# Design, synthesis and biological evaluation of a novel non-Gemini analog of UVB1 and crystal structure of its complex with the vitamin D receptor

Uxía Gómez-Bouzó, Carole Peluso-Iltis, Hugo Santalla, Lieve Verlinden, Annemieke Verstuyf, Natacha Rochel, Generosa Gómez, Yagamare Fall

► To cite this version:

Uxía Gómez-Bouzó, Carole Peluso-Iltis, Hugo Santalla, Lieve Verlinden, Annemieke Verstuyf, et al. Design, synthesis and biological evaluation of a novel non-Gemini analog of UVB1 and crystal structure of its complex with the vitamin D receptor. Bioorganic Chemistry, 2025, 157, pp.108239. 10.1016/j.bioorg.2025.108239. hal-05078960

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Design, Synthesis and Biological Evaluation of a Novel Non-Gemini
Analog of UVB1 and Crystal Structure of its Complex with the
Vitamin D Receptor

Uxía Gómez-Bouzó,¹ Carole Peluso-Iltis,²⁻⁵ Hugo Santalla,¹ Lieve Verlinden,⁶
Annemieke Verstuyf,⁶ Natacha Rochel²⁻⁵* Generosa Gómez¹* and Yagamare Fall,¹*

¹ Departamento de Química Orgánica and Instituto de Investigación Sanitaria Galicia Sur (IISGS), Campus Lagoas Marcosende, Universidad de Vigo, 36310 Vigo, Spain.

² Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, 67400, France;

³ CNRS UMR 7104, Illkirch, 67400, France;

⁴ Inserm U1258, Illkirch, 67400, France;

⁵ University of Strasbourg, Illkirch, 67400, France.

⁶ Clinical and Experimental Endocrinology, Department of Chronic Diseases and Metabolism, KU Leuven, 3000 Leuven, Belgium.

**Key words:** Calcitriol, antiproliferative agents, hypercalcemia, cancer, Gemini

*To whom correspondence should be addressed:
(YF) +34 986812323, e-mail: yagamare@uvigo.es
(GG) +34 986813679, e-mail: ggomez@uvigo.es
(NR) +33.369.48.52.93, e-mail: rochel@igbmc.fr

**Abstract:**

We synthesized **UG-650**, a novel non-Gemini analog of **UVB1** that combines structural features of **UVB1** and **MC 1288**. The evaluation of the bioactivities indicates that **UG-650** dose-dependently inhibited MCF-7 cell proliferation. In comparison to calcitriol, slightly lower concentrations of **UG-650** were needed to achieve equivalent levels of
proliferation inhibition. Structural analysis revealed that the analog forms additional
interactions stabilizing VDR active form.

# 1. INTRODUCTION

1α,25-Dihydroxyvitamin D₃ (calcitriol) (Fig. 1) is the hormonally active form of vitamin D₃ but the therapeutic utility of this hormone is hampered by its calcemic side effects [1]. There is accordingly much interest in the design and synthesis of new calcitriol analogs displaying less calcemic toxicity [2]. Among the more than 3000 synthetic calcitriol analogs, worth mentioning C-20 modified compounds such as 1α,25-dihydroxy-20-epi-vitamin D₃ [3] (MC 1288, Fig. 1) and Gemini type analog Ro-438-3582 [4] (Fig. 1) bearing a second side chain replacing the C-21 methyl group (Fig. 2), that are more active than calcitriol.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Structure of calcitriol, MC 1288, and Gemini type analog Ro-438-3582.</figcaption></figure>

Our research group developed a new synthetic procedure for Gemini analogs, using a
key sigmatropic rearrangement, providing a versatile method to introduce novel side-
chains to the vitamin D scaffold giving access to a variety of analogues with potentially
interesting biological properties. We thus synthesized and patented Gemini analog **Ro-438-3582** [5].

During the synthesis of **Ro-438-3582** we serendipitously synthesized a new Gemini type analog we coined **UVB1** (Fig. 2). **UVB1** (1,25(OH)₂-21-(3-hydroxy-3-methyl-1-methylene-butyl)vitamin D₃) carries one modified side chain in a 20-*epi* configuration) [6]. The *in vivo* biological evaluation showed that it has potent antitumoral effects over a wide range of tumor cell lines and lacks hypercalcemic activity and toxicity effects
[7]. Recent research has shown that **UVB1** exhibits antineoplastic activity in breast cancer patient-derived xenograft cells [8].

In view of these excellent biological results of **UVB1**, we thought that compound **UG-650** that contains the modified side chain of **UVB1** and a 20-*epi* methyl group, could be of interest. Each modification, 20-*epi* configuration, additional C22-methylene group and elongation of the side chain, were previously shown to enhance VDR activities [7-9].

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Structures of **UVB1** and **UG-650**.</figcaption></figure>

Here we describe the design and synthesis of the new compound **UG-650**, its binding mode to the Vitamin D nuclear receptor (VDR), and evaluation of some biological effects in comparison to calcitriol.

## 2. RESULTS AND DISCUSSION

2.1. **Design.** We anticipated that analog **UG-650** could be synthesized from alcohol **5** involving the construction of Grundmann's ketone **7** followed by Wittig-Horner coupling using phosphine oxide **8** to afford the target compound. The retrosynthetic analysis for **UG-650** is depicted in Scheme 1.
<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Retrosynthetic analysis of UG-650.</figcaption></figure>

Accordingly, chiral alcohol **5** was prepared as outlined in **Scheme 2**.

Inhoffen-Lythgoe diol was deprotected with silyl ether groups to afford 90% yield of compound **10**. TBAF selective deprotection of the primary silyl ether group of **10** gave 99% yield of alcohol **11**, which underwent a TEMPO, BAIB oxidation to give 90% yield of aldehyde **12**. Epimerization of **12** with DBU, followed by reaction with NaBH₄ afforded a mixture of alcohols **11** (46%) and **5** (43%). Recovered alcohol **11** could be oxidized again and the corresponding aldehyde epimerized to afford more quantity of target compound **5**.

<figure><img src="image_5.png" /></figure>
**Scheme 2.** Synthesis of alcohol **5**.

Alcohol **5** afforded iodine **13** via Appel reaction in 97% yield. The latter was transformed into nitrile **14** by treatment with KCN in DMF in 95% yield. DIBAL-H reduction of nitrile **14** afforded aldehyde **15** in 90% yield. Treatment of **15** with the lithium derivative of alkyne **16** afforded a mixture of epimeric propynyl alcohols **6** in 90% yield as depicted in **Scheme 3**.

<figure><img src="image_6.png" /></figure>

**Scheme 3.** Synthesis of propynyl alcohols **6**.

Oxidation of **6** followed by catalytic hydrogenation of **17** gave ketone **18** in 89% overall yield. Ketone **18** underwent the Wittig reaction to afford alkene **19** in 60% yield. TBAF deprotection of the hydroxyl groups of **19** afforded diol **20** in 90% yield. PDC oxidation and trimethylsilyl (TMS) protection of the side chain hydroxyl group afforded target ketone **7** in 81% overall yield as shown in Scheme 4.
<figure><img src="image_7.png" /><figcaption>**Scheme 4.** Synthesis of ketone 7.</figcaption></figure>

During the synthesis of ketone 7, we found out that diol 20 could be easily recrystallized in a mixture of MeOH:CH₂Cl₂ (2:1). The resulting monocrystal was analyzed by X-Ray Diffraction (Supplementary material Table S1) as shown in Figure 3. We thus confirmed unambiguously the stereochemistry at C-20 in UG-650.

<figure><img src="image_8.png" /><figcaption>**Figure 3.** ORTEP plot of the X-Ray single crystal structure of diol 20.</figcaption></figure>
With ketone 7 in hand, the stage was now set for the Wittig-Horner reaction with phosphine oxide 8 and the final desilylation to afford uneventfully 60% yield of the target compound **UG-650** (Scheme 5).

