Synthesis and Biological Evaluation of 3-Amino-4,4-Dimethyl Lithocholic Acid Derivatives as Novel, Selective, and Cellularly Active Allosteric SHP1 Activators

Abstract

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor member of the protein tyrosine phosphatase (PTP) family, negatively regulates several signaling pathways that are responsible for pathological cell processes in cancers. In this study, we report a series of 3-amino-4,4-dimethyl lithocholic acid derivatives as SHP1 activators. The most potent compounds, 5az-ba, showed low micromolar activating effects (EC₅₀: 1.54–2.10 μM) for SHP1, with 7.63–8.79-fold maximum activation and significant selectivity over the closest homologue Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) (>32-fold). 5az-ba showed potent anti-tumor effects with IC₅₀ values of 1.65–5.51 μM against leukemia and lung cancer cells. A new allosteric mechanism of SHP1 activation, whereby small molecules bind to a central allosteric pocket and stabilize the active conformation of SHP1, was proposed. The activation mechanism was consistent with the structure–activity relationship (SAR) data. This study demonstrates that 3-amino-4,4-dimethyl lithocholic acid derivatives can be selective SHP1 activators with potent cellular efficacy.

1. Introduction

No commercial PTP-targeted drug has been approved for decades. The biological and clinical discovery of PTP inhibitors is hampered by off-target profiles, poor pharmacokinetic properties, or the emergence of drug resistance. In contrast, SHP1 was found to negatively regulate various pathological processes, including hematopoietic lineage differentiation, tumorigenesis, and immune response, which has led to the development of SHP1 activators. SHP1 is localized mainly in the cytoplasm and is expressed on multiple cell types, including hematopoietic, epithelial, and mesenchymal cells. Low expression of SHP1 protein was observed in patients with hepatocellular carcinoma [1], prostate cancer [2], multiple sclerosis [3], and a variety of hematopoietic diseases, such as leukemia [4], lymphoma [5], and multiple myeloma [6], as well as myelodysplastic syndrome [7], whereas SHP1 is up-regulated in patients with nasopharyngeal carcinoma [8] and ovarian cancer [9].

SHP1 dephosphorylates the signal transducer and activator of transcription 3 (STAT3) [10], extracellular signal-regulated kinase (ERK) [11], protein kinase B (Akt) [12], and other signaling components [13,14,15,16], thereby contributing to a decrease in cancer cell proliferation and survival. SHP1 suppresses angiogenesis [17,18] and enhances the anti-tumor efficacy of chemotherapeutic agents [16,19]. SHP1 also inhibits inflammation [20] and osteoclastogenesis [21,22], suggesting that it has therapeutic potential for treating inflammation-mediated diseases and bone diseases. Thus, targeting SHP1 activation could represent a promising therapeutic approach for the above diseases and open a new avenue for PTP drug discovery.

The reported molecules with SHP1-activating effects mainly include diphenyl urea [23,24,25,26], steroid [27,28,29,30,31,32], pentacyclic triterpenoid [33,34,35], and indole scaffolds [36,37,38,39,40] (Figure 1). SC-43 (1) enhanced SHP1 activity to inactivate STAT3, thereby inhibiting the proliferation of hepatocellular carcinoma and colorectal cancer cells [23,24]. β-Sitosterol (2) elevated SHP1 activity, suppressing the subsequent nuclear factor kappa-B (NF-κB) and signal transducer and activator of transcription 1 (STAT1) signaling pathways to exert anti-inflammatory effects [27]. SHP1 activated by betulinic acid (3) induced apoptosis and enhanced the cytotoxic effects of chemotherapeutic agents in multiple myeloma cells via suppressing the STAT3 signaling pathway [33]. Thieno [2,3-b]quinoline-procaine hybrid 4 can directly activate SHP1 and inhibit activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cell proliferation via blocking the STAT3 pathway [41]. However, most of the molecules described above have been identified as inhibitors for other targets. There are still few studies on selective SHP1 activators, which limits the investigations of SHP1 as a pharmacological target for cancer treatment.

Lithocholic acid derivatives, which contain the above steroid scaffold, have been found to inhibit the growth of cancer cells [42,43,44,45]. Two lithocholic acid derivatives, ursodeoxycholic acid and obeticholic acid, are approved drugs for the treatment of primary biliary cholangitis. Furthermore, obeticholic acid is currently in clinical trials for nonalcoholic steatohepatitis (NASH) as a farnesoid X receptor (FXR) agonist. Here, we replaced the carboxyl group of lithocholic acid with a hydroxyl group, modified the C-3, C-7 positions to obtain a series of novel structures, and evaluated their biological activities for SHP1 activation.

2. Results and Discussion

We initiated our efforts by investigating C-3 substituents. Usually, compounds containing the aliphatic –NH– group have better water solubility and stronger hydrogen-bonding ability. We replaced the C-3 hydroxyl group with an amino group to afford the compounds 5aa-af (Scheme 1). Lithocholic acid (6) was subjected to esterification and oxidation via a combination of 2-iodoxybenzoic acid (IBX) and trifluoroacetic acid to afford α,β-unsaturated ketone compound 8. Compound 9 was obtained by C-4 methylation accompanied by a rearrangement of the double bond. Reductive amination of the C-3 carbonyl group afforded compound 10. Compound 5aa was prepared by reducing the C-24 carboxylate group of compound 10. The N-alkylation of compound 10 and the subsequent LiAlH₄ reduction formed compounds 5ab-af.

The SHP1-activating effects of the synthetic compounds were examined by the 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) assay. As shown in

Table 1. SHP1-activating effects of compounds 5aa-af.

Compd	R1	EC50 (μM)	Maximum Activation Fold
5aa	-NH2	19.23 ± 4.59	6.03 ± 0.87
5ab		39.15 ± 3.7	ND a
5ac		30.73 ± 3.8	ND
5ad		47.34 ± 0.34	ND
5ae		>50	ND
5af		>50	ND

^a ND: not determined.

, the compounds 5aa-ac bearing the C-3 hydrophilic amino or extended hydroxyl groups showed better activity. The C-3 alkylamino substitution of compounds 5ad-af showed a decline in the activity. These results indicated that the C-3 free amino group benefits activity.

Given that C-3 free amino substitution appeared to have a better effect on potency, we next synthesized 3-amino compounds, 5ag-ak, with different C-7 substituents (Scheme 2 and Scheme 3). Compound 5aa underwent C-3 Boc protection and C-24 TBS protection to afford compound 13, which then oxidized into compound 14 using sodium dichromate. Compound 5ag was finally produced through deprotection. Compounds 5ah-ai were prepared by C-7 oximation or the reduction of compound 5ag. Compound 10 underwent C-3 Boc protection and C-7 oxidation to afford compound 16. The reductive amination of the C-7 carbonyl group resulted in compound 17. Compound 5aj was prepared by the C-3 deprotection and C-24 reduction of compound 17. The treatment of compound 16 with NaBH₄ gave compound 19. The reaction with NaH and MeI failed to obtain a 7-methoxy substituted product but afforded 5,7-diene compound 20. Subsequent similar deprotection and reduction resulted in compound 5ak. As shown in

Table 2. SHP1-activating effects of compounds 5ag-ak.

Compd	R2	EC50 (μM)	Maximum Activation Fold
5ag	=O	33.76 ± 2.93	1.84 ± 0.11
5ah	=NOH	34.38 ± 2.47	2.13 ± 0.28
5ai	-OH	>50	ND a
5aj	-NH2	>50	ND
5akb	/	19.95 ± 3.34	7.74 ± 0.20

^a ND: not determined. ^b 5,7-diene compound.

, the C-7 substituted compounds 5ag-aj were significantly less potent than compound 5aa. 5,7-Diene compound 5ak led to only a small change in SHP1 potency compared to compound 5aa, while the maximum activation was increased 7.74-fold.

In addition, we replaced the hydroxyl group at C-24 with amide or amine to form compounds 5al-am (Scheme 4). Compound 15 was hydrolyzed into carboxylic acid 22, which then reacted with methylamine hydrochloride in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) to afford amide 23. Compound 5al was finally produced through deprotection. Compound 12 was transformed into aldehyde 24 by IBX oxidation. The subsequent reductive amination of 24 with n-propylamine and C-3 deprotection resulted in compound 5am.

We envisioned that the introduction of secondary/tertiary alcohol groups into the side chain would probably increase the activity. The compounds 5an-av were designed and synthesized, as described in Scheme 5. The treatment of compounds 24 and 15 with Grignard reagents, followed by C-3 deprotection, furnished compounds 5an-av.

To verify the influence of changing the side chain length on a compound’s biological activity, we removed two ethylene spacers between the hydroxyl group and the remainder of the scaffold to design compound 5aw (Scheme 6). Commercially available 21-hydroxy-20-methylpregn-4-en-3- one (28) was initially protected with an Ac- group, methylated with KO^tBu and MeI to furnish compound 30. The following reductive amination and C-22 deprotection resulted in compound 5aw.

Table 3. SHP1-activating effects of compounds 5al-aw.

Compd	R3	EC50 (μM)	Maximum Activation Fold
5al		40.54 ± 6.58	6.42 ± 0.28
5am		>50	ND a
5an		17.61 ± 1.75	6.33 ± 0.01
5ao		15.52 ± 4.38	6.59 ± 0.48
5ap		38.11 ± 3.76	6.65 ± 0.35
5aq		22.15 ± 3.73	6.32 ± 0.34
5ar		12.88 ± 0.75	6.94 ± 0.17
5as		22.00 ± 3.84	5.73 ± 0.32
5at		12.88 ± 0.30	6.50 ± 0.12
5au		24.40 ± 3.61	6.42 ± 0.28
5av		35.80 ± 1.92	5.90 ± 0.42
5aw	OH	35.49 ± 3.15	ND

^a ND: not determined.

shows that the replacement of the C-24 hydroxyl group with an amide or alkylamino moiety (compounds 5al-am) attenuated the activity. Most of the secondary and tertiary alcohols exerted good efficacies, with EC₅₀ values from 12.88 μM to 38.11 μM and 5.73- to 6.94-fold maximum activation. Ethyl substitution (compounds 5ao and 5at) exhibited more potent biological activity than methyl substitution (compounds 5an and 5as). The activation property of compound 5aw was decreased compared to compound 5aa. It appeared that compounds with different side chain lengths affected the activity. These data implied that the C-24 terminal position deserves further modification, and the introduction of proper groups may increase biological activity.

Urea exhibits unique hydrogen-binding capabilities and becomes an important functional group for drug–target interactions. We employed the rational design strategy of incorporating a diphenyl urea active substructure into the steroids to obtain the merged structures 5ax-ba. The syntheses of compounds 5ax-ba are outlined in Scheme 7 and Scheme 8. Treating compound 32 with isocyanate compound 33 produced diphenyl urea intermediate 34. Compound 5ax was made in one step from compounds 5aa and 34 in the presence of Et₃N. Compound 12 underwent C-24 bromination, coupling with compound 35, and then deprotection to generate compound 5ay.

