Semi-Synthetic Derivatives from a Lupane-Type Triterpene Isolated from Phoradendron wattii with Anti-Leukemia Activity

Abstract

Introduction: Leukemia is among the most common types of cancer. Currently, the main anti-leukemia treatments have significant drawbacks, which has led to the search for new and better drugs, especially those developed from natural products. Objective: The aim of this work was to perform simple modifications to a lupane-type pentacyclic triterpene isolated from Phoradendron wattii, in order to generate new semi-synthetic analogues for their possible effect against a panel of leukemia cell lines. Methods: Simple structural modifications on the triterpene 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (T2) isolated from Phoradendron wattii were performed and evaluated against a panel of leukemia cell lines and normal mononuclear cells (MNC). In addition, molecular docking analysis was used to better understand the possible mechanism of action, employing key molecular targets used in the search for compounds with anti-leukemic activity (BCL-2 and EGFR). Results: Of the all derivatives synthetized (T2a-T2f), the compound (T2e), 3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol, showed inhibition in leukemia cell lines (CCRF-CEM, REH, JURKAT, MOLT-4, and THP-1) and induction of death by apoptosis to different degrees. Moreover, it showed a minor cytotoxic effect on MNC and was found to interact with BCL-2 and EGFR in silico. Conclusion: The present work shows that simple modifications at promising sites in the lupane-type pentacyclic triterpene can generate derivatives with enhanced biological activity against leukemia cell lines. The T2e derivative is a promising candidate for future research.

Keywords

leukemia lupane-type triterpene apoptosis molecular docking

Introduction

Cancer is the second cause of death worldwide and accounted for 20 million new cases and 9.7 million deaths in 2022.^1,2 Leukemia is the 13th most frequently diagnosed cancer, and the 10th leading cause of cancer-related death worldwide and frequently affects people under 20 years of age.^2,3 The main drugs for the treatment of leukemia, such as dasatinib and imatinib, cause side effects and face challenges such as toxicity, development of cellular resistance mechanisms, and disease recurrence.^4–6 Therefore, it is necessary to search for more effective and selective new drugs against leukemia cells.

One of the most important alternatives in the search for new anticancer drugs are natural products. It is estimated that 40% of the drugs for the treatment of cancer have been inspired by natural products or semi-synthesized from a compound isolated from a natural source.⁷ For this reason, numerous studies are aimed at searching for new bioactive compounds from different natural sources, as well as in the development of structural analogues of these compounds to improve their biological activity and reduce side effects.^7,8

Pentacyclic triterpenes (PT) are compounds isolated from natural sources that exhibit various biological activities.^9–12 Although they have received significant attention in cancer research due to their antineoplastic properties,¹³ their therapeutic use is limited by their low bioavailability, which results from their poor water solubility.¹⁴

To improve the bioavailability of triterpenes, derivatives can be synthesized by binding them to cations, nitrogen compounds, counterions or by forming complexes with cyclodextrins, liposomes, carbon nanotubes and gold nanoparticles.^15–18 In addition, the introduction of functional groups such as glycosides, esters, amides, phosphates and ammonium salts has been shown to optimize both their bioavailability and biological activity.^16–18

Additionally, PT offer promising sites for obtaining new derivatives with improved biological activity. For example, the introduction of a propynoyl group at C-28 of betulin has demonstrated a strong cytotoxic effect on acute lymphoblastic leukemia (CCRF-CEM) and murine leukemia (P388) cells.¹⁹ Also, the combination of hydroxyl oxidation at C-3 and the incorporation of an acetylenic group at C-28 in betulin has enhanced its activity against the acute promyelocytic leukemia cell line (HL-60).¹⁹ Additional modifications at C-3, such as the addition of a 4-fluorophenylhydrazone molecule in betulin, have generated compounds with significant cytotoxic activity in the acute lymphoblastic leukemia cell line (MOLT-4).²⁰ On the other hand, the formation of betulinic and betulonic acid methyl esters has shown increased activity in chronic myeloid leukemia (K562).^21–23 In addition, the introduction of heterocycles such as 12,3-triazole at C-3 or at C-28 of betulinic acid has generated derivatives with enhanced cytotoxic activity in HL-60 and acute myeloid leukemia (THP-1) cell lines.^13,24

Moreover, other structural modifications of lupane-type triterpenes have also been performed at C-30. For example, oxidation at C-30 of lupeol leads to the formation of an α,β-unsaturated aldehyde on the isopropylidene fragment, generating a compound with a more cytotoxic profile in acute lymphoblastic leukemia (JURKAT) and K562 cell lines.⁴ Therefore, the presence of the formyl group, in the form of the α,β-unsaturated aldehyde, has contributed to more active compounds against leukemic cells compared to its non-functionalized analogue.^25–27

PT also have become crucial compounds in medicinal chemistry due to their versatility and broad compatibility with various biological structures and targets.¹⁸ This highlights their importance in the research and development of new antineoplastic agents.²⁸

In previous studies, the pentacyclic triterpenes 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic (T2) and 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T3), as well as betulin, isolated from Phoradendron wattii, were evaluated in leukemia cell lines.^29–31 Although T2 showed low activity in leukemia cell lines, it is one of the most abundant triterpenes in P. wattii. Its structural features, due to the presence of promising sites at C-28 and C-30, make it a good candidate for structural modifications to generate new semi-synthetic analogues for possible effect against acute lymphoblastic leukemia cell lines (CCRF-CEM, REH, JURKAT, MOLT-4) and one acute myeloid leukemia cell line (THP-1).

Materials and Methods

General Experimental Procedures

NMR spectra were recorded in CDCl₃ on a Bruker Avance 400 spectrometer. The chemical shifts are given in δ (ppm) with residual deuterated solvent as internal reference and coupling constants in Hz.

Precoated TLC silica gel 60 F254 aluminum sheets from Sigma-Aldrich were used for thin-layer chromatography (0.25 and 0.5 mm layer thickness for analytical and preparative TLC, respectively) and visualized under short (254 nm) and long (366 nm) wave-length UV light, or a spray reagent (H₂SO₄-AcOH-H₂O, 1:20:4). Column chromatography (CC) was conducted using silica gel 60 (63-200 or 40-63, particle size), TLC (2-25 µm, particle size) or Sephadex LH-20 from Sigma-Aldrich (Saint Louis, MO, USA).

