Novel 3-Dehydroteasterone Derivatives with 23,24-Dinorcholanic Side Chain and Benzoate Groups at C-22: Synthesis and Activity Evaluation by Rice Lamina Inclination Test and Bean Second-Internode Bioassay

Abstract

Herein, a new series of 3-DT analogs with benzoylated groups at C-23 are synthesized and characterized. The benzoylated groups carry the same substituents in the ortho- or para-positions. Thus, the effect of structure on activity, measured using the rice lamina inclination test (RLIT) and the bean second-internode assay (BSI), is evaluated. The RLIT results indicate that a benzoylate function at C-22 induces a strong increase in activity that depends on the position and nature of the substituent in the phenyl ring. For example, an analog with an -OAc group in the ortho-position is the most active derivative, and its activity is like that of brassinolide. A relative index is calculated using brassinolide as a positive control to compare the RLIT results with those reported previously. This analysis allows for the conclusion that benzoylated derivatives with a hydroxyl group at C-3 are much more active than the corresponding analogs with a carbonyl group in this position, and one extra alcohol group in the alkyl chain decreases RLIT activity. Finally, the results obtained with the BSI are clearly different to those obtained in the RLIT bioassay. Therefore, the application of any activity–structure relationship will always be dependent on the bioassay used to determine activity.

1. Introduction

It is well established that the biosynthesis of brassinolide occurs by two parallel routes, connected at different steps, and both lead to castasterone, which is finally converted to brassinolide. In one of these pathways (Figure 1), epimerization of the C-3 hydroxyl group of teasterone (TE) leads to thyphasterol (TY) through 3-dehydroteasterone (3-DT), which is a very short-lived biosynthetic intermediate [1,2,3,4]. Typhasterol is subsequently hydroxylated at C-2 to give castasterone (1). Finally, an oxygen atom is inserted between C-6 and C-7 to form brassinolide (2) [1,2,3,4].

The biological activities of brassinolide (2), castasterone (1), and its precursors have been examined using the rice lamina inclination test (RLIT) and wheat leaf unrolling assay. In both bioassays, brassinolide (2) and castasterone (1) exhibit much higher activities than TE, 3-DT, and TY, whose activities are almost the same [3]. These results suggest that chemical modifications of precursors that produce 1 and 2, two of the most active natural brassinosteroids, could provide a synthetic means of obtaining active compounds.

Following this line, a series of TE and 3-DT derivatives with modifications in the alkyl chain, compounds 3–10 and 11–16 in Figure 2, respectively, have been synthesized, and their bioactivity has been assessed using different bioassays [5].

More specifically, the synthesis of 3-hydroxy-6-oxo-23,24-dinorcholans with a benzoate function at C-22 (compounds 3–10, TE analogs) has recently been reported [6]. Assessments of the bioactivity of these analogs by using the RLIT indicate that, at the lowest tested concentration (1 × 10⁻⁸ M), TE analogs in which the aromatic ring is substituted in the para-position with methoxy (5), I atom (9), and CN (10) are more active than brassinolide (50–72%) and 2–3 times more active than analogs in which the substituent group is F, Cl, or Br atoms [6]. Similarly, a series of 24-Nor-22(S)-hydroxy side chains with a p-substituted benzoate function at C-23 (compounds 11–16, 3-DT analogs) has also been synthesized and evaluated using the RLIT [7]. The obtained results show that all these 3-DT analogs exhibit much lower activity than brassinolide. In addition, compound 16, i.e., an analog with p-Br in the aromatic ring, is the most active compound tested at 1 × 10⁻⁸ M [7].

Thus, by comparing the RLIT activities of these two series of compounds, it can be concluded that TE analogs are more active than 3-DT analogs. The main structural differences between these two series are in the side alkyl chain. For TE analogs, 3–10, the benzoylated groups are attached to C-22, whereas in 3-DT analogs, 11–16, these groups are attached to C-23 and leave a hydroxyl group at C-22.

Thus, considering the different effects on biological activity observed between TE analogs and 3-DT analogs, herein, the synthesis of a new series of 3-DT analogs with benzoylated groups at C-22 is described. In this way, we intend to elucidate the importance of the shortest alkyl chain with no hydroxyl group at C-22 on the biological activity of 3-DT analogs. Compounds with different substituents in the para- (19–26) and ortho-positions (27–33) of the aromatic ring are obtained, as shown in Figure 2. All obtained compounds are characterized by IR, HRMS, 1D, and 2D NMR spectroscopic techniques, and their bioactivity is evaluated by RLIT and BSI bioassays [6,7].

The results obtained herein can be used to determine and compare the effect on the bioactivity of substituents in the “ortho”- and “para”-positions of the aromatic ring, as it has already been described for other aryl or phenyl analogs of the brassinosteroid type [8,9]. Finally, a new structural requirement in the side chain for this type of BR analog is established.

2. Results and Discussion

2.1. Chemical Synthesis

Commercially available hyodeoxycholic acid (17) (Figure 2 and Scheme 1) (AK Scientific Inc.) was used as a starting material for the preparation of new 3-DT analogs 19–33. The first part of the synthetic route is obtaining alkene 38 (Scheme 1). Following a described procedure, deoxycholic acid (17) is completely oxidized with Jones reagent to produce 3,6-dioxo acid 34 with 98.0% yield [7]. IR and NMR spectroscopic data of 34 are consistent with those previously reported [7]. Subsequently, isomerization of C-5 from 5β to 5α on compounds 34 to 35 is performed under acidic conditions (HCl 2.5%, CH₃OH, reflux) according to a reported procedure, with 95.8% yield [7,10]. The structure of compound 35 is confirmed by ¹H and ¹³C and 2D NMR spectroscopy (Figures S1–S5, Supplementary Material). Thus, in the ¹H NMR spectrum, the signal observed at δ = 3.65 ppm (s, 3H) is assigned to the CH₃O group at C-24, while the signal at δ = 2.62–2.53 (m, 2H) is assigned to H-5 and H-2. In the ¹³C NMR spectrum, the signal observed at δ = 174.57 and δ = 51.47 ppm confirm the presence of a methyl ester group (-CO₂CH₃). Isomerization from 5β (compound 34) to 5α (compound 35) is shown by the change in field chemical shift of C-5 signal, from δ = 59.69 to 57.43 ppm. The saponification reaction of 35 with K₂CO₃/CH₃OH (15.0% w/v) reflux, followed by acidification (HCl 2.5% v/v), gives acid 36 with 98.4% yield. The presence of the carboxylic function is confirmed by the signal appearing at δ = 179.88 ppm in the ¹³C NMR spectrum (Figures S6–S10, Supplementary Material). Oxidative decarboxylation of the side chain in compound 36, with Pb(OAc)₄/Cu(OAc)₂/C₅H₅N/C₆H₆ under reflux, and following a reported procedure [11], leads to olefin 37 with 36.8% yield. The formation of a terminal double bond at the C-22 position is confirmed by ¹H-NMR and ¹³C-NMR spectra, and by ¹³C DEPT-135, 2D HSQC and 2D HMBC NMR experiments (Figures S11–S15, Supplementary Material). All NMR spectroscopic data registered for compound 37 are consistent with that reported in [8]. The protection of keto groups of compound 37 with an ethylene glycol/TsOH/C₆H₆ system gives the bisdioxolane derivative 38. The presence of bisdioxolane groups in compound 38 is confirmed by signals observed at δ = 3.97–3.87 ppm (m, 7H) and δ = 3.76–3.72 ppm (m, 1H) in ¹H NMR spectrum, and signals observed at δ = 65.39, 64.26, 64.16, and 64.10 ppm in the ¹³C NMR spectrum, which are assigned to four carbinol carbons of bisdioxolane groups (Figures S16–S20, Supplementary Material).

The second part of the synthetic route, shown in Scheme 2, starts with Lemieux–Johnson oxidation of 38 with OsO₄/NaIO₄/THF/H₂O to obtain aldehyde 39 with 57.1% yield [12]. ¹H and ¹³C NMR spectroscopic data obtained for 39 (Figures S21–S25, Supplementary Material) are consistent with those previously reported [13]. Reduction of aldehyde 39 with NaBH₄/THF/CH₃OH at 40 °C [14], followed by acid hydrolysis (HCl 5% v/v [6]), produces primary alcohol 18 with 80.8% yield. Primary alcohol at C-22 and both ketone functions at C-3 and C-6 are identified by ¹H and ¹³C NMR spectroscopies. Thus, in the ¹H NMR spectrum, the signals appearing at δ = 3.63 ppm (1H, dd, J = 10.6 and 3.3 Hz) and 3.38 ppm (1H, dd, J = 10.6 and 6.7 Hz) are assigned to carbinolic and diasterotopic hydrogen atoms H-22a and H-22b. In contrast, in the ¹³C NMR spectrum, signals at δ = 211.25, 209.02 and 67.76 ppm appear, which are assigned to carbon atoms of carbonyl groups C-3 and C-6, and carbinolic carbon atom C-22, respectively. Additionally, the full molecular structure is assigned by ¹³C DEPT-135, 2D HSQC and 2D HMBC NMR experiments (Figures S26–S30, Supplementary Material). Finally, benzoylation of the primary alcohol at C-22 with the corresponding benzoyl chlorides, following a reported protocol [7,11,15], gives new 3,6-dioxo BRs analogs 19–33 (3-DT derivatives) with yields ranging from 18.4% to 76.9% (Scheme 2). Compounds 19–33 are fully characterized by the IR, 1D, 2D NMR (Figures S31–S105, Supplementary Material) and HRMS (Figures S106–S121, Supplementary Material) spectroscopic techniques.