Compound 14 was selectively deprotected and brominated with CBr₄ and PPh₃ to furnish compound 38. Next, coupling with compound 34, followed by C-7 NaBH₄ reduction, yielded compound 40. Finally, removal of the Boc protecting group resulted in compound 5az. The C-3 protection and C-22 bromination of compound 5aw gave compound 42. The subsequent coupling and deprotection afforded compound 5ba.

It is evident from

Table 4. SHP1-activating effects of compounds 5ax-ba.

Compd	R1	R2	R3	EC50 (μM)	Maximum Activation Fold
5ax		H		>50	ND a
5ay	-NH2	H		2.05 ± 0.14	5.54 ± 0.24
5az	-NH2	-OH		2.10 ± 0.00	8.79 ± 0.17
5ba	-NH2	H		1.54 ± 0.24 b	7.63 ± 0.20

^a ND: not determined. ^b Poor solubility in 10 mM DMSO.

that the position in the steroid scaffold for the urea substituents specifies the SHP1 activity. With the placing of 4-chloro-3-(trifluoromethyl)phenyl urea in the C-3 position, the compound 5ax showed no activity. As the diphenyl urea group was placed in the C-24 position and the other positions were kept constant, the activating effects of compounds 5ay-ba were increased several-fold compared to those of compound 5aa. These data support the idea that the urea group probably contacts with both the hydrophilic and the hydrophobic halves of the SHP1 active site and that a flexible side chain may enhance proper orientation. Interestingly, the 7-hydroxyl compound 5az displayed 3-fold higher maximum activation than compound 5ay though their EC₅₀ values were similar. Compound 5ba showed better activity than compound 5ay, which indicates that the short length of the side chain benefits the activity. In particular, 5ba is more than 32-fold more selective for SHP1 over the closest homologue SHP2 (EC₅₀ > 50 μM). In order to ameliorate the solubility of the most potent compound, 5ba, this class of derivatives was prepared in a hydrochloride salt form for further modifications.

The structure–activity relationships (SARs) for the 3-amino-4,4-dimethyl lithocholic acid derivatives are summarized below. The presence of a primary amine at the C-3 position (5aa) seems to be important for the interaction of the compound with the binding site of SHP1. The substitutions of the aliphatic amine groups at the C-3 position (5ab-af) exhibited lower activities, and the compound with bulky phenyl urea substitution at the C-3 position (5ax) was inactive, indicating that the binding site cavity of SHP1 near the C-3 position is relatively small. The compounds with substitutions at the C-7 position (5ag-aj) had weaker potency. The primary alcohol moiety in a side chain can be replaced by other chemical groups while maintaining or increasing potency. The compounds with diphenyl urea moieties in a side chain (5ay-ba) markedly increased their potency, indicating that the hydrogen bond and hydrophobic interactions play an important role in the binding pocket.

The anti-tumor effects of compounds 5az-ba which showed higher maximum activation ability were examined by cell viability assay using acute lymphoblastic leukemia (ALL) cell line RS4;11, acute promyelocytic leukemia (APL) cell line NB4, and non-small cell lung cancer (NSCLC) cell line NCI-H1299. As shown in

Table 5. Anti-tumor effects of compounds 5az-ba.

Compd	IC50 (μM) a
	RS4;11	NB4	NCI-H1299
5az	1.65 ± 0.05	2.04 ± 0.11	2.48 ± 0.20
5ba	4.00 ± 0.22	3.74 ± 0.39	5.51 ± 0.23

^a Cell viability was assayed in cultured cells incubated with indicated concentrations of compounds for 72 h by CCK8 assay. Data are given as mean values of three independent experiments.

, 5az-ba exhibited significant cytotoxicity to effectively inhibit cancer cell viability; this was particularly the case with compound 5az against RS4;11 cells, with an IC₅₀ value of less than 2 μM.

Based on these activity data, we selected the crystal structure of SHP1 in the open conformation (PDB ID: 3PS5) and the 3-β-diastereomer of 5ba for the docking study (see Supplementary Materials). Compound 5ba binds at the central allosteric site through a network of hydrogen bonds and hydrophobic interactions (Figure 2). More importantly, this site is closest to the reported interface of three domains [46], and the binding of compounds tightens the inter-domain connection, which thereby stabilizes the active conformation of SHP1.

The binding pocket is mainly composed of a hydrophobic pocket and a hydrophilic pocket (Figure 3). The narrow hydrophobic pocket is made up of Val2, Trp4, Phe27, and Val76, with Met1 as gatekeeper. The depth of the hydrophobic pocket is approximately 10 Å, and the width is approximately 6 Å. Four hydrophilic residues, namely Arg217, Asp481, Asp483, and Lys486, embrace the larger hydrophilic pocket from one side. The hydrophilic pocket provides the interaction between the protein and the amino head of the compound. Compound 5ba possesses an L-shaped structure, and the phenyl urea tail fits well in the hydrophobic pocket (Figure 4).

As shown in Figure 5, the 4-chloro-3-(trifluoromethyl)phenyl ring occupies the hydrophobic pocket through hydrophobic interactions with Met1, Phe27, Val76, and Glu77. The urea group of 5ba forms two hydrogen bonds with Thr80 (3.7 Å) and Lys97 (2.9 Å), which could explain why the potency of 5ba increased ∼23 times than 5aw. The phenoxy group forms a hydrogen bond with Gln81 (3.1 Å) and forms a hydrophobic interaction with Thr80. The 3-amino group of the steroid parent nucleus creates two hydrogen bonds with Asn472 (1.9, 2.9 Å), which accounts for the potency of the series of newly synthesized compounds. The C-4 methyl group forms a hydrophobic interaction with Asp481. The C-21 methyl group forms a hydrophobic interaction with Pro314.

Structure modification at the C-3 position (5ab-af) dramatically attenuated the potency, suggesting that -NH₂ is the key group for activity retention. This could also be explained by the reduced hydrogen bond interactions of the amino group with Asn472. Those compounds lacking a urea group only occupy and bind the entrance of the pocket with less interactions, leading to reduced potency compared to 5ay-ba. The three methyl groups at C-4 and C-19 incur steric hindrance to the urea 3-NH of compound 5ax, making it energetically unfavorable to engage Asn472, which is consistent with the loss of potency. As shown in Figure 6A, Thr80, Gln81, and Lys97 are on the surface of the auto-inhibited SHP1, which allows the free access and binding of compounds. The long and flexible BG loop plays a pivotal role in the conformational change and activation of SH2 domain-containing proteins [46,47]. The positively charged head group of the compound may interact with the negatively charged LQDRDG motif on the BG loop of the N-SH2 domain, which causes movement of the BG loop. Then, the “switch” N-SH2 domain shifts away, moving from one side of the PTP domain to the other, leading to open conformation and thus the activation of SHP1. In contrast, no similar pockets were observed in the center of the homologue SHP2 in open conformation (Figure 6B). These observations probably explain the selectivity of 5ba for SHP1 over SHP2.

3. Materials and Methods

3.1. Chemical Synthesis

All reagents and solvents were obtained from commercial suppliers and used without further purification unless otherwise indicated. Melting points (mps) were taken in open capillaries on a WRS-2 melting point system. The ¹H NMR and ¹³C NMR spectra were recorded using TMS as the internal standard on a Bruker Ascend 400 spectrometer at 400 and 100 MHz, respectively. The ¹H NMR chemical shifts were reported in parts per million (ppm) relative to the centerline of the singlet signal of the solvent molecule (7.26 ppm for CDCl₃ or 3.31 ppm for CD₃OD). The ¹³C NMR chemical shifts were reported in ppm relative to the centerline at 77.16 ppm for CDCl₃ or at 49.00 ppm for CD₃OD. The data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), coupling constant (Hz), and integration. Evaporation was carried out in vacuo using a rotating evaporator. Silica gel chromatography was performed using silica gel (200–300 mesh). Reactions were monitored by TLC with detection by phosphomolybdic acid visualization or UV light (λ = 254 nm). All reactions involving air- or moisture-sensitive reagents were performed under a nitrogen atmosphere using anhydrous solvents. The purity of the final compounds was >95%, as deduced by ¹H NMR spectra. High-resolution mass spectroscopy (HRMS) was performed on a time-of-flight instrument with electrospray ionization (ESI) in the positive ionization mode.

3.1.1. Methyl (4R)-4-((3R,10S,13R,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (7)

TsOH^.H₂O (47.5 mg, 0.25 mmol) was added to a stirred solution of lithocholic acid 6 (1.88 g, 5 mmol) in MeOH (20 mL). The mixture was refluxed for 2 h, and TLC indicated the consumption of the starting material. MeOH was removed in vacuo, and aqueous NaHCO₃ solution was added. The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated to afford compound 7 (1.95 g, 100% yield) as a white solid. The mp was 105.3–106.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 3.65 (s, 3H), 3.62–3.59 (m, 1H), 2.38–2.30 (m, 1H), 2.24–2.17 (m, 1H), 1.96–1.93 (m, 1H), 1.87–0.96 (m, other aliphatic ring protons), 0.90 (s, 3H), 0.89 (d, J = 6.0 Hz, 3H), 0.63 (s, 3H). HRMS (ESI): calcd for C₂₅H₄₂NaO₃ [M + Na]⁺ 413.3026; found 413.3037.

3.1.2. Methyl(4R)-4-((10R,13R,17R)-10,13-dimethyl-3-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (8)

IBX (3.5 g, 12.5 mmol), TFA (0.2 mL) and a solution of compound 7 (1.95 g, 5 mmol) in DMSO (20 mL) were combined. The mixture was warmed to 50 °C, and all the solids were dissolved. After stirring for 2 h under a N₂ atmosphere, the reaction was completed, as indicated by TLC. The reaction mixture was cooled and added to an aqueous NaHSO₃ solution (20 mL). The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, and concentrated. The crude material was purified by silica gel column chromatography using PE/EtOAc (10:1, v/v) to afford compound 8 (1.02 g, 53% yield) as a white solid. The mp was 124.2–126.1 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.71 (s, 1H), 3.65 (s, 3H), 2.45–2.30 (m, 4H), 2.27–2.17 (m, 2H), 2.02–1.98 (m, 2H), 1.85–0.99 (m, other aliphatic ring protons), 0.90 (d, J = 6.4 Hz, 3H), 0.70 (s, 3H). HRMS (ESI): calcd for C₂₅H₃₈NaO₃ [M + Na]⁺ 409.2713; found 409.2732.

