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Article

Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents

by
Yanli Wang
1,2,†,
Xiaowan Huang
3,4,†,
Xiao Zhang
4,
Jingchen Wang
4,
Keyan Li
2,
Guotao Liu
2,
Kexin Lu
4,
Xiang Zhang
3,
Chengping Xie
3,
Teresa Zheng
5,
Yung-Yi Cheng
5,* and
Qiang Wang
1,2,3,4,*
1
School of Medicine, Huanghe Science and Technology College, Zhengzhou 450063, China
2
National Health Commission Key Laboratory of Birth Defect Prevention, Henan Institute of Reproductive Health Science and Technology, Zhengzhou 450002, China
3
High & New Technology Research Center, Henan Academy of Science, Zhengzhou 450002, China
4
BGI College & Henan Institute of Medical and Pharmaceutical Science, Zhengzhou University, Zhengzhou 450052, China
5
Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7568, USA
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2022, 27(15), 4738; https://doi.org/10.3390/molecules27154738
Submission received: 25 May 2022 / Revised: 20 July 2022 / Accepted: 20 July 2022 / Published: 25 July 2022
(This article belongs to the Special Issue Natural Products as Sources of New Drugs: In Memory of Professor Kuo-Hsiung Lee (1940-2021))
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Abstract

:
Allobetulin is structurally similar tobetulinic acid, inducing the apoptosis of cancer cells with low toxicity. However, both of them exhibited weak antiproliferation against several tumor cell lines. Therefore, the new series of allobetulon/allobetulin–nucleoside conjugates 9a–10i were designed and synthesized for potency improvement. Compounds 9b, 9e, 10a, and 10d showed promising antiproliferative activity toward six tested cell lines, compared to zidovudine, cisplatin, and oxaliplatin based on their antitumor activity results. Among them, compound 10d exhibited much more potent antiproliferative activity against SMMC-7721, HepG2, MNK-45, SW620, and A549 human cancer cell lines than cisplatin and oxaliplatin. In the preliminary study for the mechanism of action, compound 10d induced cell apoptosis and autophagy in SMMC cells, resulting in antiproliferation and G0/G1 cell cycle arrest by regulating protein expression levels of Bax, Bcl-2, and LC3. Consequently, the nucleoside-conjugated allobetulin (10d) evidenced that nucleoside substitution was a viable strategy to improve allobetulin/allobetulon’s antitumor activity based on our present study.

Graphical Abstract

1. Introduction

In the population of people under the age of 70, cancer is one of the leading causes of death in six-tenths of the countries in the world, according to the World Health Organization (WHO) report in 2019 [1]. Globally, over 10 million new cases and deaths occurred in 2020 [2]. Chemotherapy is usedas a first-line anticancer remedy due to this treatment’s efficacy, despite its significant adverse effects [3,4,5]. Seeking alternative medicines with acceptable or less adverse effects and promising anticancer activities are the goals of anticancer drug discovery and development. Natural products are considered sources for drug discovery, and the discovery of such products leads to promising clinical outcomes.
Pentacyclic triterpenes (PTs) from natural products, including betulin (1), betulinic acid (2), oleanolic acid (3), and ursolic acid (4) (Figure 1), have attracted much attention because of their various biological activities (e.g.,antiviral, antineoplastic, antiparasitic, antibacterial, anti-inflammatory, antiulcer, antifeedant, antidiabetic, anticarcinogenic, hepatoprotective, nephroprotective, neuroprotective, and cardioprotective activities) [6,7,8,9,10,11,12,13,14,15,16]. Their multi-target behaviorin cancer allows them to bein the forefrontofa new generation of anticancer drug candidates [17]. Betulinic acid (2), for instance, improved reactive oxygen species production, triggered mitochondrial-mediated apoptosis via the caspase-dependent signaling pathway, and was linked to the p38 and stress-activated protein (SAP) kinase/c-junN-terminal kinase (JNK) inseveral human cancer cell lines [18,19]. Allobetulin (19β,28-epoxyolenan-3-ol, 5), one extractive substance from birch bark, is structurally similar to oleanolic acid (3). Regarding the configuration of H-18, the hydrogen atom at C-18 of allobetulin is in α-configuration rather than β-configurationin oleanane-type triterpenoids [20]. Allobetulin (5) is converted from the rearrangement reaction of botulin [21,22,23]. Therefore, allobetulin (5) not only belongs to the oleanane terpenoidsbut also as a “re-arranged”betulin derivative [24]. Additionally, the bioactivities of allobetulin (5) werereportedlyantiviral [25,26], anticancer [27,28,29,30,31,32], anti-inflammatory [33], antichlamydial [34], antioxidant [35,36], and neuroprotective effects [24].
Despite allobetulin (5) exhibiting multi-bioactivity, thestrength of its antiproliferation against several tumor cell lines is insufficient at micromolar concentrations. In our previous study, anti-HIV activities of betulinic acid derivatives were increased by conjugating them with nucleosides [37]. We hypothesized that various nucleoside pharmacophores introduced into allobetulin/allobetulon via click chemistry might also improve their potency. In the present study, we designed and synthesized allobetulin/allobetulon-nucleoside conjugates, and then estimated their antineoplastic activity. Subsequently, we investigated the mechanism of action for the promising candidate.

2. Results and Discussion

2.1. Chemistry

The synthesis of the 2-propargyl substituted intermediates isshown in Scheme 1. Allobetulin (5) was obtained by Wagner–Meerwein rearrangement from betulin (1) in the presence of p-toluenesulfonic acid [24]. By Jones’ oxidation [30,37], allobetulin (5) reacted with CrO3 to produce allobetulon (6). The key intermediate (7) was obtained by the propargyl α-alkylation of allobetulon (6) reacted intheKN(SiMe3)2/Et3B system. Reduced 2α-propargyl-allobetulon was reacted with NaBH4 in isopropanol to preferentially produceanother intermediate, 2α-propargyl-allobetulin (8). Regarding the structural establishment, the NOE effect (Figure S11) between H-3 and H-23 indicated an equatorial position (β-orientation) for the OH group, and the NOE effects between H-2 and H-24/H-25 suggested an axial position (β-orientation) for H-2, and thus, an equatorial position (α-orientation) for the propargyl group. Furthermore, the spin–spin coupling constant (3JH(2),H(3)= 10.6 Hz, CDCl3) between H-3 and H-2 in the 1H-NMR spectrum of 8 (Figure S10) was consistent with axial positions for H-2 and H-3. The axial position (β-orientation) ofH-2 was also demonstrated by the X-ray diffraction determination of single crystals of compound 9c (Figure 2).The 2-propargyl allobetulon (7) and allobetulin (8) were coupled with different azides (4′-azido-2′-deoxy-2′-fluoro-β-d-arabinocytidine (AFC), 4′-azido-2′-deoxy-2′-fluoro-β-d-arabinouridine (AFU), 4′-azido-β-d-ribocytidine (AZC), 4′-azido-β-d-ribouridine (AZU), 4′-azido-2′-deoxy-β-d-ribocytidine (AdC), 4′-azido-2′-deoxy-β-d-ribouridine (AdU), and AZT) via click chemistry to produce the target compounds 9a–i and 10a–i, respectively (Scheme 2).
Finally, all the target compounds were fully characterized by 1H- and 13C-NMR, and HRMS spectra which were listed in the Supplementary Materials (Figures S1–S67). Additionally, the purity of the target compounds (≥95%) was confirmed by HPLC.

2.2. Biological Evaluation

2.2.1. Antiproliferative Activities and Structure-Activity Relationship

Synthesized allobetulon/allobetulin–nucleoside derivatives were evaluated for their antitumor activity against six human tumor cell lines by Cell Counting Kit-8 (CCK8) assay, including a human hepatoma cell line (SMMC-7721), human hepatocellular carcinoma cell line (HepG2), human gastric cancer cell line (MNK-45), human non-small cell lung cell line (A549), human colorectal cell line (SW620), and human breast cancer cell line (MCF-7). Cisplatinand oxaliplatin, belonging to the platinum-based antineoplastic chemotherapy drugs on the World Health Organization’s List of Essential Medicines, interfere with DNA replication by binding to DNA [38]. In the CCK8 assay, cisplatin and oxaliplatin were used as the positive control. Additionally, zidovudine (azidothymidine, AZT), a kind of nucleoside analog reverse-transcriptase inhibitor (NRTI), was used as a positive control in this study.As a result, the 2-propargyl allobetulon (7) and allobetulin (8) (the synthetic scaffolds for other nucleoside–allobetulin/allobetulin conjugates) exhibited weak activity against six tested cell lines (Table 1). Among these derivatives, compound 9b exhibited similar potency tooxaliplatin against MCF-7 cell line. Compared to cisplatin and oxaliplatin, compounds 9e, 10a, and 10d showed significant potency against MNK-45 and SW620 cell lines. Interestingly, compound 10d exhibited the lowest IC50 value for SMMC-7721 (5.57 μM), HepG2 (7.49 μM), MNK-45 (6.31 μM), SW620 (6.00 μM), and A549 (5.79 μM) cell lines. Allobetulon (7) exhibited lower potency than allobetulin (8) against SMMC-7721, HepG2, and A549 cell lines. Compared to the antineoplastic activities of zidovudine (>100 μM), the synthesized allobetulon/allobetulin–nucleoside derivatives had much more promising potency. Taken together, introducing various nucleosides to the scaffolds (7 and 8) could improve the antiproliferative activity against the tested cell lines. Conjugated nucleoside-substituted with fluorine glycosyl compounds (9b, 10a, 10d) presented promising antitumor activity. Consequently, compound 10d exhibitedthe most promising antitumor activity against tested human cancer cell lines.