2.2. Biological Activity

2.2.1. Bioactivity in the Rice Lamina Inclination Test (RLIT)

The biological activities of precursor 18 and new 19–33 derivatives have been evaluated by using RLIT, a highly sensitive and specific assay for BRs [6,7]. Leaf–sheath bending angles are measured in growing rice plants in the absence and presence of different concentrations of exogenously applied BR analogs. The effect of BR analogs on bending angle is calculated as described in the Material and Methods, and RLIT activities at different concentrations of 3-DT derivatives are presented in

Table 1. RLIT activities obtained by measurements of bending angles at different concentrations of 3-DT derivatives.

RLIT Activities ± (Standard Error) 1
Compounds	1 × 10−8 M	1 × 10−7 M	1 × 10−6 M
Brassinolide (2)	2.3 ± 1.4 d	3.13 ± 1.7 e	4.17 ± 1.5 f
18	-	0.13 ± 3.8 a	0.70 ± 4.2 a
19 (p-H)	1.65 ± 5.2 c	1.22 ± 4.9 c	0.48 ± 4.1 b
20 (p-CH3)	1.83 ± 8.2 c	1.83 ± 4.2 d	1.30 ± 4.9 d
27 (o-CH3)	0.52 ± 5.8 a	1.13 ± 5.2 c	1.48 ± 4.1 d
21 (p-OCH3)	0.83 ± 5.5 b	1.22 ± 4.1 c	2.70 ± 8.8 e
28 (o-OCH3)	1.87 ± 5.8 c	0.91 ± 4.1 b	0.26 ± 2.0 a
22 (p-F)	1.78 ± 5.2 c	0.87 ± 2.0 b	0.83 ± 4.5 c
29 (o-F)	0.56 ± 4.5 a	0.83 ± 2.6 b	2.52 ± 3.2 e
23 (p-Cl)	1.04 ± 3.3 b	0.74 ± 2.7 b	1.22 ± 4.1 d
30 (o-Cl)	-	0.56 ± 4.1 a	1.43 ± 4.5 d
24 (p-Br)	-	0.13 ± 1.1 a	0.13 ± 1.0 a
31 (o-Br)	1.78 ± 5.2 c	1.34 ± 2.6 d	0.70 ± 7.5 b
25 (p-I)	1.78 ± 8.2 c	1.74 ± 2.0 d	0.78 ± 2.6 c
32 (o-I)	2.0 ± 2.3 c	1.4 ± 4.9 d	0.96 ± 3.8 c
26 (p-OAc)	1.74 ± 3.8 c	1.83 ± 2.7 d	0.83 ± 5.0 c
33 (o-OAc)	2.74 ± 6.6 d	1.87 ± 4.5 e	1.04 ± 2.6 c

¹ Values are the mean of leaf–sheath bending angle, normalized to the negative control (23 ± 2.7°). Data are expressed as mean ± standard deviation of two independent experiments, each with at least eight replicates. Superscript letters indicate significant differences between treatments according to the LSD multiple comparison test (p < 0.05). (-) indicates lower values than the negative control. Brassinolide was used as a positive control.

.

By comparing the RLIT activities of 3-DT derivatives and 18, it can be concluded that a benzoate function at the C-22 position induces a strong increase in activity, which depends on the position and nature of the phenyl substituent. Additionally, experimental data show that RLIT activities are concentration-dependent, i.e., brassinolide activity increases with increasing concentration. This behavior is shown only by derivatives 21 and 29. Therefore, the analysis of structure effect on activity must be performed separately for each tested concentration.

At the lowest tested concentration, the data indicate that the highest activity corresponds to analog 33, which has an acetyl group substitution in the ortho-position of the aromatic ring, and its activity is slightly higher than that obtained for brassinolide. Interestingly, the analog with this substituent in the para-position, 26, shows slightly lower activity. A similar effect is observed for derivatives with methoxy and a Br atom as substituents (compare the activities of 21 with 28, and 24 with 31, respectively). The opposite effect, i.e., lowest activities for the ortho-substitution, are obtained for methyl (20 and 27), F (22 and 29), and Cl (23 and 30) atoms. Finally, for the I atom, the substitution position has no effect on RLIT activity (25 and 32). Thus, at the lowest tested concentration, the results obtained for the activity of 3-DT derivatives indicate that a substituent attached to the ortho- or para-positions produces different effects on activities.

The effect of increasing activity with increasing concentration has been observed only for brassinolide (2), 21, and 29. Therefore, at concentrations higher than 1 × 10⁻⁸ M, 3-DT analogs become less active than brassinolide (2). By comparing 3-DT activities at 1 × 10⁻⁷ M, it is seen that the most active analogs are 20 and 25, substituted in para-position with a methyl group and an I atom, respectively, and 26 and 33, which are substituted with an acetyl group in the para- and ortho-positions, respectively. The same comparison at 1 × 10⁻⁶ M show that 21 and 29, which are substituted with a methoxy group in the para-position and a F atom in the ortho-position, respectively, are the most active derivatives.

It is worth mentioning that the effect of substituent position is also dependent on concentration. In other words, the ratio of activities obtained for derivatives with substituents in the ortho- or para-positions changes with concentration as well. Thus, the chemical structure of 3-DT derivatives is somehow related to their RLIT activity. It has been shown that the RLIT process occurs thanks to a very complex mechanism, and therefore, it is not possible to attribute this behavior to a special step [16].

A main aim of this work is to obtain a more general relationship between the measured RLIT activity and the chemical structure of benzoylated BRs. To allow for a comparison of activity data obtained under varying experimental conditions, it is essential to include the positive control, brassinolide, in the calculation of RLIT activity. This goal is accomplished by defining a relative activity (RA), calculated by dividing the activity value of each tested compound by that of brassinolide under the same conditions (see the Experimental Section). Thus, the relative activity values obtained herein at 1 × 10⁻⁸ M are listed in

Table 2. RLIT relative activity (RA) at 1 × 10⁻⁸ M, using brassinolide (2) as a reference compound.

3-DT	Activity	TE	Activity (Ref. [6])	3-DT(C22-OH)	Activity (Ref. [7])
18 *	-	(*)	0.95	(*)	0.32
19 (p-H)	0.72	3 (p-H)	0.95	11 (p-H)	0.24
20 (p-CH3)	0.80	4 (p-CH3)	1.05	12 (p-CH3)	0.24
21 (p-OCH3)	0.36	5 (p-OCH3)	1.50	13 (p-OCH3)	-
22 (p-F)	0.77	6 (p-F)	0.73	14 (p-F)	0.47
23 (p-Cl)	0.45	7 (p-Cl)	0.55	15 (p-Cl)	0.40
24 (p-Br)	-	8 (p-Br)	0.68	16 (p-Br)	0.93
25 (p-I)	0.77	9 (p-I)	1.73
26 (p-OAc)	0.76
33 (o-OAc)	1.19

(-) No activity. (*) Precursor with no benzoylate function.

along with those calculated from data previously reported for two series of compounds, namely, TE and 3-DT (C22-OH) derivatives.

A comparison of the results obtained herein (19–25) with those described in reference [6] (3–9) indicate that derivatives with a -OH group in C-3 are much more active than derivatives with a carbonyl group in this position. This effect is observed even for precursors of benzoylated derivatives, and the only exceptions are compounds 22 and 6 that exhibit the same activity. The key role played by the hydroxyl group in this position has been already established [17,18]. On the other hand, by comparing results obtained for compounds 11–15 with 18–23, it becomes clear that an extra alcohol group in the alkyl chain decreases RLIT activity. Thus, these results represent a contribution to the understanding of how the structure of BR benzoylated analogs determine RLIT activity, but so far, it is not possible to establish a consistent structure–activity relationship.

Another important aspect to highlight is that data for analogs substituted in the ortho-positions are scarce, and benzoylated analogs with substitution with acetate group (33) exhibit interesting activity on the RLIT bioassay.

2.2.2. Bioactivity in Bean Second-Internode Bioassay (BSI)

All 3-DT derivatives, 19–33, and its synthetic precursor 18, have also been tested using the BSI bioassay. The values of the bean second-internode elongation have been obtained for BR analogs applied at 1 × 10⁻⁸ M concentration. This concentration was chosen because it has been shown that the optimal response for brassinolide in the range 1 × 10⁻⁵–1 × 10⁻¹⁰ is obtained at this concentration [19]. The results are shown in

Table 3. Activities in the bean second-internode bioassay of brassinolide (2), 18, and 3-DT derivatives 19–33 at 1 × 10⁻⁸ M.

