Discovery and synthesis of novel beesioside I derivatives with potent anti-HIV activity

Abstract

In this study, 12 known cycloartane triterpenoids (1–12) with four different skeletons isolated from the roots of Souliea vaginata were screened for the first time for in vitro anti-HIV activity using AZT as a standard. Among the compounds, beesioside I (1) showed the highest potency against HIV-1_NL4-3 with an EC₅₀ value of 2.32 μM (CC₅₀ > 40 μM). Preliminary structure-activity relationship (SAR) studies on 1 indicated that simple modification of its aglycone (13) could significantly influence the antiviral activity. Particularly, the introduction of an acyl group at the C-3 position of 13 led to significant improvement in both anti-HIV potency and selectivity index. Among all synthetically modified derivatives, compound 13g was the most potent compound with an EC₅₀ value of 0.025 μM and TI value greater than 800, comparable to those of 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB, bevirimat). Other analogues exhibited strong to weak inhibition of HIV-1 replication in MT-4 cells. The length, carboxylic terminus, and C-3′ dimethyl substitution of the C-3 side chain substantially affected the anti-HIV activity. Finally, compound 13g was an effective agent against HIV with high potential for further investigation.

Graphical Abstract

1. Introduction

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) remains a global health threat and a leading cause of death. The emergence of drug-resistant HIV compels the continuous development of novel potent agents with new mechanisms of action [1], and natural products remain an important source for the discovery of new drugs [2]. Our prior studies found that natural triterpenoids are potential antiviral agents deserving further investigation [3].

In our continuous search for novel anti-HIV constituents from natural sources, cycloartane triterpenoids isolated from the roots of Souliea vaginata were screened in vitro for anti-HIV activity. S. vaginata is a perennial herb endemic to southwest and northwest China and is commonly used to treat conjunctivitis, stomatitis, pharyngitis, and enteritis [4]. Chemical investigations on this plant revealed cycloartane triterpenoid glycosides as the main bioactive components [5–10]. Such glycosides show various biological effects, including anti-HIV, cytotoxic, anti-acetylcholinesterase, and neuroprotective activities [11–14]. In our previous investigation on this species, we isolated a series of new cycloartane triterpene glycosides [15–21]. Herein, twelve known cycloartane triterpenoids with four different skeletons, beesioside I (1), beesioside K (2), soulieoside M (3), beesioside M (4), soulieoside N (5), soulieoside R (6), soulieoside Q (7), beesioside O (8), (20S,24S)-15α-acetoxy-16β,24;20,24-diepoxy-3β-(β-D-xylopyranosyloxy)-9,19-cyclolanostane-18,25-diol (9), soulieoside S (10), soulieoside O (11), and soulieoside P (12) (Fig. 1), were evaluated against HIV-1 using AZT as standard in antiviral assays. Among the isolates, beesioside I (1) exhibited the highest potency against HIV-1_{NL 4-3} in MT 4 cells with an EC₅₀ value of 2.32 μM (CC₅₀ > 40 μM).

Fig.1.

Structures of compounds 1–12 isolated from Souliea vaginata.

Therefore, we selected 1 as a lead compound for structural modification. In addition to its high anti-HIV potency, compound 1 has three positions, C-3, C-15, and C-16, at which different substituents can be easily introduced to improve the pharmacological activity. Initially, compound 1 was hydrolyzed enzymatically to its aglycone (13). Then, the aglycone was subjected to acetylation, propionylation, oxidation and alkaline hydrolysis to give derivatives 13a–d, respectively, as shown in Scheme 1. In addition, compound 1 was also deacetylated to give 1a. Among these compounds, the 3-acetyl derivative (13a) of 13 was equipotent to 1, the 15,16-deacetyl analogue (1a) of 1 was less potent, and the aglycone 13 and 13b-d were inactive. Thus, the preliminary structure-activity relationship (SAR) studies indicated that subtle modifications of the aglycone have key influences on the antiviral activity. Particularly, the introduction of an acyl group at the C-3 position, a possible privileged sub-structural motif, could significantly improve both anti-HIV potency and selectivity index [22]. Further structural modification led to the synthesis of nine new C-3 acylated 13-derivatives (13e–m) (Scheme 2). Among these derivatives, compound 13g was the most potent with an EC₅₀ value of 0.025 μM and TI value greater than 800, comparable to those of the control 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB, bevirimat, the first HIV-1 maturation inhibitor). The eight remaining derivatives exhibited strong to weak inhibition of HIV-1 replication in MT-4 cells.

Scheme 1.

The synthetic routes to compounds 1a, 13 and 13a–d. Reagents and conditions: (i) molsin, 0.2 M Na₂HPO₄-0.1 M citric acid buffer (pH 4.0), 37 °C, 2 days; (ii) Ac₂O or Pr₂O, anhydrous pyridine, rt, overnight; (iii) PCC, anhydrous CH₂Cl₂, rt, overnight; (iv) KOH, EtOH/H₂O, 60 °C, 2 h.

Scheme 2.

Synthesis of compounds 13e–m. Reagents and conditions: (a) appropriate acid anhydride, DMAP, anhydrous pyridine, microwave 155 °C, 2 h.