The bioassays were performed inside a laminar flow hood, brand NuAire, class II, type A2. The cells were kept inside a CO₂ incubator with a water jacket and HEPA filter, brand NuAire.

Plant Material

Aerial parts of Phoradendron wattii Krug & Urb. (Santalaceae) (synonym: Phoradendron vernicosum Greenm.) were collected in March 2019 on the Hunucmá-Sisal highway (21°05038.0” N, 89°58021.4” W), Yucatán (México). Plant material was identified by Tech. Paulino Sima-Polanco (CICY). A voucher sample (PS 3220) was deposited at the U Najil Tikin Xiu herbarium of CICY. The aerial parts were dried under artificial light (50-60 °C) for three days and then ground. Finally, after having the plant material dried, it was crushed using a blade mill without cooling (Pagani, model 2030) until 33.5 kg of powdered material of a particle size # 7 were obtained.

Extraction and Isolation

The compounds 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (T2), 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T3), and betulin were isolated from the aerial parts of P. wattii according to a previous study.²⁹

Preparation of Compound T2a from T2

A solution of 50 mg (0.10 mmol) of compound T2 in tetrahydrofuran anh. (3 mL) was treated with 9.9 mg (0.26 mmol) of LiAlH₄. The reaction mixture was refluxed for 5 h. Ethyl acetate (20 mL) was slowly added after 5 h to decompose residual LiAlH₄. The crude was treated with H₂O and extracted with CH₂Cl₂ (3 × 30 mL), and the organic phase was dried with Na₂SO₄ anh., and distilled to remove the solvent.²¹ The residue was purified by CC (hexane-EtOAc, 85:15) to give 39 mg (82%) of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-ol (T2a).

3α,23-O-Isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-ol (T2a): white, amorphous powder (CHCl₃); R_f= 0.35 (hexane-EtOAc, 85:15); ¹H-NMR (CDCl₃, 400 MHz) δ 4.68 (d, J = 2.3 Hz, 1H, H-29a), 4.58 (dd, J = 2.3, 1.3 Hz, 1H, H-29b), 3.81 (d, J = 10.8 Hz, 1H, H-28a), 3.68 (d, J = 12.0 Hz, 1H, H-23a), 3.61 (pt, 1H, H-3), 3.32 (d, J = 10.8 Hz, 1H, H-28b), 3.23 (d, J = 12.0 Hz, 1H, H-23b), 2.39 (m, 1H, H-19), 1.98–1.74 (m, 7H), 1.68 (br s, 3H), 1.65–1.11 (m, 17H), 1.40 (s, 6H, ((CH₃)₂C), 1.05 (m, 1H), 1.02 (s, 3H), 1.01 (s, 3H), 0.85 (s, 3H), 0.68 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 150.6 (C-20), 109.8 (C-29), 98.2 ((CH₃)₂C), 73.2 (C-3), 68.5 (C-23), 60.7 (C-28), 50.4, 48.9, 48.0, 48.0, 43.2, 42.9, 41.3, 37.4, 37.1, 35.3, 34.1, 34.1, 33.4, 29.9, 29.4, 29.3 ((CH₃)₂C), 27.2, 25.3, 23.8, 20.8, 19.5, 19.2 ((CH₃)₂C), 17.9, 17.5 (C-24), 16.7, 16.2, 15.0; see Figures S1 and S2.

Preparation of Compound T2b from T2a

An amount of 23 mg (0.05 mmol) of compound T2a was solubilized in 3.0 mL of Ac₂O and 0.02 mL of C₅H₅N. The reaction mixture was refluxed for 3 h. Once the reaction product was obtained, it was stopped with H₂O and separated by liquid-liquid extraction with H₂O-EtOAc (1:2 × 2 and 1:1), and the organic phase was washed with HCl (5%) and brine.²¹ The organic fraction was dried over Na₂SO₄ anh., filtered, and after removing the solvent under reduced pressure, the crude reaction product was purified by CC, eluting with a mixture of hexane-EtOAc (85:15) to give 19.5 mg (78%) of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-methylene acetate (T2b).

3α,23-O-Isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-methylene acetate (T2b): white, amorphous powder (CHCl₃); R_f= 0.56 (hexane-EtOAc, 85:15); ¹H-NMR (CDCl₃, 400 MHz) δ 4.68 (d, J = 2.3 Hz, 1H, H-29a), 4.58 (dd, J = 2.3, 1.3 Hz, 1H, H29b), 4.24 (d, J = 11.0 Hz, 1H, H-28a), 3.86 (d, J = 11.0 Hz, 1H, H-28b), 3.66 (d, J = 12.0 Hz, 1H, H-23a), 3.61 (pt, 1H, H-3), 3.24 (d, J = 12.1 Hz, 1H, H-23b), 2.44 (m, 1H, H-19), 2.07 (s, 3H, (OCOCH₃), 1.98–1.73 (m, 9H), 1.67 (br s, 3H), 1.65–1.05 (m, 18H), 1.40 (s, 6H, ((CH₃)₂C), 1.01 (s, 3H), 0.85 (s, 3H), 0.68 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 171.8 (OCOCH₃), 150.4 (C-20), 110.0 (C-29), 98.2 ((CH₃)₂C), 73.2 (C-3), 68.5 (C-23), 63.0 (C-28), 50.4, 49.0, 47.9, 46.5 (OCOCH₃), 43.2, 42.9, 41.3, 37.4, 37.1, 35.3, 34.7, 34.1, 33.4, 29.9 ((CH₃)₂C), 19.8, 29.4, 27.2, 25.3, 23.8, 21.2, 20.7, 19.5, 19.2 ((CH₃)₂C), 17.9, 17.5 (C-24), 16.7, 16.2, 15.0; see Figures S3 and S4.

Preparation of Compound T2c from T2

Compound T2c was prepared from 30 mg (0.06 mmol) of compound T2 and 32.5 mg (0.30 mmol) of SeO₂ in 10 mL of EtOH anhydrous. The reaction mixture was heated under reflux for 72 h. Then, the reaction mixture was cooled and EtOH was removed under reduced pressure. The crude was treated with water (20 mL) and extracted with CH₂Cl₂ (3 × 30 mL), and the organic phase was dried with Na₂SO₄ anh., and distilled to remove the solvent.^32,33 The residue was purified by CC (hexane-EtOAc, 80:20) to give 14.4 mg (47%) of 3α,23-O-isopropylidenyl-3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T2c).