Compounds	Elongation of the Second Internode, mm ± SD
Brassinolide (2)	7.67 ± 2.35 b
18	2.67 ± 1.0 d,e
19 (p-H)	3.17 ± 0.89 d,e
20 (p-CH3)	1.67 ± 1.0 e,f
27 (o-CH3)	6.83 ± 3.0 b
21 (p-OCH3)	13.67 ± 2.90 a
28 (o-OCH3)	6.00 ± 2.0 b,c
22 (p-F)	6.17 ± 1.79 b,c
29 (o-F)	7.00 ± 2.48 b
23 (p-Cl)	3.17 ± 0.89 e,f
30 (o-Cl)	7.17 ± 2.8 b
24 (p-Br)	0.00 ± 0
31 (o-Br)	2.17 ± 0.89 d,e,f
25 (p-I)	2.50 ± 1.51 d,e
32 (o-I)	5.83 ± 2.8 b,c
26 (p-OAc)	1.83 ± 1.0 e,f
33 (o-OAc)	4.33 ± 2.48 c,d
Negative Control	1.00 ± 0.0 f

Letters indicate the level of significance according to the LSD–Fischer test; p < 0.05.

and Figure S123, Supplementary Materials.

The data listed in Table 3 indicate that, in the BSI bioassay and at the concentration of 1 × 10⁻⁸ M, analogs 22 and 27–30 show biological activity comparable to that of brassinolide, whereas analog 21 (with OCH₃ group substituent in the “para” position of the aromatic ring), exhibits biological activity that is twofold that exhibited by brassinolide (2). This is the only case in which a derivative with a substituent in the para-position is more active than the corresponding ortho-isomer. In all other cases, 27–30 and 32, the ortho-derivatives are significantly more active in the BSI bioassay.

These results are clearly different from those obtained in the RLIT bioassay, and therefore, it can be concluded that the activity of BR derivatives cannot be determined by just one bioassay. So, their application and activity–structure relationship will always be dependent on the bioassay used to determine activity.

3. Materials and Methods

3.1. General Chemicals and Methods

All reagents were purchased from commercial suppliers and used without further purification. Melting points were measured on a SMP3 apparatus (Stuart-Scientific, now Merck KGaA, Darmstadt, Germany) and left uncorrected. ¹H-, ¹³C-, ¹³C DEPT-135, gs 2D HSQC, and gs 2D HMBC NMR spectra were recorded in CDCl₃ solutions and were referenced to the residual peaks of CHCl₃ at δ = 7.26 ppm and δ = 77.00 ppm for ¹H and ¹³C, respectively, on an Avance Neo 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for ¹H and 100.6 MHz for ¹³C. Chemical shifts are reported in δ ppm, and coupling constants (J) are given in Hz. Multiplicities are reported as follows: singlet (s), doublet (d), broad doublet (bd), doublet of doublets (dd), doublet of triplets (dt), triplet (t), broad triplet (bt), quartet (q), doublet of quartet (dq), doublet of double doublets (ddd), triplet of triplets (tt), and multiplet (m). IR spectra were recorded as KBr disks in a FT-IR 6700 spectrometer (Nicolet, Thermo Scientific, San Jose, CA, USA), and frequencies are reported in cm⁻¹. High-resolution mass spectra (HRMS-ESI) were recorded in a Bruker Daltonik. The analysis for the reaction products was performed with the following relevant parameters: dry temperature, 180 °C; nebulizer, 0.4 Bar; dry gas, 4 L/min; and spray voltage, 4.5 kV in positive mode. Accurate mass measurements were performed at a resolving power: 140,000 FWHM in the range m/z 50–1300. For analytical TLC, silica gel 60 in a 0.25 mm layer was used, and TLC spots were detected by heating after spraying with 25% H₂SO₄ in H₂O. Chromatographic separations were carried out by conventional column on silica gel 60 (230–400 mesh) using hexane–EtOAc mixtures of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40 °C.

3.2. Synthesis

3.2.1. 3,6-Dioxo-5β-cholan-24-oic acid (34)

Compound 34 is prepared from hyodeoxycholic acid (17) according to a described protocol, with 98% yield. All characterization data are consistent with those reported [7].

3.2.2. Methyl 3,6-Dioxo-5α-cholan-24-oate (35)

Compound 34 (2.00 g, 5.15 mmol) is dissolved in 200 mL of 2.5% v/v HCl-MeOH and refluxed for 2 h. The end of reaction is verified by TLC. The solvent is evaporated under reduced pressure, and the residue is dissolved in EtOAc (60 mL). The solution is washed with a saturated solution of NaHCO₃ (3 × 50 mL) and water (3 × 40), and the organic layer is dried over MgSO₄. Finally, the solvent is evaporated by using a rotary evaporator. Compound 35 (1.99 g, 95.8% yield) is obtained as a colorless solid; m.p. = 105.2–106.9 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2947 and 2867 (C-H); 1736 and 1712 (C=O); 1433 (CH2-); 1384 (CH3-); 1239 and 1166 (C-O). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 3.65 (3H, s, OCH₃); 2.62–2.53 (2H, m, H-5 and H-2); 2.21 (1H, ddd, J = 18.6, 9.8 and 9.4 Hz, H-22a); 2.09 (1H, dd, J = 6.4 and 2.4 Hz, H-1α); 1.99 (1H, dd, J = 12.8 and 12.5 Hz, H-7α); 0.942 (3H, s, H-19); 0.916 (3H, d, J = 6.6 Hz, H-21); 0.678 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.23 (C-3); 209.01 (C-6); 174.57 (C-24); 57.43 (C-5); 56.49 (C-14); 55.71 (C-17); 53.34 (C-9); 51.47 (OCH₃); 46.51 (C-7); 42.98 (C-13); 41.17 (C-10); 39.27 (C-12); 38.02 (C-1); 37.93 (C-20); 37.32 (C-4); 36.93 (C-2); 35.22 (C-8); 30.98 (C-22); 30.82 (C-23); 27.85 (C-16); 23.90 (C-15); 21.59 (C-11), 18.17 (C-21); 12.50 (C-19); 11.97 (C-18) (Figures S1–S5, Supplementary Material).

3.2.3. 3,6-Dioxo-5α-cholan-24-oic Acid (36)

Compound 35 (2.00 g, 4.97 mmol) dissolved in CH₃OH (120 mL) and in presence of 15% w/v K₂CO₃ is refluxed for 1h. The end of the reaction is verified by TLC. The solvent is evaporated under reduced pressure, and the residue is dissolved in water (50 mL) and neutralized with HCl 2.5% v/v until pH = 3. EtOAc (60 mL) is added to the solution and washed with water (3 × 50), and the organic layer is dried over MgSO₄. Finally, the solvent is evaporated in a rotary evaporator. Compound 36 (1.90 g, 98.4% yield) is obtained as a colorless solid; m.p. = 196.3–198.3 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 3500–2600 (OH); 2945 and 2868 (C-H); 1710 (C=O); 1416 (CH₂-); 1384 (CH₃-); 1240 and 1165 (C-O). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 2.61–2.53 (2H, m, H-5 and H-2), 2.09 (1H, dd, J = 6.4 and 2.4 Hz, H-1α); 2.00 (1H, dd, J = 12.8 and 12.3 Hz, H-7α); 0.942 (3H, s, H-19), 0.928 (3H, d, J = 7.1 Hz, H-21), 0.681 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.47 (C-3); 209.12 (C-6); 179.88 (C-24); 57.43 (C-5); 56.48 (C-14); 55.69 (C-17); 53.33 (C-9); 46.50 (C-7); 43.00 (C-13); 41.18 (C-10); 39.27 (C-12); 38.01 (C-1); 37.93 (C-20); 37.30 (C-4); 36.92 (C-12); 35.17 (C-2); 30.91 (C-22); 30.57 (C-23); 27.84 (C-16); 23.90 (C-15); 21.59 (C-11), 18.15 (C-21); 12.51 (C-19); 11.98 (C-18) (Figures S6–S10, Supplementary Material).

3.2.4. 24-Nor-5α-chol-22-ene-3,6-dione (37)

To a solution of compound 36 (2.00 g, 5.15 mmol) in dry benzene (60 mL), Cu(OAc)₂·H₂O₂ (200 mg, 1.00 mmol) and pyridine (0.8 mL) are added. The reaction mixture is refluxed, and Pb(OAc)₄ (5.6 g, 12.6 mmol) is added in four portions every 1 h. Then, the mixture is filtered, and the solvent is evaporated under reduced pressure. The crude is diluted with EtOAc (50 mL) and washed with water (3 × 20 mL), and the organic layer is dried over MgSO₄. Then, the solvent is evaporated in vacuo, and the crude is redissolved in CH₂Cl₂ (7 mL) and chromatographed on silica gel with hexane–EtOAc mixtures of increasing polarity (19.8:0.2 → 15.8:4.2). Compound 37 (0.65 g, 36.8%) is obtained as a colorless solid; m.p. = 148.9–149.7 °C (AcOEt/hex.). IR_νmax (KBr, cm⁻¹): 3073 (CH=CH₂); 2946, 2870 and 2866 (C-H); 1712 (C=O); 1638 (C=C); 1460 (CH₂-); 1389 (CH₃-); 1248 and 1216 (C-O); 906 (CH=CH₂). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 5.65 (1H, ddd, J = 17.2, 10.2 and 8.5 Hz, H-22); 4.91 (1H, dd, J = 17.2 and 1.8 Hz, H_trans-23); 4.83 (1H, dd, J = 10.2 and 1.8 Hz, H_cis-23); 2.62–2.54 (2H, m, H-5 and H-2); 2.00 (1H, t, J = 13.0 Hz, H-7α); 1.86 (1H, ddd, J = 14.6, 10.3 and 4.2 Hz, H-8); 1.43 (1H, ddd, J = 16.7, 12.7 and 3.7 Hz, H-11β); 1.10 (1H, ddd, J = 18.0, 11.7 and 5.9 Hz, H-15); 1.03 (3H, d, J = 6.6 Hz, H-21); 0.953 (3H, s, H-19); 0.712 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃), δ (ppm): 211.23 (C-3); 209.03 (C-6); 144.75 (C-22); 111.92 (C-23); 57.47 (C-5); 56.58 (C-14); 55.34 (C-9); 53.44 (C-17); 46.55 (C-7); 42.95 (C-13); 41.22 (C-10); 41.06 (C-20); 39.22 (C-12); 38.06 (C-1); 37.96 (C-8); 37.35 (C-2); 36.96 (C-4); 28.15 (C-16); 23.95 (C-15); 21.62 (C-11); 20.03 (C-21); 12.54 (C-19); 12.17 (C-18) (Figures S11–S15, Supplementary Material).