2.2.2. Effects of Compound 10d on Apoptosis, Autophagy, and Cell Cycle Study of SMMC-7721 Human Cancer Cells

Given the promising antineoplastic activities of compound 10d against five tested human cancer cell lines, flow cytometry investigated the cell cycle distribution to determine whether compound 10d influenced cell cycle progression. SMMC-7721 cells were exposed to the five different concentrations of compound 10d (0, 1, 5, 10, 15 µM) and then subjected to flow cytometry. Cell cycle analysis showed increased accumulation of cells in the G0/G1 phase after treatment with compound 10d (Figure 3B,C). Therefore, induction of G0/G1 cell cycle arrest in SMMC-7721 cell lines implied compound 10d reduced cell proliferation by induction of G0/G1 cell cycle arrest. Some factors can trigger the G0/G1 cell cycle arrest, including apoptosis, cyclin-dependent kinase inhibition, the regulation of tumor suppressors, and autophagy. The apoptotic effect of compound 10d toward SMMC-7721 was assessed by annexin VFITC and propidium iodide (PI) staining. SMMC-7721 was treated with dose-dependent concentrations of compound 10d for 48 h and then subjected to flow cytometry analysis. As illustrated in Figure 3A, the percentage of the total proportion of apoptotic cells increased from the base value (control, 0.35%) to 46.41% for 15 µM, implying that compound 10d induced apoptosis of SMMC-7721 cells. To clarify the potential factors for that, we investigated the critical regulators of cell apoptosis by Western blot analysis. Pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 are well-known factors linked to the regulation of apoptosis. SMMC-7721 cells were treated with 10d (1, 5, 10, 15 μM) for 48 h, and we then examined the associated protein levels (Bax, Bcl-2, and GAPDH) by Western blotting. As shown in Figure 3D, the expression of the pro-apoptotic protein Bax was upregulated, and that of the anti-apoptotic protein Bcl-2 was significantly down-regulated, both in a dose-dependent manner. The balance of Bax/Bcl-2 ratio is important in determining whether cells will undergo apoptosis. The ratio of Bax to Bcl-2 was dose-dependently increased in the range of 1 to 10 µM (Figure 3E). Based on these results, compound 10d might lead to G0/G1 phase arrest in SMMC-7721 cells through apoptosis by Bax and Bcl-1 regulation. Additionally, G0/G1 phase arrest may be induced by autophagy. Therefore, the autophagy marker, LC3, was examined by Western blotting. Cells were treated with 10d (1, 5, 10, 15 μM) for 48 h, and then the expression of LC3 was measured. As shown in Figure 3F, compound 10d significantly induced the LC3 expression in a dose-dependent manner. Taken together, compound 10d dose-dependently induced antiproliferation, caused by apoptosis and autophagy, which was respectively modulated by regulating protein expression levels of Bax and Bcl-2, and LC3.

3. Materials and Methods

3.1. General Information

All reagents were purchased and used without further purification unless otherwise indicated. Progress of reactions was monitored using TLC visualized by UV lamp (254 nm) or KMnO4 developer. Column chromatography was performed using 300 mesh silica gel (Shanxi Nuotai Biological Technology Co., Ltd., Yuncheng, China). Melting points (m.p.) were measured on a Shenguang WRR melting point apparatus (Shanghai Shenguang Instrument Co., Ltd., Shanghai, China). 1H- and 13C-NMR spectra were recorded using an Agilent 400 MR (Agilent Technology, Santa Clara, CA, USA) in deuterated solvents. Chemicals shifts are reported in parts per million (δ ppm) relative to TMS or the solvent peak. Coupling constants (J) are expressed in hertz (Hz). High-resolution mass spectrometry (HRMS) analysis was performed using an Agilent 1290–6545B Q-TOF mass spectrometer (Agilent Technology, Singapore).

3.2. Procedure for the Synthesis of 2α-Propargyl Substituted Analogs

3.2.1. Synthesis of Allobetulin (5)

Betulin (2.0 g, 4.52 mmol) and p-TSA (2.0 g, 11.63 mmol) were added inCH2Cl2 (100 mL) and refluxed overnight (monitoring by TLC). We removed the solvent under vacuum and the residue was purified by column chromatography on SiO2 eluting with CH2Cl2 to afford compound 5 as a white solid (1.8 g, 4.06 mmol, 89.9%); m.p. 257–258 °C (264–266). 1H-NMR (CDCl3, 400 MHz) δ: 3.77 (d, J = 7.8 Hz, 1H), 3.52 (s, 1H), 3.43 (d, J = 7.8 Hz, 1H), 3.19 (dd, J = 11.1, 5.1 Hz, 1H), 1.71 (dt, J = 13.1, 3.6 Hz, 1H), 0.97 (s, 6H), 0.92 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.79 (s, 3H), 0.76 (s, 3H), 0.69 (d, J = 9.4 Hz, 1H).13C-NMR (CDCl3, 100 MHz) δ: 87.9, 78.9, 71.2, 55.5, 51.0, 46.8, 41.4, 40.7, 40.6, 38.9, 38.9, 37.2, 36.7, 36.2, 34.1, 33.9, 32.7, 28.8, 28.0, 27.4, 26.4, 26.4, 26.2, 24.5, 21.0, 18.2, 16.5, 15.7, 15.4, 13.5. HRMS (ESI) calcd for C30H51O2 [M + H]+ 443.3889, found 443.3884.

3.2.2. Synthesis of Allobetulon (6)

To a solution of allobetulin (1.8 g, 4.06 mmol) in acetone (100 mL) was added freshly prepared Jones’ reagent (18 mL) dropwise at 0 °C, and the solution was stirred for 2 h (monitoring by TLC). The reaction was quenched with MeOH (35 mL) and water (35 mL). The solvent was removed under vacuum, and the aqueous residue was extracted with CH2Cl2 (3 × 20 mL). We combined the organic layer and dried it with Na2SO4, then removed the solvent under vacuum to afford compound 6 as a white solid (1.68 g, 3.81 mmol, 93.9%). m.p. 224–226 °C. 1H-NMR (CDCl3, 400 MHz) δ: 3.78 (d, J = 7.8 Hz, 1H), 3.53 (s, 1H), 3.45 (d, J = 7.8 Hz, 1H), 2.57–2.36 (m, 2H), 1.94 (ddd, J = 12.5, 7.6, 4.6 Hz, 1H), 1.66 (d, J = 12.4 Hz, 1H), 1,22 (dd, J = 13.3, 4.9 Hz, 1H), 1.08 (s, 3H), 1.03 (s, 3H), 1.01 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.92 (s, 6H), 0.79 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ: 218.2, 87.9, 71.2, 55.0, 50.4, 47.3, 46.8, 41.4, 40.7, 40.5, 39.8, 37.0, 36.7, 36.3, 34.2, 34.1, 33.2, 32.7, 28.8, 26.7, 26.4, 26.4, 26.2, 24.5, 21.5, 21.0, 19.6, 16.3, 15.5, 13.4. HRMS (ESI) calcd for C30H49O2 [M + H]+ 441.3733, found 441.3727.

