), 2.06 – 1.86 (m, 5H), 1.70 – 1.55 (m, 5H), 1.50 – 1.23 (m, 8H), 1.21 – 0.99 (m, 2H), 0.91 (d, J = 6.4 Hz, 3H, Me-21), 0.86 (s, 9H, t-Bu-Si), 0.55 (s, 3H, Me-18), 0.52 – 0.35 (m, 2H, H-24), -0.08 (s, 6H, 2 x Me-Si). ¹³C NMR (101 MHz, CDCl3) d 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-1), 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, t-Bu-Si), 23.8 (CH2), 22.4 (CH2), 21.1 (CH2), 19.0 (CH3, Me-21), 16.7 (C, t-Bu-Si), 13.0 (CH2), 12.2 (CH3, Me-18), -6.0 (CH2, C-24), -6.1 (CH2, 2 x Me-Si). IR (film, cm⁻¹): 3394 (nOH), 2973, 2924, 2872, 2853, 1652, 1634, 1622, 791. HRMS ([ESI]⁺, m/z) [M+Na]⁺ calc'd for C₃₀H₅₂O₂SiNa, 495.3635; found. 495.3637. UV (96% EtOH): λmax = 265 nm (ε = 18808), λmin = 235 nm].

**21,25,26,27-Tetranor-20,23-diyne-24-trimethylsilyl-1α-hydroxy-vitamin D₃ (Sila-6).**

**Procedure as for Sila-1. Sila-6:** 78% (two steps), Rf = 0.89 (20% EtOAc/hexanes), colorless oil]. [α]D +1.5 (c1.05, CHCl3]. ¹H NMR (400 MHz, CDCl3) δ 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-1), 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), 0.93 – 0.78 (m, 2H, H-12), 0.67 (s, 3H, Me-18), 0.18 (s, 9H, 3 x Me-Si). ¹³C NMR (101 MHz, CDCl3) δ 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-1), 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, 3 x Me-Si). IR (film, cm⁻¹): 3394 (nOH), 2971, 2922, 2874, 2853, 2249, 2142, 1652, 1635, 1623, 789. HRMS ([ESI]⁺, m/z) [M+Na]⁺ calc'd for C₂₆H₃₆O₂SiNa, 431.2383; found. 431.2380. UV (96% EtOH): λmax = 265 nm (ε = 52072), λmin = 230 nm].
**Cell culture and Treatments.** All cell lines (normal human keratinocytes, HaCaT cells; human breast adenocarcinoma, MCF-7, and MDAMB-231 cells; human ovary adenocarcinoma, SKOV-3 cells; and human prostate adenocarcinoma, PC-3 cells) were obtained from ATCC-LGC (Barcelona, Spain) and were negative and periodically tested for mycoplasma. Cell lines were cultured in DMEM supplemented with charcoal-treated FCS to remove liposoluble hormones (Sigma-Aldrich, San Luis, USA) and grown in atmosphere air-CO₂ (95:5) at 37 °C. Treatments were performed after plating the cells for 24 h with 1,25D, and the sila-analogs (all at 10⁻⁷ M) dissolved in ethanol for 48 h. Ethanol was also used as a control. MDA-MB-231 cells were treated with paclitaxel (PT, 2 nM, Kabi, Spain) alone and combined with the 1,25D, or the **Sila-1-6** analogues at 10⁻⁷ M for 48 h. For three-dimensional (3D) cultures, a single-cell suspension containing 5×10³ of MDA-MB-231 cells/100 µl was placed on a 96-well plate cell repellent surface (Greiner Bio-One, Germany) for 6 days and then treated twice (days 6 and 8) with ethanol (control), 1,25D, or the sila-analogs at 10⁻⁷ M with or without paclitaxel (2 nM).

**Human VDR binding, serum calcium, and body weight evaluation.** VDR binding, calcium levels in mice, and body weight were evaluated as previously described.[59]

**Cell proliferation.** An MTT assay (Merck, Darmstadt, Germany) was used to evaluate cell proliferation in HaCat, MCF-7, SKOV-3, PC-3, and MDA-MB-231 cells (2x10⁵ cells/well) after plated in a 48-well and treated with the compounds alone and combined with chemotherapy for 48 h. Absorbance was measured at 570 nm (Mithras LB 940, Bad Wildbad, Germany). For 3D assays, the spheres were stained on day 10 with the CellTrakrackerTM Red CMTPX (Invitrogen, Carlsbad, USA) and photographed using an Olympus IX73 microscope (Olympus Iberia, Barcelona, Spain). Fluorescence intensity was quantified using the ImageJ software (National Institutes of Health, Bethesda, USA) and represented as relative units (RU).

**Real-time PCR, Western blot, and immunofluorescence.** MCF-7 and MDAMB-231 cells were treated as above described. Total RNA was isolated with TRIzol reagent (Invitrogen, Barcelona, Spain), and 1 µg of total RNA was used for cDNA synthesis. Amplification of the CYP24A1 gene (25-hydroxyvitamin D-24-hydroxylase) was
performed by real-time PCR (Eppendorf Master cycler ep realplex, Hamburg, Germany). Western blot was carried out as previously described.[59] E-cadherin (Cell Signaling, Danvers, USA), cyclin D1, p-27, and GAPDH (all from Santa Cruz Biotechnology, Dallas, USA) protein expression was quantified using ImageJ software. For immunofluorescence (IF), $1 \times 10^4$ cells/well were seeded in a 24-well plate containing coverslips pre-treated with poly-l-lysine (Sigma-Aldrich), fixed in cold absolute ethanol, blocked, and permeabilized. Cells were treated and incubated overnight with e-cadherin primary antibody (Cell Signaling) and followed by staining with Alexa-488 secondary antibody (Thermo Fisher Scientific). Samples were photographed using an Olympus IX73 microscope (Olympus Iberia).