3.1.3. Methyl(4R)-4-((10R,13R,17R)-4,4,10,13-tetramethyl-3-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (9)

Potassium tert-butoxide (1.18 g, 10.56 mmol) was added in batches to the suspension of compound 8 (1.02 g, 2.64 mmol) in absolute tert-butyl alcohol (50 mL). The mixture was stirred at r.t. for 30 min to form a clear yellow solution and then added to MeI (1.5 g, 10.56 mmol) dropwise. The reaction was stirred at r.t. for 12 h in the dark under a N₂ atmosphere, and the starting material disappeared, as monitored by TLC. Upon addition of an aqueous Na₂S₂O₃ solution (5 mL) for quenching the excess MeI, the mixture was evaporated and water (30 mL) was added. The suspension was acidified to pH < 6 with an aqueous HCl solution. The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude material was dissolved in MeOH (20 mL), and TsOH^.H₂O (47.5 mg, 0.25 mmol) was added. The mixture was refluxed for 2 h, and the MeOH was removed in vacuo. An aqueous NaHCO₃ solution was added, and it was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using PE/EtOAc (10:1, v/v) to obtain compound 9 (547 mg, 50% yield) as a white solid. The mp was 133.6–135.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.55–5.53 (m, 1H), 3.66 (s, 3H), 2.59–2.43 (m, 2H), 2.39–2.31 (m, 1H), 2.26–2.18 (m, 1H), 2.13–2.06 (m, 1H), 2.02–1.97 (m, 2H), 1.90–1.76 (m, 2H), 1.68–1.02 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.84 (s, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₂₇H₄₂NaO₃ [M+Na]⁺ 437.3026; found 437.3059.

3.1.4. Methyl(4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (10)

Compound 9 (547 mg, 1.32 mmol) was dissolved in a saturated NH₄OAc/EtOH solution (20 mL). It was added to NaBH₃CN (166 mg, 2.64 mmol) and NH₃^.H₂O (0.8 mL). The mixture was refluxed for 12 h under a N₂ atmosphere, and TLC indicated the consumption of the starting material. The solvents were removed in vacuo and water (20 mL) was added. The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH/NH₃^.H₂O (100:3:0.5, v/v/v) to obtain compound 10 (412 mg, 75% yield) as a white solid. The mp was 145.0–147.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.55–5.51 (m, 1H), 3.65 (s, 3H), 2.38–2.31 (m, 2H), 2.25–2.17 (m, 1H), 2.11–2.04 (m, 1H), 1.99–1.96 (m, 1H), 1.86–1.77 (m, 2H), 1.73–1.69 (m, 1H), 1.63–0.95 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H).

3.1.5. (4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5aa)

LiAlH₄ (11 mg, 0.3 mmol) was added in batches at 0 °C to a dry THF solution (20 mL) of compound 10 (116 mg, 0.28 mmol). The mixture was stirred at r.t. for 12 h under a N₂ atmosphere. Water (11 μL) was added to quench the reaction, then a 15% NaOH solution (11 μL) and then water (33 μL). The suspension was filtrated, and the solid residue was washed with THF (10 mL). The solvents were removed in vacuo, and water (30 mL) was added to the residue. The mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH/NH₃^.H₂O (100:5:0.5, v/v/v) to obtain compound 5aa (81 mg, 75% yield) as a white solid. The mp was 163.5–166.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.55–5.52 (m, 1H), 3.59–3.57 (m, 2H), 2.37–2.33 (m, 1H), 2.10–2.05 (m, 1H), 2.00–1.97 (m, 1H), 1.87–1.80 (m, 1H), 1.73–1.70 (m, 1H), 1.65–0.89 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.52, 119.58, 63.52, 58.37, 57.43, 56.02, 51.22, 42.33, 41.01, 39.91, 37.79, 37.04, 35.71, 32.80, 32.05, 30.93, 29.54, 28.43, 28.35, 27.73, 24.29, 23.76, 21.38, 20.65, 18.81, 12.00. HRMS (ESI) calcd for C₂₆H₄₆NO [M+H]⁺ 388.3501; found 388.3585.

3.1.6. Methyl(4R)-4-((10R,13R,17R)-3-((2-hydroxyethyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11a)

Potassium carbonate (166 mg, 1.2 mmol) and 2-bromoethanol (82 mg, 0.66 mmol) were added to a solution of compound 10 (249 mg, 0.6 mmol) in DMF (10 mL), and the mixture was heated to 80 °C and stirred for 2 h under a N₂ atmosphere. When the reaction was completed, as indicated by TLC, water (30 mL) was added to the residue. The mixture was filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH/NH₃^.H₂O (100:5:0.5, v/v/v) to obtain compound 11a (168 mg, 61% yield) as a white solid. The mp was 165.4–166.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.54 (m, 1H), 3.65 (s, 3H), 3.62–3.51 (m, 2H), 3.00–2.95 (m, 1H), 2.67–2.61 (m, 1H), 2.38–2.31 (m, 1H), 2.24–0.87 (m, other aliphatic ring protons), 0.65 (s, 3H). HRMS (ESI) calcd for C₂₉H₅₀NO₃ [M + H]⁺ 460.3785; found 460.3795.

3.1.7. Methyl(4R)-4-((10R,13R,17R)-3-((3-hydroxypropyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11b)

Compound 11b was synthesized in a 63% yield as a white solid using a similar procedure to that in 3.1.6. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.54 (m, 1H), 3.82 (t, J = 5.2 Hz, 2H), 3.65 (s, 3H), 3.59–3.47 (m, 1H), 3.18–3.13 (m, 1H), 2.69–2.63 (m, 1H), 2.38–2.30 (m, 2H), 2.25–2.17 (m, 2H), 2.10–0.87 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₀H₅₂NO₃ [M + H]⁺ 474.3942; found 474.3973.

3.1.8. Methyl(4R)-4-((10R,13R,17R)-3-((3-chloropropyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11c)

Compound 11c was synthesized in a 47% yield as a white solid using a similar procedure to that in 3.1.6, and the reaction temperature was r.t. The mp was 119.7–121.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.53 (m, 1H), 3.65 (s, 3H), 3.63 (t, J = 6.4 Hz, 2H), 2.95–2.88 (m, 2H), 2.61–2.56 (m, 1H), 2.39–2.31 (m, 1H), 2.25–2.17 (m, 1H), 2.11–2.04 (m, 1H), 1.99–0.87 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₀H₅₁ClNO₂ [M + H]⁺ 492.3603; found 492.3646.

3.1.9. Methyl(4R)-4-((10R,13R,17R)-4,4,10,13-tetramethyl-3-(propylamino)-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11d)

Compound 11d was synthesized in an 86% yield as a white solid using a similar procedure to that in 3.1.6. ¹H-NMR (400 MHz, CDCl₃) δ 5.57–5.53 (m, 1H), 3.66 (s, 3H), 2.83–2.76 (m, 1H), 2.54–2.48 (m, 1H), 2.39–2.31 (m, 1H), 2.25–2.20 (m, 1H), 2.11–2.05 (m, 2H), 2.00–1.97 (m, 1H), 1.88–0.89 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₀H₅₂NO₂ [M+H]⁺ 458.3993; found 458.4004.

3.1.10. Methyl(4R)-4-((10R,13R,17R)-3-(azetidin-1-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11e)

Compound 11e was synthesized in a 73% yield as a white solid using a similar procedure to that in 3.1.6. The mp was 173.7–175.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.54 (m, 1H), 3.65 (s, 3H), 3.60–3.63 (m, 4H), 2.38–2.31 (m, 1H), 2.25–0.84 (m, other aliphatic ring protons), 0.65 (s, 3H). HRMS (ESI): calcd for C₃₀H₅₀NO₂ [M + H]⁺ 456.3836; found 456.3853.

3.1.11. (4R)-4-((10R,13R,17R)-3-((2-hydroxyethyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5ab)

Compound 5ab was synthesized in a 68% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 184.3–187.5 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 3:1) δ 5.54–5.52 (m, 1H), 3.66–3.56 (m, 2H), 3.52–3.48 (m, 2H), 2.87–2.81 (m, 1H), 2.60–2.54 (m, 1H), 2.09–2.01 (m, 2H), 2.00–1.96 (m, 1H), 1.83–1.78 (m, 1H), 1.77–1.71 (m, 2H), 1.62–0.84 (m, other aliphatic ring protons), 0.64 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 3:1) δ 150.56, 119.88, 65.41, 63.12, 60.80, 57.54, 56.19, 51.27, 50.24, 42.42, 40.73, 40.00, 37.38, 37.11, 35.87, 32.82, 32.13, 31.03, 29.35, 28.44, 27.52, 24.89, 24.36, 24.33, 21.40, 20.78, 18.76, 12.00. HRMS (ESI): calcd for C₂₈H₅₀NO₂ [M + H]⁺ 432.3836; found 432.3840.

3.1.12. (4R)-4-((10R,13R,17R)-3-((3-hydroxypropyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5ac)

Compound 5ac was synthesized in a 62% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 183.5–186.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.57–5.55 (m, 1H), 3.82 (t, J = 5.2 Hz, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.18–3.13 (m, 1H), 2.69–2.63 (m, 1H), 2.11–2.06 (m, 2H), 2.02–1.98 (m, 1H), 1.88–0.87 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.46, 119.85, 65.74, 64.88, 63.70, 57.44, 56.03, 51.15, 49.39, 42.35, 40.72, 39.91, 37.51, 36.98, 35.71, 32.76, 31.99, 31.36, 30.94, 29.53, 28.42, 27.81, 24.98, 24.27, 21.35, 20.71, 18.81, 12.02. HRMS (ESI): calcd for C₂₉H₅₁NNaO₂ [M + Na]⁺ 468.3812; found 468.3805.

3.1.13. (4R)-4-((10R,13R,17R)-3-((3-chloropropyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5ad)

Compound 5ad was synthesized in a 79% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 162.7–165.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.52 (m, 1H), 3.66–3.60 (m, 4H), 2.96–2.92 (m, 1H), 2.64–2.59 (m, 1H), 2.10–0.91 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.98, 119.55, 64.98, 63.70, 57.45, 56.02, 51.17, 45.44, 43.42, 42.35, 41.02, 39.92, 37.70, 36.99, 35.71, 33.29, 32.76, 31.98, 30.97, 29.52, 28.42, 27.61, 24.90, 24.81, 24.27, 21.40, 20.70, 18.80, 12.02. HRMS (ESI): calcd for C₂₉H₅₁ClNO [M + H]⁺ 464.3654; found 464.3676.

3.1.14. (4R)-4-((10R,13R,17R)-4,4,10,13-tetramethyl-3-(propylamino)-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5ae)

Compound 5ae was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 147.5–150.4 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.53 (m, 1H), 3.62–3.59 (m, 2H), 2.82–2.76 (m, 1H), 2.54–2.47 (m, 1H), 2.10–2.05 (m, 2H), 2.01–1.98 (m, 1H), 1.84–1.80 (m, 2H), 1.79–1.74 (m, 1H), 1.62–0.87 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.71, 119.74, 65.25, 63.68, 57.45, 56.03, 51.16, 50.50, 42.35, 40.77, 39.92, 37.53, 37.00, 35.71, 32.77, 31.99, 30.94, 29.53, 28.42, 27.58, 24.99, 24.32, 24.27, 22.82, 21.39, 20.70, 18.81, 12.02, 11.89. HRMS (ESI): calcd for C₂₉H₅₂NO [M + H]⁺ 430.4043; found 430.4068.