3.2.3. Synthesis of 2α-Propargyl-Allobetulon (7)

Compound 6 (1.68 g, 3.81 mmol) was dissolved in DME (80 mL); then 1 M solution of KN(SiMe3)2 (25 mL, 25 mmol) was added under a nitrogen atmosphere. After 30 min of stirring at room temperature, 1M Et3B (27 mL, 27 mmol) in THF was added, and the mixture was stirred for 90 min. Then, a solution of propargyl bromide (2.7 mL, 32 mmol) was added. The reaction mixture was stirred for 6 h under nitrogen (monitoring by TLC), neutralized with 3M HCl (aq), and diluted with water (200 mL). After extraction with EtOAc (3 × 80 mL), the organic layers were combined, washed with saturated NaHCO3 and dried over Na2SO4. The solvent was removed under vacuum and the residue was purified by column chromatography on SiO2 via elution with petroleum ether/EtOAc (20/1). Compound 7 was obtained as a white powder (1.18 g, 2.46 mmol, 64.6%). m.p. 184–186 °C. 1H-NMR (CDCl3, 400 MHz) δ: 3.78 (d, J = 7.1 Hz, 1H), 3.53 (s, 1H), 3.45 (d, J = 7.8 Hz, 1H), 2.88 (ddt, J = 10.0, 8.4, 5.2 Hz, 1H), 2.62 (ddd, J = 17.1, 4.4, 2.7 Hz, 1H), 2.37 (dd, J = 12.9, 5.6 Hz, 1H), 2.21 (ddd, J = 17.1, 8.3, 2.6 Hz, 1H), 1.97 (t, J = 2.7 Hz, 1H), 1.15 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.80 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ: 215.7, 87.9, 83.0, 71.2, 69.4, 57.4, 50.6, 48.3, 46.8, 46.7, 41.4, 41.3, 40.8, 40.7, 37.5, 36.7, 36.2, 34.1, 33.6, 32.7, 28.8, 26.4, 26.3, 26.2, 25.0, 24.5, 21.6, 21.3, 19.5, 19.2, 16.5, 15.8, 13.4. HRMS (ESI) calcd for C33H51O2 [M + H]+ 479.3889, found 479.3874.

3.2.4. Synthesis of 2α-Propargyl-Allobetulin (8)

Compound 7 (1.18 g, 2.46 mmol) was dissolved in isopropanol (100 mL), NaBH4 (186.1 mg, 4.92 mmol) was added, and the mixture was stirred at room temperature overnight (monitoring by TLC). HCl (3M, 40 mL) was dropwise added under 0 °C. The solvent was removed under vacuum, and the residue was extracted with EtOAc (3 × 60 mL); the combined organic layer was washed with saturated NaHCO3 and dried over Na2SO4. We removed the solvent under reduced pressure, and the residue was purified by column chromatography on SiO2 eluting with petroleum ether/EtOAc (15/1) to afford compound 8 as a white solid (638.6 mg, 1.33 mmol, 54.1%). m.p. 230–232 °C. 1H-NMR (CDCl3, 400 MHz) δ: 3.77 (dd, J = 7.8, 1.6 Hz, 1H), 3.53 (s, 1H), 3.44 (d, J = 7.8 Hz, 1H), 3.03 (dd, J = 10.6, 6.3 Hz, 1H), 2.46–2.31 (m, 2H), 2.01 (t, J = 2.7 Hz, 1H), 1.86 (dd, J = 12.8, 3.8 Hz), 1.83–1.73 (m, 1H), 1.69–1.61 (m, 1H), 1.16–1.06 (m, 1H), 0.99 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.80 (s, 3H), 0.70 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ: 87.9, 83.0, 81.4, 71.3, 70.0, 55.5, 51.0, 46.8, 44.9, 41.5, 40.8, 40.6, 39.1, 37.4, 36.7, 36.3, 34.8, 34.1, 33.8, 32.7, 28.8, 28.3, 26.4, 26.3, 24.5, 22.3, 21.0, 18.4, 17.3, 16.2, 15.7, 13.5. HRMS (ESI) calcd for C33H53O2 [M + H]+ 481.4046, found 481.4038.

3.3. General Procedure for Click Reactions

3.3.1. Method A

First, 200 µL of freshly prepared CuSO4 solution (1 M) and copper powder (0.1 mmol) were added into a solution of the alkyne (0.30 mmol) and azide (0.20 mmol) in 15 mL ethanol. The resulting mixture was stirred at 45 °C for 48 h until the conversion of azide was completed (monitoring by TLC). The solvent was removed under reduced pressure, and the crude residue was purified by column chromatography on SiO2 (5–25% MeOH in CH2Cl2).

3.3.2. Method B

Azide (0.2 mmol) and alkyne (0.30 mmol) were dissolved in 15 mL t-BuOH/H2O (1:1, v:v); then DIPEA (80 μL, 0.48 mmol) was added and stirred for 20 min at 45 °C under nitrogen protection. A solution of CuI (50 mg, 0.26 mmol) in CH3CN (1 mL) was added, and the resulting mixture was stirred at 45 °C for 48 h until conversion of azide was completed (monitoring by TLC). The solvent was removed under reduced pressure, and the crude residue was purified by column chromatography on SiO2 (6–20% MeOH in CH2Cl2).

3.3.3. Method C

Azide (0.2 mmol) and alkyne (0.30 mmol) were dissolved in 15 mL t-BuOH/H2O (1:1, v:v); then 400 µL fresh prepared sodium ascorbate solution (1 M, 0.4 mmol) and 200 μL CuSO4 solution (1M, 0.2 mmol) were added in. The resulting mixture was stirred at 40 °C for 48 h until the conversion of azide was completed (monitoring by TLC). The solvent was removed under reduced pressure, and the crude residue was purified by column chromatography on SiO2 (12–15% MeOH in CH2Cl2).

3.4. Procedure for the Preparation of Compounds 9a–10i

3.4.1. 2α-{1N[1-(2-deoxy-2β-fluoro-β-d-arabinopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9a)

Method B, yield 54.9%, m.p.: decomposition at 200 °C.1H-NMR (MeOH-d4, 400 MHz) δ: 7.93 (d, J = 7.4 Hz, 1H), 7.88 (s, 1H), 6.81 (dd, J = 12.1, 4.4 Hz, 1H), 5.99 (brs, 1H), 5.34 (dt, J = 54.0, 4.6 Hz, 1H), 4.77 (dd, J = 20.4, 4.2 Hz, 1H), 4.33 (d, J = 12.6 Hz, 1H), 4.24 (d, J = 12.2 Hz, 1H), 3.79 (d, J = 7.7 Hz, 1H), 3.55 (s, 1H), 3.47 (d, J = 7.8 Hz, 1H), 3.26–3.10 (m, 2H), 2.63 (dd, J = 14.3, 6.8 Hz, 1H), 2.08 (dd, J = 13.0, 5.0 Hz, 1H), 1.15 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.05 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.7, 167.9, 157.9, 146.7, 143.4, 124.1, 99.0 (d, J = 6.6 Hz), 96.4 (d, J = 193.2 Hz), 89.7, 86.7 (d, J = 15.7 Hz), 76.4 (d, J = 25.4 Hz), 72.3, 63.4, 59.0, 52.0, 49.6, 48.2, 48.1, 43.6, 42.7, 42.1, 42.0, 38.8, 37.7, 37.3, 35.7, 34.9, 33.9, 29.3, 27.6, 27.5, 27.2, 27.0, 25.7, 24.9, 22.5, 22.1, 20.4, 16.9, 16.4, 13.9.HRMS (ESI) calcd for C42H62FN6O6 [M + H]+ 765.4715, found 765.4702, calcd forC42H61FN6O6Na [M + Na]+ 787.4534, found 787.4521.

3.4.2. 2α-{1N[1-(2-deoxy-2β-fluoro-β-d-arabinopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9b)

Method C; yield 52.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.91 (dd, J = 8.2, 1.1 Hz, 1H), 7.87 (s, 1H), 6.79 (dd, J = 10.5, 5.5 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 5.38 (dt, J = 54.2, 5.2 Hz, 1H), 4.84 (dd, J = 22.2, 4.9 Hz, 1H), 4.34–4.22 (m, 2H), 3.79 (d, J = 7.8 Hz, 1H), 3.55 (s, 1H), 3.48 (d, J = 7.8 Hz, 1H), 3.26–3.10 (m, 2H), 2.64 (dd, J = 14.4, 7.0 Hz, 1H), 2.08 (dd, J = 12.9, 5.2 Hz, 1H), 1.15 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.05 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100MHz) δ: 218.6, 165.9, 152.0, 146.8, 143.1, 124.0, 102.8, 98.4 (d, J = 8.8 Hz), 96.2 (d, J = 193.7 Hz), 89.7, 85.4 (d, J = 16.9 Hz), 76.3 (d, J = 24.8 Hz), 72.3, 63.0, 59.0, 52.0, 49.6, 48.3, 48.1, 43.5, 42.7, 42.1, 42.0, 38.8, 37.7, 37.3, 35.7, 34.9, 33.9, 29.3, 27.6, 27.5, 27.2, 27.0, 25.7, 24.9, 22.5, 22.1, 20.4, 16.9, 16.4, 13.9.HRMS (ESI) calcd for C42H61FN5O7 [M + H]+ 766.4555, found 766.4539, calcd forC42H60FN5O7Na[M + Na]+ 788.4374, found 788.4359.