2. Results and discussion

2.1. Chemistry

Compounds 1–12 were isolated from S. vaginata similarly to our previous reported studies [15–21]. Scheme 1 outlines the general synthetic procedure leading to six derivatives 1a, 13 and 13a–d including two new ones (13b–c). Compound 1 was hydrolyzed to afford its aglycone 13, which was acylated separately with Ac₂O or Pr₂O in anhydrous pyridine to give 13a–b. In addition, compound 13 was oxidized using pyridinium chlorochromate (PCC) to afford 13c and hydrolyzed to 13d. Furthermore, compound 1 was also treated with KOH to give 1a. Scheme 2 shows the synthetic route to new compounds 13e–m. In a solution of anhydrous pyridine containing 4-dimethylaminopyridine, compound 13 was esterified with different acid anhydrides, including succinic, methylsuccinic, 2,2-dimethylsuccinic, glutaric, 3-methylglutaric, 2,2-dimethylglutaric, 3,3-dimethylglutaric, 3,3-tetramethyleneglutaric, and diglycolic, to furnish the corresponding 3-O-acyl derivatives (13e–m), respectively. To drive the reaction to completion, the triterpenoids were treated with at least 10-fold molar excess of each acid anhydride, and the recovery of aglycone was generally 15–20%.

2.2. Evaluation of anti-HIV activity

All 12 known compounds isolated from S. vaginata and the 15 synthetic derivatives of 1, including 11 new ones, were evaluated for anti-HIV activity by determining the inhibitory effects on virus replication in MT4 lymphocytes infected by HIV-1 NL4-3 Nanoluc-sec virus. Table 1 shows the anti-HIV results for the tested compounds with AZT as positive control. Compound 1 showed the best potency among the 12 isolated constituents. All four active compounds (1, 2, 4, 10) are tetracyclic cycloartane triterpenoids with a pendant tetrahydrofuran at C-17; thus, this structure might play a role in the anti-HIV activity. However, the presence or location of oxygen bridges, as well as the substituents in the side chain, also influenced the antiviral activity. Two (1, 2) of three (1–3) compounds with an oxygen bridge between C-18 and C-24 were quite active, while only compound 4 was significantly active among four compounds (4–7) without this oxygen bridge. With an oxygen bridge between C-16 and C-24, compounds 8 and 9 were inactive, while compound 10, which also has a glucosylated hydroxyl group at C-25, showed good activity (EC₅₀ 3.76 μM). Compounds 11 and 12 with the tetrahydrofuran fused to ring D were inactive. Acetylation of hydroxyl groups at C-15 or C-16 in ring D led to enhanced anti-HIV activity (compare 1, 15-OAc, 16-OAc: EC₅₀ 2.32 μM vs 2, 15-OH, 16-OAc: EC₅₀ 3.65 μM and 4, 15-OAc, 16-OH: EC₅₀ 4.85 μM vs 5, 15-OH, 16-OH: inactive).

Table 1.

In vitro anti-HIV data of compounds 1–12 ^a

Compd	EC50 (μM) NL4-3 b	CC50 (μM) MT4 c	TI e	Compd	EC50 (μM) NL4-3 b	CC50 (μM) MT4 c	TI e
1	2.32 ± 0.46	> 40	> 17.2	7	> 10	> 40	-
2	4.65 ± 1.15	> 40	> 8.6	8	> 10	> 40	-
3	*- d	> 40	-	9	*- d	> 40	-
4	4.85 ± 1.40	> 40	> 8.2	10	3.76 ± 1.4	35.4 ± 3.2	9.4
5	*- d	> 40	-	11	> 10	> 40	-
6	> 10	> 40	-	12	> 30	> 30	-
AZT	0.015 ± 0.0041	77.5 ± 10.1	5170

^aCompounds were evaluated by using a multicycle viral replication assay using HIV-1 NL4-3 Nanoluc-sec virus.

^bEC₅₀: 50% HIV inhibitory concentration (mean ± SD of 3 tests).

^cCC₅₀: 50% cytotoxic concentration

^d*-: no selective anti-HIV activity (CC₅₀/EC₅₀ < 5).

^eTI: CC₅₀/EC₅₀

To explore the effect of the sugar group at C-3, compound 1 was hydrolyzed to afford its aglycone (13), followed by acylation and hydrolysis. As seen in Table 2, although compounds 13 and 13b-c were inactive, 13a and 1 were equipotent. These results indicated that a sugar moiety at C-3 is dispensable but proper modification at C-3 is essential for anti-HIV activity. In addition, the antiviral activity was reduced due to deacetylation at C-15 and C-16 (compare 1a vs 1), again supporting the importance of acetyl groups at these positions, as mentioned above in the results for 1–12. Compound 13d without sugar or acetyl group on the hydroxyls at C-3, −15 and −16 was inactive. Thus, it is likely that the combination of ester functional groups at C-3 in ring A and C-15 or C-16 in ring D can significantly enhance anti-HIV activity. Furthermore, we postulated that a C-3 acyl group is critical for the anti-HIV activity and consequently synthesized and investigated additional C-3 acylated 1-derivatives to confirm our hypothesis and develop more potent anti-HIV agents. Compound 13g containing a 3′,3′-dimethylsuccinyl moiety was the most potent anti-HIV compound (EC₅₀ 0.025 μM, TI > 800), values that are comparable to those of the control 3-O-(3′-,3′-dimethylsuccinyl)-betulinic acid (DSB, bevirimat, the first HIV-1 maturation inhibitor) (Table 2). Bevirimat inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. The potent HIV-1 maturation inhibitor bevirimat dimeglumine (MPC-4326, formerly PA-457) reached an advanced stage of drug development [23]. The results suggest that this ester group is a privileged sub-structure that can play an important role in improving anti-HIV potency of multiple compound classes. Furthermore, compound 13g was also evaluated for activity against bevirimat-resistant HIV viruses NL4-3-V370A. However, no significant effects were observed, which suggested that C-3 modification alone is not enough for activity against bevirimat-resistant HIV viruses. In addition, compounds 13j (4′,4′-dimethylglutaryl ester) and 13m (diglycolyl ester) also exhibited significantly improved activity with EC₅₀ values of 0.048 and 0.12 μM, respectively. Meanwhile, the remaining seven derivatives exhibited strong to weak inhibition of the replication of HIV-1 in MT-4 cells, leading to the following SAR correlations. 1) Addition of a carboxylic terminus improved the anti-HIV potency (compare 13b, propionyl ester vs 13e, succinyl ester). 2) Addition of methyl groups in the ester increased potency remarkably (compare 13e, succinyl vs 13f, 3′-methylsuccinyl vs 13g, 3′,3′-dimethylsuccinyl). 3) The placement of the methyl groups was important (compare 13j, 4′,4′-dimethylglutaryl ester, vs 13k, 3′,3′-dimethylglutaryl ester). These findings suggested that the proper length, a carboxylic terminus, and C-3′ dimethyl substitution in the C-3 side chain can substantially contribute to enhanced anti-HIV activity (Fig. 2).