3.1.15. (4R)-4-((10R,13R,17R)-3-(azetidin-1-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5af)

Compound 5af was synthesized in a 77% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 174.9–177.6 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.53–5.51 (m, 1H), 3.60 (t, J = 6.4 Hz, 2H), 3.37 (dd, J = 11.2, 4.4 Hz, 2H), 3.27 (dd, J = 11.2, 4.4 Hz, 2H), 2.09–1.97 (m, 4H), 1.87–1.84 (m, 2H), 1.78–1.75 (m, 1H), 1.64–0.86 (m, other aliphatic ring protons), 0.65 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.70, 119.22, 72.83, 63.70, 57.46, 56.02, 55.32 (2C), 51.30, 42.32, 41.75, 39.92, 37.68, 37.02, 35.70, 32.87, 31.99, 30.90, 29.53, 28.92, 28.42, 26.06, 24.26, 21.38, 20.70, 20.08, 18.81, 17.99, 12.01. HRMS (ESI): calcd for C₂₉H₅₀NO [M + H]⁺ 428.3887; found 428.3866.

3.1.16. Tert-butyl((10R,13R,17R)-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (12)

Boc₂O (524 mg, 2.4 mmol) and Et₃N (404 mg, 4 mmol) were added to a solution of compound 5aa (776 mg, 2 mmol) in dry DCM (30 mL), and the reaction was stirred at r.t. for 2 h; TLC indicated the consumption of the starting material. Water (30 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with aqueous NaOH solution, brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in 5 mL of EtOAc to obtain compound 12 (945 mg, 97% yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 5.58–5.55 (m, 1H), 4.44 (d, J = 10.0 Hz, 1H), 3.61 (t, J = 6.0 Hz, 2H), 3.32–3.27 (m, 1H), 2.09–2.04 (m, 1H), 2.00–1.97 (m, 1H), 1.86–1.79 (m, 1H), 1.73–0.92 (m, other aliphatic ring protons), 0.92 (d, J = 6.0 Hz, 3H), 0.66 (s, 3H).

3.1.17. Tert-butyl((10R,13R,17R)-17-((R)-5-((tert-butyldimethylsilyl)oxy)pentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (13)

TBSCl (226 mg, 1.5 mmol) was added to a solution of compound 12 (366 mg, 0.75 mmol) and imidazole (204 mg, 3 mmol) in dry DMF (8 mL). The reaction mixture was stirred at 80 °C for 6 h under a N₂ atmosphere, and the starting material disappeared, as monitored by TLC. The mixture was added with an aqueous NaOH solution (20 mL) and extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in 5 mL of EtOAc to obtain compound 13 (451 mg, 100% yield) as a white solid. The mp was 183.4–185.1 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.56–5.54 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.33–3.27 (m, 1H), 2.09–1.97 (m, 2H), 1.85–1.78 (m, 1H), 1.73–0.86 (m, other aliphatic ring protons), 0.66 (s, 3H), 0.04 (s, 6H).

3.1.18. Tert-butyl((10R,13R,17R)-17-((R)-5-((tert-butyldimethylsilyl)oxy)pentan-2-yl)-4,4,10,13-tetramethyl-7-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (14)

Na₂Cr₂O₇^.2H₂O (243 mg, 0.81 mmol) was added to a solution of compound 13 (406 mg, 0.74 mmol), N-hydroxyphthalimide (241 mg, 1.48 mmol), and HOAc (0.2 mL) in acetone (30 mL). The reaction was heated to 50 °C and stirred for 12 h under a N₂ atmosphere, and then, another quantity of Na₂Cr₂O₇^.2H₂O (110 mg, 0.37 mmol) was added. The reaction was continued for 6 h. When the reaction was completed, as indicated by TLC, the mixture was concentrated and purified by silica gel column chromatography using PE/EtOAc (5:1, v/v) to obtain compound 14 (419 mg, 92% yield) as a white solid. mp 190.5–192.6 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.95 (s, 1H), 4.48 (d, J = 10.0 Hz, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.49–3.44 (m, 1H), 2.31–2.29 (m, 1H), 2.22–2.17 (m, 1H), 2.03–1.01 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.88 (s, 9H), 0.67 (s, 3H), 0.04 (s, 6H). HRMS (ESI): calcd for C₃₇H₆₅NNaO₄Si [M + Na]⁺ 638.4575; found 638.4576.

3.1.19. (10R,13R,17R)-3-amino-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-7H-cyclopenta[a]phenanthren-7-one (5ag)

An aqueous 4N HCl solution (5 mL) was added to the solution of compound 14 (167 mg, 0.32 mmol) in THF (5 mL). The reaction was refluxed for 2 h, and TLC indicated the consumption of the starting material. The suspension was basified to pH > 8 with aqueous NaOH solution. The solvent THF was removed in vacuo, and water (30 mL) was added. The mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated to obtain compound 5ag (121 mg, 94% yield) as a white solid. The mp was 151.3–153.7 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.93 (s, 1H), 3.57 (t, J = 6.4 Hz, 2H), 2.53–2.49 (m, 1H), 2.30–2.26 (m, 1H), 2.22–2.17 (m, 1H), 2.02–1.99 (m, 1H), 1.88–1.85 (m, 1H), 1.80–1.76 (m, 1H), 1.66–1.02 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.67 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 203.23, 176.64, 124.91, 63.48, 57.21, 54.82, 52.00, 50.80, 45.20, 43.40, 42.10, 38.95, 38.77, 36.54, 35.67, 32.00, 29.52, 28.67, 27.88, 26.66, 26.48, 23.20, 20.91, 19.59, 18.92, 12.04. HRMS (ESI): calcd for C₂₆H₄₄NO₂ [M + H]⁺ 402.3367; found 402.3372.

3.1.20. (10R,13R,17R)-3-amino-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-7H-cyclopenta[a]phenanthren-7-oxime (5ah)

NH₂OH^.HCl (35 mg, 0.5 mmol) and NaOAc (136 mg, 1 mmol) was added to a solution of compound 5ag (40 mg, 0.1 mmol) in EtOH (5 mL). The mixture was refluxed for 2 h. When the reaction was completed, as indicated by TLC, the solvent EtOH was removed in vacuo, and an aqueous NaOH solution (10 mL) was added. The mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in MeCN to obtain compound 5ah (39 mg, 93% yield) as a white solid. The mp was 218.3–219.9 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 10:1) δ 6.86 (s, 1H), 3.53 (t, J = 6.4 Hz, 2H), 2.46–2.42 (m, 1H), 2.31–2.27 (m, 1H), 2.21–2.18 (m, 1H), 2.00–1.97 (m, 1H), 1.80–0.82 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 10:1) δ 163.70, 157.64, 110.97, 63.30, 57.53, 54.83, 51.89, 51.67, 43.17, 41.96, 38.97, 38.57, 37.50, 36.79, 35.59, 31.94, 29.31, 28.42, 27.59, 27.32, 26.95, 23.38, 20.68, 19.91, 18.90, 12.25. HRMS (ESI): calcd for C₂₆H₄₅N₂O₂ [M + H]⁺ 417.3476; found 417.3486.

3.1.21. (10R,13R,17R)-3-amino-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-7-ol (5ai)

NaBH₄ (19 mg, 0.5 mmol) was added to the solution of compound 5ag (40 mg, 0.1 mmol) in MeOH (10 mL). The mixture was stirred at r.t. for 2 h under a N₂ atmosphere. When the reaction was completed, as indicated by TLC, HOAc (0.1 mL) was added, and the solvent was removed in vacuo. An aqueous NaOH solution (10 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in EtOAc to obtain compound 5ai (37 mg, 73% yield) as a white solid. The mp was 171.4–173.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.52–5.48 (m, 1H), 3.81–3.79 (m, 1H), 3.51 (t, J = 6.4 Hz, 2H), 2.40–2.37 (m, 1H), 2.06–2.03 (m, 1H), 1.88–1.02 (m, other aliphatic ring protons), 0.97 (d, J = 5.6 Hz, 3H), 0.73 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 153.20, 123.49, 73.77, 63.02, 57.80, 56.31, 55.37, 49.98, 42.63, 40.46, 39.84, 39.47, 37.09, 36.96, 35.57, 31.90, 29.16, 28.46, 27.28 (2C), 25.98, 23.23, 21.08, 20.63, 18.63, 11.71. HRMS (ESI): calcd for C₂₆H₄₆NO₂ [M + H]⁺ 404.3523; found 404.3548.

3.1.22. Methyl(4R)-4-((10R,13R,17R)-3-((tert-butoxycarbonyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (15)

Compound 15 was synthesized in a 99% yield as a white solid using a similar procedure to that in 3.1.16. The mp was 190.6–191.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.53 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.65 (s, 3H), 3.32–3.27 (m, 1H), 2.39–2.31 (m, 1H), 2.25–2.17 (m, 1H), 2.09–2.04 (m, 1H), 1.88–1.96 (m, 1H), 1.85–0.95 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₂H₅₃NNaO₄ [M + Na]⁺ 538.3867; found 538.3880.

3.1.23. Methyl(4R)-4-((10R,13R,17R)-3-((tert-butoxycarbonyl)amino)-4,4,10,13-tetramethyl-7-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (16)

Compound 16 was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.18. ¹H-NMR (400 MHz, CDCl₃) δ 5.95 (s, 1H), 4.48 (d, J = 9.6 Hz, 1H), 3.65 (s, 3H), 3.49–3.44 (m, 1H), 2.38–2.30 (m, 2H), 2.25–2.17 (m, 2H), 2.02–1.99 (m, 1H), 1.91–1.89 (m, 1H), 1.81–1.05 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₃₂H₅₂NO₅ [M + H]⁺ 530.3840; found 530.3835.

3.1.24. Methyl(4R)-4-((10R,13R,17R)-7-amino-3-((tert-butoxycarbonyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (17)

Compound 17 was synthesized in a 74% yield as a white solid using a similar procedure to that in 3.1.4. and was used for the next reaction without further purification.

3.1.25. Methyl(4R)-4-((10R,13R,17R)-3,7-diamino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (18)

Compound 17 (160 mg, 0.3 mmol) was dissolved in an anhydrous EtOAc/HCl solution (10 mL). The mixture was stirred at r.t. for 4 h. Water (20 mL) was added, and the suspension was basified to pH > 8 with an aqueous NaOH solution. The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated to obtain compound 18 (118 mg, 92% yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 5.42–5.40 (m, 1H), 3.65 (s, 3H), 3.03 (dd, J = 8.0, 2.8 Hz, 1H), 2.40–2.32 (m, 2H), 2.25–2.19 (m, 1H), 1.99–0.98 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₂₇H₄₆N₂NaO₂ [M + Na]⁺ 453.3451; found 453.3447.