3α,23-O-Isopropylidenyl-3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T2c): white, amorphous powder (CHCl₃); R_f= 0.27 (hexane-EtOAc, 85:15); ¹H-NMR (CDCl₃, 400 MHz) δ 9.52 (s, 1H, CHO), 6.29 (s, 1H, H-29a), 5.92 (s, 1H, H-29b), 3.65 (d, J = 12.2 Hz, 1H, H-23a)), 3.60 (pt, 1H, H-3), 3.34 (m, 1H, H-19), 3.23 (d, J = 12.2 Hz, 1H, H-23b), 2.33–1.65 (m, 10H), 1.59–1.15 (m, 15H), 1.40 (s, 6H, ((CH₃)₂C), 0.97 (s, 3H), 0.92 (s, 3H), 0.84 (s, 3H), 0.67 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 195.1 (C-30), 181.1 (C-28), 156.5 (C-20), 134.1 (C-29), 98.2 ((CH₃)₂C), 73.2 (C-3), 68.5 (C-23), 56.6, 50.3, 43.2, 42.6, 41.0, 41.0, 38.4, 37.1, 37.0, 35.3, 34.2, 33.4, 32.3, 32.1, 29.9, 29.8, 29.4 ((CH₃)₂C), 27.4, 23.8, 20.8, 19.5 ((CH₃)₂C), 17.9, 17.4 (C-24), 16.7, 16.3, 14.9; see Figures S5 and S6.

Preparation of Compound T2d and T2e from T2a

Compound T2d was prepared from 48 mg (0.10 mmol) of compound T2a and 54.4 mg (0.50 mmol) of SeO₂ in 10 mL of EtOH anh., following the same procedure described for the preparation of T2c. The residue was purified by CC (hexane-EtOAc, 50:50) to give 24.2 mg (49%) of 3α,23-O-isopropylidenyl-3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol (T2d) and 10.2 mg (22%) of the subproduct 3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol (T2e).

3α,23-O-Isopropylidenyl-3α,23-dihydroxy-30-oxolup-20 (29)-en-28-ol (T2d): white, amorphous powder (CHCl₃); R_f= 0.65 (hexane-EtOAc, 50:50); ¹H-NMR (CDCl₃, 400 MHz) δ 9.50 (s, 1H, CHO), 6.29 (s, 1H, H-29a), 5.92 (s, 1H, H-29b), 3.80 (d, J = 10.8 Hz, 1H, H-28a), 3.65 (d, J = 12.0 Hz, 1H, H-23a), 3.60 (pt, 1H, H-3), 3.36 (d, J = 10.8 Hz, 1H, H-28b), 3.23 (d, J = 12.0 Hz, 1H, H-23b), 2.76 (m, 1H, H-19), 2.26–2.13 (m, 1H), 1.94–1.78 (m, 4H), 1.77–1.03 (m, 19H), 1.39 (s, 6H, ((CH₃)₂C), 0.92 (m, 1H), 1.00 (s, 3H), 0.99 (s, 3H), 0.83 (s, 3H), 0.68 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 195.1 (C-30), 157.2 (C-20), 133.3 (C-29), 98.2 ((CH₃)₂C), 73.1 (C-3), 68.5 (C-23), 60.4 (C-28), 51.9, 50.2, 48.2, 47.8, 43.1, 42.8, 41.3, 37.1, 37.0, 35.3, 34.1, 34.0, 33.3, 32.7, 29.4 ((CH₃)₂C), 29.3, 27.8, 27.1, 23.8, 20.8, 19.4 ((CH₃)₂C), 17.9, 17.4 (C-24), 16.6, 16.2, 14.9; see Figures S7 and S8.

3α,23-Dihydroxy-30-oxolup-20(29)-en-28-ol (T2e): white, amorphous powder (CHCl₃); R_f= 0.23 (hexane-EtOAc, 50:50); ¹H-NMR (CDCl₃, 400 MHz) δ 9.50 (s, 1H, CHO), 6.30 (s, 1H, H-29a), 5.93 (s, 1H, H-29b), 3.80 (d, J = 10.8 Hz, 1H, H-28a), 3.66 (pt, 1H, H-3), 3.53 (d, J = 11.3 Hz, 1H, H-23a), 3.39 (d, J = 11.3 Hz, 1H, H-23b), 3.36 (d, J = 10.8 Hz, 1H, H-28b), 2.77 (m, 1H, H-19), 2.22–2.11 (m, 1H), 2.00–1.82 (m, 6H), 1.76–1.03 (m, 19H), 0.91 (m, 1H), 1.01 (s, 3H), 0.98 (s, 3H), 0.84 (s, 3H), 0.67 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 195.1 (C-30), 157.2 (C-20), 133.4 (C-29), 71.5 (C-3), 60.4 (C-28), 52.0 (C-23), 50.2, 48.2, 43.0, 42.8, 41.1, 40.6, 37.2, 37.1, 36.3, 34.0, 33.9, 33.1, 32.7, 29.8, 29.3, 27.8, 27.0, 26.6, 20.9, 18.1 (C-24), 17.9, 16.3, 16.1, 14.9; see Figures S9 and S10.

Preparation of Compound T2f from T2

An amount of 15.3 mg (0.03 mmol) of compound T2 and 110 mg (0.79 mmol) of K₂CO₃ were solubilized in 0.5 mL of CH₃I and 1.0 mL of Me₂CO anhydrous. The reaction was carried out under constant stirring for 72 h at room temperature. Once the reaction product was obtained, it was separated by liquid-liquid extraction with H₂O-EtOAc (1:2 × 2 and 1:1), and the organic phase was dried with Na₂SO₄ anh., and distilled to remove the solvent.³¹ The residue was purified by CC (hexane-Me₂CO, 95:5) to give 11.5 mg (75%) of methyl ester of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (T2f).