**Biochemistry, Crystallization, and Structure Determination.** The cDNA encoding His-tagged zVDR LBD (156-453) was cloned into pET28b. The recombinant proteins were produced in Escherichia coli BL21 DE3 grown ON at 18 °C after induction with 1 mM IPTG at an OD600 of ~0.7. The recombinant proteins were produced in Escherichia coli BL21 DE3, grown ON at 18 °C after induction with 1 mM IPTG (OD600 of ~0.7). Soluble proteins were purified on Ni Hitrap FFcrude column (Cytiva), followed by His tag removal by thrombin cleavage and by size exclusion chromatography on HiLoad Superdex 75 column (Cytiva) equilibrated in Tris 20 mM pH7, NaCl 200 mM, TCEP 1 mM. The proteins were concentrated to 3-7 mg/mL with an Amicon Ultra 30 kDa MWCO and incubated with a 2-fold excess of ligand and a 3-fold excess of the coactivator NCOA2 NR2 (KHKILHRLLQDSS) peptide. Crystals of VDR-Sila-1 were obtained at 293K in $(NH_4)_2SO_4$ 1.5 M, Tris 0.1 M pH 8 and were mounted in a fiber loop and flash-cooled under a nitrogen flux after cryoprotection with $(NH_4)_2SO_4$ 2.2 M, Tris 0.1 M pH 8. Crystals of VDR-Sila-3 were obtained at 293K in NaAcetate 2.5 M and were mounted in a fiber loop and flash-cooled under a nitrogen flux after cryoprotection with NaAcetate 3 M. Crystals of VDR-Sila-4,5,6 were obtained at 293K in NaAcetate 2.5 M, Hepes 0.1 M pH 7.5 and were mounted in a fiber loop and flash-cooled under a nitrogen flux after cryoprotection with NaAcetate 3 M, Hepes 0.1 M pH 7.5. Data collection from a single frozen crystal was performed at 100 K on the ID30b at ESRF (France) for Sila-1,3,4 complexes and on the PX2A at SOLEIL (France) for Sila-5,6 complexes. The raw data were processed with XDS[60] and scaled with AIMLESS[61] programs. The crystals belong to the space group $P\bar{6}_522$, with one LBD complex per asymmetric unit. The structures were solved and refined
using Phenix[62], Buster[63] and iterative model building using COOT.[64]
Crystallographic refinement statistics are presented in Supporting Information Table S2.

## ASSOCIATED CONTENT

**Supporting Information.**

Additional supplemental figures and tables, synthetic procedures, and purity HPLC traces and characterization data for compounds can be found in SI appendix (PDF).

Molecular formula strings (CSV) with biochemical source data (CSV).

**Accession Codes.** Atomic coordinates for the X-ray structure of zVDR LBD-Sila-1 (PDB 9GY8), zVDR LBD-Sila-3 (PDB 9GYJ), zVDR LBD-Sila-4 (PDB 9GYC), zVDR LBD-Sila-5 (PDB 9GYA) and zVDR LBD-Sila-6 (PDB 9GYK) are available from the RCSB Protein Data Bank.

## AUTHOR INFORMATION

**Corresponding Authors:**

José E. Rodríguez-Borges

E-mail: jrborges@fc.up.pt

Román Pérez-Fernández

E-mail: roman.perez.fernandez@usc.es

Natacha Rochel

E-mail: rochel@igbmc.fr

Antonio Mouriño

E-mail: antonio.mourino@usc.es

## Author contributions

J. L. and S. S. contributed equally to this work.

## ACKNOWLEDGEMENTS

This work received support and help from FCT/MCTES (PTDC/BIA-MIB/29059/2017, LA/P/0008/2020
DOI 10.54499/LA/P/0008/2020, UIDP/50006/2020 DOI 10.54499/UIDP/50006/2020, and
UIDB/50006/2020 DOI 10.54499/UIDB/50006/2020), through national funds. I.E.S.-D. thanks FCT for
funding through the Individual Call to Scientific Employment Stimulus
(2020.02311.CEECIND/CP1596/CT0004 DOI 10.54499/2020.02311.CEECIND/CP1596/CT0004).
Funding from Xunta de Galicia (GRC/ED431B/2021/004 to A.M. and R.P.-F. and MICIN/AEI/PID2021-
127394OB-100 to R.P.F.) is gratefully acknowledged. The authors thank Dishman Netherlands BV for
the generous gift of vitamin D₂. We thank A. McEwen for help with X-ray data collection, and the staff of
PX2 beam line of SOLEIL synchrotron and of ID30 beam line of ESRF synchrotron for assistance during
X-ray data collection. The research was also supported by French Infrastructure for Integrated Structural
Biology (FRISBI) [ANR-10-INBS-05]; Instruct-ERIC; Agence Nationale de la Recherche under the
program Investissements d'Avenir [ANR-10-LABX-0030-INRT, ANR-10-IDEX-0002-02].

ABBREVIATIONS USED

1,25D₃, 1α,25-dihydroxyvitamin D₃; LBD, ligand-binding domain; VDR, vitamin D receptor.

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