3.1.26. (4R)-4-((10R,13R,17R)-3,7-diamino-4,4,10,13-tetramethyl-tetradeca hydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5aj)

Compound 5aj was synthesized in a 66% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 178.7–180.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.40–5.35 (m, 1H), 3.55 (t, J = 4.0 Hz, 2H), 3.03–3.00 (m, 1H), 2.38–2.34 (m, 1H), 1.99–1.96 (m, 1H), 1.89–1.82 (m, 1H), 1.77–0.95 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 152.40, 124.81, 63.21, 58.04, 56.93, 55.37, 54.59, 51.07, 43.07, 41.74, 40.76, 39.79, 37.45, 36.80, 35.66, 32.08, 29.55, 28.66, 28.19, 27.39, 26.41, 23.68, 21.82, 20.89, 18.82, 12.02.

3.1.27. Methyl(4R)-4-((10R,13R,17R)-3-((tert-butoxycarbonyl)amino)-7-hydroxy-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (19)

Compound 19 was synthesized in a 92% yield as a white solid using a similar procedure to that in 3.1.21. The mp was 198.3–200.4 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.51 (d, J = 2.0 Hz, 1H), 4.45 (d, J = 10.0 Hz, 1H), 3.91–3.89 (m, 1H), 3.65 (s, 3H), 3.35–3.29 (m, 1H), 2.38–2.31 (m, 1H), 2.25–2.17 (m, 1H), 1.99–1.00 (m, other aliphatic ring protons), 0.91 (d, J = 6.0 Hz, 3H), 0.67 (s, 3H). HRMS (ESI): calcd for C₃₂H₅₃NNaO₅ [M + Na]⁺ 554.3816; found 554.3831.

3.1.28. (4R)-4-((10R,13R,17R)-3-((tert-butoxycarbonyl)amino)-4,4,10,13-tetramethyl-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (20)

Sixty percent NaH (30 mg, 0.75 mmol) and MeI (107 mg, 0.75 mmol) were added to a solution of compound 19 (133 mg, 0.25 mmol) in dry DMF (10 mL). The mixture was stirred at r.t. for 2 h under a N₂ atmosphere, and the starting material disappeared. An aqueous Na₂S₂O₃ solution (5 mL) was added to quench the excess MeI. Water (30 mL) was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in 5 mL of EtOH to obtain compound 20 (76 mg, 61% yield) as a white solid and used for the next reaction without further purification.

3.1.29. (4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (21)

Compound 21 was synthesized in a 92% yield as white solid using a similar procedure to that in 3.1.25 and used for the next reaction without further purification.

3.1.30. (4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-ol (5ak)

Compound 5ak was synthesized in a 92% yield as a white solid using a similar procedure to that in 3.1.5. The mp was 179.5–181.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 6.21 (d, J = 10.0 Hz, 1H), 5.63 (d, J = 10.0 Hz, 1H), 3.61 (t, J = 6.4 Hz, 2H),2.47–2.36 (m, 2H), 2.33–2.25 (m, 1H), 2.00–1.97 (m, 1H), 1.89–0.95 (m, other aliphatic ring protons), 0.88 (s, 3H), 0.76 (s, 3H), 0.65 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 146.41, 126.76, 126.66, 125.27, 63.61, 59.86, 55.99, 55.90, 50.35, 43.72, 38.10, 37.15, 36.74, 36.16, 34.74, 31.76, 29.45, 28.78, 28.21, 27.53, 24.94, 19.54, 19.28, 19.00, 16.08, 13.13. HRMS (ESI): calcd for C₂₆H₄₄NO [M + H]⁺ 386.3417; found 386.3429.

3.1.31. (4R)-4-((10R,13R,17R)-3-((tert-butoxycarbonyl)amino)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (22)

An aqueous 2N NaOH solution (1 mL) was added to the solution of compound 15 (516 mg, 1.0 mmol) in MeOH (5 mL). The reaction was stirred at r.t. for 2 h, and TLC indicated the consumption of the starting material. Water (20 mL) was added, and the suspension was acidified to pH < 6 with aqueous HCl solution. The solvent MeOH was removed in vacuo. The mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated to obtain compound 22 (470 mg, 94% yield) as a white solid. The mp was 243.1–245.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.57–5.52 (m, 1H), 4.45 (d, J = 10.0 Hz, 1H), 3.33–3.27 (m, 1H), 2.43–2.35 (m, 1H), 2.29–2.21 (m, 1H), 2.09–2.05 (m, 1H), 2.00–1.97 (m, 1H), 1.89–1.77 (m, 2H), 1.73–0.92 (m, other aliphatic ring protons), 0.67 (s, 3H). HRMS (ESI): calcd for C₃₁H₅₁NNaO₄ [M + Na]⁺ 524.3710; found 524.3726.

3.1.32. Tert-butyl((10R,13R,17R)-4,4,10,13-tetramethyl-17-((R)-5-(methylamino)-5-oxopentan-2-yl)-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (23)

MeNH₂^.HCl (27 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol), EDCI (77 mg, 0.4 mmol), and DIPEA (52 mg, 0.4 mmol) were added to a solution of compound 22 (100 mg, 0.2 mmol) in DMF (10 mL). The reaction mixture was stirred at r.t. for 12 h under a N₂ atmosphere, and the starting material disappeared, as monitored by TLC. Water (30 mL) was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH (30:1, v/v) to obtain compound 23 (85 mg, 83% yield) as a white solid. The mp was 228.8–230.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.60–5.58 (m, 1H), 5.54–5.51 (m, 1H), 4.44 (d, J = 10.0 Hz, 1H), 3.30–3.25 (m, 1H), 2.78 (d, J = 4.4 Hz, 3H), 2.26–2.19 (m, 1H), 2.08–2.00 (m, 2H), 1.98–1.95 (m, 1H), 1.87–0.95 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.65 (s, 3H). HRMS (ESI): calcd for C₃₂H₅₄N₂NaO₃ [M + Na]⁺ 537.4027; found 537.4039.

3.1.33. (4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-methylpentanamide (5al)

Compound 5al was synthesized in a 93% yield as a white solid using a similar procedure to that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.69–5.65 (m, 1H), 3.28–3.26 (m, 2H), 2.86–2.83 (m, 1H), 2.66 (s, 3H), 2.19–0.97 (m, other aliphatic ring protons), 0.92 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 177.31, 148.94, 122.93, 60.46, 58.55, 57.15, 52.23, 43.39, 40.95, 39.86, 37.84, 37.55, 36.87, 33.99, 33.60, 33.31, 32.01, 29.20, 27.79, 26.33, 25.13, 25.03, 24.58, 21.64, 21.63, 18.86, 12.32. HRMS (ESI): calcd for C₂₇H₄₇N₂O [M + H]⁺ 415.3683; found 415.3668.

3.1.34. Tert-butyl((10R,13R,17R)-4,4,10,13-tetramethyl-17-((R)-5-oxopentan-2-yl)-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (24)

IBX (616 mg, 2.2 mmol) was added to a solution of compound 12 (976 mg, 2 mmol) in DMSO (10 mL). The mixture was stirred at r.t. for 2 h under a N₂ atmosphere. When the reaction was completed, as indicated by TLC, the reaction mixture was added to an aqueous NaHSO₃ solution (20 mL). The mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography using PE/EtOAc (10:1, v/v) to obtain compound 24 (602 mg, 62% yield) as a white solid. The mp was 173.3–174.8 °C. ¹H-NMR (400 MHz, CD₃OD) δ 9.75 (s, 1H), 5.56–5.53 (m, 1H), 4.43 (d, J = 9.6 Hz, 1H), 3.31–3.2 (m, 1H), 2.47–2.40 (m, 1H), 2.38–2.29 (m, 1H), 2.09–2.04 (m, 1H), 1.99–1.96 (m, 1H), 1.90–0.95 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₁H₅₁NNaO₃ [M + Na]⁺ 508.3761; found 508.3770.

3.1.35. Tert-butyl((10R,13R,17R)-4,4,10,13-tetramethyl-17-((R)-5-(propylamino)pentan-2-yl)-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (25)

NaBH(OAc)₃ (85 mg, 0.4 mmol) at 0 °C was added to a solution of compound 24 (92 mg, 0.2 mmol) and n-propylamine (18 mg, 0.3 mmol) in DCM (10 mL). The mixture was stirred at r.t. for 2 h under a N₂ atmosphere, and the starting material disappeared. Water (30 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH/NH₃^.H₂O (100:5:0.5, v/v/v) to obtain compound 25 (80 mg, 75% yield) as a white solid. ¹H-NMR (400 MHz, CD₃OD) δ 5.55–5.51 (m, 1H), 4.43 (d, J = 9.6 Hz, 1H), 3.30–3.27 (m, 1H), 2.55 (t, J = 7.2 Hz, 4H), 2.08–2.1.97 (m, 2H), 1.87–1.78 (m, 1H), 1.72–0.88 (m, other aliphatic ring protons), 0.65 (s, 3H). HRMS (ESI): calcd for C₃₄H₆₁N₂O₂ [M + H]⁺ 529.4728; found 529.4728.

3.1.36. (10R,13R,17R)-4,4,10,13-tetramethyl-17-((R)-5-(propylamino)pentan-2-yl)-tetradecahydro-1H-cyclopenta[a]phenanthren-3-amine (5am)

The compound 5am was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.25. mp >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.63–5.57 (m, 1H), 2.66 (t, J = 7.2 Hz, 4H), 2.42–2.39 (m, 1H), 2.13–2.08 (m, 1H), 2.07–2.04 (m, 1H), 1.91–1.84 (m, 1H), 1.80–1.77 (m, 1H), 1.69–0.94 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 151.19, 121.08, 59.59, 58.71, 57.29, 52.58, 51.96, 50.68, 43.40, 41.50, 41.11, 38.66, 38.01, 36.99, 34.49, 33.76, 32.15, 29.33, 28.05, 27.70, 26.05, 25.19, 24.46, 22.60, 21.77, 21.66, 19.16, 12.35, 11.80. HRMS (ESI): calcd for C₂₉H₅₃N₂ [M + H]⁺ 429.4203; found 429.4237.

3.1.37. Tert-butyl((10R,13R,17R)-17-((2R)-5-hydroxyhexan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (26a)

A 1M MeMgCl solution in THF (1.1 mL, 1.1 mmol) was added dropwise at 0 °C to a dry THF solution (20 mL) of compound 24 (243 mg, 0.5 mmol) under a N₂ atmosphere. The mixture was stirred at r.t. for 12 h under a N₂ atmosphere. When the reaction was completed, as indicated by TLC, an aqueous NH₄Cl solution (2 mL) was added, and the solvents were removed in vacuo. Water (30 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using DCM/MeOH (30:1, v/v) to obtain compound 26a (130 mg, 52% yield) as a white solid. The mp was 200.8–202.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.57–5.55 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.77–3.71 (m, 1H), 3.33–3.27 (m, 1H), 2.10–2.04 (m, 1H), 2.00–1.97 (m, 1H), 1.88–1.81 (m, 1H), 1.73–0.83 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₂H₅₅NNaO₃ [M + Na]⁺ 524.4074; found 524.4083.