Methyl ester of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (T2f): white, amorphous powder (CHCl₃); R_f= 0.56 (hexane-Me₂CO, 9.8:0.2); ¹H-NMR (CDCl₃, 400 MHz) δ 4.73 (d, J = 2.4 Hz, 1H, H-29a), 4.59 (dd, J = 2.4, 1.4 Hz, 1H, H-29b), 3.66 (s, 3H, (OCH₃)), 3.65 (d, J = 12.1 Hz, 1H, H-23a), 3.60 (pt, 1H, H-3), 3.23 (d, J = 12.1 Hz, 1H, H-23b), 2.99 (m, 1H, H-19), 2.29–2.12 (m, 2H), 1.95–1.70 (m, 4H), 1.68 (br s, 3H), 1.65–1.11 (m, 17H), 1.40 (s, 6H ((CH₃)₂C), 1.05 (m, 1H), 0.99 (s, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.68 (s, 3H); ¹³C-NMR (CDCl₃, 100 MHz) δ 176.9 (C-28), 150.8 (C-20), 109.7 (C-29), 98.2 ((CH₃)₂C), 73.2 (C-3), 68.6 (C-23), 56.7, 51.4 (OCH₃)), 50.5, 49.6, 47.1, 43.2, 42.6, 41.1, 38.4, 37.1, 35.3, 34.3, 33.4, 32.4, 30.8, 29.8, 29.4 ((CH₃)₂C), 25.7, 23.8, 20.8, 19.5 (CH₃)₂C), 17.9, 17.5 (C-24), 16.7, 16.2, 15.0; see Figures S11 and S12.

Leukemia Cell Lines

Acute myeloid leukemia cell line (THP-1, ATCC TIB-202) and acute lymphoblastic leukemia cell lines (CCRF-CEM, ATCC CCL-19; JURKAT CLONE E6-1, ATCC TIB-152; MOLT-4, ATCC CRL-1582; REH, ATCC CRL-8286) from the American Type Culture Collection (ATCC) were provided by María Antonieta Chávez González, from Unidad de Investigación Médica en Enfermedades Oncológias, UMAE Hospital de Oncología, Centro Médico Nacional Siglo XXI, IMSS. All procedures were approved by the Ethics and Scientific Committee at IMSS. Acute lymphoblastic leukemia cell lines and THP-1 cell line were maintained with RPMI 1640 medium (Roswell Park Memorial Institute medium) at 10% FBS (fetal bovine serum); 1% penicillin-streptomycin was added and incubated at an atmosphere of culture with 95% humidity and 5% CO₂ at 37 °C. All evaluations were performed between 3 and 4 cell passages.

Compounds and Controls

The compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL. In all cases, medium with DMSO (0.1%) was used as a negative control, and dasatinib was used as positive control. Normal mononuclear cells (MNC) were used as normal cell control. All tests were performed in triplicate.

Bioassay of Viability in Leukemic Cell Lines

A total of 1 × 10⁵ cells/well were cultured into 96-well plates and treated with different concentrations of compounds (0.01, 0.1, 1.0, and 10 µg/mL) for 48 h. At the end of this time, the cells were collected and stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 500 ng/mL for 15 min in darkness, according to manufacturer instructions. The samples were analyzed by flow cytometry with a FACSVerse flow cytometer (BD Bioscience, San Jose, CA, USA). The IC₅₀ obtained in this analysis (µg/mL) was changed to µM.

Viability Test in Normal Mononuclear Cells (MNC)

Normal mononuclear cells were obtained from normal human peripheral blood monocytes. MNC were collected according to institutional guidelines, including written informed consent from each donor. Collection procedures were approved by Ethics and Scientific Committee of the Mexican Institute of Social Security (IMSS, ethics approval number CNIC R-2021-785-025). The MNC were purified with FicollPaque Plus (Pharmacia Biotech, Uppsala, Sweden) by centrifugation at 400 × g at room temperature for 30 min according to the manufacturer's protocol. Once the cells were obtained, they were resuspended in RPMI medium) with 10% FBS, and they were counted using a hemocytometer, previously stained with a trypan blue solution, verifying the viability of 95%.³⁴

A total of 1 × 10⁵ cells/well were cultured into 96-well plates and treated with different concentrations of compounds (12.5, 25, 50, and 100 µg/mL) for 48 h. RPMI medium with 10% FBS was used as culture medium for cell growth. At the end of this time, the cells were collected, washed with phosphate buffer saline (PBS), and stained with DAPI at 500 ng/mL for 15 min in darkness, according to manufacturer instructions. The samples were analyzed by flow cytometry with a FACSVerse flow cytometer (BD Bioscience, San Jose, CA, USA). The IC₅₀ obtained in this analysis (µg/mL) was changed to µM.

Apoptosis Assay

Cells (1 × 10⁵ cells/well) were cultured in 96-well plates and treated with compounds at two different concentrations (½ and 1 × IC₅₀), to be later incubated for 48 h. After this time, cells were washed with PBS and stained with annexin V-FITC (BD Bioscience) and incubated in the dark for 15 min; subsequently, the cells were washed with PBS and stained with DAPI at 500 ng/mL for 15 min in darkness. Then they were analyzed by flow cytometry with a FACSVerse flow cytometer (BD Bioscience, San Jose, CA, USA).

In Silico Studies

The ADME (absorption, distribution, metabolism and excretion) properties of compounds, such as molecular weight (MW), hydrogen bond acceptor (nHBA), hydrogen bond donors (nHBD), lipophilicity (cLogP), rotatable bonds (nROTB), and topological polar surface area (TPSA), and pharmacokinetic parameters were predicted using SwissADME.³⁵

Protein target structures for molecular docking studies were retrieved from RCSB Protein Data Bank,³⁶ selecting BCL-2 in complex with a small molecule inhibitor targeting BCL-2 BH3 domain (N-(6-{4-[(4’-chlorobiphenyl-2-yl)methyl]piperazin-1-yl}-1,1-dioxide-1,2-benzothiazol-3-yl)-4-{[(2R)-4-dimethylamino)-1-(phenylsulfanyl) butan-2-yl]amino}-3-nitrobenzenesulfonamide) (PDB-ID: 4IEH) and Epidermal Growth Factor Receptor tyrosine kinase domain with erlotinib as an inhibitor (PDB-ID: 1M17). Molecular docking studies were performed using software Autodock4 (v.4.2).³⁷ Protein and ligands were prepared using Autodock Tools (ADT, v.1.5.7) to obtain proper inputs for Autodock4. The polar hydrogen charges of Gasteiger-type were assigned, and the nonpolar hydrogens were merged with the carbon atoms. All the protein was considered as a rigid body and the ligands being flexible. All the torsions and rotable bonds in the ligands were defined. The grid box for ligands were centered at the co-crystalized inhibitor, with dimensions of 70 × 70 × 70 with a spacing of 0.375 Å. The search was performed with the Lamarckian Genetic Algorithm as is implemented in Autodock4 code. The population of 150 individuals was mutated with a mutation rate of 0.02 and evolved for 10 generations. The number of the docking runs was 100. The best binding mode was selected based on the lowest energy binding and more populated cluster. Molecular graphics and analyses were performed with UCSF ChimeraX v1.8.^38,39

Analysis of Results

All cytometric data were analyzed using FlowJoTM v. 10.9 software. Results of viability were calculated by GraphPad Prism software version 9. IC₅₀ values (the concentration of the compound that causes 50% of cell proliferation inhibition) were obtained from dose-response curves using GraphPad Prism software version 9. The experiments were performed in triplicate. The data were expressed as means ± SEM. Statistical significance was calculated with a one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test, applying a p < 0.05 significance level.