3.4.3. 2α-{1N[1-(2-deoxy-2α-fluoro-β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9c)

Method B; yield: 45.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 8.35 (d, J = 7.9 Hz, 1H), 7.93 (s, 1H), 6.46 (dd, J = 17.4, 1.4 Hz, 1H), 6.15 (d, J = 7.9 Hz, 1H), 5.36 (ddd, J = 53.3, 5.2, 1.6 Hz, 1H), 4.97 (dd, J = 20.5, 5.2 Hz, 1H), 4.33 (d, J = 12.2 Hz, 1H), 4.06 (d, J = 12.1 Hz, 1H), 3.79 (d, J = 7.8 Hz, 1H), 3.54 (s, 1H), 3.48 (d, J = 7.9 Hz, 1H), 3.25–3.08 (m, 2H), 2.71–2.56 (m, 1H), 2.09 (dd, J = 12.7, 5.0 Hz, 1H), 1.15 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.7, 161.8, 148.8, 147.1, 146.7, 124.1, 100.7, 95.4, 93.5 (d, J = 191.1 Hz, 1H), 92.9 (d, J = 35.5 Hz), 89.7, 72.3, 71.8 (d, J = 16.2 Hz, 1H), 64.3, 59.0, 52.0, 49.6, 48.2, 48.1, 43.5, 42.7, 42.1, 42.0, 38.8, 37.6, 37.3, 35.7, 34.9, 33.8, 29.3, 27.6, 27.5, 27.2, 27.0, 25.7, 24.9, 22.5, 22.1, 20.4, 16.9, 16.4, 13.9.HRMS (ESI) calcd for C42H61FN6O6Na [M + Na]+ 787.4534, found 787.4521.

3.4.4. 2α-{1N[1-(2-deoxy-2α-fluoro-β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9d)

Method C; yield: 48.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.94 (d, J = 8.1 Hz, 1H), 7.92 (s, 1H), 6.41 (dd, J = 18.6, 2.4 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 5.38 (ddd, J = 53.4, 5.3, 2.5 Hz, 1H), 5.00 (dd, J = 18.8, 5.3 Hz, 1H), 4.28 (d, J = 12.2 Hz, 1H), 4.05 (d, J = 12.2 Hz, 1H), 3.79 (d, J = 7.9 Hz, 1H), 3.55 (s, 1H), 3.48 (d, J = 7.8 Hz, 1H), 3.25–3.09 (m, 2H), 2.70–2.56 (m, 1H), 2.09 (dd, J = 12.9, 5.2 Hz, 1H), 1.14 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.7, 166.1, 152.0, 146.5, 143.8, 124.1, 103.4, 100.4, 93.4 (d, J = 189.6 Hz), 92.7 (d, J = 35.7 Hz), 89.7, 72.3 (d, J = 15.7 Hz), 72.3, 64.8, 59.0, 52.0, 49.6, 48.2, 48.1, 43.5, 42.7, 42.1, 42.0, 38.8, 37.6, 37.3, 35.7, 34.9, 33.9, 29.3, 27.6, 27.5, 27.2, 27.0, 25.7, 24.9, 22.5, 22.1, 20.4, 16.9, 16.4, 14.0.HRMS (ESI) calcd for C42H61FN5O7 [M + H]+ 766.4555, found 766.4545, calcd forC42H60FN5O7Na[M + Na]+ 788.4374, found 788.4362.

3.4.5. 2α-{1N[1-(β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9e)

Method A; yield: 67.9%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 8.00 (d, J = 7.4 Hz, 1H), 7.95 (s, 1H), 6.32 (d, J = 4.9 Hz, 1H), 5.98 (brs, 1H), 4.68–4.53 (m, 2H), 4.43 (d, J = 11.9 Hz, 1H), 3.98 (d, J = 11.9 Hz, 1H), 3.79 (d, J = 7.9 Hz, 1H), 3.55 (s, 1H), 3.47 (d, J = 7.8 Hz, 1H), 3.29–3.07 (m, 2H), 2.60 (dd, J = 14.2, 7.1 Hz, 1H), 2.10 (dd, J = 12.9, 5.1 Hz, 1H), 1.14 (s, 3H), 1.08 (s, 3H), 1.06 (s, 3H), 1.06 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.6, 167.8, 158.6, 146.1, 143.7, 124.2, 101.0, 97.0, 93.3, 89.7, 74.3, 73.9, 72.3, 65.9, 59.0, 52.0, 49.6, 48.3, 48.1, 43.6, 42.7, 42.1, 42.0, 38.8, 37.7, 37.3, 35.7, 34.9, 33.9, 29.3, 27.6, 27.5, 27.2, 27.1, 25.7, 24.9, 22.6, 22.1, 20.4, 16.9, 16.4, 14.0.HRMS (ESI) calcd for C42H62N6O7Na [M + Na]+ 785.4578, found 785.4566.

3.4.6. 2α-{1N[1-(β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9f)

Method A; yield: 70.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 8.03 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 6.33 (d, J = 5.1 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 4.65–4.56 (m, 2H), 4.44 (d, J = 11.9 Hz, 1H), 3.96 (d, J = 11.9 Hz, 1H), 3.79 (d, J = 7.8 Hz, 1H), 3.55 (s, 1H), 3.47 (d, J = 7.8 Hz, 1H), 3.27–3.09 (m, 2H), 2.61 (dd, J = 14.3, 6.9 Hz, 1H), 2.10 (dd, J = 13.0, 5.2 Hz, 1H), 1.14 (s, 3H), 1.08 (s, 3H), 1.06 (s, 3H), 1.06 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.6, 166.0, 152.6, 146.2, 142.8, 124.1, 103.7, 101.0, 91.3, 89.7, 74.5, 74.0, 72.3, 66.0, 59.0, 52.0, 49.6, 48.3, 48.1, 43.5, 42.7, 42.1, 42.0, 38.8, 37.6, 37.3, 35.7, 34.9, 33.8, 29.4, 27.6, 27.5, 27.2, 27.1, 25.7, 25.0, 22.6, 22.1, 20.4, 16.9, 16.4, 14.0.HRMS (ESI) calcd for C42H62N5O8 [M + H]+ 764.4598, found 764.4580, calcd forC42H61N5O8Na [M + Na]+ 786.4418, found 786.4405.

3.4.7. 2α-{1N[1-(2-deoxy-β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9g)

Method B; yield: 63.0%; m.p.: decomposition at 200 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.86 (d, J = 7.2 Hz, 1H), 7.81 (s, 1H), 7.24 (brs, 2H), 6.60 (t, J = 5.0 Hz, 1H), 5.85 (br, 1H), 5.62 (t, J = 5.8 Hz, 1H), 5.49 (d, J = 5.3 Hz, 1H), 4.68 (m, 1H), 4.21 (dd, J = 12.1, 5.7 Hz, 1H), 3.95 (dd, J = 12.0, 5.9 Hz, 1H), 3.63 (d, J = 7.8 Hz, 1H), 3.40 (s, 1H), 3.34 (d, J = 7.8 Hz, 1H), 3.16 (m, 1H), 3.07 (dd,J = 14.9, 4.5 Hz, 1H), 2.48 (dd, J = 14.9, 7.9 Hz, 1H), 2.20–2.35 (m, 2H), 2.01 (dd, J = 12.8, 5.4 Hz, 1H), 1.05 (s, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.76 (s, 3H). 13C-NMR (DMSO-d6, 100 MHz) δ: 215.7, 165.7, 154.7, 144.0, 141.5, 121.9, 109.5, 99.3, 86.7, 86.1, 70.5, 70.2, 62.3, 56.7, 49.7, 47.7, 46.2, 46.1, 41.2, 40,8, 40.3, 40.1, 37.7, 37.0, 36.0, 35.9, 33.7, 33.1, 32.4, 28.7, 25.9, 25.8, 25.8, 25.1, 24.2, 21.3, 20.7, 18.7, 15.9, 15.4, 13.2.HRMS (ESI) calcd for C42H62N6O6Na [M + Na]+ 769.4629, found 769.4611.