Table 2.

Anti-HIV data of synthetic derivatives 1a, 13, 13a–13e ^a

Compd	EC50 (μM) NL4-3 b	CC50 (μM) MT4 c	TI e	Compd	EC50 (μM) NL4-3 b	CC50 (μM) MT4 c	TI e
1a	7.62 ± 2.61	> 40	> 5.2	13g	0.025 ± 0.0095	> 20	> 800
13	*- d	> 40	-	13h	*- d	> 20	-
13a	2.44 ± 0.62	> 40	> 16.4	13i	*- d	> 20	-
13b	*- d	> 40	-	13j	0.048 ± 0.015	> 5	> 104
13c	*- d	> 30	-	13k	*- d	> 20	-
13d	> 40	> 40	-	13l	1.59 ± 0.44	> 20	> 12.6
13e	7.70 ± 2.3	> 40	> 5.2	13m	0.12 ± 0.04	> 5	> 41.7
13f	1.11 ± 0.34	> 20	> 18.0
DSB	0.026 ± 0.007	15.9 ± 1.4	612

^aCompounds were evaluated by using a multicycle viral replication assay using HIV-1 NL4-3 Nanoluc-sec virus.

^bEC₅₀: 50% HIV inhibitory concentration (mean ± SD of 3 tests).

^cCC₅₀: 50% cytotoxic concentration

^d*-: no selective anti-HIV activity (CC₅₀/EC₅₀ < 5).

^eTI: CC₅₀/EC₅₀

Fig. 2.

Graphical depiction of the general SAR for anti-HIV activity of 1 and its analogues

3. Conclusion

To discover potent antiviral natural products, 12 known cycloartane triterpenoids with four different skeletons isolated from the roots of S. vaginata [24] were assessed in vitro against HIV-1_{NL 4-3} using AZT as standard for the first time. The SAR results suggested that further structural modification at the C-3 position of the aglycone (13) of 1 could be beneficial. Consequently, a series of 3-acylated triterpenoid derivatives were synthesized and investigated for anti-HIV activity with DSB as reference. Among these derivatives, compound 13g showed the most potent anti-HIV activity (approximately 100-fold more potent than 1 comparable to the first-in-class HIV maturation inhibitor bevirimat). Our findings suggest that the introduction of a dimethylsuccinyl ester at C-3 of the aglycone can greatly improve anti-HIV potency and that compound 13g is a potential and promising antiviral agent meriting further investigation.

4. Experimental protocols

4.1. General information

All chemical reagents and solvents were used as received from Sigma-Aldrich or other commercial source. ¹H and ¹³C NMR spectra were measured in pyridine-d₅ on an Inova 400 MHz (Agilent Technologies, Palo Alto, CA, USA) or Bruker AV III 600 NMR spectrometer (Bruker, Billerica, German) with TMS as internal standard. Mass spectrometry was performed on a Shimadzu LCMS-2020 mass spectrometer coupled with an ESI source (Shimadzu Corporation, Tokyo, Japan). Melting points were determined on an X-4B apparatus (Shanghai Precision Instruments Co., Ltd, Shanghai, China) and are uncorrected. Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). Thin-layer chromatography was performed on Sorbtech aluminum backed silica XG TLC plates (Sorbent Technologies, Norcross, GA, USA). To purify all semisynthetic compounds, silica gel chromatograph was carried out on a Biotage Isolera One flash chromatograph system with prepacked Redi Sep Rf Si gel column (Teledyne ISCO, NE, USA).

4.2. (20S,24S)-15β, 16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol (13)

Colorless needles, 55.4% yield, m.p. 188–190 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −1.1 (c 0.12, MeOH); NMR and MS data of 13 agreed with those published in the literature [24].

4.3. General procedure for synthesis of compounds 13a–b

Compound 13 (0.017 mmol) in anhydrous pyridine (1.0 ml) was acylated separately with acetic (0.3 ml) and propionic (0.3 ml) anhydride to give 13a and 13b, respectively.

4.3.1. (20S, 24S)-15β, 16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-25-hydroxy-3β-yl acetate (13a)

Colorless needles, 59.8% yield, m.p. 214–216 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −1.6 (c 0.15, MeOH); NMR spectroscopic and MS data of 13a agreed with those published in the literature [24].