3.2.5. 3,6-Didioxolan-24-nor-5α-cholan-22-ene (38)

To a solution of compound 37 (1.00 g, 2.92 mmol) in C₆H₆ (80 mL), ethylene glycol (7.2 mL, 129.9 mmol) and TsOH (140 mg, 0.81 mmol) are added. The solution is refluxed for 3 h, using a Dean-Stark apparatus. Later, a saturated solution of NaHCO₃ (80 mL) is added to quench the reaction and dissolved with EtOAc (60 mL). The organic layer is washed with water (2 × 25 mL) and dried over MgSO₄. Finally, the solvent is evaporated in vacuo. Compound 38 (1.15 g, 91.3% yield) is obtained as a colorless solid; m.p. = 108.3–109.4 °C (EtOAc). IR_νmax (KBr, cm⁻¹): 2961, 2930, 2905, 2875 and 2862 (C-H); 1639 (C=C); 1459 (CH₂-); 1355 (CH₃-); 1256, 1229, 1198, 1180, 1106, 1090 and 1042 (C-O); 906 (CH=CH₂). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 5.64 (1H, ddd, J = 17.2, 10.2 and 8.5 Hz, H-22); 4.88 (1H, dd, J = 17.1 and 1.7 Hz, H_trans-23); 4.80 (1H, dd, J = 10.1 and 1.7 Hz, H_cis-23); 3.96–3.83 (7H, m, dioxolane); 3.77–3.71 (1H, m, dioxolane); 2.10–2.01 (1H, m, H-20); 1.95 (1H, dt, J = 12.5 and 3.2 Hz, H-12α); 1.08–0.979 (3H, m, H-15, H-7 and H-5); 1.01 (3H, d, J = 6.6 Hz, H-21); 0.937 (3H, s, H-19); 0.793 (1H, dd, J = 12.1 and 4.1 Hz, H-17); 0.688 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃), δ (ppm): 145.24 (C-22); 111.52 (C-23); 109.70 (C-3 and C-6); 65.41; 64.28; 64.17 and 64.12 (dioxolane); 56.00 (C-5); 55.40 (C-14); 53.32 (C-9); 49.56 (C-17); 42.58 (C-13); 41.18 (C-20 and C-7); 39.83 (C-12); 37.23 (C-1); 36.83 (C-10); 33.38 (C-8); 31.02 (C-2); 29.23 (C-4); 28.33 (C-16); 24.16 (C-15); 21.00 (C-11); 20.08 (C-21); 13.52 (C-19); 12.22 (C-18) (Figures S16–S20, Supplementary Material).

3.2.6. 3,6-Didioxolan-23,24-dinor-5α-cholan-22-al (39)

To a solution of compound 38 (600mg, 1.39 mmol) in THF (30 mL), H₂O (18 mL) and NaIO₄ (900 mg, 4.21 mmol) are added at room temperature. Later, 10% v/v OsO₄-H₂O (0.7 mL) is added under inert gas atmosphere (N₂). After 6 h, a solution of 5% w/v Na₂SO₃-H₂O (5 mL) is added and stirred for 15 min (TLC). THF is evaporated under reduced pressure, and the resulting mixture is diluted with EtOAc (50 mL), washed with water (2 × 10 mL), and dried over MgSO₄. Finally, the solvent is evaporated under reduced pressure, and the crude is chromatographed on silica gel with hexane–EtOAc mixtures of increasing polarity (19.8:0.2 → 10.2:9.8). Compound 39 (344 mg, 57.1%) was obtained as a colorless solid; m.p. = 148.9–149.7 °C (AcOEt/hex.). IR_νmax (KBr, cm⁻¹): 2946 and 2913 (C-H); 2871 and 2707 (CHO); 1713 (C=O); 1460 (CH₂-); 1390 (CH₃-); 1261, 1241 and 1167 (C-O). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 9.54 (1H, d, J = 3.3 Hz, H-22); 3.96–3.86 (7H, m, dioxolane); 3.76–3.71 (1H, m, dioxolane); 2.38–2.29 (1H, m, H-20); 1.91 (1H, dt, J = 12.5 and 3.3 Hz, H-12α); 1.87–1.79 (1H, m, H-16); 1.17–0.997 (3H, m, H-15, H-7 and H-5); 1.10 (3H, d, J = 7.0 Hz, H-21); 0.934 (3H, s, H-19); 0.797 (1H, dd, J = 11.3 and 4.0 Hz, H-17); 0.706 (3H, s, H-18) [18]. ¹³C NMR (100.6 MHz, CDCl₃), δ (ppm): 205.08 (C-22); 109.71 and 109.54 (C-3 and C-6); 65.38; 64.24; 64.16 and 64.07 (dioxolane); 55.22 (C-5); 53.25 (C-17); 50.95 (C-14); 49.51 (C-9); 49.43 (C-20); 43.19 (C-13); 41.14 (C-7); 39.44 (C-12); 37.19 (C-1); 36.81 (C-10); 33.34 (C-8); 30.98 (C-2); 29.19 (C-4); 26.94 (C-16); 24.50 (C-15); 20.93 (C-11); 13.49 (C-19); 13.38 (C-21); 12.39 (C-18) [18] (Figures S21–S25, Supplementary Material).

3.2.7. 22-Hydroxy-23,24-dinor-5α-cholan-3,6-dione (18)

Compound 39 (690 mg, 1.60 mmol) is dissolved in THF (15 mL) and MeOH (30 mL) with slow stirring. Then, NaBH₄ (40 mg, 1.06 mmol) is added in two portions (each 1 h). The reaction reduction is verified by TLC (2 h). Later, acetone (5 mL) and acid water (pH = 3) (15 mL) are added, and the solvents are evaporated under reduced pressure. The resulting residue is diluted with EtOAc (30 mL), washed with water (2 × 10 mL), and dried over MgSO₄. Subsequently, the compound is dissolved in acetone (60 mL) and hydrochloric acid (37%) (1 mL), and H₂O (10 mL) is added at 50 °C. The end of the reaction is verified by TLC (3 h). Then, acetone is evaporated under reduced pressure. The resulting compound is diluted with CH₂Cl₂ (10 mL), washed with water (2 × 15 mL), and dried over MgSO₄. Finally, the solvent is evaporated in vacuo. Compound 18 (445 mg, 80.5%) is obtained as a colorless solid; m.p. = 172.4–174.1 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 3571–3200 (OH); 2972, 2940, 2905 and 2861 (CH-); 1714 (C=O); 1460 (CH₂-); 1385 (CH₃-); 1270 and 1239 (C-O). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 3.63 (1H, dd, J = 10.6 and 3.2 Hz, H-22a); 3.38 (1H, dd, J = 10.6 and 6.6 Hz, H-22b); 2.63–2.54 (2H, m, H-5 and H-2); 2.00 (1H, dd, J = 13.2 and 12.7 Hz, H-7α); 1.45 (1H, dd, J = 12.7 and 3.7 Hz, H-11b); 1.14 (1H, ddd, J = 17.7, 11.9 and 6.0 Hz, H-15); 1.05 (3H, d, J = 6.6 Hz, H-21); 0.952 (3H, s, H-19); 0.706 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.25 (C-3); 209.02 (C-6); 67.76 (C-22); 57.46 (C-5); 56.30 (C-14); 53.38 (C-9); 52.29 (C-17); 46.55 (C-7); 43.04 (C-13); 41.20 (C-10); 39.17 (C-12); 38.56 (C-20); 38.04 (C-1); 37.99 (C-8); 37.34 (C-2); 36.95 (C-4); 27.49 (C-16); 24.06 (C-15); 21.61 (C-11); 16.69 (C-21); 12.53 (C-19); 12.06 (C-18) (Figures S26–S30, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₂H₃₄O₃: 347.2581 [M + H]⁺; found 347.2590 [M + H]⁺ (Figure S106, Supplementary Material).

3.2.8. 3,6-Dioxo-23,24-dinor-5α-cholan-(4-substituted or 2-substituted)-benzoate-22-yl (19–33)

General procedure: Compound 18 is dissolved in CH₂Cl₂ (20 mL) with slow stirring, and after that, pyridine (py, 0.3 mL), 4-dimetilaminopyiridine (DMAP) (15 mg), and p-PhCOCl or o-PhCOCl are added at room temperature. The end of the reaction is verified by TLC (3 h). Then, 5 mL of acid water (pH = 4) is added and stirred for 30 min. The crude is extracted with CH₂Cl₂ (2 × 20 mL) and washed with acid water (3 × 25 mL) until pH = 4. The organic layer is dried over MgSO₄ and filtered, and the solvent is evaporated under reduced pressure. The crude is redissolved in CH₂Cl₂ (5–10 mL) and chromatographed on silica gel with hexane–EtOAc mixtures of increasing polarity. Finally, the selected fractions are chromatographed on Sephadex LH-20 with hexane:CH₂Cl₂:MeOH (2:1:1).