<figure><img src="image_9.png" /><figcaption>**Scheme 5.** Synthesis of target analog **UG-650**.</figcaption></figure>

**2.2. Binding mode of UG-650 to VDR ligand binding domain.** Nano differential scanning fluorimetry analysis confirmed the binding of **UG-650** compound (Fig. 4A) to the zebrafish (z)VDR ligand binding domain (LBD), a surrogate of the human vitamin D receptor [10-11]. Furthermore, **UG-650** promotes the interaction of NCOA1 coactivator peptide that encompasses one nuclear receptor LXXLL interacting motif, similarly as calcitriol (Fig. 4A). The binding mode of **UG-650** to VDR was determined by X-ray crystallography analysis of the complex formed by **UG-650**, the zVDR LBD and the NCoA2 NR2 peptide. The overall structure is highly homologous to the VDR-calcitriol structure with a root mean square deviation of 0.24 Å over 239 residues when comparing the Cα atoms for the two complexes. Superposition of the VDR LBP in the presence of the analog and calcitriol shows similar positioning of the ligands (Fig. 4B). The 20-epi configuration, the longer side chain and the additional methylene group in **UG-650**, induce a tilt of the D-ring and position the 17β-chain on the opposite side of the pocket. Superposition with MC1288 complex [12] indicates that the side chain of the 2 ligands have similar positions (Fig. 4C). The positions of the 3 hydroxyl groups are similar as in the VDR-calcitriol complex. The distances between 1-OH and 25-OH were found to be 13.2 Å and 13.1 Å for **UG-650** and calcitriol, respectively. Those between 3-OH and 25-OH were 15.6 Å and 15.4 Å, respectively. The hydrogen bonds with the 3 hydroxyl groups of **UG-650** are similar to those of 1,25D₃ as well as the interactions between the A-, Seco B-, and C/D- rings and VDR. Differences are
observed around the side chain (Fig. 4D and Supplementary material Table S2). New or
increased contacts are formed between the carbon atom of the additional methylene
group and Leu258 and Val328. As a consequence of the different path of the ligand side
chain, the C21 methyl group forms increased interaction with Met300 and the geminal
methyl group with Leu440. In contrary, calcitriol side chain forms increased
interactions with Leu337 and Val444.

<figure><img src="image_10.png" /></figure>

**Figure 4.** Binding mode of **UG-650** to zVDR. (A) Thermal stability of the purified zVDR LBD alone and upon binding to the ligands in absence or presence of the NCOA1 NR2 coactivator peptide. (B-D) Crystal structure of zVDR-**UG-650** complex. (B) Hydrogen bonds formed by compounds **UG-650** (green) and calcitriol (PDB 2HC4) (violet) are shown by red dotted lines. (C) Superposition of **UG-650** (green), calcitriol (violet) and MC1288 (PDB 1IE9) (pink). (D) Details of the interactions mediated **UG-650** (green) or calcitriol (violet) with residues of the zVDR LBD at a 4 Å distance cutoff. Dashed lines correspond to interactions formed by **UG-650**. Residues forming increased interactions with **UG-650** or calcitriol are shown in green or violet and underlined.

**2.3. Biological Evaluation of UG-650.**
Calcitriol is a known growth inhibitor and differentiation inducer of several cancer cell lines and its molecular mechanism of antiproliferation in estrogen receptor positive human breast adenocarcinoma MCF-7 cells is well established [13]. The antiproliferative activity of **UG-650** was evaluated in MCF-7 cells and was compared with the activity of calcitriol and expressed relative to proliferation of vehicle (EtOH)-stimulated cells (Fig. 5). **UG-650** dose-dependently inhibited MCF-7 cell proliferation. In comparison to calcitriol, slightly lower concentrations of **UG-650** were needed to achieve equivalent levels of proliferation inhibition. Consequently, the concentration to achieve half-maximal inhibition of cell proliferation (EC₅₀) is lower for **UG-650** (5 x 10⁻⁹ M) than for calcitriol (3 x 10⁻⁸ M). Notably, a previously published compound **EB1231**, a 20-epi vitamin D analog with a side chain extended by one carbon atom but lacking the methylene group at C23, was also more potent than calcitriol in inhibiting cell proliferation (human U937 histiocytic lymphoma cells, mouse primary T-cells) by 2 orders of magnitude [9]. Although a direct head-to-head comparison is not possible, introduction of an additional methylene group at C23, as present in **UG-650**, seems to decrease the biological activity.

<figure><img src="image_11.png" /></figure>

**Figure 5.** UG-650 exerts a dose-dependent inhibition of MCF-7 proliferation. MCF-7 cells were treated for 72 h with vehicle (EtOH), or increasing doses of calcitriol or UG-650, after which [³H]thymidine incorporation was measured. \* p < 0.05, \*\*\* p < 0.0001 versus vehicle treatment. \$\$ p < 0.01, \$\$\$ p < 0.0001 versus the same concentration of calcitriol.
Mechanistically, we demonstrated that calcitriol and the analog **UG-650** exerted their antiproliferative effects by significantly inducing protein levels of the vitamin D receptor (VDR) in MCF-7 cells (Fig. 6). Both calcitriol and **UG-650** induced VDR protein levels to the same extent. Interestingly, treatment with the analog **UG-650** also reduced protein abundance of cyclin-dependent kinase 2 (CDK2), an important cell cycle regulatory protein, whereas this reduction was not observed after treatment with calcitriol (Fig. 6). These data demonstrate that **UG-650** exhibits a greater antiproliferative activity compared to calcitriol.

<figure><img src="image_12.png" /></figure>

**Figure 6.** UG-650 significantly induces VDR protein levels and downregulates CDK2 protein levels in MCF-7 cells. (A) MCF-7 cells were treated for 48 h with vehicle (EtOH), calcitriol (3 x 10⁻⁸ M), or UG-650 (3 x 10⁻⁸ M). Total protein lysates were extracted and used for Western blotting to visualize protein levels of VDR, CDK2, and Actin. (B) Protein bands were quantified, and the protein levels of VDR and CDK2 were normalized to Actin. Data were analyzed with one-way ANOVA and significant differences are indicated. * p < 0.05

We next investigated whether calcitriol and the analog **UG-650** induced apoptosis of MCF-7 cells (Fig. 7). However, although more than in vehicle-stimulated cells, very few apoptotic cells were visible after a 72 h incubation period with calcitriol or **UG-650**. Moreover, no differences were observed between calcitriol and **UG-650**, illustrating that these compounds mainly slow down cell proliferation without inducing

<figure><img src="image_13.png" /></figure>
programmed cell death.

**Figure 7. UG-650 is not a strong inducer of apoptotic cell death.** MCF-7 cells were treated for 72 h with vehicle (EtOH), calcitriol ($3 \times 10^{-8}$ M), or **UG-650** ($3 \times 10^{-8}$ M). Apoptotic cells are stained red. Scale bar: 100 µm.

Because MCF-7 cells showed very little migration in the scratch-wound assay (Supplementary material Fig. S1), we performed scratch-wound assays in the mouse pre-osteoblast MC3T3-E1 cell line. Serum-starved, vehicle-treated MC3T3-E1 cells (Supporting Fig. S2, left panel) migrated over a longer distance than serum-starved MC3T3-E1 cells that were treated with calcitriol or with **UG-650** (Fig. 8 and Supplementary material Fig. S2, middle and right panel). No differences between calcitriol- or **UG-650**-treated cells were observed.