4.3.2. (20S,24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-25-hydroxy-3β-yl propionate (13b)

Colorless needles, 56.2% yield; m.p. 219–221 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −8.8 (c 0.10, MeOH); ¹H NMR (400 MHz, pyridine-d5) δ_H 0.20 (1H, d, J = 4.0 Hz, H-19), 0.51 (1H, d, J = 4.0 Hz, H-19), 0.60 (1H, q, J = 11.6 Hz, H-6a), 0.90 (3H, s, H₃-28), 0.93 (3H, s, H₃-29), 1.10 (2H, m, H-1a,7a), 1.15 (3H, t, J = 7.6 Hz, H-3'), 1.16 (1H, m, H-11a), 1.20 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.31 (1H, m, H-6b), 1.47 (1H, m, H-1b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.61 (1H, m, H-8), 1.67 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.97 (3H, m, H-2b,11b,22a), 2.02 (1H, m, H-7b), 2.08 (1H, m, H-23a), 2.12 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.38 (2H, q, J = 7.6 Hz, H-2'), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (1H, m H-12b), 2.99 (1H, m, H-22b), 4.51 (1H, d, J = 13.2 Hz, H-18a), 4.60 (1H, d, J = 13.2 Hz, H-18b), 4.80 (1H, dd, J = 11.6, 4.4 Hz, H-3), 5.69 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.6 (C-6), 21.6 (2×COCH₃ 25.8 (C-26), 25.9 (C-27), 26.0 (C-11), 26.2 (C-28), 26.6 (C-7), 27.5 (C-2), 27.7 (C-10), 28.2 (C-12), 28.5 (C-2'), 31.3 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 38.6 (C-22), 40.0 (C-4), 46.1 (C-13), 47.1 (C-5), 47.3 (C-8), 51.8 (C-14), 56.5 (C-17), 66.7 (C-18), 73.1 (C-25), 75.5 (C-16), 80.4 (C-3), 82.4 (C-15), 87.2 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 174.2 (C-1'); ESIMS m/z 645 [M + H]⁺.

4.4. (20S, 24S)-15β, 16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-25-hydroxy-3-one (13c)

To a solution of 13 (10 mg, 0.017 mmol) in CH₂Cl₂ (1 mL) was added PCC (4.0 mg, 0.019 mmol). The reaction mixture was stirred for 6 h at room temperature and then chromatographed on silica gel eluting with a hexane-acetone gradient to give 13c (0.01 mmol, 58.8%) as a colorless solid. m.p. 248–250 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −12.5 (c 0.10, MeOH); ¹H NMR (400 MHz, pyridine-d5) δ_H 0.40 (1H, d, J = 4.0 Hz, H-19), 0.67 (1H, d, J = 4.0 Hz, H-19), 0.72 (1H, q, J = 12.0 Hz, H-6a), 1.01 (3H, s, H₃-29), 1.14 (3H, s, H₃-28), 1.19 (3H, s, H₃-30), 1.28 (3H, m, H-5,7a,11a), 1.30 (3H, s, H₃-21), 1.41 (1H, m, H-1a), 1.47 (1H, m, H-6b), 1.56 (3H, m, H-8,11b,12a), 1.57 (3H, s, H₃-27), 1.68 (3H, s, H₃-26), 1.78 (1H, m, H-1b), 1.97 (1H, m, H-22a), 2.05 (1H, m, H-7b), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.15 (3H, s, COCH₃), 2.37 (1H, m, H-2a), 2.67 (1H, m, H-2b), 2.73 (1H, d, J = 11.6 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.54 (1H, d, J = 13.2 Hz, H-18a), 4.61 (1H, d, J = 13.2 Hz, H-18b), 5.70 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.6, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 20.5 (C-9), 21.2 (C-6), 21.6 (2×COCH₃), 22.9 (C-28), 26.0 (C-27), 26.2 (C-26), 26.7 (C-7,11), 27.7 (C-10), 28.2 (C-12), 31.2 (C-19), 31.3 (C-23), 32.8 (C-21), 33.9 (C-1), 37.5 (C-2), 38.6 (C-22), 46.0 (C-13), 47.3 (C-5), 48.4 (C-8), 50.5 (C-4), 51.9 (C-14), 56.6 (C-17), 66.8 (C-18), 73.1 (C-25), 75.4 (C-16), 82.3 (C-15), 87.2 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 215.0(C-3); ESIMS m/z 587 [M + H]⁺.

4.5. General procedure for synthesis of compounds 1a and 13d

Compound 1 (15 mg, 0.021 mmol) and 13 (12 mg, 0.02 mmol) were treated separately with 2.5% KOH in EtOH (2 mL) at room temperature overnight followed by chromatography to give 1a (0.012, 57.1%) and 13d (0.012 mmol, 60.0% yield), respectively.

4.5.1. (20S,24S)-18,24;20,24-Diepoxy-9,19-cyclolanostane-3β, 15β, 16β,25-tetraol 3-O-β-D-xylopyrano-side (1a)