3,6-Dioxo-23,24-dinor-5α-cholan-benzoate-22-yl (19)

Compound 19 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (90 mg, 0.26 mmol), CH₂Cl₂ (20 mL), py (0.3 mL), DMAP (15 mg) and PhCOCl (0.3 mL, 2.58 mmol, d = 1.21 g/mL). Compound 19 (52 mg, 44.4% yield) is obtained as a colorless solid; m.p. = 208.0–209.1 °C (EtOAc/hex.). IR_νmax (KBr, cm⁻¹): 2945, 2900 and 2868 (CH-); 1716 (C=O); 1449 (CH₂-); 1393 (CH₃-); 1277 and 1108 (C-O); 718 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 8.04 (2H, d, J = 8.3 Hz, H-2′ and H-6′); 7.57 (1H, t, J = 7.4 Hz, H-4′); 7.45 (2H, d, J = 7.4 Hz, H-3′ and H-5′); 4.32 (1H, dd, J = 10.8 and 3.4 Hz, H-22a); 4.07 (1H, dd, J = 10.8 and 6.9 Hz, H-22b); 2.63–2.57 (2H, m, H-5 and H-2); 2.02 (1H, dd, J = 13.0 and 12.6 Hz, H-7α); 1.16 (1H, ddd, J = 17.8, 11.9 and 5.9 Hz, H-15); 1.13 (3H, d, J = 6.6 Hz, H-21); 0.964 (3H, s, H-19); 0.751 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.20 (C-3); 208.91 (C-6); 166.70 (Ar-CO₂); 132.86 (C-4′); 130.46 (C-1′); 129.49 (C-2′ and C-6′); 128.36 (C-3′ and C-5′); 69.77 (C-22); 57.48 (C-5); 56.26 (C-14); 53.39 (C-9); 52.82 (C-17); 46.52 (C-7); 43.17 (C-13); 41.19 (C-10); 39.20 (C-12); 38.07 (C-1); 37.98 (C-8); 37.39 (C-2); 36.96 (C-4); 35.94 (C-20); 27.54 (C-16); 24.07 (C-15); 21.64 (C-11); 17.31 (C-21); 12.55 (C-19); 12.08 (C-18) (Figures S31–S35, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₈O₄: 451.2843 [M + H]⁺; found 451.2847 [M + H]⁺ (Figure S107, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-methyl)-benzoate-22-yl (20)

Compound 20 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (20 mL), py (0.2 mL), DMAP (15 mg) and p-CH₃-PhCOCl (0.7 mL, 5.30 mmol, d = 1.17 g/mL). Compound 20 (67 mg, 50.0% yield) is obtained as a colorless solid; m.p. = 199.3–200.9 °C (EtOAc/hex.). IR_νmax (KBr, cm⁻¹): 2947 and 2868 (CH-); 1714 (C=O); 1610 (C=C); 1274 and 1178 (C-O); 755 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.92 (2H, d, J = 8.2 Hz, H-2′ and H-6′); 7.24 (2H, d, J = 8.2 Hz, H-3′ and H-5′); 4.29 (1H, dd, J = 10.7 and 3.4 Hz, H-22a); 4.04 (1H, dd, J = 10.7 and 7.1 Hz, H-22b); 2.61–2.57 (2H, m, H-5 and H-2); 2.41 (3H, s, Ar-CH₃); 2.01 (1H, t, J = 12.7 Hz, H-7α); 1.12 (3H, d, J = 6.6 Hz, H-21); 0.962 (3H, s, H-19); 0.746 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.18 (C-3); 208.91 (C-6); 166.75 (Ar-CO₂); 143.50 (C-4′); 129.50 (C-3′ and C-5′); 129.05 (C-2′ and C-6′); 127.71 (C-1′); 69.55 (C-22); 57.46 (C-5); 56.24 (C-14); 53.36 (C-9); 52.82 (C-17); 46.50 (C-7); 43.15 (C-13); 41.17 (C-10); 39.18 (C-12); 38.05 (C-1); 37.95 (C-8); 37.33 (C-2); 36.94 (C-4); 35.93 (C-20); 27.52 (C-16); 24.06 (C-15); 21.63 (Ar-CH₃); 21.61 (C-11); 17.29 (C-21); 12.53 (C-19); 12.06 (C-18) (Figures S36–S40, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₀H₄₀O₄: 465.2999 [M + H]⁺; found 465.3012 [M + H]⁺ (Figure S108, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-methoxy)-benzoate-22-yl (21)

Compound 21 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (149 mg, 0.43 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and p-OCH₃-PhCOCl (970 mg, 5.67 mmol). Compound 21 (38 mg, 18.4% yield) is obtained as a colorless solid; m.p. = 206.2–207.6 °C (EtOAc/hex.). IR_νmax (KBr, cm⁻¹): 2947, 2894 and 2862 (CH-); 1709 (C=O); 1604 and 1511 (C=C); 1259 and 1164 (C-O); 852 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.98 (2H, d, J = 8.9 Hz, H-2′ and H-6′); 6.92 (2H, d, J = 8.9 Hz, H-3′ and H-5′); 4.27 (1H, dd, J = 10.7 and 3.4 Hz, H-22a); 4.02 (1H, dd, J = 10.7 and 7.1 Hz, H-22b); 3.85 (3H, s, Ar-OCH₃); 2.60–2.56 (2H, m, H-5 and H-2); 2.00 (1H, t, J = 12.7 Hz, H-7α); 1.11 (3H, d, J = 6.6 Hz, H-21); 0.952 (3H, s, H-19); 0.737 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.24 (C-3); 208.95 (C-6); 166.45 (Ar-CO₂); 163.26 (C-4′); 131.47 (C-2′ and C-6′); 122.85 (C-1′); 113.57 (C-3′ and C-5′); 69.44 (C-22); 57.43 (C-5); 56.23 (C-14); 55.39 (Ar-OCH₃); 53.35 (C-9); 52.83 (C-17); 46.48 (C-7); 43.13 (C-13); 41.16 (C-10); 39.17 (C-12); 38.02 (C-1); 37.94 (C-8); 37.31 (C-2); 36.92 (C-4); 35.92 (C-20); 27.50 (C-16); 24.04 (C-15); 21.60 (C-11); 17.29 (C-21); 12.51 (C-19); 12.04 (C-18) (Figures S41–S45, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₀H₄₀O₅: 481.2949 [M + H]⁺; found 481.2951 [M + H]⁺ (Figure S109, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-fluoro)-benzoate-22-yl (22)

Compound 22 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and p-F-PhCOCl (0.3 mL, 2.54 mmol, d = 1.34 g/mL). Compound 22 (80 mg, 59.2% yield) is obtained as a colorless solid; m.p. = 197.3–198.5 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2965, 2944 and 2865 (CH-); 1716 (C=O); 1604 (C=C); 1281 and 1155 (C-O); 763 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 8.04 (2H, dd, J = 8.7 and 5.5 Hz, H-2′ and H-6′); 7.11 (2H, t, J = 8.7 Hz, H-3′ and H-5′); 4.30 (1H, dd, J = 10.7 and 3.4 Hz, H-22a); 4.04 (1H, dd, J = 10.7 and 7.1 Hz, H-22b); 2.61–2.56 (2H, m, H-5 and H-2); 2.01 (1H, t, J = 12.7 Hz, H-7α); 1.11 (3H, d, J = 6.6 Hz, H-21); 0.955 (3H, s, H-19); 0.740 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.16 (C-3); 208.87 (C-6); 165.70 (Ar-CO₂); 165.67 (d, ¹J_C-F = 253.6 Hz, C-4′); 131.98 (d, ³J_C-F = 10.1 Hz, C-2′ and C-6′); 126.66 (d, ⁴J_C-F = 3.0 Hz, C-1′); 115.47 (d, ²J_C-F = 22.1 Hz, C-3′ and C-5′); 69.87 (C-22); 57.44 (C-5); 56.22 (C-14); 53.34 (C-9); 52.79 (C-17); 46.47 (C-7); 43.15 (C-13); 41.15 (C-10); 39.17 (C-12); 38.03 (C-1); 37.93 (C-8); 37.31 (C-2); 36.92 (C-4); 35.89 (C-20); 27.51 (C-16); 24.04 (C-15); 21.60 (C-11); 17.27 (C-21); 12.51 (C-19); 12.05 (C-18) (Figures S46–S50, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇FO₄: 469.2749 [M + H]⁺; found 469.2755 [M + H]⁺ (Figure S110, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-chloro)-benzoate-22-yl (23)