Results

In the present study and based on previous studies about cytotoxic effects of betulin and T3, which are attributed to the presence of specific groups in their structure, such as the alcohol group at C-28 of betulin and the formyl group at C-30 of T3, in the form of α,β-unsaturated aldehyde (Scheme 1).^14,19,30 A series of T2 analogues were generated by minor structural changes to be evaluated in a panel of leukemia cell lines.

Scheme 1.

Chemical structures of T2, T3, and betulin.

The structural modification of T2 was carried out by a series of reactions focused on C-28 and C-30 (Scheme 2). It started with the reduction of C-28 leading to the derivative 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-ol (T2a) with 82% yield. This compound was used as starting material for further structural modifications.

Scheme 2.

Semi-synthesis of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic derivatives (T2a-T2f).

Subsequently, from T2a an acetylation reaction was carried out to obtain the derivative 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-methylene acetate (T2b) with 78% yield. Furthermore, from the parent T2 compound, the derivative 3α,23-O-isopropylidenyl-3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T2c) was obtained by the allylic oxidation reaction on C-30 in 47% yield.

The allylic oxidation reaction on C-30 of T2a afforded the derivative 3α,23-O-isopropylidenyl-3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol (T2d) with 49% yield and the by-product 3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol (T2e) with 22% yield. Finally, the methyl ester of 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (T2f) was synthesized by methylation of T2 with 75% yield. All compounds were characterized by ¹H and ¹³C-NMR experiments (Supplemental Material).

The effect on the viability of leukemic cells was evaluated using different concentrations of T2, T3, betulin, and T2 derivatives, compared to dasatinib (Table 1). Of particular interest were the derivatives T2c, T2d and T2e, which presented significant activity in the five cell lines evaluated, with IC₅₀ values lower than that of the original compound T2, which has no activity. Furthermore, semi-synthetic compounds showed similar effects to dasatinib.

Table 1.

The Mean Inhibitory Concentrations of Triterpenes and Derivatives Evaluated Against Leukemia Cell Lines and Peripheral Blood Mononuclear Cells.

Compound	IC₅₀ µM (SI)
	CCRF-CEM	REH	JURKAT	MOLT-4	THP-1	MNC
T2	N/A	N/A	N/A	N/A	N/A	N/A
T2a	N/A	153.13 ± 2.0	N/A	N/A	N/A	N/A
T2b	N/A	N/A	N/A	N/A	N/A	N/A
T2c	17.86 ± 0.9 (13.15)	24.41 ± 0.4 (9.62)	13.80 ± 1.1 (17.03)	3.41 ± 0.1 (68.92)	17.52 ± 0.2 (13.41)	235.02 ± 3.1
T2d	8.44 ± 0.8 (179.20)	18.68 ± 0.2 (80.97)	6.08 ± 0.5 (248.77)	2.30 ± 0.1 (657.62)	7.87 ± 0.2 (192.18)	1512.53 ± 5.2
T2e	13.13 ± 0.6 (140.00)	17.07 ± 0.2 (107.69)	7.78 ± 0.3 (236.28)	10.00 ± 0.2 (183.83)	14.99 ± 0.1 (122.63)	1838.33 ± 4.3
T2f	N/A	N/A	N/A	N/A	N/A	N/A
T3	27.36 ± 0.9 (19.48)	46.74 ± 1.6 (11.40)	62.27 ± 2.3 (8.56)	N/A	N/A	532.98 ± 6.1
Betulin	N/A	19.96 ± 1.2 (113.16)	54.11 ± 10.2 (41.74)	32.36 ± 1.3 (69.79)	19.71 ± 0.1 (114.59)	>2258.71
Dasatinib	10.76 ± 1.2	5.43 ± 0.3	5.26 ± 0.5	2.63 ± 0.1	4.66 ± 0.3	N/A

MNC: normal mononuclear cells; SI: selectivity index (ratio of IC₅₀ in the MNC line over IC₅₀ in the leukemia cell line); N/A: no active.

On the other hand, T3 showed better selectivity against the CCRF-CEM and REH cell lines than the JURKAT cell line. In contrast, betulin showed good selectivity in four leukemia cell lines.

The safety of the compounds was measured by evaluating the effect on normal mononuclear cells (MNC). When compounds T2c-T2e were evaluated on MNC at the maximum concentration (100 µg/mL), they showed a non-significant effect, with a high selectivity (SI >10) on leukemia cell lines. On the other hand, the other compounds showed no activity at the maximum concentration.

Because T2c-T2e and betulin reduced cell viability and have IC₅₀ values ≤20 µΜ, their possible mode of action was evaluated by determining the apoptotic effect at 48 h at the concentration of ½ IC₅₀ and IC₅₀ obtained in the viability assay in the different cell lines, also evaluating the positive control at different concentrations. Double staining was used to quantify the rate of cell death by apoptosis (annexin V-FITC as positive) and other death processes (DAPI as positive).The results obtained were normalized to 100% from the negative control.

For the REH cell line (Figure 1), death by apoptosis was 93.29 ± 1.29% in the positive control, while for the active compounds at ½ IC₅₀ were: 95.81 ± 2.17% (T2d), 64.24 ± 2.01% (T2e), and 7.56 ± 1.67% (betulin). When the IC₅₀ values were used, the apoptotic rate for betulin changed slightly to 16.48 ± 1.52%, while for T2e it increased at 98.07 ± 1.10%. It should be noted that the apoptotic rate for T2d (97.03 ± 1.44%) changed a little.

Figure 1.