White solid; m.p. 256–258 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −12 (c 0.18, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.20 (1H, d, J = 4.0 Hz, H-19), 0.52 (1H, d, J = 4.0 Hz, H-19), 0.72 (1H, q, J =12.0, H-6a), 1.02 (3H, s, H₃-30), 1.05 (1H, m, H-7a), 1.16 (2H, m, H-1a,11a), 1.22 (3H, s, H₃-29), 1.26 (1H, m, H-7b), 1.28 (3H, s, H₃-28), 1.29 (1H, m, H-5), 1.36 (1H, m, H-6b), 1.54 (1H, m, H-12a), 1.55 (3H, s, H₃-21), 1.58 (1H, m, H-1b), 1.60 (1H, dd, J = 12.0, 4.8 Hz, H-8), 1.69 (3H, s, H₃-27), 1.73 (3H, s, H₃-26), 1.90 (1H, m, H-23a), 1.97 (1H, m, H-22a), 2.00 (1H, m, H-11b), 2.06 (1H, m, H-2a), 2.40 (1H, m, H-23b), 2.61 (1H, d, J = 10.8 Hz, H-17), 2.75 (1H, m, H-2b), 2.94 (1H, m, H-12b), 2.99 (1H, m, H-22b), 3.30 (1H, dd, J = 12.0, 4.2 Hz, H-3), 3.40 (1H, d, J = 10.8, 8.0 Hz, H-16), 3.70 (1H, t, J = 10.8 Hz, H-5'), 4.02 (1H, m, H-2'), 4.15 (1H, t, J = 8.4 Hz, H-3′), 4.22 (1H, m, H-4′), 4.34 (1H, dd, J = 11.4, 5.4 Hz, H-5'), 4.36 (1H, d, J = 8.0 Hz, H-15), 4.45 (1H, d, J = 13.2 Hz, H-18a), 4.56 (1H, d, J = 13.2 Hz, H-18b), 4.84 (1H, d, J = 7.6 Hz, H-1'); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.1 (C-30), 15.7 (C-29), 20.0 (C-9), 21.1 (C-6), 26.1 (C-28), 26.0 (C-26,27), 26.8 (C-7), 27.0 (C-11), 27.8 (C-10), 28.8 (C-12), 30.4 (C-23), 31.1 (C-2), 32.2 (C-19), 32.7 (C-1), 33.5 (C-21), 38.6 (C-22), 41.6 (C-4), 46.3 (C-13), 47.8 (C-5), 49.0 (C-8), 51.0 (C-14), 58.4 (C-17), 67.3 (C-18,5'), 71.5 (C-4'), 73.2 (C-25), 75.8 (C-2'), 76.9 (C-16), 78.8 (C-3'), 84.4 (C-15), 88.4 (C-20), 88.7 (C-3), 107.8 (C-1'), 114.0 (C-24); ESIMS m/z 637 [M + H]⁺.

4.5.2. (20S, 24S)-18,24;20,24-Diepoxy-9,19-cyclolanostane-3β, 15β, 16β, 25-tetraol (13d)

White solid, m.p. 158–160 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −1.6 (c 0.17, MeOH); The NMR and MS data of 13d agreed with those published in the literature [24].

4.6. General procedure for synthesis of compounds 13e–m

A solution of 13 (0.06 mmol) with DMAP (1 equiv) and an appropriate acid anhydride (10 equiv) in anhydrous pyridine (10 ml) was stirred at 155 °C for 2 h in a microwave oven (Biotage). The reaction mixture was diluted with EtOAc and neutralized with HCl (1 N) and then extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO₄, and concentrated in vacuum to afford the crude product, followed by chromatography on a silica gel column and recrystallization to give 13e–m.

4.6.1. (20S,24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-succinate (13e)

Colorless needles, 39.3% yield; m.p. 203–205 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −11.8 (c 0.12, MeOH); ¹H NMR (600 MHz, pyridine-d₅) δ_H 0.17 (1H, d, J = 4.0 Hz, H-19), 0.48 (1H, d, J = 4.0 Hz, H-19), 0.55 (1H, q, J = 12.0 Hz, H-6a), 0.95 (3H, s, H₃-28), 0.96 (3H, s, H₃-29), 1.09 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.22 (1H, m, H-5), 1.28 (3H, s, H₃-21), 1.30 (1H, m, H-7b), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.68 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.91 (1H, m, H-11b), 1.96 (2H, m, H-2b, 22a), 2.07 (1H, m, H-23a), 2.12 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.72 (1H, d, J = 11.4 Hz, H-17), 2.78 (1H, m, H-23b), 2.92 (2H, m, H-3'), 2.96 (4H, m, H-2',12b,22b), 4.49 (1H, d, J = 13.0 Hz, H-18a), 4.57 (1H, d, J = 13.0 Hz, H-18b), 4.87 (1H, dd, J = 11.4, 4.8 Hz, H-3), 5.68 (1H, d, J = 9.0 Hz, H-15), 5.94 (1H, dd, J = 11.4, 9.0 Hz, H-16); ¹³C NMR (pyridine-d₅, 150 MHz) δ_C 15.8 (C-29,30), 19.5 (C-9), 20.6 (C-6), 21.6 (2×COCH₃), 26.0 (C-26,27), 26.2 (C-11), 26.5 (C-28), 27.5 (C-2), 27.7 (C-10,12), 28.2 (C-2'), 30.7 (C-23), 31.2 (C-19), 31.7 (C-1), 32.1 (C-21), 32.8 (C-3'), 38.6 (C-22), 40.0 (C-4), 46.0 (C-13), 47.1 (C-5), 47.3 (C-8), 51.8 (C-14), 56.5 (C-17), 66.7 (C-18), 73.0 (C-25), 75.4 (C-16), 80.7 (C-3), 82.3 (C-15), 87.1 (C-20), 114.6 (C-24), 171.0 (COCH3), 171.3 (COCH3), 173.0 (C-1', 4'); ESIMS m/z 689 [M + H]₊.