3.1.38. Tert-butyl((10R,13R,17R)-17-((2R)-5-hydroxyheptan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (26b)

Compound 26b was synthesized in a 57% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 187.3–188.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.47–5.45 (m, 1H), 4.45 (d, J = 9.2 Hz, 1H), 3.54–3.52 (m, 1H), 3.48–3.46 (m, 1H), 2.09–1.99 (m, 3H), 1.88–1.82 (m, 1H), 1.74–0.82 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₃H₅₇NNaO₃ [M + Na]⁺ 538.4231; found 538.4248.

3.1.39. Tert-butyl((10R,13R,17R)-17-((2R)-5-hydroxyoctan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (26c)

Compound 26c was synthesized in a 49% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 173.1–174.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.47–5.45 (m, 1H), 4.45 (d, J = 9.2 Hz, 1H), 3.54–3.53 (m, 1H), 3.52–3.49 (m, 1H), 2.10–1.99 (m, 3H), 1.88–1.82 (m, 1H), 1.66–0.82 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₄H₅₉NNaO₃ [M + Na]⁺ 552.4387; found 552.4389.

3.1.40. Tert-butyl((10R,13R,17R)-17-((2R)-5-hydroxy-6-methylheptan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (26d)

Compound 26d was synthesized in a 45% yield as a white solid using a similar procedure to that in 3.1.37. and was used for the next reaction without further purification.

3.1.41. Tert-butyl((10R,13R,17R)-17-((2R)-5-hydroxyoct-7-en-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (26e)

Compound 26e was synthesized in a 48% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 191.4–192.5 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.88–5.78 (m, 1H), 5.56–5.53 (m, 1H), 5.15–5.11 (m, 2H), 4.44 (d, J = 10.0 Hz, 1H), 3.60–3.59 (m, 1H), 3.33–3.27 (m, 1H), 2.35–2.28 (m, 1H), 2.18–0.86 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₄H₅₇NNaO₃ [M + Na]⁺ 550.4231; found 550.4247.

3.1.42. (5R)-5-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)hexan-2-ol (5an)

Compound 5an was synthesized in a 92% yield as a white solid using a similar procedure to that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.70–5.65 (m, 1H), 3.68–3.62 (m, 1H), 2.78–2.75 (m, 1H), 2.16–2.11 (m, 1H), 2.07–2.04 (m, 1H), 1.93–0.95 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 149.56, 122.44, 69.19, 60.25, 58.62, 57.33, 52.35, 43.35, 41.03, 40.30, 37.89, 37.14, 36.70, 33.66, 33.19, 32.06, 29.27, 27.88, 25.42, 25.16, 24.89, 23.65, 23.40, 21.70, 21.65, 19.27, 12.37. HRMS (ESI): calcd for C₂₇H₄₈NO [M + H]⁺ 402.3730; found 402.3752.

3.1.43. (6R)-6-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)heptan-3-ol (5ao)

Compound 5ao was synthesized in a 91% yield as a white solid using a similar procedure as that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.72–5.64 (m, 1H), 3.39–3.35 (m, 1H), 2.89–2.86 (m, 1H), 2.17–2.12 (m, 1H), 2.07–2.04 (m, 1H), 1.96–0.93 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 148.87, 122.87, 74.46, 60.44, 58.50, 57.27, 52.17, 43.28, 40.91, 39.81, 37.79, 37.52, 37.15, 34.29, 33.56, 33.04, 31.95, 31.03, 30.72, 29.23, 27.79, 25.09, 24.54, 21.65, 21.58, 19.29, 12.35, 10.42. HRMS (ESI): calcd for C₂₈H₄₉NNaO [M + Na]⁺ 438.3706; found 438.3692.

3.1.44. (7R)-7-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)octan-4-ol (5ap)

Compound 5ap was synthesized in a 92% yield as a white solid using a similar procedure to that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.72–5.65 (m, 1H), 3.48–3.46 (m, 1H), 2.83–2.80 (m, 1H), 2.17–2.12 (m, 1H), 2.07–2.04 (m, 1H), 1.92–0.93 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 149.25, 122.72, 72.83, 60.36, 58.62, 57.36, 52.33, 43.35, 41.02, 40.80, 40.50, 40.10, 37.88, 37.73, 37.22, 37.00, 34.93, 33.63, 33.11, 32.05, 29.29, 27.82, 25.15, 24.92, 21.67, 19.97, 19.30, 14.51, 12.34. HRMS (ESI): calcd for C₂₉H₅₁NNaO [M + Na]⁺ 452.3863; found 452.3856.

3.1.45. (6R)-6-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylheptan-3-ol (5aq)

Compound 5aq was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.71–5.66 (m, 1H), 3.22–3.20 (m, 1H), 2.79 (d, J = 11.2 Hz, 1H), 2.16–2.12 (m, 1H), 2.08–2.04 (m, 1H), 1.91–0.90 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 149.36, 122.62, 78.11, 60.32, 58.63, 57.48, 52.34, 43.37, 41.03, 40.16, 37.88, 37.78, 37.36, 34.87, 33.65, 33.36, 32.06, 31.67, 29.32, 27.84, 25.17, 24.91, 21.68, 21.65, 19.56, 19.36, 18.00, 17.53, 12.37. HRMS (ESI): calcd for C₂₉H₅₂NO [M + H]⁺ 430.4043; found 430.4068.

3.1.46. (7R)-7-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)oct-1-en-4-ol (5ar)

Compound 5ar was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 250.4–252.6 °C. ¹H-NMR (400 MHz, CD₃OD) δ 5.91–5.81 (m, 1H), 5.68–5.63 (m, 1H), 5.06 (d, J = 15.6 Hz, 1H), 5.03 (d, J = 8.4 Hz, 1H), 3.54–3.52 (m, 1H), 2.63 (d, J = 11.2 Hz, 1H), 2.27–2.19 (m, 2H), 2.17–2.09 (m, 1H), 2.07–2.04 (m, 1H), 1.91–0.95 (m, other aliphatic ring protons), 0.72 (s, 3H). ¹³C-NMR (100 MHz, CD₃OD) δ 150.11, 136.54, 121.98, 117.16, 72.74, 60.01, 58.66, 57.37, 52.44, 43.36, 43.09, 41.06, 40.72, 38.15, 37.93, 37.23, 34.23, 33.70, 33.01, 32.09, 29.31, 27.93, 26.23, 25.18, 24.72, 21.73, 21.66, 19.32, 12.37. HRMS (ESI): calcd for C₂₉H₅₀NO [M + H]⁺ 428.3887; found 428.3886.

3.1.47. Tert-butyl((10R,13R,17R)-17-((R)-5-hydroxy-5-methylhexan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (27a)

Compound 27a was synthesized in a 68% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 220.4–223.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.51 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.30–3.26 (m, 1H), 2.09–2.04 (m, 1H), 2.00–1.97 (m, 1H), 1.89–1.80 (m, 1H), 1.73–0.95 (m, other aliphatic ring protons), 0.92 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₃H₅₇NNaO₃ [M + Na]⁺ 538.4231; found 538.4234.

3.1.48. Tert-butyl((10R,13R,17R)-17-((R)-5-ethyl-5-hydroxyheptan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (27b)

Compound 27b was synthesized in a 64% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 242.2–243.7 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.53 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.33–3.26 (m, 1H), 2.09–2.04 (m, 1H), 2.00–1.97 (m, 1H), 1.90–1.80 (m, 1H), 1.73–0.96 (m, other aliphatic ring protons), 0.92 (d, J = 6.0 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H), 0.84 (t, J = 7.2 Hz, 3H), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₅H₆₁NNaO₃ [M + Na]⁺ 566.4544; found 566.4538.

3.1.49. Tert-butyl((10R,13R,17R)-17-((R)-5-hydroxy-5-propyloctan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (27c)

Compound 27c was synthesized in a 55% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 205.2–207.6 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.57–5.54 (m, 1H), 5.29 (d, J = 10.0 Hz, 1H), 3.75–3.69 (m, 1H), 2.18–2.14 (m, 1H), 2.10–2.05 (m, 1H), 1.99–1.97 (m, 1H), 1.89–1.81 (m, 1H), 1.70–0.91 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₃₇H₆₅NNaO₃ [M + Na]⁺ 594.4857; found 594.4842.

3.1.50. Tert-butyl((10R,13R,17R)-17-((R)-5-allyl-5-hydroxyoct-7-en-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (27d)

Compound 27d was synthesized in a 61% yield as a white solid using a similar procedure to that in 3.1.37. The mp was 181.5–183.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.89–5.79 (m, 2H), 5.56–5.53 (m, 1H), 5.13 (d, J = 8.0 Hz, 2H), 5.10 (d, J = 16.0 Hz, 2H), 4.43 (d, J = 10.0 Hz, 1H), 3.33–3.27 (m, 1H), 2.20 (d, J = 7.2 Hz, 4H), 2.09–2.04 (m, 1H), 1.99–1.96 (m, 1H), 1.88–1.81 (m, 1H), 1.73–0.96 (m, other aliphatic ring protons), 0.91 (d, J = 6.4 Hz, 3H), 0.65 (s, 3H).

3.1.51. (5R)-5-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylhexan-2-ol (5as)

Compound 5as was synthesized in a 93% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 215.2–217.6 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 5:1) δ 5.47–5.43 (m, 1H), 2.34–2.31 (m, 1H), 2.00–1.96 (m, 1H), 1.90–1.87 (m, 1H), 1.75–1.69 (m, 1H), 1.66–1.63 (m, 1H), 1.53–0.80 (m, other aliphatic ring protons), 0.56 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 5:1) δ 149.37, 120.11, 70.95, 58.20, 57.19, 55.61, 50.94, 42.09, 40.20, 39.78, 39.64, 37.23, 36.76, 35.85, 32.54, 30.68, 30.13, 28.77, 28.43, 28.08, 27.29, 26.63, 24.05, 23.58, 21.07, 20.42, 18.58, 11.72. HRMS (ESI): calcd for C₂₈H₅₀NO [M + H]⁺ 416.3887; found 416.3898.

3.1.52. (6R)-6-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-3-ethylheptan-3-ol (5at)

Compound 5at was synthesized in a 93% yield as a white solid using a similar procedure to that in 3.1.25. The mp was >300 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 2:1) δ 5.47–5.45 (m, 2H), 2.47–2.43 (m, 1H), 1.99–1.92 (m, 1H), 1.87–1.84 (m, 1H), 1.76–0.74 (m, other aliphatic ring protons), 0.70 (t, J = 7.2 Hz, 3H), 0.69 (t, J = 7.2 Hz, 3H), 0.52 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 2:1) δ 148.45, 120.73, 74.68, 58.55, 57.08, 55.59, 50.78, 42.03, 39.52, 39.49, 36.80, 36.63, 36.03, 33.79, 32.42, 30.77, 30.58, 30.47, 29.08, 28.10, 27.08, 25.25, 23.98, 23.71, 20.95, 20.35, 18.56, 11.61, 7.43, 7.32. ESI-MS (m/z): 444.65 (M + H)⁺.