3.4.8. 2α-{1N[1-(2-deoxy-β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9h)

Method C; yield: 59.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.99 (d, J = 8.1 Hz, 1H), 7.83 (s, 1H), 6.68 (dd, J = 7.2, 5.2 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.86 (m, 1H), 4.37 (d,J = 12.1 Hz, 1H), 4.09 (d, J = 12.1 Hz, 1H), 3.79 (d, J = 7.9 Hz, 1H), 3.55 (s, 1H), 3.47 (d, J = 7.8 Hz, 1H), 3.25–3.09 (m, 2H), 2.68–2.52 (m, 2H), 2.43 (dt, J = 13.8, 7.0 Hz, 1H), 2.08 (dd, J = 12.9, 5.3 Hz, 1H), 1.14 (s, 3H), 1.08 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.7, 166.2, 152.2, 146.4, 143.1, 123.9, 103.2, 101.7, 89.7, 87.9, 73.1, 72.3, 64.6, 59.0, 52.0, 48.2, 48.1, 43.6, 42.7, 42.1, 42.0, 38.9, 38.8, 37.7, 37.3, 35.7, 34.9, 33.9, 29.3, 27.6, 27.5, 27.2, 27.1, 25.7, 24.9, 22.6, 22.1, 20.4, 16.9, 16.4, 14.0.HRMS (ESI) calcd for C42H62N5O7 [M + H]+ 748.4649, found 748.4635, calcd forC42H61N5O7Na [M + Na]+ 770.4469, found 770.4454.

3.4.9. 2α-{1N[1-(2,3-dideoxy-β-d-ribopentafuranosyl)thymine-3-yl]-1H-1,2,3-triazole-4-yl}-allobetulon (9i)

Method A; yield: 78.0%; m.p.: 193–195 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.92 (d, J = 1.2 Hz, 1H), 7.87 (brs, 1H), 6.47 (t, J = 6.4 Hz, 1H), 5.40 (dt, J = 8.5, 5.5 Hz, 1H), 4.34 (dt, J = 5.6, 3.0 Hz, 1H), 3.90 (dd, J = 12.2, 3.0 Hz, 1H), 3.78 (d, J = 6.5 Hz, 1H), 3.77 (dd, J = 15.4, 3.1 Hz, 1H), 3.55 (s, 1H), 3.47 (d, J = 7.9 Hz, 1H), 3.27–3.18 (m, 1H), 3.13 (dd, J = 14.4, 5.0 Hz, 1H), 2.97–2.84 (m, 1H), 2.72 (ddd, J = 14.2, 8.5, 6.3 Hz, 1H), 2.60 (dd, J = 14.2, 5.8 Hz, 1H), 2.11 (dd, J = 12.9, 5.1 Hz, 1H), 1.90 (d, J = 1.1 Hz, 3H), 1.16 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.82 (s, 3H). 13C-NMR (MeOH-d4, 100 MHz) δ: 218.3, 166.4, 152.3, 147.9, 146.2, 138.3, 124.1, 111.7, 89.6, 86.7, 86.5, 72.2, 62.2, 60.9, 59.0, 52.0, 49.6, 48.7, 48.1, 43.4, 42.7, 42.1, 42.0, 39.1, 38.9, 37.7, 37.3, 35.7, 34.9, 33.9, 29.4, 27.6, 27.5, 27.2, 25.7, 25.0, 22.6, 22.1, 20.4, 17.0, 16.5, 14.0, 12.7.HRMS (ESI) calcd for C44H64N5O6 [M + H]+ 746.4857, found 746.4846, calcd forC44H63N5O6Na [M + Na]+ 768.4676, found 768.4665.

3.4.10. 2α-{1N[1-(2-deoxy-2β-fluoro-β-d-arabinopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10a)

Method B; yield: 58.7%; m.p.: decomposition at 200 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.90 (s, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.33 (brs, 1H), 7.30 (brs, 1H), 6.76 (dd, J = 7.3, 5.6 Hz, 1H), 6.23 (d, J = 5.0 Hz, 1H), 5.85 (t, J = 5.6 Hz, 1H), 5.80 (d, J = 7.3 Hz, 1H), 5.32 (dt, J = 55.3, 5.6 Hz, 1H), 4.72 (dt, J = 25.0, 4.6 Hz, 1H), 4.58 (d, J = 6.4 Hz, 1H), 4.23–4.06 (m, 2H), 3.62 (d, J = 7.5 Hz, 1H), 3.39 (s, 1H), 3.33 (d, J = 7.5 Hz, 1H), 3.16 (d, J = 12.8 Hz, 1H), 2.71 (dd, J = 10.2, 6.5 Hz, 1H), 2.29 (dd, J = 14.4, 9.8 Hz, 1H), 1.90–1.75 (m, 1H), 1.63 (d, J = 11.7 Hz, 1H), 0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H), 0.75 (s, 3H), 0.75 (s, 3H), 0.72 (s, 3H), 0.52 (t, J = 12.8 Hz, 1H). 13C-NMR (DMSO-d6, 100 MHz) δ: 165.6, 154.7, 144.9, 141.8, 122.1, 95.5 (d, J = 10.2 Hz), 94.7 (d, J = 191.4 Hz), 94.2, 86.7, 83.0, 80.4, 74.2 (d, J = 24.8 Hz), 70.2, 60.9, 55.2, 50.4, 46.1, 44.4, 40.9, 40.2, 40.1, 39.0, 36.8, 36.0, 35.9, 35.4, 33.7, 33.3, 32.4, 28.8, 28.5, 28.5, 25.9, 25.9, 25.8, 24.2, 20.5, 18.1, 16.9, 16.6, 15.4, 13.3.HRMS (ESI) calcd for C42H64FN6O6 [M + H]+ 767.4871, found 767.4860.

3.4.11. 2α-{1N[1-(2-deoxy-2β-fluoro-β-d-arabinopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10b)

Method C; yield: 59.9%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.92 (d, J = 8.3 Hz, 1H), 7.89 (s, 1H), 6.81 (dd, J = 10.2, 5.5 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 5.41 (dt, J = 54.5, 5.3 Hz, 1H), 4.86 (dd, J = 22.4, 5.0 Hz, 1H), 4.30 (s, 2H), 3.77 (d, J = 7.8 Hz, 1H), 3.54 (s, 1H), 3.46 (d, J = 7.8 Hz, 1H), 3.24–3.11 (m, 1H), 2.83 (d, J = 10.7 Hz, 1H), 2.54 (dd, J = 14.2, 9.0 Hz, 1H), 2.06–1.88 (m, 1H), 1.70 (dd, J = 12.9, 2.8 Hz, 1H), 0.98 (s, 6H), 0.93 (s, 3H), 0.91 (s, 3H), 0.83 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74 (d, J = 9.3 Hz, 1H), 0.62 (t, J = 12.6 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 165.9, 152.0, 147.0, 143.1, 123.8, 102.8, 98.3 (d, J = 8.6 Hz), 96.22 (d, J = 193.9 Hz), 89.6, 85.4 (d, J = 16.7 Hz), 83.1, 76.2 (d, J = 25.1 Hz, 1H), 72.3, 62.9, 57.2, 52.4, 48.1, 46.3, 42.7, 41.9, 41.9, 40.5, 38.5, 37.7, 37.3, 37.2, 35.7, 35.1, 33.9, 29.8, 29.4, 29.1, 27.6, 27.6, 27.2, 25.0, 22.3, 19.7, 17.8, 17.1, 16.4, 14.1.HRMS (ESI) calcd for C42H63FN5O7 [M + H]+ 768.4712, found 768.4700.

3.4.12. 2α-{1N[1-(2-deoxy-2α-fluoro-β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10c)

Method B; yield: 50.9%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.96 (s, 1H), 7.95 (d, J = 7.4 Hz, 1H), 6.38 (dd, J = 19.0, 1.7 Hz, 1H), 5.94 (d, J = 7.6 Hz, 1H), 5.32 (ddd, J = 53.9, 5.1, 2.0 Hz, 1H), 5.01 (dd, J = 19.7, 5.3 Hz, 1H), 4.29 (d, J = 12.2 Hz, 1H), 4.10 (d, J = 12.1 Hz, 1H), 3.77 (d, J = 7.9 Hz, 1H), 3.54 (s, 1H), 3.46 (d, J = 7.8 Hz, 1H), 3.18 (dd, J = 14.4, 3.1 Hz, 1H), 2.83 (d, J = 10.8 Hz, 1H), 2.54 (dd, J = 14.4, 8.9 Hz, 1H), 2.02–1.88 (m, 1H), 1.72, (dd, J = 13.1, 3.3 Hz, 1H), 0.99 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74 (d, J = 9.5 Hz, 1H), 0.63 (t, J = 12.7 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 168.2, 157.9, 146.7, 144.5, 124.0, 100.4, 96.8, 94.12 (d, J = 35.1 Hz), 93.78 (d, J = 189.4 Hz), 89.7, 83.1, 72.4 (d, J = 17.5 Hz), 72.3, 65.0, 57.2, 52.5, 48.2, 46.3, 42.8, 42.0, 41.9, 40.5, 38.5, 37.7, 37.3, 37.3, 35.7, 35.1, 33.9, 29.8, 29.3, 29.1, 27.6, 27.6, 27.2, 25.0, 22.3, 19.7, 17.8, 17.1, 16.3, 14.0.HRMS (ESI) calcd for C42H63FN6O6Na [M + Na]+ 789.4691, found 789.4676.