4.6.2. (RS-3′-20S, 24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β, 25-diol 3-O-3′-methylsuccinate (13f)

Colorless needles, 38.6% yield; m.p. 210–212 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −20.2 (c 0.12, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.18 (1H, d, J = 3.6 Hz, H-19), 0.49 (1H, d, J = 3.6 Hz, H-19), 0.58 (1H, q, J = 12.0 Hz, H-6a), 0.94 (3H, d, J = 4.8 Hz, H₃-3'), 0.96 (6H, s, H₃-28,29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.27 (3H, s, H₃-21), 1.30 (1H, m, H-7b), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.53 (3H, s, H₃-27), 1.55 (1H, m, H-12a), 1.59 (1H, m, H-8), 1.64 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.11 (3H, s, COCH₃), 2.13 (3H, s, COCH₃), 2.66 (1H, m, H-2'a), 2.71 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.90 (1H, m, H-2'b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 3.09 (1H, m, H-3'a), 3.26 (1H, m, H-3'b), 4.48 (1H, d, J = 13.2 Hz, H-18a), 4.57 (1H, d, J = 13.2 Hz, H-18b), 4.83 (1H, dd, J = 11.2, 3.6 Hz, H-3), 5.65 (1H, d, J = 8.8 Hz, H-15), 5.92 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 18.0 (CH₃-3') 19.6 (C-9), 20.6 (C-6), 21.6 (2×COCH₃ 26.0 (C-26,27), 26.2 (C-11,28), 26.6 (C-7), 27.5 (C-2), 27.8 (C-10), 28.3 (C-12), 31.2 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 35.2 (C-3'), 37.4 (C-2'), 38.6 (C-22), 40.0 (C-4), 46.1 (C-13), 47.1 (C-5), 47.4 (C-8), 51.8 (C-14), 56.6 (C-17), 66.7 (C-18), 73.1 (C-25), 75.5 (C-16), 80.6 (C-3), 82.8 (C-15), 87.2 (C-20), 114.6 (C-24), 171.0 (COCH3), 171.3 (COCH3), 172.4 (C-1'), 175.7 (C-4'); ESIMS m/z 703 [M + H]⁺.

4.6.3. (20S, 24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β, 25-diol 3-O-3′,3′-dimethylsuccinate (13g)

Colorless needles, 37.2% yield; m.p. 221–223 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −12.0 (c 0.10, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.17 (1H, d, J = 4.0 Hz, H-19), 0.48 (1H, d, J = 4.0 Hz, H-19), 0.55 (1H, q, J = 12.0 Hz, H-6a), 0.97 (6H, s, H₃-28,29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.28 (3H, s, H₃-21), 1.31 (2H, m, H-6b,7b), 1.47 (1H, m, H-1b), 1.54 (1H, m, H-12a), 1.55 (3H, s, H₃-27), 1.56 (6H, s, H₃-3'), 1.59 (1H, m, H-8), 1.66 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.12 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.89 (1H, d, J = Hz, H-2'a), 2.96 (1H, m, H-12b), 2.98 (1H, d, J = 15.6 Hz, H-2'b), 2.99 (1H, m, H-22b), 4.49 (1H, d, J = 13.2 Hz, H-18a), 4.58 (1H, d, J = 13.2 Hz, H-18b), 4.86 (1H, dd, J = 12.0, 4.0 Hz, H-3), 5.67 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.6 (C-6), 21.6 (2×COCH₃, 26.0 (3'-2×CH₃), 26.2 (C-26,27), 26.3 (C-11), 26.6 (C-7,28), 27.5 (C-2), 27.8 (C-10), 28.3 (C-12), 31.2 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 38.6 (C-22), 40.0 (C-4), 41.2 (C-3'), 45.6 (C-2'), 46.1 (C-13), 47.2 (C-5), 47.4 (C-8), 51.8 (C-14), 56.5 (C-17), 66.7 (C-18), 73.1 (C-25), 75.5 (C-16), 80.8 (C-3), 82.4 (C-15), 87.2 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 171.9 (C-1'), 179.7 (C-4'); ESIMS m/z 717 [M + H]⁺, 739 [M + Na]⁺.

4.6.4. (20S, 248)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-glutarate (13h)

Colorless needles, 38.2% yield; m.p. 209–211 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −9.8 (c 0.15, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.19 (1H, d, J = 3.2 Hz, H-19), 0.50 (1H, d, J = 3.2 Hz, H-19), 0.56 (1H, q, J = 12.0 Hz, H-6a), 0.92 (6H, s, H₃-28, 29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.30 (1H, m, H-7b), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.65 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b, 22a), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.26 (2H, m, H₃-3'), 2.64 (4H, m, H-2',4'), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.50 (1H, d, J = 13.2 Hz, H-18a), 4.59 (1H, d, J = 13.2 Hz, H-18b), 4.83 (1H, dd, J = 11.6, 4.0 Hz, H-3), 5.69 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyndme-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.6 (C-6), 21.6 (2×COCH₃). 21.7 (C-3'), 26.0 (C-26,27), 26.2 (C-11,28), 26.6 (C-7), 27.5 (C-2), 27.8 (C-10), 28.3 (C-12), 31.2 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 34.4 (C-2'), 34.6 (C-4'), 38.6 (C-22), 40.0 (C-4), 46.1 (C-13), 47.1 (C-5), 47.4 (C-8), 51.8 (C-14), 56.6 (C-17), 66.7 (C-18), 73.1 (C-25), 75.5 (C-16), 80.6 (C-3), 82.4 (C-15), 87.3 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 173.3 (C-1'), 175.8 (C-5'); ESIMS m/z 703 [M + H]⁺.