Compound 23 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (92 mg, 0.27 mmol), CH₂Cl₂ (20 mL), py (0.2 mL), DMAP (15 mg) and p-Cl-PhCOCl (0.6 mL, 4.70 mmol, d = 1.37 g/mL). Compound 23 (37 mg, 28.7% yield) is obtained as a colorless solid; m.p. = 210.1–211.5 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2946 (CH-); 1715 (C=O); 1592 (C=C); 1274 and 1116 (C-O); 762 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.97 (2H, d, J = 8.6 Hz, H-2′ and H-6′); 7.42 (2H, d, J = 8.6 Hz, H-3′ and H-5′); 4.31 (1H, dd, J = 10.7 and 3.5 Hz, H-22a); 4.05 (1H, dd, J = 10.8 and 7.2 Hz, H-22b); 2.61–2.58 (2H, m, H-5 and H-2); 2.01 (1H, t, J = 12.7 Hz, H-7α); 1.12 (3H, d, J = 6.6 Hz, H-21); 0.964 (3H, s, H-19); 0.748 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.19 (C-3); 208.90 (C-6); 165.83 (Ar-CO₂); 139.31 (C-4′); 130.88 (C-2′ and C-6′); 128.87 (C-1′); 128.72 (C-3′ and C-5′); 70.01 (C-22); 57.48 (C-5); 56.25 (C-14); 53.37 (C-9); 52.81 (C-17); 46.50 (C-7); 43.18 (C-13); 41.18 (C-10); 39.19 (C-12); 38.06 (C-1); 37.96 (C-8); 37.34 (C-2); 36.95 (C-4); 35.91 (C-20); 27.54 (C-16); 24.06 (C-15); 21.62 (C-11); 17.28 (C-21); 12.54 (C-19); 12.07 (C-18) (Figures S51–S55, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇ClO₄: 485.2453 [M + H]⁺; found 485.2440 [M + H]⁺ (Figure S111, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-bromo)-benzoate-22-yl (24)

Compound 24 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and p-Br-PhCOCl (380 mg, 1.73 mmol). Compound 24 (90 mg, 58.9% yield) is obtained as a colorless solid; m.p. = 224.1–225.8 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2951, 2899 and 2866 (CH-); 1714 (C=O); 1588 (C=C); 1271 and 1175 (C-O); 761 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.89 (2H, d, J = 8.6 Hz, H-2′ and H-6′); 7.58 (2H, d, J = 8.6 Hz, H-3′ and H-5′); 4.31 (1H, dd, J = 10.8 and 3.5 Hz, H-22a); 4.05 (1H, dd, J = 10.8 and 7.2 Hz, H-22b); 2.61–2.57 (2H, m, H-5 and H-2); 2.01 (1H, t, J = 12.7 Hz, H-7α); 1.11 (3H, d, J = 6.6 Hz, H-21); 0.963 (3H, s, H-19); 0.747 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.17 (C-3); 208.88 (C-6); 165.95 (Ar-CO₂); 131.71 (C-2′ and C-6′); 131.03 (C-3′ and C-5′); 129.34 (C-1′); 127.97 (C-4′); 70.03 (C-22); 57.47 (C-5); 56.25 (C-14); 53.37 (C-9); 52.81 (C-17); 46.50 (C-7); 43.19 (C-13); 41.18 (C-10); 39.19 (C-12); 38.06 (C-1); 37.96 (C-8); 37.34 (C-2); 36.95 (C-4); 35.90 (C-20); 27.54 (C-16); 24.06 (C-15); 21.62 (C-11); 17.28 (C-21); 12.54 (C-19); 12.08 (C-18) (Figures S56–S60, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇BrO₄: 531.1932 [M + H]⁺; found 531.1940 [M + H]⁺ (Figure S112, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-iodo)-benzoate-22-yl (25)

Compound 25 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl2 (25 mL), py (0.3 mL), DMAP (15 mg) and p-I-PhCOCl (370 mg, 1.39 mmol). Compound 25 (45 mg, 27.0% yield) is obtained as a colorless solid; m.p. = 245.6–247.6 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2949, 2899 and 2865 (CH-); 1712 (C=O); 1583 (C=C); 1283, 1266 and 1180 (C-O); 759 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.80 (2H, d, J = 8.4 Hz, H-3′ and H-5′); 7.73 (2H, d, J = 8.4 Hz, H-2′ and H-6′); 4.30 (1H, dd, J = 10.8 and 3.3 Hz, H-22a); 4.05 (1H, dd, J = 10.8 and 7.2 Hz, H-22b); 2.62–2.56 (2H, m, H-5 and H-2); 2.41 (3H, s, Ar-CH₃); 2.01 (1H, t, J = 12.7 Hz, H-7α); 1.11 (3H, d, J = 6.6 Hz, H-21); 0.960 (3H, s, H-19); 0.743 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.14 (C-3); 208.86 (C-6); 166.17 (Ar-CO₂); 137.72 (C-3′ and C-5′); 130.94 (C-2′ and C-6′); 129.90 (C-1′); 100.61 (C-4′); 70.01 (C-22); 57.46 (C-5); 56.24 (C-14); 53.36 (C-9); 52.80 (C-17); 46.49 (C-7); 43.17 (C-13); 41.17 (C-10); 39.19 (C-12); 38.05 (C-1); 37.94 (C-8); 37.33 (C-2); 36.94 (C-4); 35.89 (C-20); 27.53 (C-16); 24.05 (C-15); 21.61 (C-11); 17.27 (C-21); 12.53 (C-19); 12.07 (C-18) (Figures S61–S65, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇IO₄: 577.1809 [M + H]⁺; found 577.1800 [M + H]⁺ (Figure S113, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(4-acetoxy)-benzoate-22-yl (26)

Compound 26 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (110 mg, 0.32 mmol), CH₂Cl₂ (25mL), py (0.5 mL), DMAP (23 mg) and p-CH₃COO-PhCOCl (300 mg, 1.52 mmol). Compound 26 (52 mg, 63.8% yield) is obtained as a colorless solid; m.p. = 179.5–180.8 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2966, 2944 and 2865 (CH-); 1763 and 1715 (C=O); 1603 (C=C); 1276, 1192, 1159 and 1113 (C-O); 763 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 8.06 (2H, d, J = 8.8 Hz, H-2′ and H-6′); 7.17 (2H, d, J = 8.8 Hz, H-3′ and H-5′); 4.30 (1H, dd, J = 10.7 and 3.4 Hz, H-22a); 4.07 (1H, dd, J = 10.7 and 7.0 Hz, H-22b); 2.61–2.57 (2H, m, H-5 and H-2); 2.32 (3H, s, COCH3); 2.01 (1H, t, J = 12.8 Hz, H-7α); 1.12 (3H, d, J = 6.6 Hz, H-21); 0.960 (3H, s, H-19); 0.744 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.17 (C-3); 208.90 (C-6); 168.90 (COCH₃); 165.85 (Ar-CO₂); 154.21 (C-4′); 131.07 (C-2′ and C-6′); 128.02 (C-1′); 121.60 (C-3′ and C-5′); 69.87 (C-22); 57.46 (C-5); 56.24 (C-14); 53.37 (C-9); 52.78 (C-17); 46.50 (C-7); 43.15 (C-13); 41.18 (C-10); 39.18 (C-12); 38.05 (C-1); 37.96 (C-8); 37.33 (C-2); 36.94 (C-4); 35.91 (C-20); 27.52 (C-16); 24.05 (C-15); 21.62 (C-11); 21.13 (COCH₃); 17.29 (C-21); 12.53 (C-19); 12.07 (C-18) (Figures S66–S70, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₁H₄₀O₆: 509.2898 [M + H]⁺; found 509.2897 [M + H]⁺ (Figure S114, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-methyl)-benzoate-22-yl (27)

Compound 27 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-CH₃-PhCOCl (0.5 mL, 3.85 mmol, d = 1.19 g/mL). Compound 27 (54 mg, 40.3% yield) is obtained as a colorless solid; m.p. = 203.2–205.5 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2932 and 2854 (CH-); 1714 (C=O); 1264 and 1130 (C-O); 741 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.91 (1H, d, J = 8.4 and 1.5 Hz, H-6′); 7.40 (1H, ddd, J = 7.5, 7.5 and 1.3 Hz, H-4′); 7.27–7.23 (2H, m, H-5′ and H-3′); 4.30 (1H, dd, J = 10.8 and 3.2 Hz, H-22a); 4.04 (1H, dd, J = 10.8 and 7.0 Hz, H-22b); 2.62–2.54 (2H, m, H-5 and H-2); 2.61 (3H, s, CH₃-Ar); 2.01 (1H, dd, J = 12.7 and 12.7 Hz, H-7α); 1.19–1.15 (1H, m, H-15); 1.13 (3H, d, J = 6.6 Hz, H-21); 0.962 (3H, s, H-19); 0.747 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.16 (C-3); 208.89 (C-6); 167.76 (Ar-CO₂); 140.11 (C-2′); 131.88 (C-4′); 131.70 (C-5′); 130.48 (C-6′); 129.85 (C-1′); 125.68 (C-3′); 69.65 (C-22); 57.47 (C-5); 56.28 (C-14); 53.37 (C-9); 52.76 (C-17); 46.51 (C-7); 43.13 (C-13); 41.18 (C-10); 39.20 (C-12); 38.06 (C-1); 37.97 (C-8); 37.34 (C-2); 36.95 (C-4); 35.87 (C-20); 27.54 (C-16); 24.05 (C-15); 21.85 (CH₃-Ar); 21.63 (C-11); 17.44 (C-21); 12.53 (C-19); 12.08 (C-18) (Figures S71–S75, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₀H₄₀O₄: 465.2999 [M + H]⁺; found 465.3000 [M + H]⁺ (Figure S115, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-methoxy)-benzoate-22-yl (28)