3.4.13. 2α-{1N[1-(2-deoxy-2α-fluoro-β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10d)

Method C; yield: 54.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.95 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 6.41 (dd, J = 18.6, 2.0 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 5.39 (ddd, J = 53.4, 5.1, 2.3 Hz, 1H), 5.01 (dd, J = 18.7, 5.3 Hz, 1H), 4.29 (d, J = 12.2 Hz, 1H), 4.07 (d, J = 12.2 Hz, 1H), 3.78 (d, J = 7.8 Hz, 1H), 3.53 (s, 1H), 3.46 (d, J = 7.7 hz, 1H), 3.19 (dd, J = 14.1, 2.7 Hz, 1H), 2.83 (d, J = 10.8 Hz, 1H), 2.53 (dd, J = 14.5, 9.1 Hz, 1H), 2.02–1.86 (m, 1H), 1.72 (dd, J = 13.0, 2.9 Hz, 1H), 0.99 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.75 (d, J = 9.2 Hz, 1H), 0.63 (t, J = 12.5 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 166.0, 152.0, 146.8, 143.8, 124.0, 103.5, 100.4, 93.4 (d, J = 190.1 Hz), 92.7 (d, J = 35.4 Hz), 89.7, 83.1, 72.3, 72.3 (d, J = 16.2 Hz), 64.9, 57.2, 52.5, 48.2, 46.3, 42.8, 42.0, 41.9, 40.5, 38.6, 37.7, 37.4, 37.3, 35.7, 35.1, 33.9, 29.8, 29.3, 29.1, 27.6, 27.6, 27.2, 25.0, 22.3, 19.7, 17.8, 17.1, 16.3, 14.0.HRMS (ESI) calcd for C42H63FN5O7 [M + H]+ 768.4712, found 768.4702.

3.4.14. 2α-{1N[1-(β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10e)

Method B; yield: 56.9%; m.p.: decomposition at 200 °C; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.82 (d, J = 7.5 Hz, 1H), 7.79 (s, 1H), 7.33 (brs, 1H), 7.29 (brs, 1H), 6.22 (d, J = 5.7 Hz, 1H), 5.81 (d, J = 7.4 Hz, 1H), 5.69 (t, J = 5.9 Hz, 1H), 5.42 (d, J = 8.4 Hz, 1H), 5.40 (d, J = 9.3 Hz, 1H), 4.54 (d, J = 6.7 Hz, 1H), 4.44–4.36 (m, 2H), 4.25 (dd, J = 11.8, 6.3 Hz, 1H), 3.83 (dd, J = 11.8, 5.5 Hz, 1H), 3.62 (d, J = 7.9 Hz, 1H), 3.38 (s, 1H), 3.31 (d, J = 7.9 Hz, 1H), 3.21–3.11 (m, 1H), 2,71 (dd,J = 10.5, 6.9 Hz, 1H), 2.26 (dd, J = 14.4, 9.8 Hz, 1H), 1.88–1.74 (m, 1H), 1.66 (dd, J = 12.9, 2.6 Hz, 1H), 0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H), 0.76 (s, 3H), 0.74 (s, 3H), 0.71 (s, 3H), 0.54 (t, J = 12.4 Hz, 1H). 13C-NMR (DMSO-d6, 100 MHz) δ: 165.6, 155.3, 144.4, 142.0, 121.9, 98.4, 94.8, 90.0, 86.7, 80.5, 72.5, 71.7, 70.2, 64.1, 55.2, 50.3, 46.1, 44.5, 40.8, 40.2, 40.1, 39.0, 36.7, 36.0, 35.8, 35.7, 33.7, 33.3, 32.4, 28.7, 28.6, 28.5, 25.9, 25.8, 25.8, 24.2, 20.5, 18.1, 16.9, 16.6, 15.4, 13.3.HRMS (ESI) calcd for C42H64N6O7Na [M + Na]+ 787.4734, found 787.4716.

3.4.15. 2α-{1N[1-(β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10f)

Method A; yield: 52.2%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 8.03 (d, J = 8.1 Hz, 1H), 7.93 (s, 1H), 6.36 (d,J = 5.5 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 4.66–4.56 (m, 2H), 4.46 (d, J = 11.9 Hz, 1H), 3.98 (d, J = 11.9 Hz, 1H), 3.77 (d, J = 7.8 Hz, 1H), 3.54 (s, 1H), 3.46 (d, J = 7.8 Hz, 1H), 3.24–3.12 (m, 1H), 2.83 (d, J = 10.7 Hz, 1H), 2.52 (dd, J = 14.2, 9.1 Hz, 1H), 2.05–1.87 (m, 1H), 1.79–1.67 (m, 1H), 0.99 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74 (d, J = 9.4 Hz, 1H), 0.64 (t, J = 12.7 Hz, 1H). 13C-NMR (MeOH-d4, 100MHz) δ: 166.0, 152.6, 142.8, 103.7, 101.1, 91.3, 89.7, 83.0, 74.6, 74.0, 72.3, 65.9, 57.2, 52.4, 48.1, 46.4, 42.7, 41.9, 41.9, 40.6, 38.5, 37.7, 37.3, 37.3, 35.7, 35.1, 33.9, 29.9, 29.4, 29.1, 27.6, 27.6, 27.2, 25.0, 22.3, 19.7, 17.9, 17.2, 16.3, 14.1.HRMS (ESI) calcd for C42H64N5O8 [M + H]+ 766.4755, found 766.4740.

3.4.16. 2α-{1N[1-(2-deoxy-β-d-ribopentafuranosyl)cytosine-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10g)

Method B; yield: 60.1%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 8.05 (d, J = 7.5 Hz, 1H), 7.87 (s, 1H), 6.69 (t, J = 5.9 Hz, 1H), 5.95 (brs, 1H), 4.84 (t, J = 6.8 Hz, 1H), 4.40 (d, J = 12.0 Hz, 1H), 4.12 (d, J = 12.1 Hz, 1H), 3.77 (d, J = 7.9 Hz, 1H), 3.54 (s, 1H), 3.46 (d, J = 7.8 Hz, 1H), 3.17 (dd, J = 14.1, 2.5 Hz, 1H), 2.83 (d, J = 10.8 Hz, 1H), 2.60–2.39 (m, 3H), 2.05–1.89 (m, 1H), 1.71 (dd, J = 13.0, 2.9 Hz, 1H), 0.99 (s, 6H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74 (d, J = 9.4 Hz, 1H), 0.63 (t, J = 12.7 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 167.8, 158.1, 146.6, 143.1, 123.8, 101.8, 96.6, 89.7, 89.0, 83.1, 73.0, 72.3, 64.6, 57.2, 52.5, 48.2, 46.3, 42.8, 42.0, 41.9, 40.5, 39.6, 38.6, 37.7, 37.3, 37.3, 35.7, 35.1, 33.9, 29.8, 29.3, 29.1, 27.6, 27.6, 27.2, 24.9, 22.3, 19.7, 17.8, 17.1, 16.3, 14.0.HRMS (ESI) calcd for C42H64N6O6Na [M + Na]+ 771.4785, found 771.4767.