4.6.5. (3′RS,20S,24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-3′-methylglutarate (13i)

Colorless needles, 36.5% yield; m.p. 215–217 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −22.4 (c 0.10, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.21 (1H, d, J = 3.2 Hz, H-19), 0.52 (1H, d, J = 3.2 Hz, H-19), 0.58 (1H, q, J = 12.0 Hz, H-6a), 0.94 (6H, s, H₃-28, 29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.25 (3H, d, J = 6.4 Hz, H₃-3'), 1.29 (3H, s, H₃-21), 1.30 (2H, m, H-7b), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.65 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.50 (1H, m, H-3'), 2.70 (4H, m, H-2',4'), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.50 (1H, d, J = 13.2 Hz, H-18a), 4.59 (1H, d, J = 13.2 Hz, H-18b), 4.83 (1H, dd, J = 11.6, 4.0 Hz, H-3), 5.69 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.5 (C-3'), 20.6 (C-6), 21.6 (2×COCH₃), 26.0 (C-26,27), 26.2 (C-11,28), 26.6 (C-7), 27.5 (C-2), 27.8 (C-10), 28.3 (C-12), 31.2 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 38.6 (C-22), 40.0 (C-4), 41.9 (C-2'), 42.0 (C-4'), 46.1 (C-13), 47.1 (C-5), 47.4 (C-8), 51.8 (C-14), 56.6 (C-17), 66.7 (C-18), 73.1 (C-25), 75.5 (C-16), 80.6 (C-3), 82.4 (C-15), 87.3 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 172.8 (C-1'), 175.3 (C-5'); ESIMS m/z 717 [M + H]⁺, 739 [M + Na]⁺.

4.6.6. (20S, 24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β, 25-diol 3-O-4',4'-dimethylglutarate (13j)

Colorless needles, 35.7% yield; m.p. 228–230 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −13.6 (c 0.15, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.18 (1H, d, J = 3.2 Hz, H-19), 0.49 (1H, d, J = 3.2 Hz, H-19), 0.60 (1H, q, J = 12.0 Hz, H-6a), 0.91 (6H, s, H₃-28,29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.18 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.30 (2H, m, H-7b), 1.38 (6H, s, H₃-3'), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.54 (3H, s, H₃-27), 1.55 (1H, m, H-12a), 1.59 (1H, m, H-8), 1.65 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.27 (2H, m, H-3'), 2.72 (2H, m, H-2'), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.74 (2H, s, H-4'), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.50 (1H, d, J = 13.2 Hz, H-18a), 4.59 (1H, d, J = 13.2 Hz, H-18b), 4.83 (1H, dd, J = 11.2, 4.0 Hz, H-3), 5.67 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.9 (C-29,30), 19.7 (C-9), 20.8 (C-6), 21.8 (2×COCH₃), 26.0 (4'-2×CH₃), 26.1 (C-26,27), 26.3 (C-11), 26.4 (C-28), 27.6 (C-2), 27.9 (C-10), 28.4 (C-12), 31.4 (C-23), 31.8 (C-19,2'), 32.3 (C-1), 33.0 (C-21), 36.6 (C-3'), 38.7 (C-22), 40.2 (C-4), 42.4 (C-4'), 46.2 (C-13), 47.3 (C-5), 47.5 (C-8), 52.0 (C-14), 56.7 (C-17), 66.9 (C-18), 73.3 (C-25), 75.6 (C-16), 80.8 (C-3), 82.5 (C-15), 87.3 (C-20), 114.8 (C-24), 171.2 (COCH₃), 171.6 (COCH₃), 174.0 (C-1'), 180.4 (C-5'); ESIMS m/z 731 [M + H]⁺, 753 [M + Na]⁺.

4.6.7. (20S,24S)-15β, 16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β, 25-diol 3-O-3′,3′-dimethylglutarate (13k)

Colorless needles, 39.8% yield; m.p. 226–228 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −5.9 (c 0.11, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.21 (1H, d, J = 3.2 Hz, H-19), 0.52 (1H, d, J = 3.2 Hz, H-19), 0.60 (1H, q, J = Hz, H-6a), 0.96 (6H, s, H₃-28, 29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.20 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.30 (2H, m, H-7b), 1.39 (6H, s, H₃-3'), 1.43 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.67 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.72 (2H, m, H-2'), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.74 (2H, m, H-4'), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.50 (1H, d, J = 12.8 Hz, H-18a), 4.59 (1H, d, J = 12.8 Hz, H-18b), 4.83 (1H, dd, J = 11.6, 4.4 Hz, H-3), 5.69 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.7 (C-6), 21.6 (2×COCH₃), 26.1 (C-26,27), 26.2 (C-11), 26.3 (C-28), 26.6 (C-7), 27.8 (3'-2×CH₃), 28.3 (C-2), 28.4 (C-10), 28.5 (C-12), 31.3 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 33.1 (C-3'), 38.6 (C-22), 39.9 (C-4), 46.1 (C-13), 46.2 (C-2'), 46.3 (C-4'), 47.2 (C-5), 47.4 (C-8), 51.9 (C-14), 56.6 (C-17), 66.8 (C-18), 73.1 (C-25), 75.5 (C-16), 80.5 (C-3), 82.4 (C-15), 87.2 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 172.3 (C-1'), 174.9 (C-5'); ESIMS m/z 731 [M + H]⁺, 753 [M + Na]⁺.

4.6.8. (20S,24S)-15β,16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-3′,3′-tetramethyleneglutarate (13l)