<figure><img src="image_14.png" /></figure>

**Figure 8. UG-650 inhibits migration of mouse MC3T3-E1 (pre)osteoblasts.** Scratches were made in confluent monolayers of serum-starved MC3T3-E1 cells that were treated with vehicle (EtOH), calcitriol ($3 \times 10^{-8}$ M), or **UG-650** ($3 \times 10^{-8}$ M). Cell migration distance was analyzed after 48 h.

**2.4. CONCLUSION**

In summary, we described the synthesis of a novel analog, **UG-650**, that combines structural features of **UVB1** and **MC 1288**, and carries one modified side chain in a 20-*epi* configuration. We showed that **UG-650** mediates biological effects similar to calcitriol. Structural analysis of the complex with VDR LBD revealed that **UG-650** maintain the anchoring interactions formed by calcitriol and form new or increased interactions that stabilize VDR active conformation.
### 3. EXPERIMENTAL SECTION

**3.1. Chemistry.** Solvents were purified and dried by standard procedures before use. Melting points are uncorrected. ¹H-NMR and ¹³C-NMR spectra were recorded with a Bruker ARX-400 spectrometer (400 MHz for ¹H-NMR, 100.61 MHz for ¹³C NMR) using TMS as internal standard (chemical shifts in δ values, *J* in Hz). Flash chromatography (FC) was performed on silica gel (Merck 60, 230-400 mesh); analytical TLC was performed on plates precoated with silica gel (Merck 60 F254, 0.25mm). Mass spectra (ESI-MS) spectroscopy was recorded using Bruker FTMS APEXIII. IR spectra were recorded on a JASCO FT/I(R)-6100 spectrophotometer.

#### Synthesis of (8β)-Des-A,B-8,22-bis[(tert-butyldimethyl)silyloxy]-23,24-dinorcholane (10)

<figure><img src="image_15.png" /></figure>

To a solution of **Inhoffen-Lythgoe diol** (2.0 g, 9.42 mmol, 1.0 equiv.) in anhydrous DMF (10.0 mL) at rt was added Im (2.6 g, 37.68 mmol, 4.0 equiv.), DMAP (172 mg, 1.41 mmol, 15 mol%) and TBSCl (4.3 g, 28.25 mmol, 3.0 equiv.), and the solution was stirred at this temperature for 4 days. Then, the mixture reaction was quenched by addition of H₂O (15 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 15 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by FCC (SiO₂; using hexane) affording the product as a colorless oil (4.3 g, 90%).

**Compound 10:** ¹H-NMR (400 MHz, CDCl₃): δ 4.06 – 4.03 (m, 1H, H-8), 3.61 (dd, *J* = 9.6, 3.3 Hz, 1H, CH₂-22), 3.32 (dd, *J* = 9.6, 7.3 Hz, 1H, CH₂-22), 2.05 – 1.96 (m, 1H, H-20), 1.94 – 1.47 (m, 5H), 1.46 – 1.22 (m, 5H), 1.22 – 1.04 (m, 2H), 1.01 (d, *J* = 6.5
Hz, 3H, CH₃-21), 0.97 (s, 3H, CH₃-18), 0.93 (s, 18H, CH₃-'Bu), 0.06 (s, 6H, CH₃-Si), 0.07 (s, 3H, CH₃-Si), 0.06 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 69.7 (CH-8), 68.1 (CH₂-22), 53.7 (CH-17), 53.2 (CH-14), 42.4 (C-13), 41.0 (CH₂), 38.9 (CH-20), 34.8 (CH₂), 27.1 (CH₂), 26.3 (CH₃-'Bu), 26.1 (CH₃-'Bu), 23.5 (CH₂), 18.6 (C-'Bu), 18.3 (C-'Bu), 18.0 (CH₂), 17.2 (CH₃-21), 14.1 (CH₃-18), -4.5 (CH₃-Si), -4.9 (CH₃-Si), -5.1 (CH₃-Si), -5.1 (CH₃-Si); IR (ATR, cm⁻¹): ν 2949, 2930, 2855, 1473, 1456, 1259, 1252, 1096, 1022, 853, 776; MS (ESI): m/z (%) 441.4 ([M + H]⁺, 100), 309.3 ([M - OTBS]⁺, 5); HRMS (ESI): m/z calculated for C₂₅H₅₃O₂Si₂ [M + H]⁺ 441.3578, found 441.3572; TLC (SiO₂; 10% EtOAc/hexane): R_f = 0.89.

**Synthesis of (8β)-Des-A,B-8-[(tert-butyldimethyl)silyloxy)]-23,24-dinorcholan-22-ol**
(11)

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

To a solution of **10** (3.8 g, 8.61 mmol, 1.0 equiv.) in anhydrous THF (12.0 mL) at rt was added TBAF (1.0 M in THF, 9.5 mL, 9.47 mmol, 1.1 equiv.) and the resulting mixture was stirred at this temperature for 21 h. The solvent was removed *in vacuo* and the crude material was purified by FCC (SiO₂; using 30% EtOAc/hexane) affording the product as a colorless oil (2.8 g, 99%).

**Compound 11:** ¹H-NMR (400 MHz, CDCl₃): δ 4.00 (q, *J* = 2.6 Hz, 1H, H-8), 3.62 (dd, *J* = 10.5, 3.2 Hz, 1H, CH₂-22), 3.36 (dd, *J* = 10.5, 6.8 Hz, 1H, CH₂-22), 1.94 (dt, *J* = 12.5, 3.0 Hz, 1H, H-20), 1.89 - 1.72 (m, 2H), 1.71 - 1.64 (m, 1H), 1.60 - 1.50 (m, 2H), 1.40 - 1.30 (m, 4H), 1.29 - 1.18 (m, 2H), 1.17 - 1.08 (m, 2H), 1.01 (d, *J* = 6.6 Hz, 3H, CH₃-21), 0.93 (s, 3H, CH₃-18), 0.88 (s, 9H, CH₃-'Bu), 0.01 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 69.5 (CH-8), 68.1 (CH₂-22), 53.3 (CH-17), 53.0 (CH-14), 42.3 (C-13), 40.7 (CH₂), 38.4 (CH-20), 34.8 (CH₂), 27.0 (CH₂), 26.0 (CH₃-'Bu), 23.3 (CH₂), 18.2 (C-'Bu), 17.8 (CH₂), 16.8 (CH₃-21), 13.9 (CH₃-18), -4.7
(CH₃-Si), -5.0 (CH₃-Si); **IR** (ATR, cm⁻¹): ν 3337, 2949, 2930, 2856, 1471, 1360, 1252, 1164, 1003, 835, 773; **MS** (ESI): *m/z* (%) 349.3 ([M + Na]⁺, 100), 327.3 ([M + H]⁺, 15); **HRMS** (ESI): *m/z* calculated for C₁₉H₃₈NaO₂Si [M + Na]⁺ 349.2539, found 349.2533; **TLC** (SiO₂; 20% EtOAc/hexane): R_f = 0.44.

**Synthesis of (8β)-Des-A,B-8-[(tert-butyldimethyl)silyloxy)]-23,24-dinorcholan-22-al (12)**

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

To a solution of **11** (531 mg, 1.63 mmol, 1.0 equiv.) in CH₂Cl₂ (16.0 mL) at rt was added BAIB (603 mg, 1.96 mmol, 1.2 equiv.) and TEMPO (38 mg, 0.24 mmol, 15 mol%), and the solution was stirred at this temperature for 19 h. The solvent was removed *in vacuo* and the crude material was dissolved in ¹BuOMe (15 mL) and washed with 10% w/v aq. Na₂S₂O₃ solution (2x 10 mL) and sat. aq. NaHCO₃ solution (2x 10 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using 3% EtOAc/hexane) affording the product as a colorless oil (474 mg, 90%).