(A) T2d and T2e induced apoptosis in the REH cell line. Betulin, T2d, and T2e were evaluated at ½ IC₅₀ and IC₅₀ exposed for 48 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of REH cultures, treated with vehicle control (DMSO), positive control (dasatinib), betulin, T2d, and T2e for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

Under the same conditions, the apoptotic rate for the CCRF-CEM cell line (Figure 2) was 94.84 ± 1.07% in the positive control. Apoptotic rates at ½ IC₅₀ of the most active compounds were 82.50 ± 0.90% (T2c), 96.05 ± 0.68% (T2d), and 89.96 ± 0.85% (T2e). Only T2d showed an effect similar to the positive control. At IC₅₀, increased apoptosis was observed for T2c (97.35 ± 0.61%) and T2e (98.19 ± 0.77%), similar to the positive control, whereas for T2d, the apoptosis rate was slightly increased (97.23 ± 0.88).

Figure 2.

(A) T2c, T2d, and T2e induced apoptosis in the CCRF-CEM cell line. T2c, T2d, and T2e were evaluated at ½ IC₅₀ and IC₅₀ exposed for 48 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of CCRF-CEM cultures, treated with vehicle control (DMSO), positive control (dasatinib), T2c, T2d, and T2e for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

When evaluated on THP-1 cell line (Figure 3) the positive control showed 32.14 ± 3.15% of apoptosis. Of the evaluated compounds, only T2e exhibited an effect at ½ IC₅₀ of 23.44 ± 2.11%. When the IC₅₀ values were used, death by apoptosis was clearly higher in three active compounds: 34.95 ± 2.76% (T2c), 59.53 ± 1.47% (T2e), and 39.66 ± 2.46% (betulin) than the positive control. The THP-1 cell line is clearly less sensitive to the compounds evaluated than the CCRF-CEM cell line.

Figure 3.

(A) Betulin and T2c-T2e were evaluated at ½ IC₅₀ and IC₅₀ exposed for 48 h in the THP-1 cell line. **p < 0.01, ****p < 0.0001 were compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of THP-1 cultures, treated with vehicle control (DMSO), positive control (dasatinib), betulin, T2c, T2d, and T2e for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

For the MOLT-4 cell line (Figure 4), the apoptotic rate was 96.13 ± 0.71% for the positive control, while for T2c-T2e at ½ IC₅₀ no effect was observed. Although a slight change in apoptotic effect at IC₅₀ was observed for T2c (10.88 ± 1.25%) and T2d (12.04 ± 1.85%), and only T2e (63.23 ± 2.82%) showed a notable change. These results highlight that T2c-T2e have a minimal effect on the MOLT-4 cell line.

Figure 4.

(A) T2c-T2e were evaluated at ½ IC₅₀ and IC₅₀ exposed for 48 h in the MOLT-4 cell line. *p < 0.05, ***p < 0.001, ****p < 0.0001 were compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of MOLT-4 cultures, treated with vehicle control (DMSO), positive control (dasatinib), T2c, T2d, and T2e for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

When tested against the JURKAT cell line (Figure 5) the positive control showed 97.10 ± 0.11% apoptosis. Of the evaluated compounds at ½ IC₅₀, T2d (61.19 ± 3.03%) and T2e (60.67 ± 0.50%) presented similar apoptotic rates, while T2c (42.01 ± 0.46%) was lower. Nevertheless, when IC₅₀ values were calculated, a significant increase was observed for the three compounds: T2c (96.64 ± 1.15%), T2d (95.67 ± 0.60%), and T2e (97.15 ± 0.23%). These results indicated that the JURKAT cell line is clearly more sensitive to T2c-T2e.

Figure 5.

(A) T2c, T2d, and T2e induced apoptosis in the JURKAT cell line. T2c, T2d, and T2e were evaluated at ½ IC₅₀ and IC₅₀ exposed for 48 h. ****p < 0.0001 was compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of JURKAT cultures, treated with vehicle control (DMSO), positive control (dasatinib), T2c, T2d, and T2e for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

Under the same conditions, only the effect of T2c was evaluated to generate apoptotic effects in MNC, because it generated a slight effect on these cells according to the viability assay (Figure 6). At 20 µM concentration, it was observed that T2c presented the highest percentage of viable cells 83.30 ± 1.18%, compared to the positive control (7.43 ± 0.16%). On the other hand, when evaluating the T2c derivative at 40 µM, a slight increase in apoptotic rates of 45.52 ± 0.87% was observed. These results demonstrate that T2c had minimal effect on normal cells and induced varying degrees of apoptotic death in leukemic cells.

Figure 6.

(A) T2c was evaluated at 20 and 40 µM exposed for 48 h in MNC. ****p < 0.0001 were compared with the positive control. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc analysis. (B) Representative dot plots of MNC cultures, treated with vehicle control (DMSO), positive control (dasatinib), and T2c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic; Q3: Early apoptotic; Q4: Living cells. Apoptotic cell population in red box.

Based on the above results, it was decided to perform a molecular docking analysis using key molecular targets involved in apoptotic processes. Among these are members of the BCL-2 family, which are related to the control and regulation of apoptotic mitochondrial events (eg, the anti-apoptotic protein BCL-2); these proteins block apoptosis through the intrinsic pathway and are found at elevated levels in cancers, especially blood cancers, thus representing an important molecular target.^40–42 On the other hand, the vascular endothelial growth factor receptor epidermal (EGFR) is also a relevant target. In leukemia, EGFR is deregulated, which plays an important role as it is involved in key signaling pathways responsible for cell survival and proliferation. In addition, it has downstream signaling pathways associated with the BCL-2 family. As a result, EGFR kinase activity is a promising therapeutic target in the treatment of leukemia.^43–45

For molecular docking analyses, the crystal structure of BCL-2 was used in complex with a small molecule inhibitor targeting the interaction of the BH3 domain of BCL-2 (Figure 7) and the EGFR tyrosine kinase domain with erlotinib as inhibitor (Figure 8). The derivatives (T2c-T2e), with affinity energies in the range of −8.26 to −8.80 kcal/mol (Table 2), were found to be less affine for the anti-apoptotic protein BCL-2 than the precursor T2 (−9.25 kcal/mol). Compared to betulin (−8.57 kcal/mol), the T2e derivative presented a better affinity with an energy of −8.80 kcal/mol. As for the EGFR kinase domain, the T2e derivative was found to be more affine than betulin (−9.42 kcal/mol) and its precursor T2 (−8.16 kcal/mol). These scores suggest the implication of these molecular targets in the different effects observed during the experimental analysis, where T2d and T2e derivatives obtained lower IC₅₀ values than T2c in the different leukemia cell lines.