Colorless needles, 33.2% yield; m.p. 247–249 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −12.1 (c 0.14, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.21 (1H, d, J = 3.6 Hz, H-19), 0.52 (1H, d, J = 3.6 Hz, H-19), 0.58 (1H, q, J = 12.0 Hz, H-6a), 0.98 (6H, s, H₃-28, 29), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.20 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.30 (2H, m, H-7b), 1.47 (1H, m, H-6b), 1.48 (1H, m, H-1b), 1.55 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.67 (3H, s, H₃-26), 1.69 (5H, m, H-2a, 1"a, 2"a, 3"a, 4"a), 1.96 (5H, m, H-11b, 1"b, 2"b, 3"b, 4"b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.12 (3H, s, COCH₃), 2.14 (3H, s, COCH₃), 2.73 (1H, d, J = 11.2 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (5H, m, H-12b,2',4'), 2.99 (1H, m, H-22b), 4.50 (1H, d, J = 12.8 Hz, H-18a), 4.59 (1H, d, J = 12.8 Hz, H-18b), 4.85 (1H, dd, J = 11.6, 4.4 Hz, H-3), 5.67 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 11.2, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.6 (C-9), 20.7 (C-6), 21.6 (2×COCH₃), 24.9 (C-1",4"), 26.0 (C-26,27), 26.2 (C-11,28), 26.6 (C-7), 27.6 (C-2), 27.8 (C-10), 28.3 (C-12), 31.2 (C-23), 31.7 (C-19), 32.2 (C-1), 32.8 (C-21), 38.7 (C-22), 38.8 (C-3"), 39.9 (C-4), 43.3 (C-1",4"), 43.6 (C-2'), 43.8 (C-4'), 46.1 (C-13), 47.2 (C-5), 47.4 (C-8), 51.8 (C-14), 56.5 (C-17), 66.8 (C-18), 73.1 (C-25), 75.5 (C-16), 80.5 (C-3), 82.4 (C-15), 87.2 (C-20), 114.7 (C-24), 171.0 (COCH₃), 171.3 (COCH₃), 172.6 (C-1'), 175.2 (C-5'); ESIMS m/z 757 [M + H]⁺, 779 [M + Na]⁺.

4.6.9. (20S,24S)-15β, 16β-Diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-diglycolate(13m)

Colorless needles, 42.6% yield; m.p. 197–199 °C (MeOH); ${[\alpha ]}_{\mathrm{D}}^{20}$ −1.9 (c 0.16, MeOH); ¹H NMR (400 MHz, pyridine-d₅) δ_H 0.19 (1H, d, J = 4.0 Hz, H-19), 0.52 (1H, d, J = 4.0 Hz, H-19), 0.55 (1H, q, J = 12.0 Hz, H-6a), 0.90 (3H, s, H₃-29), 0.92 (3H, s, H₃- 28), 1.11 (1H, m, H-7a), 1.12 (1H, m, H-1a), 1.16 (1H, m, H-11a), 1.21 (3H, s, H₃-30), 1.23 (1H, m, H-5), 1.29 (3H, s, H₃-21), 1.31 (2H, m, H-6b,7b), 1.47 (1H, m, H-1b), 1.54 (1H, m, H-12a), 1.56 (3H, s, H₃-27), 1.59 (1H, m, H-8), 1.68 (3H, s, H₃-26), 1.69 (1H, m, H-2a), 1.96 (1H, m, H-11b), 1.97 (2H, m, H-2b,22a), 2.08 (1H, m, H-23a), 2.13 (3H, s, COCH₃), 2.15 (3H, s, COCH₃), 2.73 (1H, d, J = 11.6 Hz, H-17), 2.79 (1H, m, H-23b), 2.96 (1H, m, H-12b), 2.99 (1H, m, H-22b), 4.49 (1H, d, J = 13.2 Hz, H-18a), 4.58 (1H, d, J = 13.2 Hz, H-18b), 4.70 (2H, s, H-3'), 4.71 (2H, s, H-2'), 4.90 (1H, dd, J = 12.0, 4.0 Hz, H-3), 5.69 (1H, d, J = 8.8 Hz, H-15), 5.94 (1H, dd, J = 6, 8.8 Hz, H-16); ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 15.8 (C-29,30), 19.7 (C-9), 20.6 (C-6), 21.6 (2×COCH₃), 26.0 (C-26,27), 26.2 (C-11,28), 26.6 (C-7), 27.5 (C-2), 27.9 (C-10), 28.3 (C-12), 31.2 (C-23), 31.6 (C-19), 32.1 (C-1), 32.8 (C-21), 38.6 (C-22), 40.1 (C-4), 46.1 (C-13), 47.1 (C-5), 47.3 (C-8), 51.7 (C-14), 56.6 (C-17), 66.8 (C-18), 68.9 (C-2'), 69.0 (C-3'), 73.0 (C-25), 75.5 (C-16), 81.3 (C-3), 82.3 (C-15), 87.2 (C-20), 114.7 (C-24), 170.7 (C-1'), 171.0 (COCH₃), 171.3 (COCH₃), 173.2 (C-4'); ESIMS m/z 727 [M + Na]⁺.

4.7. Biological experiments

4.7.1. Anti-HIV assay

A previously described HIV-1 infectivity assay was used in the experiments [25]. HIV-1 NL4-3 Nanoluc-sec virus infection of MT4 cells was carried out in 96-well plates in the presence of various concentrations of compounds. HIV-1 NL4-3 Nanoluc-sec virus is a reporter virus possessing a secNluc insertion as a reporter gene (Promega Cat.# N1021). The viral replication thus can be monitored by measuring the luciferase activity using Promega Nano-Glo Luciferase Assay System.

4.7.2. Cytotoxicity Assay

A CellTiter-Glo® Luminescent cytotoxicity assay (Promega) was utilized to measure the cytotoxicity of compounds. MT4 cells were cultured in the presence of various concentrations of the compounds for 3 days. Cytotoxicity of the compounds was measured by following the protocol provided by the manufacturer.

Highlights.

• The first anti-HIV study was run on 12 cycloartane triterpenes from Souliea vaginata.

• Beesioside I exhibited the best potency against HIV-1_{NL 4-3} (EC₅₀ 2.32 μM).

• Eleven new and five known cycloartane triterpenoid derivatives were synthesized.

• Among them, compound 13g was the most potent (anti-HIV EC₅₀ 0.025 μM, TI > 800).