**Compound 12:** ¹H-NMR (400 MHz, CDCl₃): δ 9.56 (d, *J* = 3.2 Hz, 1H, CHO), 4.01 (q, *J* = 2.4 Hz, 1H, H-8), 2.41 - 2.28 (m, 1H, H-20), 1.95 - 1.74 (m, 2H), 1.73 - 1.54 (m, 2H), 1.47 - 1.13 (m, 8H), 1.08 (d, *J* = 6.8 Hz, 3H, CH₃-21), 0.95 (s, 3H, CH₃-18), 0.87 (s, 9H, CH₃-¹Bu), 0.00 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 205.5 (CHO), 69.2 (CH-8), 52.5 (CH-20), 51.8 (CH-17), 49.3 (CH-14), 42.7 (C-13), 40.5 (CH₂), 34.5 (CH₂), 26.3 (CH₂), 25.9 (CH₃-¹Bu), 23.4 (CH₂), 18.1 (C-¹Bu), 17.7 (CH₂), 14.2 (CH₃-21), 13.5 (CH₃-18), -4.7 (CH₃-Si), -5.1 (CH₃-Si); **IR** (ATR, cm⁻¹): ν 2951, 2929, 2857, 1726, 1459, 1373, 1252, 1164, 923, 850, 774; **MS** (ESI): *m/z* (%) 347.2 ([M + Na]⁺, 100), 325.3 ([M + H]⁺, 64); **HRMS** (ESI): *m/z* calculated for
$C_{19}H_{36}NaO_2Si [M + Na]^+ 347.2339$, found 347.2377; **TLC** ($SiO_2$; 20% EtOAc/hexane):
$R_f = 0.79$.

Synthesis of (8β)-(20R)-Des-*A*,*B*-8-[(tert-butyldimethyl)silyloxy]-23,24-dinorcholan-22-ol (5)

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

To a solution of **12** (218 mg, 0.67 mmol, 1.0 equiv.) in anhydrous THF (2.5 mL) at rt was added DBU (120 µL, 0.80 mmol, 1.2 equiv.) dropwise and the solution was refluxed for 20 h. Then the mixture was allowed to reach rt, was added Et₂O (7 mL) and the layers were separated. The organic phase was washed with 1.0 M aq. HCl solution (7 mL), H₂O (7 mL) and sat. aq. NaCl solution (7 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated *in vacuo*. To a solution of the crude material from the prior reaction in dry MeOH (4.5 mL) at 0 °C was added NaBH₄ (125 mg, 3.36 mmol, 5.0 equiv.) and the solution was stirred at this temperature for 5 min. Then, the mixture reaction was quenched by addition of H₂O (5 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 8 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by several FCCs ($SiO_2$; using 2-5% EtOAc/hexane) affording the alcohol **5** in the first fractions as a colorless oil (101 mg, 46%) and the alcohol **11** in the last fractions as colorless oil (96 mg, 43%) whose physical constants and spectroscopic data virtually coincide with those described for this compound in the previous section.

**Compound 1.58:** ¹H-NMR (400 MHz, CDCl₃): δ 4.00 (q, *J* = 2.6 Hz, 1H, H-8), 3.71 (dd, *J* = 10.7, 3.6 Hz, 1H, CH₂-22), 3.44 (dd, *J* = 10.6, 7.0 Hz, 1H, CH₂-22), 1.89 – 1.72 (m, 3H), 1.71 – 1.50 (m, 3H), 1.41 – 1.06 (m, 8H), 0.93 (m, 6H, CH₃-18, CH₃-21), 0.88 (s, 9H, CH₃'-Bu), 0.01 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz,
CDCl₃): δ 69.5 (CH-8), 67.0 (CH₂-22), 53.2 (CH-20), 53.1 (CH-17), 42.1 (C-13), 40.3 (CH₂), 37.6 (CH-14), 34.6 (CH₂), 26.9 (CH₂), 26.0 (CH₃'-Bu), 23.0 (CH₂), 18.2 (C'-Bu), 17.8 (CH₂), 16.7 (CH₃-21), 14.2 (CH₃-18), -4.7 (CH₃-Si), -5.0 (CH₃-Si); **IR** (ATR, cm⁻¹): ν 3337, 2951, 2930, 2886, 1471, 1360, 1252, 1164, 1003, 835, 773; **MS** (ESI): *m/z* (%) 349.3 ([M + Na]⁺, 100), 327.2 ([M + H]⁺, 15); **HRMS** (ESI): *m/z* calculated for C₁₉H₃₈NaO₂Si [M + Na]⁺ 349.2539, found 349.2533; **TLC** (SiO₂; 30% EtOAc/hexane): R_f = 0.60.

## Synthesis of (8β)-(20R)-Des-*A*,*B*-8-[(tert-butyldimethyl)silyloxy]-22-iodo-23,24-dinorcholane (13)

<figure><img src="image_19.png" /></figure>

To a solution of **5** (105 mg, 0.32 mmol, 1.0 equiv.) in anhydrous THF (3.5 mL) at rt was added PPh₃ (126 mg, 0.48 mmol, 1.5 equiv.) and Im (65 mg, 0.96 mmol, 3.0 equiv.). Then, the mixture was cooled to 0 °C, added I₂ (113 mg, 0.45 mmol, 1.4 equiv.), allowed to reach rt and left standing for 1 h. The mixture reaction was quenched with sat. aq. NaHCO₃ solution (4 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 5 mL) and extracts were washed with 10% w/v aq. Na₂S₂O₃ solution (2x 4 mL), dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using hexane) affording the product as a colorless oil (135 mg, 97%).

**Compound 13:** ¹H-NMR (400 MHz, CDCl₃): δ 3.99 (q, *J* = 2.7 Hz, 1H, H-8), 3.46 (dd, *J* = 9.6, 3.0 Hz, 1H, CH₂-22), 3.36 (dd, *J* = 9.6, 6.3 Hz, 1H, CH₂-22), 1.87 - 1.73 (m, 2H), 1.70 - 1.63 (m, 1H), 1.59 - 1.49 (m, 1H), 1.46 - 1.14 (m, 9H), 0.94 (d, *J* = 6.4 Hz, 3H, CH₃-21), 0.91 (s, 3H, CH₃-18), 0.88 (s, 9H, CH₃'-Bu), 0.01 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 69.4 (CH-8), 55.3 (CH-17), 52.8 (CH-14), 42.1 (C-13), 40.6 (CH₂), 36.3 (CH-20), 34.4 (CH₂), 27.1 (CH₂), 25.9 (CH₃'-Bu),
22.9 (CH₂), 21.6 (CH₃-21), 19.9 (CH₂-22), 18.2 (C-`tBu`), 17.8 (CH₂), 14.3 (CH₃-18), -4.6 (CH₃-Si), -5.0 (CH₃-Si); **IR** (ATR, cm⁻¹): v 2950, 2927, 2854, 1461, 1376, 1251, 1166, 1084, 1070, 1018, 954, 923, 873, 851, 835, 791, 773, 687; **MS** (ESI): m/z (%) 459.2 ([M + Na]⁺, 100), 437.2 ([M + H]⁺, 20); **HRMS** (ESI): m/z calculated for C₁₉H₃₇InaOSi [M + Na]⁺ 459.1626, found 459.1623; **TLC** (SiO₂; 20% EtOAc/hexane): R_f = 0.94.

Synthesis of (8β)-(20S)-Des-*A*,*B*-8-[(tert-butyldimethyl)silyloxy)]-23,24-dinorcholane-22-carbonitrile (14)

<figure><img src="image_20.png" /></figure>

To a solution of **13** (264 mg, 0.61 mmol, 1.0 equiv.) in anhydrous DMF (5.5 mL) at rt was added NaCN (89 mg, 1.81 mmol, 3.0 equiv.) and the solution was stirred at this temperature for 3 h. The mixture reaction was quenched by addition of H₂O (5 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 7 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by FCC (SiO₂; using 3% EtOAc/hexane) affording the product as a colorless oil (192 mg, 95%).