3.4.17. 2α-{1N[1-(2-deoxy-β-d-ribopentafuranosyl)uracil-4-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10h)

Method C; yield: 63.0%; m.p.: decomposition at 200 °C; 1H-NMR (MeOH-d4, 400 MHz)δ: 7.99 (d, J = 8.1 Hz, 1H), 7.85 (s, 1H), 6.70 (dd, J = 7.1, 5.2 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.85 (m, 1H), 4.38 (d, J = 12.1 Hz, 1H), 4.11 (d, J = 12.1 Hz, 1H), 3.78 (d, J = 7.8 Hz, 1H), 3.54 (s, 1H), 3.46 (d, J = 7.8 Hz, 1H), 3.17 (dd, J = 14.3, 3.1 Hz, 1H), 2.83 (d, J = 10.8 Hz, 1H), 2.67–2.49 (m, 2H), 2.45 (dt,J = 13.8, 7.0 Hz, 1H), 2.03–1.87 (m, 1H), 1.71 (dd, J = 13.0, 3.3 Hz, 1H), 0.99 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74 (d, J = 9.1 Hz, 1H), 0.63 (t, J = 12.6 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 166.3, 152.3, 146.6, 143.1, 123.7, 103.2, 101.6, 89.7, 88.0, 83.1, 73.1, 72.3, 64.6, 57.2, 52.5, 48.2, 46.3, 42.8, 42.0, 41.9, 40.5, 38.9, 38.5, 37.7, 37.3, 37.3, 35.7, 35.1, 33.9, 29.8, 29.3, 29.1, 27.6, 27.6, 27.2, 24.9, 22.3, 19.7, 17.8, 17.1, 16.3, 14.0.HRMS (ESI) calcd for C42H64N5O7 [M + H]+ 750.4806, found 750.4790.

3.4.18. 2α-{1N[1-(2,3-dideoxy-β-d-ribopentafuranosyl)thymine-3-yl]-1H-1,2,3-triazole-4-yl}-allobetulin (10i)

Method B; yield: 78.2%; m.p.: 192–193 °C; 1H-NMR (MeOH-d4, 400 MHz) δ: 7.91 (s, 1H), 7.87 (s, 1H), 6.48 (t, J = 6.4 Hz, 1H), 5.40 (dt, J = 8.5, 5.6 Hz, 1H), 4.35 (dt, J = 5.7, 3.0 Hz, 1H), 3.91 (dd, J = 12.2, 2.9 Hz, 1H), 3.78 (d, J = 7.5 Hz, 1H), 3.77 (dd, J = 12.3, 3.2 Hz, 1H), 3.54 (s, 1H), 3.47 (d, J = 7.8 Hz, 1H), 3.19 (dd,J = 14.5, 2.6 Hz, 1H), 2.93 (dt, J = 12.5, 6.4 Hz, 1H), 2.82 (d, J = 10.8 Hz, 1H), 2.74 (ddd, J = 14.3, 8.5, 6.2 Hz, 1H), 2.51 (dd, J = 14.5, 9.2 Hz, 1H), 1.91 (s, 3H), 1.70 (dd, J = 13.1, 3.3 Hz, 1H), 0.99 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.75 (d, J = 9.1 Hz, 1H), 0.65 (t, J = 12.7 Hz, 1H). 13C-NMR (MeOH-d4, 100 MHz) δ: 166.5, 152.4, 148.2, 138.4, 123.8, 111.8, 89.7, 86.8, 86.5, 83.1, 72.3, 62.2, 60.9, 57.2, 52.5, 48.2, 46.5, 42.8, 42.0, 41.9, 40.5, 39.1, 38.5, 37.7, 37.4, 37.3, 35.7, 35.1, 33.9, 29.9, 29.3, 29.1, 27.6, 27.6, 27.2, 24.9, 22.4, 19.7, 17.8, 17.1, 16.3, 14.0, 12.6.HRMS (ESI) calcd for C43H66N5O6 [M + H]+ 748.5013, found 748.5004, calcd for C43H65N5O6Na [M + Na]+ 770.4833, found 770.4814.

3.5. X-ray Structure of Compound 9c

Colorless, block-like, single crystals of compound 7c were obtained after recrystallization from CH2Cl2 and methanol. A crystal of dimensions 0.15 × 0.1 × 0.09 mm was selected to collect a room temperature (293K) X-ray crystallographic dataset. The data were collected on a Gemini E diffractometer (Agilent Technology, Oxyford, UK) with graphite monochromated Cu Kα radiation (λ = 1.54184 Å).

3.6. Cell Culture

HepG2, MNK-45, MCF-7, SW620, and A549 cell lines were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). SMMC-7721 cell line was purchased from BeNa Culture Collection (Beijing, China). MNK-45, SMMC-7721, and SW620 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Solarbio, Beijing, China); andHepG2, MCF-7,and A549 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Solarbio, Beijing, China).All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere.

3.7. Cell Viability Assay

Cell viability was measured by the CCK-8 assay. Confluent cells in the well-state were cultured in 96-well plates (5–10 × 104 cells/mL). After cells were attached to the plate, compounds with various concentrations were applied at 37 °C for 48 h. Then, the medium containing drugs was replaced with 10% CCK-8 solution prepared by using the fresh serum-free medium. After incubation at 37 °C for 30 min, the medium was transferred to the 96-well plates and measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm.

3.8. Flow Cytometry Assay

Flow cytometry analysis was applied for apoptosis detection. Firstly, SMMC-7721 cells were adjusted to 2 × 105/mL, inoculated into a six-well plate, and placed in an incubator at 37 °C containing 5% CO2 saturated humidity overnight. After the cells were fully attached to the plate; 1, 5, 10, and 15 μM of 8d were administrated to the cells for 48 h. Cells were collected and stained with Annexin V-FITC and PI. Subsequently, flow cytometry was used for detection.
Flow cytometry analysis was applied for cell cycle detection. Firstly, SMMC-7721 cells were adjusted to 2 × 105/mL, inoculated into a six-well plate, and placed in an incubator at 37 °C containing 5% CO2 in saturated humidity overnight. After the cells were fully attached to the plate, 1, 5, 10, and 15 μM of 8d were administrated to the cells for 24 h. After cells were collected and fixed with 70% ethanol, PI was applied to stain the cells; subsequently, onboard testing by flow cytometry (Cytoflex S (Beckman Coulter, Brea, CA, USA)) was conducted.

3.9. Western Blot Analysis

Cells were treated with different concentrations of 10d for 48 h, and then were harvested, and total protein was extracted using lysis buffer (Solarbio, Beijing, China). Equal lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes (Solarbio, Beijing, China). Subsequently, the membranes were blocked with 5% nonfat milk in TBST (50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20) for 2 h and incubated with the following primary antibodies at 4 °C overnight: LC3 (Protrintech Group, Wuhan, China), Bcl-2, GAPDH, and Bax (SAB, Beijing, China). In sequence, the membranes were washed and probed with goat anti-rabbit IgG/HRP (Beijing Biosynthesis Biotechnology Co., Ltd.) at room temperature for 2 h. The signals were detected by ECL Plus Hypersensitive luminescence solution (Solarbio, Beijing, China) and an ECL system (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China). The quantitative analysis of mean pixel density was performed by the ImageJ® software.

3.10. Statistical Analysis

All experiments were performed at least three times, and statistical analysis was performed by using Microsoft Excel. Data were presented as mean ± SD, and statistical significance was determined by ANOVA with the post hoc test. The p-value < 0.05 indicated a statistically significant difference.

4. Conclusions

The new series of allobetulon/allobetulin–nucleoside conjugates (9a–10i) were synthesized, and their antitumor activities were evaluated. Among them, compounds 9b, 9e, 10a, and 10d showed promising antiproliferative activity in six tested cell lines, compared to zidovudine, cisplatin, and oxaliplatin. Regarding the structure–activity relationship, introducing nucleosides to the scaffolds (7 and 8) can improve their potency. However, their potency did not significant correspond to their substituted types of nucleotide base. Based on their antiproliferative activity, compound 10d can be considered a promising candidate for further investigation. We investigated the potential mechanism for compound 10d. Compound 10d dose-dependently induced cell apoptosis and autophagy in SMMC cells, resulting in antiproliferation and G0/G1 cell cycle arrest by regulating protein expression levels of Bax, Bcl-2, and LC3. Consequently, the nucleoside-conjugated allobetulin (10d) evidenced that nucleoside substitution is an available strategy for improving allobetulon/allobetulin antitumor activity based on our present study.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27154738/s1, Figures S1–S68: NMR and HRMS spectra for compounds 9a–10i.

Author Contributions

Conceptualization, Q.W.; methodology, G.L., Y.-Y.C., and Q.W.; validation, Y.W. and X.H.; investigation, Y.W., X.H., X.Z. (Xiao Zhang), J.W., K.L. (Keyan Li), K.L. (Kexin Lu), X.Z. (Xiang Zhang) and C.X.; resources, Y.W. and Q.W.; data curation, Y.W., X.H., X.Z.(Xiao Zhang) and J.W.; writing—original draft preparation, Y.W. and X.H.; writing—review and editing, T.Z., Y.-Y.C. and Q.W.; supervision, Y.-Y.C. and Q.W.; project administration, Q.W.; funding acquisition, Q.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Research Project of Henan Province (222102310400), the Basal Research Fund of Henan Academy of Sciences (220602063), and the Basal Research Fund of the National Health Commission Key Laboratory of Birth Defect Prevention (JBKY2022012).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this work are available in the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 9a–10i are available from the authors.