3.1.53. (7R)-7-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-4-propyloctan-4-ol (5au)

Compound 5au was synthesized in a 92% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 242.6–244.9 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.62–5.60 (m, 1H), 2.89–2.86 (m, 1H), 2.11–2.07 (m, 1H), 2.00–1.98 (m, 2H), 1.84–1.78 (m, 2H), 1.66–1.60 (m, 2H), 1.49–0.91 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 148.14, 121.47, 74.73, 59.83, 57.29, 55.81, 50.88, 42.32, 42.00, 41.73, 39.77, 39.26, 36.89, 36.68, 36.26, 35.50, 32.67, 30.84, 29.85, 29.45, 28.39, 28.07, 25.36, 24.29, 24.08, 21.27, 20.66, 18.94, 16.91, 16.82, 14.89, 11.99. ESI-MS (m/z): 472.71 (M + H)⁺.

3.1.54. (7R)-4-allyl-7-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)oct-1-en-4-ol (5av)

Compound 5av was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 235.5–237.4 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.89–5.79 (m, 2H), 5.56–5.53 (m, 1H), 5.13 (d, J = 8.0 Hz, 2H), 5.15 (d, J = 16.0 Hz, 2H), 2.47–2.43 (m, 1H), 2.20 (d, J = 7.2 Hz, 4H), 2.10–2.05 (m, 1H), 1.99–1.96 (m, 1H), 1.87–1.82 (m, 1H), 1.74–0.94 (m, other aliphatic ring protons), 0.90 (d, J = 6.4 Hz, 3H), 0.65 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃) δ 150.07, 133.96, 133.93, 119.91, 118.73, 118.64, 73.74, 58.62, 57.39, 55.80, 51.12, 43.93, 43.71, 42.31, 40.68, 39.84, 37.56, 36.99, 36.21, 35.52, 32.77, 30.90, 29.38, 28.44, 27.78, 27.58, 24.29, 24.06, 21.35, 20.63, 18.92, 11.97. HRMS (ESI): calcd for C₃₂H₅₄NO [M + H]⁺ 468.4200; found 468.4200.

3.1.55. (2S)-2-((10R,13S,17R)-4,4,10,13-tetramethyl-3-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)propylacetate (29)

Ac₂O (286 mg, 2.8 mmol), Et₃N (1 g, 10 mmol), and DMAP (12 mg, 0.1 mmol) were added to a solution of 21-hydroxy-20-methylpregn-4-en-3-one 28 (717 mg, 2 mmol) in dry DCM (30 mL), and the mixture was stirred at r.t. for 12 h under a N₂ atmosphere; TLC indicated the consumption of the starting material. Water (30 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with aqueous NaOH solution, brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in 5 mL of EtOAc to obtain compound 29 (780 mg, 97% yield) as a white solid, which was used for the next reaction without further purification.

3.1.56. (10R,13S,17R)-17-((S)-1-hydroxypropan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one (30)

Compound 30 was synthesized in a 67% yield as a white solid using a similar procedure to that in 3.1.3., except for the esterification procedure, and was used for the next reaction without further purification.

3.1.57. (2S)-2-((10R,13S,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)propylacetate (31)

Compound 31 was synthesized in a 76% yield as a white solid using a similar procedure to that in 3.1.4. The mp was 198.3–199.9 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.55 (m, 1H), 4.07 (dd, J = 10.8, 3.2 Hz, 1H), 3.76 (dd, J = 10.8, 3.2 Hz, 1H), 2.42–2.38 (m, 1H), 2.12–2.06 (m, 1H), 2.04 (s, 3H), 2.00–1.97 (m, 1H), 1.83–0.89 (m, other aliphatic ring protons), 0.68 (s, 3H). HRMS (ESI): calcd for C₂₆H₄₃NNaO₂ [M + Na]⁺ 424.3186; found 424.3199.

3.1.58. (2S)-2-((10R,13S,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)propan-1-ol (5aw)

Compound 5aw was synthesized in a 90% yield as a white solid using a similar procedure to that in 3.1.31. The mp was 170.6–173.8 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 5:1) δ 5.47–5.45 (m, 1H), 3.52–3.49 (m, 1H), 3.21–3.16 (m, 1H), 2.29–2.25 (m, 1H), 2.03–1.96 (m, 1H), 1.93–1.90 (m, 1H), 1.77–1.70 (m, 1H), 1.67–1.63 (m, 1H), 1.57–0.76 (m, other aliphatic ring protons), 0.60 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 5:1) δ 149.84, 119.76, 67.37, 58.02, 56.99, 52.35, 51.00, 42.25, 40.56, 39.56, 38.77, 37.44, 36.82, 32.58, 30.75, 27.73, 27.40, 27.31, 24.23, 23.52, 21.14, 20.45, 16.60, 11.82. HRMS (ESI) calcd for C₂₄H₄₂NO [M + H]⁺ 360.3261; found 360.3267.

3.1.59. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-hydroxyphenyl)urea (34)

Compound 33 (443 mg, 2 mmol) was added to a solution of compound 32 (218 mg, 2 mmol) in dry DCM (10 mL). The reaction was stirred at r.t. for 10 min, and the starting material disappeared. Water (30 mL) was added, and the mixture was extracted with DCM (3 × 30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and recrystallized in 2 mL of MeCN to obtain compound 34 (635 mg, 99% yield) as a brown solid. The mp was 232.1–233.6 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 5:1) δ 7.74 (d, J = 2.0 Hz, 1H), 7.53 (dd, J = 8.4, 2.0 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H). HRMS (ESI): calcd for C₁₄H₁₁ClF₃N₂O₂ [M + H]⁺ 331.0456; found 331.0451.

3.1.60. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-((10R,13R,17R)-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)urea (5ax)

Compound 5ax was synthesized in a 96% yield as a white solid using a similar procedure to that in 3.1.59. The mp was 221.1–223.7 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 3:1) δ 7.72 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 10.0 Hz, 1H), 5.54–5.50 (m, 1H), 3.49 (t, J = 4.8 Hz, 2H), 3.47–3.40 (m, 1H), 2.07–2.00 (m, 1H), 1.98–1.94 (m, 1H), 1.85–1.76 (m, 1H), 1.72–0.86 (m, other aliphatic ring protons), 0.63 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 3:1) δ 156.05, 149.73, 139.29, 131.82, 128.50 (q, J = 31.2 Hz), 123.94, 122.81 (q, J = 228.0 Hz), 122.31, 120.48, 117.27 (d, J = 5.8 Hz), 63.13, 57.41, 56.41, 56.18, 51.08, 42.39, 40.54, 39.91, 37.62, 36.87, 35.88, 32.73, 32.11, 31.03, 29.37, 28.43, 27.89, 26.43, 24.80, 24.32, 21.36, 20.72, 18.74, 11.97. ¹⁹F-NMR (375 MHz, CDCl₃:CD₃OD = 3:1) δ−63.05. HRMS (ESI): calcd for C₃₄H₄₈ClF₃N₂NaO₂ [M + Na]⁺ 631.3249; found 631.3247.

3.1.61. Tert-butyl((10R,13R,17R)-17-((R)-5-bromopentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (35)

PPh₃ (131 mg, 0.5 mmol), imidazole (33 mg, 0.5 mmol), and CBr₄ (150 mg, 0.45 mmol) were added to a solution of compound 12 (146 mg, 0.3 mmol) in dry DMF (10 mL). The reaction was stirred at r.t. for 2 h, and TLC indicated the consumption of the starting material. Upon the addition of MeOH (0.2 mL) for quenching, water (30 mL) was added and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel column chromatography using PE/EtOAc (50:1, v/v) to obtain compound 35 (126 mg, 76% yield) as a white solid. The mp was 192.3–194.7 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.59–5.57 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.43–3.35 (m, 2H), 3.33–3.27 (m, 1H), 2.11–2.04 (m, 1H), 2.00–1.97 (m, 1H), 1.92–1.83 (m, 2H), 1.80–0.95 (m, other aliphatic ring protons), 0.92 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H).

3.1.62. Tert-butyl((10R,13R,17R)-17-((R)-5-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)pentan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (36)

Compound 36 was synthesized in a 68% yield as a white solid using a similar procedure to that in 3.1.6 and used for the next reaction without further purification.

3.1.63. 1-(4-(((4R)-4-((10R,13R,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentyl)oxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (5ay)

Compound 5ay was synthesized in a 94% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 192.3–194.5 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 5:1) δ 7.65 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.54–5.50 (m, 1H), 3.84 (t, J = 7.6 Hz, 2H), 2.36–2.32 (m, 1H), 2.00–1.97 (m, 1H), 1.88–1.79 (m, 2H), 1.71–0.86 (m, other aliphatic ring protons), 0.66 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 5:1) δ 155.68, 153.57, 149.85, 138.40, 131.76, 131.02, 128.43 (d, J = 31.2 Hz), 124.57, 122.78 (q, J = 271.9 Hz), 122.65, 122.09, 119.89 (2C), 117.59 (d, J = 4.4 Hz), 115.02 (2C), 68.96, 58.15, 57.30, 55.89, 51.06, 42.24, 40.60, 39.77, 37.48, 36.89, 35.59, 32.66, 32.09, 30.80, 28.29, 27.49, 27.38, 25.91, 24.18, 23.62, 21.22, 20.53, 18.65, 11.88. ¹⁹F-NMR (375 MHz, CDCl₃:CD₃OD = 5:1) δ -62.77. HRMS (ESI): calcd for C₄₀H₅₄ClF₃N₃O₂ [M + H]⁺ 700.3851; found 700.3900.

3.1.64. Tert-butyl((10R,13R,17R)-17-((R)-5-hydroxypentan-2-yl)-4,4,10,13-tetramethyl-7-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (37)

Compound 37 was synthesized in a 96% yield as a white solid using a similar procedure to that in 3.1.19., and the reaction temperature was r.t. The mp was 225.5–227.2 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.94 (s, 1H), 4.49 (d, J = 10.0 Hz, 1H), 3.60 (t, J = 6.0 Hz, 2H), 3.49–3.43 (m, 1H), 2.31–2.26 (m, 1H), 2.22–2.17 (m, 1H), 2.03–2.00 (m, 1H), 1.89–1.85 (m, 1H), 1.81–1.00 (m, other aliphatic ring protons), 0.93 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₃₁H₅₁NNaO₄ [M + Na]⁺ 524.3710; found 524.3728.

3.1.65. Tert-butyl((10R,13R,17R)-17-((R)-5-bromopentan-2-yl)-4,4,10,13-tetramethyl-7-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (38)

Compound 38 was synthesized in a 72% yield as a white solid using a similar procedure to that in 3.1.61. The mp was 226.3–228.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.95 (s, 1H), 4.48 (d, J = 10.0 Hz, 1H), 3.49–3.42 (m, 1H), 3.40–3.32 (m, 2H), 2.31–2.28 (m, 1H), 2.23–2.17 (m, 1H), 2.02–1.09 (m, other aliphatic ring protons), 0.93 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₃₁H₅₀BrNNaO₃ [M + Na]⁺ 586.2866; found 586.2864.