**Compound 14:** ¹H-NMR (400 MHz, CDCl₃): δ 4.00 (q, *J* = 2.6 Hz, 1H, H-8), 2.48 – 2.31 (m, 2H, CH₂-22), 1.88 – 1.70 (m, 4H), 1.71 – 1.60 (m, 1H), 1.60 – 1.47 (m, 1H), 1.46 – 1.23 (m, 7H), 1.05 (d, *J* = 6.7 Hz, 3H, CH₃-21), 0.90 (s, 3H, CH₃-18), 0.87 (s, 9H, CH₃-*tBu*), 0.00 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 118.9 (CN), 69.3 (CH-8), 55.1 (CH-17), 52.8 (CH-14), 42.0 (C-13), 40.3 (CH₂), 34.2 (CH₂), 31.9 (CH-14), 27.1 (CH₂), 25.9 (CH₃-*tBu*), 24.1 (CH₂-22), 22.9 (CH₂), 19.7 (CH₃-20), 18.1 (C-*tBu*), 17.6 (CH₂), 14.1 (CH₃-18), -4.7 (CH₃-Si), -5.1 (CH₃-Si); **IR** (ATR, cm⁻¹): v 2951, 2928, 2855, 1471, 1251, 1020, 834, 772; **MS** (ESI):
$m/z$ (%) 336.3 ([M + H]$^+$, 100), 337.3 (29); **HRMS** (ESI): $m/z$ calculated for $C_{19}H_{38}NOSi [M + H]^+$ 336.2717, found 336.2712; **TLC** ($SiO_2$; 10% EtOAc/hexane): $R_f = 0.76$.

**Synthesis of (8β)-(20S)-Des-A,B-8-[(tert-butyldimethyl)silyloxy)]-24-norcholan-23-αl (15)**

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

To a solution of **14** (131 mg, 0.39 mmol, 1.0 equiv.) in anhydrous THF (8.0 mL) at -78 °C was added DIBAL-H (1.0 M in hexane, 584 µL, 0.58 mmol, 1.5 equiv.) dropwise and the solution was stirred at this temperature for 30 min and after was worked up according to Fieser method. The mixture was allowed to warm up to rt and was added tBuOMe (5 mL), $H_2O$ (1 mL) and the mixture was left standing until the formation of a translucid gel. Subsequently, was added 4.0 M aq. NaOH solution (1 mL), $H_2O$ (1 mL) and the mixture was left standing until the formation of white solid. The solvent was dried over $Na_2SO_4$, filtered, and concentrated in vacuo. The crude material was purified by FCC ($SiO_2$; using 3% EtOAc/hexane) affording the product as a colorless oil (119 mg, 90%).

**Compound 15:** ¹H-NMR (400 MHz, $CDCl_3$): δ 9.73 (dd, *J* = 3.7, 1.3 Hz, 1H, CHO), 4.00 (q, *J* = 2.7 Hz, 1H, H-8), 2.64 (dd, *J* = 15.9, 3.0 Hz, 1H, $CH_2-22$), 2.26 – 2.12 (m, 1H, $CH_2-22$), 2.08 – 1.94 (m, 1H), 1.89 – 1.74 (m, 2H), 1.72 – 1.63 (m, 1H), 1.47 – 1.02 (m, 9H), 0.95 (s, 3H, $CH_3-18$), 0.91 (d, *J* = 6.6 Hz, 3H, $CH_3-21$), 0.89 (s, 9H, $CH_3-tBu$), 0.01 (s, 3H, $CH_3-Si$), 0.00 (s, 3H, $CH_3-Si$); ¹³C-NMR (101 MHz, $CDCl_3$): δ 203.6 (CHO), 69.4 (CH-8), 56.3 (CH-17), 53.1 (CH-14), 50.1 ($CH_2-22$), 42.3 (C-13), 40.8 ($CH_2$), 34.4 ($CH_2$), 30.4 (CH-20), 27.2 ($CH_2$), 26.0 ($CH_3-tBu$), 23.0 ($CH_2$), 19.9 ($CH_3-21$), 18.2 ($C-tBu$), 17.8 ($CH_2$), 14.3 ($CH_3-18$), -4.6 ($CH_3-Si$), -5.0 ($CH_3-Si$); **IR** (ATR,
cm⁻¹): v 2950, 2928, 2855, 1707, 1471, 1252, 1023, 836, 774; **MS** (ESI): *m/z* (%) 337.3
([M - H]⁺, 45), 223.2 (100); **HRMS** (ESI): *m/z* calculated for C₂₀H₃₇O₂Si [M - H]⁺
337.2557, found 337.2567; **TLC** (SiO₂; 10% EtOAc/hexane): R_f = 0.64.

**Synthesis of (8β)-(20S)-Des-A,B-8-[(tert-butyldimethyl)silyloxy]-23-(3'-methyl-3'-[(trimethyl)silyloxy]but-1'-ynyl)-24-norcholan-23-ol (6)**

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

To a solution of alkyne **16** (246 mg, 1.57 mmol, 3.1 equiv.) in anhydrous THF (8.0 mL) at -78 °C was added *n*-BuLi (2.5 M in hexane, 576 µL, 1.44 mmol, 2.9 equiv.) dropwise and the solution was stirred at 0 °C for 2 h. Then, **15** (169 mg, 0.50 mmol, 1.0 equiv.) was added in anhydrous THF (12.0 mL) and the resulting mixture was stirred at 0 °C for 1 h. The mixture reaction was quenched with sat. aq. NH₄Cl solution (10 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 10 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using 5 % EtOAc/hexane) affording the product as a colorless oil (223 mg, 90%).

**Compound 6:** ¹H-NMR (400 MHz, CDCl₃): δ 4.43 (dd, *J* = 9.3, 4.5 Hz, 1H, H-23), 3.99 (q, *J* = 2.8 Hz, 1H, H-8), 2.05 - 1.96 (m, 2H), 1.87 - 1.62 (m, 4H), 1.48 (s, 6H, CH₃-4', CH₃-5'), 1.44 - 1.21 (m, 8H), 1.16 - 1.07 (m, 2H), 0.95 (s, 3H, CH₃-18), 0.88 (s, 9H, CH₃-'Bu), 0.85 (d, *J* = 6.5 Hz, 3H, CH₃-21), 0.19 (s, 9H, CH₃-Si), 0.01 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 90.6 (C-1'), 83.6 (C-2'), 69.6 (CH-8), 66.7 (C-3'), 61.7 (CH-23), 57.1 (CH-17), 53.2 (CH-14), 43.9 (CH₂-22), 42.4 (C-13), 41.0 (CH₂), 34.6 (CH₂), 33.2 (CH₃-4', CH₃-5'), 32.4 (CH-20), 27.1 (CH₂), 26.0 (CH₃-'Bu), 23.0 (CH₂), 18.8 (CH₃-21), 18.2 (C-'Bu), 17.8 (CH₂), 14.2 (CH₃-18), 2.1 (CH₃-Si), -4.6 (CH₃-Si), -5.0 (CH₃-Si); *IR* (ATR, cm⁻¹): v 3422, 2952, 2930, 2857,
1471, 1462, 1377, 1360, 1249, 1164, 1081, 1036, 923, 839, 774; **MS** (ESI): *m/z* (%) 517.4 ([M + Na]⁺, 58), 273.2 ([M + H – TBSOH - TMSOH]⁺, 49), 255.2 ([M - OH – TBSOH - TMSOH]⁺, 100); **HRMS** (ESI): *m/z* calculated for C₂₈H₅₄NaO₃Si₂ [M + Na]⁺ 517.3504, found 517.3501; **TLC** (SiO₂; 10% EtOAc/hexane): R_f = 0.37/0.53.