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Molecules 27 04738 g001 550
Figure 1. Structures of natural pentacyclic triterpenes.
Figure 1. Structures of natural pentacyclic triterpenes.
Molecules 27 04738 g001
Molecules 27 04738 sch001 550
Scheme 1. Synthesis of 2-propargyl-substituted allobetulin and allobetulon. Reagents and conditions: (a) CH2Cl2, p-TSA, reflux; (b) CrO3, H2SO4, acetone, 0 °C, 2 h; (c) KN(SiMe3)2, Et3B, propargyl bromide, DME, rt, N2, 6 h; (d) NaBH4, isopropanol, rt.
Scheme 1. Synthesis of 2-propargyl-substituted allobetulin and allobetulon. Reagents and conditions: (a) CH2Cl2, p-TSA, reflux; (b) CrO3, H2SO4, acetone, 0 °C, 2 h; (c) KN(SiMe3)2, Et3B, propargyl bromide, DME, rt, N2, 6 h; (d) NaBH4, isopropanol, rt.
Molecules 27 04738 sch001
Molecules 27 04738 g002 550
Figure 2. X-ray crystallographic structure of compound 9c.
Figure 2. X-ray crystallographic structure of compound 9c.
Molecules 27 04738 g002
Molecules 27 04738 sch002 550
Scheme 2. Synthesis of allobetulin–nucleoside hybrids via click chemistry.
Scheme 2. Synthesis of allobetulin–nucleoside hybrids via click chemistry.
Molecules 27 04738 sch002
Molecules 27 04738 g003a 550Molecules 27 04738 g003b 550
Figure 3. Effects of compound 10d on cell apoptosis, cell cycle, and the expression of apoptotic and autophagic proteins. (A,B) Flow cytometry analysis of SMMC-7721 cells after treatment with compound 10d at different concentrations for 48 h. (C) Quantitative data analysis for the number of cells (% of total) in G0/G1, S, and G2/M phases for different treatment concentrations for 24 h. (D) Western blot analysis of Bcl-2, Bax, and LC3 protein expression levels in SMMC-7721 cell treated with compound 10d for different concentration for 48 h. (E) The Bax:Bcl-2 protein ratio in SMMC-7721 cells treated with different concentrations of compound 10d for 48 h. (F) The relative expression rates of LC3 protein in SMMC-7721 cells treated with different concentrations of compound 10d for 48 h. Values are mean ± SD (n = 3). * p < 0.05, ** p < 0.01.
Figure 3. Effects of compound 10d on cell apoptosis, cell cycle, and the expression of apoptotic and autophagic proteins. (A,B) Flow cytometry analysis of SMMC-7721 cells after treatment with compound 10d at different concentrations for 48 h. (C) Quantitative data analysis for the number of cells (% of total) in G0/G1, S, and G2/M phases for different treatment concentrations for 24 h. (D) Western blot analysis of Bcl-2, Bax, and LC3 protein expression levels in SMMC-7721 cell treated with compound 10d for different concentration for 48 h. (E) The Bax:Bcl-2 protein ratio in SMMC-7721 cells treated with different concentrations of compound 10d for 48 h. (F) The relative expression rates of LC3 protein in SMMC-7721 cells treated with different concentrations of compound 10d for 48 h. Values are mean ± SD (n = 3). * p < 0.05, ** p < 0.01.
Molecules 27 04738 g003aMolecules 27 04738 g003b
Table
Table 1. Antineoplastic activities of compounds 7a–8i, betulin, zidovudine, cisplatin, and oxaliplatin.
Table 1. Antineoplastic activities of compounds 7a–8i, betulin, zidovudine, cisplatin, and oxaliplatin.
Compd.IC50 (μM)
SMMC-7721HepG2MNK-45SW620MCF-7A549
7>100>100>100>100>100>100
9a20.95 ± 0.8920.04 ± 0.4042.91 ± 6.3065.90 ± 9.0926.75 ± 1.4222.86 ± 0.59
9b10.73 ± 0.8010.33 ± 1.1011.77 ± 1.6125.08 ± 6.169.57 ± 1.2612.42 ± 0.32
9c11.96 ± 1.0812.49 ± 0.9713.67 ± 3.1549.23 ± 0.3713.17 ± 0.8412.45 ± 1.12
9d15.14 ± 2.6713.63 ± 1.9813.39 ± 2.6147.67 ± 0.5348.89 ± 1.1513.14 ± 1.65
9e9.48 ± 2.3914.90 ± 2.666.46 ± 1.1011.80 ± 0.0927.14 ± 0.268.54 ± 0.72
9f18.93 ± 0.5515.71 ± 2.8621.19 ± 2.7351.96 ± 5.9984.17 ± 3.5019.49 ± 1.33
9g12.08 ± 2.3212.58 ± 2.4813.29 ± 2.60>10050.52 ± 2.108.74 ± 0.63
9h9.10 ± 2.2012.56 ± 0.818.50 ± 1.7548.75 ± 2.2315.57 ± 4.1025.32 ± 3.30
9i9.47 ± 1.8612.07 ± 1.7211.54 ± 1.2749.23 ± 1.9720.58 ± 3.0513.16 ± 2.62
864.96 ± 6.7687.73 ± 2.96>100>100>10062.96 ± 3.68
10a13.97 ± 2.4312.05 ± 1.138.01 ± 1.757.06 ± 0.4721.99 ± 0.329.95 ± 1.46
10b11.82 ± 1.4625.84 ± 4.1729.09 ± 1.9524.73 ± 3.8420.46 ± 1.4011.18 ± 1.61
10c22.26 ± 1.6052.32 ± 6.2022.48 ± 0.8931.85 ± 1.53>10039.86 ± 1.54
10d5.57 ± 0.787.49 ± 0.716.31 ± 1.646.00 ± 1.7012.32 ± 1.885.79 ± 1.00
10e15.35 ± 1.6120.48 ± 1.1925.79 ± 1.2715.32 ± 1.5545.79 ± 5.1017.96 ± 1.32
10f26.24 ± 1.8814.40 ± 1.4712.06 ± 3.9727.28 ± 0.4017.58 ± 2.9811.80 ± 0.65
10g54.74 ± 3.3940.95 ± 2.1314.63 ± 5.0266.62 ± 4.63>10017.75 ± 4.75
10h38.43 ± 4.8839.36 ± 3.9329.36 ± 1.8279.63 ± 2.6761.24 ± 6.4767.81 ± 2.66
10i10.07 ± 2.3411.33 ± 1.4512.50 ± 2.7539.66 ± 5.1143.07 ± 6.2011.18 ± 2.57
betulin82.9 ± 7.08>10055.50 ± 7.5083.70 ± 9.0530.6 ± 2.7087.39 ± 10.75
Zidovudine>100>100>100>100>100>100
cisplatin10.96 ± 1.3516.56 ± 1.7119.59 ± 1.8540.60 ± 5.6827.63 ± 2.3014.21 ± 2.80
oxaliplatin>10018.30 ± 1.6517.58 ± 1.2922.67 ± 1.717.41 ± 3.8745.89 ± 2.56
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Wang, Y.; Huang, X.; Zhang, X.; Wang, J.; Li, K.; Liu, G.; Lu, K.; Zhang, X.; Xie, C.; Zheng, T.; et al. Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents. Molecules 2022, 27, 4738. https://doi.org/10.3390/molecules27154738

AMA Style

Wang Y, Huang X, Zhang X, Wang J, Li K, Liu G, Lu K, Zhang X, Xie C, Zheng T, et al. Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents. Molecules. 2022; 27(15):4738. https://doi.org/10.3390/molecules27154738

Chicago/Turabian Style

Wang, Yanli, Xiaowan Huang, Xiao Zhang, Jingchen Wang, Keyan Li, Guotao Liu, Kexin Lu, Xiang Zhang, Chengping Xie, Teresa Zheng, and et al. 2022. "Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents" Molecules 27, no. 15: 4738. https://doi.org/10.3390/molecules27154738

APA Style

Wang, Y., Huang, X., Zhang, X., Wang, J., Li, K., Liu, G., Lu, K., Zhang, X., Xie, C., Zheng, T., Cheng, Y. -Y., & Wang, Q. (2022). Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents. Molecules, 27(15), 4738. https://doi.org/10.3390/molecules27154738

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