Figure 7.

Binding modes of T2c in the BCL-2 protein. (A) Crystal structure of BCL-2 in complex with a small molecule inhibitor targeting BCL-2 BH3 domain interaction, (B) compound T2, (C) compound T2c, (D) compound T2d, (E) compound T2e, (F) compound T3, (G) betulin, (H) compound T2c docked to BCL-2 binding site. The co-crystallized inhibitor (re-docking RMSD: 3.676 Å) is shown as yellow sticks, (I) BCL2–T2c interactions include hydrogen bonds (blue dash), hydrophobic interactions (green dash).

Figure 8.

Binding modes of T2c in the EGFR tyrosine kinase domain. (A) Crystal structure of EGFR tyrosine kinase in complex with erlotinib, (B) compound T2, (C) compound T2c, (D) compound T2d, (E) compound T2e, (F) compound T3, (G) betulin, (H) T2c docked to EGFRTyrKin binding site. The co-crystallized erlotinib (re-docking RMSD: 0.932 Å) is shown as orange sticks, (I) EGFRTyrKin–T2c interactions include hydrogen bonds (blue dash), hydrophobic interactions (green dash).

Table 2.

Docking Score for Compounds T2, T2c-T2e, T3, and Betulin.

Compound	Score (kcal/mol)
Compound	BCL-2	EGFR (TK Domain)
T2	−9.25	−8.16
T2c	−8.70	−8.75
T2d	−8.52	−9.51
T2e	−8.80	−10.72
T3	−8.26	−9.04
Betulin	−8.57	−9.42
Co-crystalized inhibitor	−12.95	−8.07

As a complementary study, we submitted the molecular structures of the six compounds to in silico analysis via the SwissADME platform to predict their physicochemical and pharmacokinetic properties (Table 3).³⁵

Table 3.

In Silico Predicted Physicochemical and Pharmacokinetic Parameters of Compounds T2, T2c-T2e, T3, and Betulin.

		Compounds
		T2	T2c	T2d	T2e	T3	Betulin
Molecular Weight (g/mol)		512.76	526.75	512.76	472.7	486.68	442.72
Physicochemical Parameters	nHBA	4	5	4	4	5	2
	nHBD	1	1	1	3	3	2
	cLogP	6.65	5.84	6.09	4.89	4.69	6.39
	nROTB	2	3	3	4	4	2
	TPSA (Å²)	55.76	72.83	55.76	77.76	94.83	40.46
Pharmacokinetic Parameters	GI absorption	Low	Low	Low	High	High	Low
	CYP1A2 inhibitor	No	No	No	No	No	No
	CYP2C19 inhibitor	No	No	No	No	No	No
	CYP2C9 inhibitor	No	No	No	No	No	No
	CYP2D6 inhibitor	No	No	No	No	No	No
	CYP3A4 inhibitor	No	No	No	No	No	No

nHBA: num. H-bond acceptor; nHBD: num. H-bond donors; cLogP: lipophilicity (consensus Log P_o/w); nROTB: num. rotatablebonds; TPSA: topological polar surface area; GI absorption: gastrointestinal absorption; CYP1A2: cytochrome P450 1A2; CYP2C19: cytochrome P450 2C19; CYP2C9: cytochrome P450 2C9; CYP2D6: cytochrome P450 2D6; CYP3A4: cytochrome P450 3A4.

Lipinski's rule of six for compound selection was applied as an initial approach. Only compounds T2e, T3, and betulin complied with all the rules, with acceptable values for molecular weight (≤ 500), hydrogen bond donors (nHBA ≤ 5), hydrogen bond acceptors (nHBD ≤ 10), lipophilicity (expressed in cLogP ≤ 5), molar refractivity (TPSA ≤ 140 Å) and rotatable bonds (nROTB ≤ 10).⁴⁶ On the other hand, although T2c and T2d are predicted to have low passive gastrointestinal uptake, they would not inhibit some of the key CYP450 isoforms (Table 3).³⁵ Together with the results obtained in MNC, suggest that they are good candidates for further studies.

Discussion

Natural products and molecules based on their structures have aroused great interest in cancer research and therapy.⁷ Triterpenes, compounds isolated from natural sources, present diverse biological activities, being of great interest in cancer research due to their antineoplastic properties.¹³ In addition, they represent a key scaffold to generate new derivatives with greater biological activity, achieving a wide structural diversity through chemical modifications in their C-3, C-17, C-20, and C-28 positions, which can enhance their effects on cancer cells.^14,47

In this context, to improve the biological activity of the lupane-type triterpene T2 in leukemia cell lines, a series of analogues were designed in the present study by structural modification at the C-28 and C-30 positions. As could be observed, of all the compounds tested, compounds T2c, T2d, and T2e containing in their structure an α,β-unsaturated aldehyde group at C-30 exhibited the best activities in the panel of cell lines tested, with a further increase due to the reduction of the carboxylic acid at C-28 (T2d).

During the process of structural modification of compound T2 at the C-30 position, it was possible to isolate and identify a reaction by-product, the compound 3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol (T2e, 22%).

Importantly, this molecule is similar to the compound 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T3), except for the hydroxymethylene group at C-28. It is worth mentioning that the compound T3 has already been evaluated in leukemia cell lines K562 and HL-60 showing inhibition of cell proliferation and cell death by apoptosis.³⁰ Compound T2e exhibited a stronger effect on the five cell lines tested than compound T2. From these differences in biological activities, interaction with enzymes or receptors could be the main mechanism of action involved. Additionally, compound T2e contains a formyl group at C-30 in its structure as an α,β-unsaturated aldehyde. This oxidized group apparently induces a more selective effect against leukemia cell lines than against solid tumors.²⁵

The α,β-unsaturated carbonyl groups, such as aldehydes or ketones, induce a cytotoxic effect through the formation of Michael-type adducts, interacting with thiols in proteins and peptides, which impacts cancer progression and drug resistance.^4,48 In addition, these compounds can induce apoptosis through the mitochondrial pathway, a key cell death pathway and a promising therapeutic target in cancer treatment.⁴⁸