Synthesis of (8β)-(20S)-Des-*A*,*B*-8-[(tert-butyldimethyl)silyloxy)]-23-(3'-methyl-3'-[(trimethyl)silyloxy]but-1'-ynyl)-24-norcholan-23-one (17)

<figure><img src="image_23.png" /></figure>

To a solution of **6** (44 mg, 0.09 mmol, 1.0 equiv.) in CH₂Cl₂ (1.5 mL) at rt was added BAIB (45 mg, 0.13 mmol, 1.4 equiv.) and TEMPO (3 mg, 14 µmol, 15 mol%), and the solution was stirred at this temperature for 5 h. The solvent was removed *in vacuo* and the crude material was dissolved in ¹BuOMe (5 mL) and washed with 10% w/v aq. Na₂S₂O₃ solution (2x 5 mL) and sat. aq. NaHCO₃ solution (2x 5 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using 2% EtOAc/hexane) affording the product as a colorless oil (42 mg, 96%).

**Compound 17:** ¹H-NMR (400 MHz, CDCl₃): δ 3.99 (q, *J* = 2.7 Hz, 1H, H-8), 2.86 (dd, *J* = 15.0, 3.3 Hz, 1H, CH₂-22), 2.23 (dd, *J* = 14.9, 10.5 Hz, 1H, CH₂-22), 2.14 – 2.00 (m, 1H), 1.88 – 1.74 (m, 3H), 1.70 – 1.63 (m, 1H), 1.53 (s, 6H, CH₃-4', CH₃-5'), 1.42 – 1.21 (m, 6H), 1.19 – 1.02 (m, 2H), 0.96 (s, 3H, CH₃-18), 0.90 – 0.86 (m, 12H, CH₃-¹Bu, CH₃-21), 0.20 (s, 9H, CH₃-Si), 0.00 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 188.1 (C=O), 95.9 (C-1'), 82.3 (C-2'), 69.4 (CH-8), 66.6 (C-3'), 56.4 (CH-17), 53.0 (CH-14), 52.0 (CH₂-22), 42.2 (C-13), 40.8 (CH₂), 34.4 (CH₂), 32.4 (CH₃-4', CH₃-5'), 31.8 (CH-20), 27.1 (CH₂), 25.9 (CH₃-¹Bu), 22.9 (CH₂), 19.4 (CH₃-21), 18.2 (C-¹Bu), 17.7 (CH₂), 14.3 (CH₃-18), 2.0 (CH₃-Si), -4.7 (CH₃-Si), -5.0 (CH₃-
Si); **IR** (ATR, cm⁻¹): v 2953, 2929, 2856, 2214, 1676, 1471, 1462, 1378, 1360, 1250, 1166, 1086, 1037, 1023, 960, 923, 837, 774; **MS** (ESI): *m/z* (%) 493.4 ([M + H]⁺, 61), 403.3 ([M - OTMS]⁺, 56), 271.2 ([M + H - TBSOH - TMSOH], 48), 149.0 (100); **HRMS** (ESI): *m/z* calculated for C₂₈H₅₃O₃Si₂ [M + H]⁺ 493.3528, found 493.3519; **TLC** (SiO₂; 10% EtOAc/hexane): R_f = 0.72.

**Synthesis of (8β)-(20S)-Des-A,B-8-[(tert-butyldimethyl)silyloxy)]-23-(3'-methyl-3'-[(trimethyl)silyloxy]butyl)-24-norcholan-23-one (18)**

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

To a solution of **17** (136 mg, 0.28 mmol, 1.0 equiv.) in hexane (6.0 mL) at rt was added Pd (10 wt. % on C, 45 mg, 42 µmol, 15 mol%) and the resulting mixture was hydrogenated under normal pressure for 4 h. Then the catalyst was filtered, and the resulted liquids are concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using 2% EtOAc/hexane) affording the product as a yellow oil (126 mg, 93%).

**Compound 18:** ¹H-NMR (400 MHz, CDCl₃): δ 3.99 (q, *J* = 2.7 Hz, 1H, H-8), 3.00 – 2.65 (m, 1H), 2.48 – 2.39 (m, 1H), 2.17 – 2.03 (m, 1H), 1.92 – 1.61 (m, 6H), 1.43 (s, 3H, CH₃-4'/5'), 1.40 – 1.22 (m, 8H), 1.21 (s, 3H, CH₃-4'/5'), 1.13 – 1.01 (m, 2H), 0.95 (s, 3H, CH₃-18), 0.88 (s, 9H, CH₃-'Bu), 0.84 – 0.78 (m, 3H, CH₃-21), 0.18 – 0.04 (m, 9H, CH₃-Si), 0.00 (s, 3H, CH₃-Si), -0.01 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 212.0 (C=O), 73.1 (C-4'), 69.3 (CH-8), 57.0 (CH-17), 53.1 (CH-14), 52.2 (CH₂), 49.7 (CH₂), 42.2 (C-13), 40.9 (CH₂), 38.2 (CH₂), 34.5 (CH₂), 31.9 (CH-20), 30.0 (CH₃-4', CH₃-5'), 27.1 (CH₂), 25.9 (CH₃-'Bu), 23.0 (CH₂), 19.4 (CH₃-21), 18.2 (C-'Bu), 17.8 (CH₂), 14.3 (CH₃-18), 2.6 (CH₃-Si), 2.5 (CH₃-Si), -4.8 (CH₃-Si), -5.1 (CH₃-Si); **IR** (ATR, cm⁻¹): v 2953, 2926, 2854, 1714, 1463, 1378, 1365, 1250, 1166, 1085, 1038, 1052, 838, 774; **MS** (ESI): *m/z* (%) 497.4 ([M + H]⁺, 90), 407.3 ([M - OTMS]⁺, 100);
**HRMS** (ESI): *m/z* calculated for C₂₈H₅₇O₃Si₂ [M + H]⁺ 497.3841, found 497.3852;
**TLC** (SiO₂; 10% EtOAc/hexane): R_f = 0.69.

Synthesis of of (8β)-(20S)-Des-*A*,*B*-8-[(tert-butyldimethyl)silyloxy)]-23-methylydene-23-(3'-methyl-3'-[(trimethyl)silyloxy]butyl)-24-norcholane (19)

<figure><img src="image_25.png" /></figure>

To a solution of methyltriphenylphosphonium bromide (246 mg, 1.57 mmol, 10.0 equiv.) in anhydrous THF (3.0 mL) at -78 °C was added *n*-BuLi (2.5 M in hexane, 250 µL, 0.63 mmol, 4.0 equiv.) dropwise and the solution was stirred at 0 °C for 1 h. Then, was added **18** (78 mg, 0.16 mmol, 1.0 equiv.) in anhydrous THF (5.0 mL) and the resulting mixture was stirred at 0 °C for 6 h. The mixture reaction was quenched with sat. aq. NH₄Cl solution (10 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 10 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated *in vacuo*. The crude material was purified by FCC (SiO₂; using 0.5 % EtOAc/hexane) affording the product as a yellow oil (51 mg, 70%).