Compound 28 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-CH₃O-PhCOCl (0.4 mL, 2.70 mmol, d = 1.15 g/mL). Compound 28 (88 mg, 63.4% yield) is obtained as a colorless solid; m.p. = 182.4–183.4 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2948 and 2866 (CH-); 1712 (C=O); 1598 and 1582 (C=C); 1253 and 1167 (C-O); 757 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.92 (1H, d, J = 7.9 and 1.9 Hz, H-6′); 7.47 (1H, ddd, J = 7.5, 7.5 and 1.8 Hz, H-4′); 7.00–6.96 (2H, m, H-5′ and H-3′); 4.30 (1H, dd, J = 10.8 and 3.3 Hz, H-22a); 4.02 (1H, dd, J = 10.8 and 7.1 Hz, H-22b); 3.90 (3H, s, OCH₃); 2.62–2.54 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 12.5 and 12.5 Hz, H-7α); 1.20–1.15 (1H, m, H-15); 1.12 (3H, d, J = 6.6 Hz, H-21); 0.957 (3H, s, H-19); 0.735 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.18 (C-3); 208.90 (C-6); 167.39 (Ar-CO₂); 159.39 (C-2′); 133.41 (C-4′); 131.54 (C-6′); 120.31 (C-1′); 120.06 (C-5′); 111.97 (C-3′); 69.65 (C-22); 57.46 (C-5); 56.30 (C-14); 55.86 (OCH₃); 53.38 (C-9); 52.71 (C-17); 46.51 (C-7); 43.12 (C-13); 41.17 (C-10); 39.20 (C-12); 38.04 (C-1); 37.96 (C-8); 37.33 (C-2); 36.94 (C-4); 35.86 (C-20); 27.46 (C-16); 24.06 (C-15); 21.62 (C-11); 17.26 (C-21); 12.52 (C-19); 12.07 (C-18) (Figures S76–S80, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₀H₄₀O₅: 481.2949 [M + H]⁺; found 481.2942 [M + H]⁺ (Figure S116, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-fluoro)-benzoate-22-yl (29)

Compound 29 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-F-PhCOCl (0.2 mL, 1.68 mmol, d = 1.33 g/mL). Compound 29 (64 mg, 47.3% yield) is obtained as a colorless solid; m.p. = 222.3–224.1 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2953 and 2867 (CH-); 1718 (C=O); 1612 (C=C); 1259 and 1159 (C-O); 769 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.94 (1H, dd, J = 7.6 and 1.7 Hz, H-4′); 7.55–7.49 (1H, m, H-5′); 7.21 (1H, t, J = 7.6 Hz, H-6′); 7.13 (1H, dd, J = 10.6, and 8.6 Hz, H-3′); 4.33 (1H, dd, J = 10.7 and 3.1 Hz, H-22a); 4.08 (1H, dd, J = 10.7 and 6.9 Hz, H-22b); 2.62–2.54 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 12.7 and 12.7 Hz, H-7α); 1.20–1.16 (1H, m, H-15); 1.13 (3H, d, J = 6.9 Hz, H-21); 0.960 (3H, s, H-19); 0.742 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.19 (C-3); 208.91 (C-6); 164.71 (d, ³J_C-F = 3.9 Hz, Ar-CO₂); 161.93 (d, ¹J_C-F = 259.8 Hz, C-2′); 134.36 (d, ³J_C-F = 8.8 Hz, C-4′); 132.09 (C-5′); 123.93 (d, ³J_C-F = 3.8 Hz, C-6′); 118.93 (d, ²J_C-F = 9.9 Hz, C-1′); 116.97 (d, ²J_C-F = 22.8 Hz, C-3′); 70.20 (C-22); 57.47 (C-5); 56.26 (C-14); 53.37 (C-9); 52.56 (C-17); 46.51 (C-7); 43.11 (C-13); 41.18 (C-10); 39.16 (C-12); 38.06 (C-1); 37.97 (C-8); 37.34 (C-2); 36.95 (C-4); 35.81 (C-20); 27.47 (C-16); 24.04 (C-15); 21.62 (C-11); 17.23 (C-21); 12.53 (C-19); 12.06 (C-18) (Figures S81–S85, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇FO₄: 469.2749 [M + H]⁺; found 469.2745 [M + H]⁺ (Figure S117, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-chloro)-benzoate-22-yl (30)

Compound 30 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-Cl-PhCOCl (0.2 mL, 1.58 mmol, d = 1.38 g/mL). Compound 30 (64 mg, 45.7% yield) is obtained as a colorless solid; m.p. = 204.8–206.2 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2945 and 2904 (CH-); 1713 (C=O); 1595 (C=C); 1307 and 1137 (C-O); 763 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.83 (1H, dd, J = 7.7 and 1.6 Hz, H-6′); 7.46 (1H, dd, J = 8.2 and 1.3 Hz, H-3′); 7.42 (1H, ddd, J = 8.2, 7.0 and 1.6 Hz, H-4′); 7.32 (1H, ddd, J = 7.5, 7.3 and 1.6 Hz, H-5′); 4.35 (1H, dd, J = 10.8 and 3.3 Hz, H-22a); 4.09 (1H, dd, J = 10.8 and 7.0 Hz, H-22b); 2.63–2.54 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 12.8 and 12.8 Hz, H-7α); 1.21–1.16 (1H, m, H-15); 1.14 (3H, d, J = 6.6 Hz, H-21); 0.961 (3H, s, H-19); 0.742 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.18 (C-3); 208.90 (C-6); 166.01 (Ar-CO₂); 133.58 (C-2′); 132.45 (C-4′); 131.41 (C-6′); 131.08 (C-3′); 130.42 (C-1′); 126.57 (C-5′); 70.54 (C-22); 57.48 (C-5); 56.28 (C-14); 53.37 (C-9); 52.57 (C-17); 46.51 (C-7); 43.13 (C-13); 41.18 (C-10); 39.18 (C-12); 38.06 (C-1); 37.97 (C-8); 37.34 (C-2); 36.95 (C-4); 35.83 (C-20); 27.52 (C-16); 24.05 (C-15); 21.63 (C-11); 17.38 (C-21); 12.54 (C-19); 12.07 (C-18) (Figures S86–S90, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇ClO₄: 485.2453 [M + H]⁺; found 485.2451 [M + H]⁺ (Figure S118, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-bromo)-benzoate-22-yl (31)

Compound 31 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (130 mg, 0.38 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-Br-PhCOCl (0.4 mL, 3.06 mmol, d = 1.68 g/mL). Compound 31 (104 mg, 52.4% yield) is obtained as a colorless solid; m.p. = 202.5–204.7 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2945, 2904 and 2861 (CH-); 1713 (C=O); 1590 (C=C); 1305 and 1134 (C-O); 759 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.78 (1H, dd, J = 7.5 and 2.0 Hz, H-6′); 7.66 (1H, dd, J = 7.7 and 1.3 Hz, H-3′); 7.37 (1H, ddd, J = 7.7, 7.7 and 1.3 Hz, H-4′); 7.32 (1H, ddd, J = 7.5, 7.5 and 2.0 Hz, H-5′); 4.34 (1H, dd, J = 10.7 and 3.2 Hz, H-22a); 4.08 (1H, dd, J = 10.7 and 6.9 Hz, H-22b); 2.62–2.54 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 13.0 and 13.0 Hz, H-7α); 1.22–1.16 (1H, m, H-15); 1.13 (3H, d, J = 6.6 Hz, H-21); 0.957 (3H, s, H-19); 0.738 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.16 (C-3); 208.88 (C-6); 166.42 (Ar-CO₂); 134.33 (C-3′); 132.51 (C-1′); 132.44 (C-5′); 131.26 (C-6′); 127.14 (C-4′); 121.51 (C-2′); 70.59 (C-22); 57.45 (C-5); 56.26 (C-14); 53.34 (C-9); 52.56 (C-17); 46.49 (C-7); 43.13 (C-13); 41.16 (C-10); 39.16 (C-12); 38.04 (C-1); 37.95 (C-8); 37.33 (C-2); 36.94 (C-4); 35.81 (C-20); 27.52 (C-16); 24.03 (C-15); 21.61 (C-11); 17.39 (C-21); 12.53 (C-19); 12.06 (C-18) (Figures S91–S95, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇BrO₄: 531.1932 [M + H]⁺; found 531.1936 [M + H]⁺ (Figure S119, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-iodo)-benzoate-22-yl (32)

Compound 32 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-I-PhCOCl (755 mg, 2.83 mmol). Compound 32 (128 mg, 76.9% yield) is obtained as a colorless solid; m.p. = 202.9–204.1 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2943, 2903 and 2861 (CH-); 1713 (C=O); 1584 (C=C); 1300, 1249 and 1130 (C-O); 755 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 7.99 (1H, dd, J = 8.0 and 0.9 Hz, H-3′); 7.78 (1H, dd, J = 7.7 and 1.6 Hz, H-6′); 7.41 (1H, ddd, J = 7.7, 7.7 and 1.1 Hz, H-5′); 7.15 (1H, ddd, J = 8.0, 7.7 and 1.6 Hz, H-4′); 4.34 (1H, dd, J = 10.8 and 3.3 Hz, H-22a); 4.07 (1H, dd, J = 10.8 and 7.2 Hz, H-22b); 2.62–2.54 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 12.9 and 12.9 Hz, H-7α); 1.20–1.15 (1H, m, H-15); 1.13 (3H, d, J = 6.6 Hz, H-21); 0.955 (3H, s, H-19); 0.737 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.15 (C-3); 208.88 (C-6); 166.68 (Ar-CO₂); 141.30 (C-3′); 135.44 (C-1′); 132.52 (C-4′); 130.78 (C-6′); 127.87 (C-5′); 93.96 (C-2′); 70.56 (C-22); 57.44 (C-5); 56.24 (C-14); 53.33 (C-9); 52.61 (C-17); 46.48 (C-7); 43.14 (C-13); 41.16 (C-10); 39.15 (C-12); 38.03 (C-1); 37.94 (C-8); 37.32 (C-2); 36.93 (C-4); 35.81 (C-20); 27.54 (C-16); 24.03 (C-15); 21.60 (C-11); 17.41 (C-21); 12.52 (C-19); 12.07 (C-18) (Figures S96–S100, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₂₉H₃₇IO₄: 577.1809 [M + H]⁺; found 577.1815 [M + H]⁺ (Figure S120, Supplementary Material).