3.1.66. Tert-butyl((10R,13R,17R)-17-((R)-5-(4-(3-(4-chloro-3-(trifluoromethyl) phenyl)ureido)phenoxy)pentan-2-yl)-4,4,10,13-tetramethyl-7-oxo-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (39)

Compound 39 was synthesized in a 77% yield as a white solid using a similar procedure to that in 3.1.6. The mp was 223.1–224.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 7.86 (s, 1H), 7.55 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.47 (s, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 5.93 (s, 1H), 4.58 (d, J = 10.0 Hz, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.47–3.41 (m, 1H), 2.28–2.18 (m, 2H), 2.05–1.08 (m, other aliphatic ring protons), 0.95 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). HRMS (ESI): calcd for C₄₅H₅₉ClF₃N₃NaO₅ [M + Na]⁺ 836.3988; found 836.3964.

3.1.67. Tert-butyl((10R,13R,17R)-17-((R)-5-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)pentan-2-yl)-7-hydroxy-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (40)

Compound 40 was synthesized in a 94% yield as a white solid using a similar procedure to that in 3.1.21. The mp was 248.7–249.4 °C. ¹H-NMR (400 MHz, CDCl₃) δ 7.84 (s, 1H), 7.55–7.53 (m, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 5.46–5.44 (m, 1H), 4.59 (d, J = 10.0 Hz, 1H), 3.85–3.83 (m, 1H), 3.80 (t, J = 6.0 Hz, 2H), 3.28–3.22 (m, 1H), 1.99–0.86 (m, other aliphatic ring protons), 0.66 (s, 3H). HRMS (ESI): calcd for C₄₅H₆₁ClF₃N₃NaO₅ [M + Na]⁺ 838.4150; found 838.4134.

3.1.68. 1-(4-(((4R)-4-((10R,13R,17R)-3-amino-7-hydroxy-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentyl)oxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (5az)

Compound 5az was synthesized in a 96% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 198.1–199.7 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 20:1) δ 7.65 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 5.73 (d, J = 4.4 Hz, 1H), 3.88–3.82 (m, 3H), 2.36–2.32 (m, 1H), 1.96–1.94 (m, 1H), 1.85–0.91 (m, other aliphatic ring protons), 0.93 (d, J = 6.0 Hz, 3H), 0.64 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 20:1) δ 155.43, 154.73, 153.56, 138.41, 131.65, 131.07, 128.31 (d, J = 31.3 Hz), 124.38, 122.71 (q, J = 271.1 Hz), 122.54, 122.14, 121.77 (2C), 117.50, 114.90 (2C), 68.85, 65.78, 57.75, 55.57, 49.22, 43.33, 41.88, 40.59, 38.96, 37.89, 37.02, 36.59, 35.53, 32.01, 28.28, 27.45, 27.03, 25.78, 24.15, 23.41, 20.00, 19.69, 18.57, 11.43. ¹⁹F-NMR (375 MHz, CDCl₃:CD₃OD = 20:1) δ -62.73. HRMS (ESI): calcd for C₄₀H₅₄ClF₃N₃O₃ [M + H]⁺ 716.3800; found 716.3761.

3.1.69. Tert-butyl((10R,13S,17R)-17-((S)-1-hydroxypropan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (41)

Compound 41 was synthesized in a 98% yield as a white solid using a similar procedure to that in 3.1.16. The mp was 213.0–214.3 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.52 (m, 1H), 4.44 (d, J = 9.6 Hz, 1H), 3.65–3.62 (m, 1H), 3.37–3.26 (m, 2H), 2.10–2.05 (m, 1H), 2.00–1.97 (m, 1H), 1.86–1.77 (m, 1H), 1.69–0.85 (m, other aliphatic ring protons), 0.68 (s, 3H). HRMS (ESI): calcd for C₂₉H₄₉NNaO₃ [M + Na]⁺ 482.3605; found 482.3615.

3.1.70. Tert-butyl((10R,13S,17R)-17-((S)-1-bromopropan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (42)

Compound 42 was synthesized in a 78% yield as a white solid using a similar procedure to that in 3.1.61. The mp was 232.2–234.8 °C. ¹H-NMR (400 MHz, CDCl₃) δ 5.56–5.53 (m, 1H), 4.43 (d, J = 10.0 Hz, 1H), 3.51–3.49 (m, 1H), 3.35–3.31 (m, 2H), 2.10–2.05 (m, 1H), 1.97–1.94 (m, 1H), 1.88–1.86 (m, 1H), 1.73–0.85 (m, other aliphatic ring protons), 0.68 (s, 3H). HRMS (ESI): calcd for C₂₉H₄₈BrNNaO₂ [M + Na]⁺ 544.2761; found 544.2759.

3.1.71. Tert-butyl((10R,13S,17R)-17-((S)-1-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)propan-2-yl)-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)carbamate (43)

Compound 43 was synthesized in an 86% yield as a white solid using a similar procedure to that in 3.1.6 and used for the next reaction without further purification.

3.1.72. 1-(4-((2S)-2-((10R,13S,17R)-3-amino-4,4,10,13-tetramethyl-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)propoxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (5ba)

Compound 5ba was synthesized in an 86% yield as a white solid using a similar procedure to that in 3.1.25. The mp was 197.1–198.6 °C. ¹H-NMR (400 MHz, CDCl₃:CD₃OD = 10:1) δ 7.65 (s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 5.52–5.50 (m, 1H), 3.81 (dd, J = 8.8, 2.4 Hz, 1H), 3.55 (dd, J = 8.8, 8.0 Hz, 1H), 2.35–2.31 (m, 1H), 2.08–1.97 (m, 2H), 1.80–1.77 (m, 2H), 1.70–0.80 (m, other aliphatic ring protons), 0.68 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃:CD₃OD = 10:1) δ 155.86, 153.64, 149.71, 138.39 (d, J = 2.5 Hz), 131.64, 130.90, 128.32 (d, J = 31.2 Hz), 124.41, 122.71 (q, J = 271.4 Hz), 122.57, 121.88, 119.81 (2C), 117.51 (d, J = 5.2 Hz), 114.90 (2C), 73.42, 58.05, 56.92, 52.61, 50.97, 42.35, 40.48, 39.53, 37.38, 36.80, 36.46, 32.55, 30.75, 27.78, 27.36, 27.18, 24.21, 23.50, 21.11, 20.44, 17.25, 11.84. ¹⁹F-NMR (375 MHz, CDCl₃:CD₃OD = 10:1) δ -62.78. HRMS (ESI): calcd for C₃₈H₅₀ClF₃N₃O₂ [M + H]⁺ 672.3538; found 672.3574.

3.2. SHP1 and SHP2 Enzyme Assay

The proteins SHP1 and SHP2, containing a GST-tag, were expressed in E. coli. 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) was used as the fluorogenic substrate. Fluorescence data were collected by the Envision (PerkinElmer, Waltham, MA, USA) plate reader, using excitation and emission wavelengths of 358 and 455 nm, respectively. A sigmoidal dose-response curve was fitted to the data, and the EC₅₀ values were calculated using GraphPad Prism. In the data processing, the fitting and curves were calculated using nonlinear regression, and the fold value of the compound was calculated using the software GraphPad Prism 8. The folds were obtained according to the equation: Y = Bottom + (Top-Bottom)/(1 + 10^((LogEC₅₀-X) × HillSlope)). The data represent mean values ± standard error of the mean (SEM) of eight-point experiments, each performed in triplicates from three independent experiments.

3.3. CCK8 Cell Viability Assay

Acute lymphoblastic leukemia (ALL) cell line RS4;11 (2.5 × 10⁴ cells per well), acute promyelocytic leukemia (APL) cell line NB4 (1 × 10⁴ cells per well), and non-small cell lung cancer (NSCLC) cell line NCI-H1299 (1.5 × 10³ cells per well) were seeded into 96-well plates. After treatment with different concentrations of drugs, the cells were incubated in quadruple for 72 h. Thereafter, a 10 μL CCK8 solution was added to each well. After a 3 h incubation at 37 °C, the plates were read for absorbance at 450 nm and 650 nm using a SpectraMax Molecular Devices microplate reader. The final data were calibrated by OD450 nm-OD650 nm. The inhibition rates of proliferation were calculated with the following equation:

Inhibition ratio = (OD_DMSO-OD_Compd)/(OD_DMSO-OD_blank) × 100%.

(1)

The concentrations of the compounds that inhibited cell growth by 50% (IC₅₀) were calculated using GraphPad Prism version 5.0. NVP-2 was used as a positive control.

3.4. Molecular Docking

The Glide (Maestro 10.2) package of Schrödinger suite 2015 (https://www.schrodinger.com, accessed on 1 September 2015) was employed for the molecular docking investigation in SP mode. The SHP1 crystal structure (open conformation, PDB ID: 3PS5, 3.10 Å of resolution) was downloaded from the Protein Data Bank (https://www.rcsb.org, accessed on 1 March 2021). The receptors and ligands were prepared using the Protein Preparation Wizard and LigPrep modules embedded in the Schrödinger suite, respectively. All water molecules and sulfate ions were removed, and hydrogen atoms were added. The optimized geometry of the ligands with minimum energy was attained using an OPLS 2005 force field. The grid box was set to search all over the protein for binding sites. All the other parameters were adjusted as a default. The graphics were generated by PyMOL 2.6.0 (https://pymol.org/2, accessed on 1 November 2022) and UCSF Chimera 1.16.

3.5. Statistical Analysis

The data are presented as the mean ± SEM unless otherwise noted. Statistical significance was calculated using the two-tailed Student’s t-test; a p-value < 0.05 was considered statistically significant. Statistical analysis was conducted using GraphPad Prism.

4. Conclusions

In summary, a new series of 3-amino-4,4-dimethyl lithocholic acid derivatives were designed, synthesized, and evaluated for SHP1 activation ability. The SAR data presented herein demonstrated that the introduction of a free amino group at the C-3 position was essential for its potency. The primary alcohol moiety in the side chain can be replaced by other chemical groups while maintaining or significantly increasing potency. A large, >9-fold increase in potency was observed with the hybrid of compound 5aa and the diphenyl urea group. Combining these observations led to the synthesis of the diphenyl urea hybrid molecules 5az-ba, which proved to be highly potent SHP1 activators, with EC₅₀ values of 2.10 and 1.54 μM, respectively. The compounds 5az-ba also showed good selectivity in the SHP2 activation assay. In the in vitro cellular assays, 5az-ba showed potent activities with IC₅₀ values up to 1.65 μM against acute lymphoblastic leukemia cell line RS4;11, acute promyelocytic leukemia cell line NB4, and lung cancer cell line NCI-H1299. The molecular modeling study revealed that compound 5ba gains hydrogen bond interactions with Thr80, Gln81, Lys97, and Asn472. The deeper and closer binding of the compound to the central allosteric pocket of SHP1 may explain the stronger activation. All of the studies presented here support the proposal that 5az-ba may provide good references for the development of new anticancer drugs targeting SHP1 activation, and they deserve further research.