**Compound 19:** ¹H-NMR (400 MHz, CDCl₃): δ 4.61 (s, 1H, H₂C=C-23), 4.64 (s, 1H, H₂C=C-23), 4.03 – 3.96 (m, 1H, H-8), 2.50 (d, *J* = 10.9 Hz, 1H), 2.09 – 1.92 (m, 3H), 1.86 – 1.72 (m, 2H), 1.70 – 1.45 (m, 7H), 1.42 – 1.24 (m, 6H), 1.23 (s, 6H, CH₃-4', CH₃-5'), 0.96 (s, 3H, CH₃-18), 0.89 (s, 9H, CH₃'-Bu), 0.74 (d, *J* = 6.0 Hz, 3H, CH₃-21), 0.11 (s, 9H, CH₃-Si), 0.01 (s, 3H, CH₃-Si), 0.00 (s, 3H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 149.8 (C-23), 110.0 (H₂C=C-23), 73.9 (C-3'), 69.6 (CH-8), 57.5 (CH-17), 53.2 (CH-14), 43.3 (CH₂), 43.2 (CH₂), 42.3 (C-13), 41.0 (CH₂), 34.4 (CH₂), 32.9 (CH-20), 30.1 (CH₂), 30.0 (CH₃-4'/5'), 30.0 (CH₃-4'/5'), 27.2 (CH₂), 26.0 (CH₃'-Bu), 23.1 (CH₂), 18.5 (CH₃-21), 18.2 (C'-Bu), 17.9 (CH₂), 14.4 (CH₃-18), 2.7 (CH₃-Si), -4.6 (CH₃-Si), -5.0 (CH₃-Si); *IR* (ATR, cm⁻¹): ν 2952, 2929, 2856, 1471, 1462, 1377, 1364,
1250, 1166, 1038, 1023, 837, 774; **MS** (ESI): *m/z* (%) 405.4 ([M - OTMS]<sup>+</sup>, 100), 273.3 ([M + H - TBSOH - TMSOH]<sup>+</sup>, 37); **HRMS** (ESI): *m/z* calculated for C<sub>29</sub>H<sub>59</sub>O<sub>2</sub>Si<sub>2</sub> [M + H]<sup>+</sup> 495.4048, found 495.4022; **TLC** (SiO<sub>2</sub>; 10% EtOAc/hexane): R<sub>f</sub> = 0.91.

Synthesis of (20S)-Des-*A*,*B*-23-methylydene-23-(3'-methyl-3'-[(trimethyl)silyloxy]butyl)-24-norcholan-8-one (7)

<figure><img src="image_26.png" /></figure>

To a solution of **19** (43 mg, 0.09 mmol, 1.0 equiv.) in anhydrous THF (2.5 mL) at rt was added TBAF (1.0 M in THF, 270 µL, 0.27 mmol, 3.0 equiv.) and the solution was refluxed for 5 days. The solvent was removed *in vacuo* and to a solution of the crude material from the prior reaction in dry CH<sub>2</sub>Cl<sub>2</sub> (2.0 mL) at rt was added PDC (101 mg, 0.27 mmol, 3.0 equiv.) and the solution was stirred at this temperature for 4 h. The solvent was removed *in vacuo* and to a solution of the crude material from the prior reaction in anhydrous THF (2.0 mL) at rt was added TMS-Im (132 µL, 0.90 mmol, 10.0 equiv.) and the solution was stirred at this temperature for 12 h. The mixture was diluted with hexane (5 mL) and washed with H<sub>2</sub>O (3x 5 mL). The organic phase was dried over Na<sub>2</sub>SO<sub>4</sub>, filtered, and concentrated *in vacuo* to give the product as a colorless oil (27 mg, 81%), which was used in the next step without any further purification due to it was spectroscopically pure.

**Compound 7:** ¹H-NMR (400 MHz, CDCl₃): δ 4.75 (s, 1H, H₂C=C-23), 4.67 (s, 1H, H₂C=C-23), 2.48 - 2.43 (m, 1H), 2.33 - 2.21 (m, 4H), 2.13 - 1.82 (m, 5H), 1.82 - 1.36 (m, 9H), 1.22 (s, 6H, CH₃-4', CH₃-5'), 0.79 (d, *J* = 5.5 Hz, 3H, CH₃-21), 0.68 (s, 3H, CH₃-18), 0.10 (s, 9H, CH₃-Si); ¹³C-NMR (101 MHz, CDCl₃): δ 212.3 (C=O), 149.0 (C-23), 110.5 (H₂C=C-23), 73.8 (C-3'), 62.1 (CH-14), 57.1 (CH-17), 50.1 (C-13), 43.5 (CH₂), 43.1 (CH₂), 41.1 (CH₂), 39.1 (CH₂), 32.8 (CH-20), 30.1 (CH₃-4'/5'), 30.0 (CH₂),
29.9 (CH₃-4'/5'), 26.9 (CH₂), 24.2 (CH₂), 19.1 (CH₃-21), 18.3 (CH₂), 13.1 (CH₃-18), 2.7 (CH₃-Si); **IR** (ATR, cm⁻¹): ν 2956, 2933, 2874, 1708, 1642, 1459, 1379, 1364, 1248, 1213, 1172, 1152, 1134, 1040, 867, 836, 752, 681; **MS** (ESI): *m/z* (%) 401.3 ([M + Na]⁺, 11), 289.3 [(M − OTMS]⁺, 100); **HRMS** (ESI): *m/z* calculated for C₂₃H₄₂NaO₂Si [M + Na]⁺ 401.2840, found 401.2846; **TLC** (SiO₂; 10% EtOAc/hexane): *R*<sub>f</sub> = 0.46.

**Synthesis of (20S)-23-Homo-23-methylydenecalcitriol (UG-650)**

<figure><img src="image_27.png" /></figure>

To a solution of the phosphine oxide 8 (413 mg, 0.71 mmol, 10.0 equiv.) in anhydrous THF (2.0 mL) at -78 °C was added *n*-BuLi (2.5 M in hexane, 257 µL, 0.64 mmol, 9.0 equiv.) dropwise and the red solution was stirred at this temperature for 1 h. Then, 7 (27 mg, 0.07 mmol, 1.0 equiv.) was added in anhydrous THF (1.5 mL) and the resulting mixture was stirred at this temperature in the absence of light for 16 h. The mixture reaction was quenched with sat. aq. NH₄Cl solution (5 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (3x 5 mL) and the combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. To a solution of the crude material from the prior reaction in anhydrous THF (2.0 mL) at rt was added TBAF (1.0 M in THF, 360 µL, 0.36 mmol, 5.0 equiv.) and the solution was stirred at this temperature for 26 h. The solvent was removed in vacuo and the crude material was purified by FCC (SiO₂; using 80% EtOAc/hexane) affording the product as a colorless oil (18 mg, 60%).

The purity of **UG-650** was assessed by HPLC-DAD equipped with a UV-1 absorbance detector 254 nm and a silica column (250 × 4.6 mm, 5 µm particle size; isopropanol/hexanes10:90; flow rate = 4 mL/min) and was identified as >95%.
**Compound UG-650:** ¹H-NMR (400 MHz, CDCl₃): 6.35, 6.01 (2 d, *J*<sub>AB</sub> = 11.2 Hz, 2H, H-6, H-7), 5.31 (s, 1H, Z-H-19), 4.98 (s, 1H, *E*-H-19), 4.75 (s, 1H, H₂C=C-23), 4.67 (s, 1H, H₂C=C-23), 4.41 (dd, *J* = 8.0, 4.3 Hz, 1H, H-1), 4.23 - 4.18 (m, 1H, H-3), 2.81 (dd, *J* = 12.1, 4.2 Hz, 1H), 2.63 – 2.50 (m, 2H), 2.48 (d, *J* = 10.1 Hz, 1H), 2.34 – 2.26 (m, 2H), 2.07 – 1.83 (m, 10H), 1.71 – 1.46 (m, 14H), 1.32 – 1.28 (4H), 1.22 (s, 6H, CH₃-27/28), 0.76 (d, *J* = 5.6 Hz, 3H, CH₃-21), 0.57 (s, 3H, CH₃-18); ¹³C-NMR (101 MHz, CDCl₃): δ 149.0 (C-23), 147.8 (C-8), 142.9 (C-5), 133.3 (C-10), 124.9 (CH-6), 117.3 (CH-7), 111.8 (CH₂-19), 110.6 (H₂C=C-23), 71.1 (C-3'), 70.8 (CH-1), 66.9 (CH-3), 57.1 (CH-17), 56.4 (CH-14), 53.1 (CH₂), 46.0 (C-13), 45.3 (CH₂), 43.4 (CH₂), 43.0 (CH₂), 42.0 (CH₂), 40.7 (CH₂), 33.4 (CH-20), 30.2 (CH₂), 29.4 (CH₃-26/27), 29.2 (CH₃-26/27), 29.2 (CH₂), 27.7 (CH₂), 27.1 (CH₂), 23.7 (CH₂), 22.3 (CH₂), 20.7 (CH₂), 18.4 (CH₃-21), 12.6 (CH₃-18); IR (ATR, *cm*⁻¹): ν 3443, 3372, 3215, 2520, 1462, 1376, 1220, 1125, 943, 773, 652; MS (ESI): *m/z* (%) 465.3 ([M + Na]⁺, 19), 443.4 ([M + H]⁺, 30), 425.3 ([M – OH]⁺, 84), 186.22 (100); HRMS (ESI): *m/z* calculated for C₂₉H₄₀NaO₃ [M + Na]⁺ 465.3339, found 465.3329; TLC (SiO₂; 50% EtOAc/hexane): *R*<sub>f</sub> = 0.10.