3,6-Dioxo-23,24-dinor-5α-cholan-(2-acetoxy)-benzoate-22-yl (33)

Compound 33 is obtained by using the general procedure described in 3.2.8 and under the following conditions: 18 (100 mg, 0.29 mmol), CH₂Cl₂ (25 mL), py (0.3 mL), DMAP (15 mg) and o-CH₃COO-PhCOCl (260 mg, 1.31 mmol). Compound 33 (82 mg, 55.9% yield) is obtained as a colorless solid; m.p. = 148.2–149.2 °C (CH₂Cl₂). IR_νmax (KBr, cm⁻¹): 2947 and 2867 (CH-); 1770 and 1716 (C=O); 1606 (C=C); 1293, 1259, 1192 and 1078 (C-O); 752 (CH=CH). ¹H NMR (400.1 MHz, CDCl₃) δ (ppm): 8.00 (1H, dd, J = 7.8 and 1.5 Hz, H-6′); 7.57 (1H, ddd, J = 8.0, 7.8 and 1.6 Hz H-4′); 7.32 (1H, ddd, J = 7.8, 7.8 and 0.8 Hz, H-5′); 7.11 (1H, ddd, J = 8.0, 7.8 and 0.8 Hz, H-3′); 4.27 (1H, dd, J = 10.7 and 3.3 Hz, H-22a); 4.00 (1H, dd, J = 10.7 and 7.4 Hz, H-22b); 2.63–2.55 (2H, m, H-5 and H-2); 2.01 (1H, dd, J = 12.6 and 12.6 Hz, H-7α); 1.17 (1H, ddd, J = 12.5, 11.8 and 5.9 Hz, H-15); 1.11 (3H, d, J = 6.6 Hz, H-21); 0.962 (3H, s, H-19); 0.740 (3H, s, H-18). ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm): 211.16 (C-3); 208.88 (C-6); 169.69 (CH₃CO); 150.79 (C-2′); 133.78 (C-4′); 131.48 (C-6′); 125.97 (C-5′); 123.86 (C-3′); 123.39 (C-1′); 69.88 (C-22); 57.48 (C-5); 56.23 (C-14); 53.37 (C-9); 52.75 (C-17); 46.51 (C-7); 43.19 (C-13); 41.19 (C-10); 39.19 (C-12); 38.07 (C-1); 37.97 (C-8); 37.35 (C-2); 36.96 (C-4); 35.88 (C-20); 27.53 (C-16); 24.07 (C-15); 21.63 (C-11); 21.07 (CH₃CO); 17.28 (C-21); 12.55 (C-19); 12.08 (C-18) (Figures S101–S105, Supplementary Material). HRMS-ESI (positive mode): m/z calculated for C₃₁H₄₀O₆: 509.2898 [M + H]⁺; found 509.2899 [M + H]⁺ (Figure S121, Supplementary Material).

3.3. Bioactivity Measurements

3.3.1. Rice Lamina Inclination Test (RLIT)

The bioactivity of BR analogs (19–33 and precursor 18) was evaluated by RLIT [20] using a modified version of a previously described procedure [7]. Seeds of a local rice (Oryza sativa L) cultivar of the Zafiro variety (provided by INIA-QUILAMAPU, Chillán, Chile) were sterilized, sown, and grown to the second internode of the lamina. Once 8 cm segments were obtained, they were placed in Petri dishes containing sterile distilled water (60 mL). Standard dissolutions of the BR analogs were prepared by dissolving pure compounds in 1 mL of THF and diluting them with water (0.01:50) to reach analog concentration of approximately 1 × 10⁻⁵ M. Then, different volumes of these dissolutions were added to obtain final concentrations of 1 × 10⁻⁸, 1 × 10⁻⁷, and 1 × 10⁻⁶ M. Brassinolide (2) (APExBIO; Houston, TX, USA) was used as a positive control at the same tested concentrations, while an aqueous solution of tetrahydrofuran (0.002%) was used as a negative control. After 72 h of incubation in the dark, the angle of inclination of the slice, between the leaf and the sheath, was measured. Images were taken with a microscope, including camera software (Leica Microsystem EZ4HD, Leica Application Suite X, Wetzlar, Germany). Each treatment consisted of 8 independent replicates. The effect of BR analogs on rice was assessed by comparing bending angles in the absence and presence of BRs. The measured bending angles (ba) are shown in Figure S122, and the differences between BRs and negative control are given in Table S1. For the sake of comparison, BR RLIT activities were calculated by normalizing these differences with respect to the negative control (Equation (1)).

$$\mathrm{Activity}(\mathrm{RLIT})={\displaystyle \frac{\left[\mathrm{ba}\right(\mathrm{BRs} \mathrm{analogs})-\mathrm{ba}(\mathrm{negative} \mathrm{control}\left)\right]}{\left[\mathrm{ba}\right(\mathrm{negative} \mathrm{control}\left)\right]}}$$

(1)

Finally, to obtain the relative activity with respect to the positive control, the values obtained from equation 1 were divided by the corresponding activity of brassinolide.

$$\mathrm{Relative} \mathrm{activity}={\displaystyle \frac{\mathrm{Activity}\left(\mathrm{RLIT}\right)}{\mathrm{Activity}\left(\mathrm{brassinolide}\right)}}$$

(2)

The data were subjected to an analysis of variance and an LSD–Fischer mean comparison test (p < 0.05) using InfoStat software version 2020 (Córdoba, Argentina).

3.3.2. Bean Second-Internode Bioassay

The bean second-internode test for compounds 18 and 19–33 was performed following the procedure described in [21], with some modifications [7]. Bean (Phaseolus vulgaris L., cv. Pinto) seeds were sown, and plants were grown under controlled conditions into a plant growth chamber until the second internode was 1–2 mm long. Then, the bract was removed from the base of the second internode, and standard dissolutions of each treatment (10 µL, 1 × 10⁻⁸ M) and TWEEN^® 20 (AMRESCO, Solon, OH, USA) (2 µL) were applied. Control plants were treated with water and TWEEN^® 20 only. After 5 days, the difference between the length of the second internode of treated and control plants was measured. Data were subjected to an analysis of variance and an LSD–Fischer mean comparison test (p < 0.05) using InfoStat software version 2020 (Córdoba, Argentina).

4. Conclusions

A new series of 23,24-dinorcholanic 3,6-dioxo derivative analogs (compounds 19–33) have been synthesized and fully characterized by their physical and spectroscopic properties. An assessment of their biological activity by using RLIT and BSI bioassays has been performed. RLIT data indicate that the incorporation of benzoylate function in the C-22 position induces an interesting increase in the angle opening at all tested concentrations. This activity depends on the position and nature of the phenyl substituent and is also dependent on concentration. At the lowest tested concentration, 1 × 10⁻⁸ M, analog 33 (o-OAc) exhibits the highest activity, i.e., slightly higher than that obtained for brassinolide. Thus, the data suggest that the chemical structure of the benzoate substituent on 3-DT derivatives is a key factor in determining their RLIT activity, but there it is not enough information to establish a clear correlation with activity. However, a comparison of the RLIT results obtained herein with those previously reported for other series of benzoylated analogs allows us to obtain a clearer structure–activity relationship. For example, 3-DT analogs carrying an extra alcohol group in the alkyl chain (C-22) exhibit lower activity than the 3-DT analogs studied herein. On the other hand, benzoylated BR analogs with a -OH group at C-3 are much more active than the respective derivatives with carbonyl group in this position. However, in both series, the activity increases for benzoylated groups substituted with -OCH₃ and I atom in the para-position. This effect has been attributed to the increasing size of these BRs derivatives. Thus, it would be interesting to evaluate the activity of benzoylated TE derivatives with the substituent in the ortho-position.

Interestingly, ortho-derivatives are significantly more active in the bean second-internode elongation bioassay, except derivative 21 (p-OCH₃), which is even more active than brassinolide and all other analogs. These results are clearly different from those obtained in the RLIT bioassay, and therefore, it can be concluded that the activity of BR derivatives cannot be determined by just one bioassay. So, their application and activity–structure relationship will always be dependent on the bioassay used to determine activity.

Finally, it is important to emphasize that the synthesis of 33, the most active 3-DT analog, is performed with a total reaction yield of 17% and using hyodeoxycholic acid, a secondary bile acid that is commercially available, as the starting material. This means that the synthesis described herein could be scaled for a potential application of this analog.