**3.2. Evaluation of biological activity.**

The human breast carcinoma (MCF-7) cell line was obtained from the American Tissue Culture Company (Rockville, MD). Cells were seeded in 96-well plates (10,000 cells/well) in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM glutamax, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Fisher Scientific). The following day, cells were incubated for 72 h with increasing concentrations of calcitriol, or **UG-650**. Thereafter, 1 µCi [methyl-³H]thymidine (ICN Biomedicals, Costa Mesa, CA) was added to the cultures and cells were semi-automatically harvested on filterplates after an additional 4 h of incubation (GF/C Filter and Filtermate Universal Harvester, Packard Instrument, Meriden, CT). Counting was performed using a microplate scintillation counter (Topcount, Packard).

MCF-7 cells (700,000) were seeded in 6 cm dishes and incubated the next day with vehicle (EtOH), calcitriol (3 x 10⁻⁸ M) or **UG-650** (3 x 10⁻⁸ M) for 48 h. Cells were washed and scraped on ice in PBS and lysed in RIPA extraction buffer (50 mM Tris-HCl pH=8; 150 mM NaCl; 0.1% sodium dodecyl sulphate; 1% NP40; 0.5% sodium deoxycholate) supplemented with 1x Halt™ protease inhibitor cocktail (Thermo Fisher
Scientific). Protein pellets were sonicated and supernatants were collected after centrifugation (14,000 rpm for 10 min at 4°C). Protein concentration was determined with the BCA method according the instructions of the manufacturer (Pierce™ BCA Protein Assay kit, Thermo Fisher Scientific). Total cell proteins (20 µg) were separated by 4-12% Bis-Tris SDS-Page (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Amersham™ Protan®, GE Healthcare). Membranes were blocked with 5% bovine serum albumin (BSA) for 60 min and overnight incubated at 4°C with primary antibodies diluted in TRIS-buffered saline containing 0.1% Tween20 (anti-cyclin dependent kinase 2, 1/1000; anti-vitamin D receptor, 1/1000; Cell Signaling Technology). The next day, membranes were washed, and incubated for 1 h at room temperature with the peroxidase conjugated goat-anti-rabbit secondary antibody (1/5000, Dako). Protein bands were visualized with enhanced chemiluminescence (Western lightning, ECL PRO, Perkin Elmer) and quantified with the Odyssey XF imaging system (LI-COR).

To detect apoptosis, MCF-7 cells (62,500) were seeded in Lab-Tek II chamber slides (Nunc, Thermo Fisher Scientific) and incubated the next day with vehicle (EtOH), calcitriol (3 x 10⁻⁸ M) or **UG-650** (3 x 10⁻⁸ M) for 72 h. Apoptosis was detected with the *in situ* cell death detection kit TMR Red (Roche) according to the instructions of the manufacturer and cells were counterstained with Hoechst 33258 (1 µg/µl, Sigma). Images were acquired on a Axio Imager M2 (Zeiss). Staining of a negative control (without TUNEL reaction mixture) and a positive control (cells treated for 6 h with 100 µM cycloheximide) was included and pictures are shown in Supporting figure S3.

Cell migration was investigated in scratch-wound assays. MCF-7 cells (400,000) and MC3T3-E1 cells (200,000) were seeded in 48-well dishes in medium containing 10% FBS. The day after seeding, cell layers reached confluency and cells were transferred to medium containing 0.2% FBS with vehicle (EtOH), calcitriol (3 x 10⁻⁸ M) or **UG-650** (3 x 10⁻⁸ M). After 24 h, scratches were made in the confluent cell layers with a pipet tip and pictures were taken on a Primovert microscope (Zeiss) equipped with an AxioCam |CM1 camera after 48 h. Cells were seeded in triplicate, and 3 images per well were taken, resulting in a total of 9 images analyzed for each experimental condition. Cell migration distance was measured using ImageJ software.

### 3.3. Biochemistry.
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. 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.

**3.4. Thermal Unfolding, Differential Scanning Fluorimetry (nanoDSF).**

Fluorescence based thermal experiments were performed using Prometheus NT.48 (NanoTemper Technologies, Germany) with capillaries containing 10 µL zVDR LBD at 3.2 mg/mL in absence and presence of 2 equivalent ligands and/or 4 equivalents of NCoA1 NR2 peptide (RHKILHRLLQEGSPS). The temperature was increased by a rate of 1 °C/min from 20 to 95 °C and the fluorescence at emission wavelengths of 330 nm and 350 nm was measured. NanoTemper PR.Stability Analysis v1.0.2 was used to fit the data and to determine the melting temperatures Tm. Triplicates of each sample were made.

**3.5. Crystallization and structure determination.**

The concentrated protein was incubated with a 2-fold excess of ligand and a 3-fold excess of the NCoA2 coactivator peptide (KHKILHRLLQDSS). The crystallization experiments were carried out by hanging drop vapor diffusion at 290 K by mixing equal volume of protein-ligand complex and of reservoir solution ((NH4)2SO4 1.5 M, BisTris 0.1 M pH 7). The crystals of the complex were flash cooled in liquid nitrogen. X-ray data were collected on the Proxima2 beamline at Soleil synchrotron. The raw data were processed with XDS [14] and scaled with AIMLESS [15]. The structures were solved and refined using Buster [16] and Phenix [17] and iterative model building using COOT [18]. Crystallographic refinement statistics are presented in Supplementary material Table S3.

**Appendix A. Supplementary material**

**ACKNOWLEDGEMENTS**
This work was supported financially by the Xunta de Galicia (ED431C2017/70).
Authors would like to acknowledge the use of scientific and technical services
of Servicio de Seguridad Alimentaria y Desarrollo Sostenible from Centro de Apoio
Científico e Tecnolóxico á Investigación (CACTI-Universidade de Vigo). U.G.B. is
grateful to Xunta de Galicia for predoctoral fellowship. The authors thank I. Janssens
for excellent technical help and would like to acknowledge the Flanders Research
Foundation (FWO; G0D0120N and G081723N). We thank A. McEwen for help in X-
ray data collection, and the staff of PX2 beam line of SOLEIL synchrotron for
assistance during X-ray data collection. The research was also supported by Agence
Nationale de la Recherche (ANR-ANR-21-CE17-0009), by the 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].

TOC GRAPHIC

<figure><img src="image_28.png" /></figure>

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