There are numerous reports on the anticancer activity of lupane-type triterpenes.^49–51 Triterpenes, such as betulinic acid, betulin, lupeol and 23-hydroxybetulinic acid, show anticancer activity by inhibiting the proliferation of several cell lines, including leukemic ones.^24,52 Their main mechanism of action is the induction of apoptosis, through loss of mitochondrial membrane potential,⁵³ DNA fragmentation,⁵³ release of proapoptotic proteins such as Bax, cytochrome c and Smac,^53,54 activation of caspases, production of reactive oxygen species and glutathione depletion.^55,56 In addition, these compounds have been found to arrest the cell cycle in the G1/S phase.³⁰

The selective inhibitory activity of betulinic acid is of interest for the study of other lupane-skeleton triterpenes, as well as to determine their mode of action. In a previous study, structural modification of the triterpene 3α,24-dihydroxylup-20(29)-en-28-oic acid by the introduction of a methyl group at C-3 generated the derivative 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid with enhanced antileukemic activity in K562 and HL60 cell lines.³¹ In addition, this derivative was found to promote apoptosis by generating ROS and altering mitochondrial membrane potential. Molecular docking analyses revealed that compound 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid has an affinity against BCL-2 and EGFR proteins.³¹

Due to the reports described on lupane-type triterpenes and the possible antileukemic potential of T2c-T2e derivatives, a more detailed study of these compounds together with betulin was performed, since they demonstrated significant cytotoxicity in leukemia cell lines (≤ 20 µM). To evaluate whether the induced cell death occurred by apoptosis, their mechanism of action was analyzed by determining the apoptotic effect at 48 h at ½ IC₅₀ and IC₅₀ concentrations, using annexin V binding as a marker. Annexin V is useful for identifying apoptotic cells as it binds specifically and with high affinity to phosphatidylserine residues.^57,58 During apoptosis, phosphatidylserine translocate from the inner to the outer layer of the plasma membrane.⁵⁸ Flow cytometry was used to quantify the percentage of apoptotic cells.

In the present results, it was observed that compounds T2c-T2e showed different apoptotic rates depending on the cell line involved. In particular, at IC₅₀, compounds T2c-T2e showed a stronger effect on CCRF-CEM (T2c, 97.35 ± 0.61%; T2d, 97.23 ± 0.88; T2e, 98.19 ± 0.77%) and JURKAT (T2c, 96.64 ± 1.15%; T2d, 95.67 ± 0.60%; T2e, 97.15 ± 0.23%) cell lines. For the REH cell line, compounds T2d (97.03 ± 1.44%) and T2e (98.07 ± 1.10%) induced high apoptosis rates. However, for MOLT-4, and THP-1 lines, only T2e generated the highest apoptotic rate at IC₅₀ (59.53 ± 1.47%, and 63.23 ± 2.82% respectively), which seems to indicate that for these cell lines compounds T2c and T2d have another type of mechanism of action, since it has been reported that triterpenes can exert their biological effects through different mechanisms. For example, a previous study demonstrated that compound T3 induces apoptosis and cell cycle arrest in the G2/M phase in K562 and HL-60 cells.³⁰ In addition, in silico studies revealed its interaction with the CDK1/cyclin B/Csk2 complex, an essential mitotic promoter.³⁰

On the other hand, betulin at IC₅₀ has a mild apoptotic effect on REH (16.48 ± 1.52%) and THP-1 (39.66 ± 2.46%) cells. However, it is documented to have a low bioavailability, which prevents it from producing the desired therapeutic effect under in vivo conditions. Therefore, the introduction of a phosphate group can improve various properties such as bioavailability and solubility, as well as enhanced biological activity.⁵⁵

Moreover, pentacyclic triterpenes, such as betulin and betulinic acid, have been shown to inhibit proliferation in leukemic cells and to have effects on the BCL-2 family of proteins, which are involved in the control and regulation of mitochondrial apoptotic events, suggests the usefulness of molecular docking analyses on the BCL-2 protein using T2c-T2e derivatives.^40–42 These derivatives were found to be less affine for the antiapoptotic protein BCL-2 (T2c, −8.70 kcal/mol; T2d, −8.52 kcal/mol; T2e, −8.80 kcal/mol) than its precursor T2 (−9.25 kcal/mol). Compared to betulin (−8.57 kcal/mol), the T2e derivative showed a better affinity. However, T2c-T2e derivatives presented better affinity than T3 (−8.26 kcal/mol) compound. The present molecular docking results suggest that interaction with BCL-2 may be involved in the biological activities observed in the evaluated cell lines.

In addition, EGFR was selected because it is considered a promising therapeutic target. It plays an essential role in cell survival and proliferation and has a downstream signaling pathway associated with the BCL-2 family, and it has been shown that it is dysregulated in leukemia.^43–45 In the present results, the T2c (−8.75 kcal/mol), T2d (−9.51 kcal/mol), and T2e (−10.72 kcal/mol) derivatives exhibited higher affinity than their precursor molecule (T2, −8.16 kcal/mol) upon docking with EGFR and were even more affine than the co-crystallized inhibitor (−8.07 kcal/mol). In particular, the T2e derivative was found to be more binding at EGFR than betulin (−9.42 kcal/mol), T3 (−9.04 kcal/mol) and its precursor T2. This result could have been due to the formation of three interactions through hydrogen bonds with residues LEU764, ARG817, and GLU738. On the other hand, these interactions may be key in the observed effects of T2e, since they did not occur with the other compounds analyzed.

Another derivative that appears interesting for further studies is T2c derivative, which showed cytotoxic activity against leukemia cell lines, and it exhibited no significant activity against normal mononuclear cells (IC₅₀= 235.02 ± 3.1). This derivative could covalently be bind to proteins due to its Michael-type acceptor, which explains its cytotoxicity. To improve its selectivity, it is suggested to substitute it with a hydrazone or azine group. Previous studies have shown that this modification in 30-oxobetulinic acid generates derivatives selective for leukemic cells (CCRF-CEM) without affecting non-malignant fibroblasts.⁵⁹ In summary, the design and structural modification of the T2c derivative could be directed towards introducing azine groups to improve its selectivity and reduce cytotoxicity in normal cells.

As can be seen from the results, compounds T2c, T2d showed a significant effect on leukemic cells. However, they have low water solubility (Log P of 5.84 and 6.09 respectively), and do not meet all the parameters of Lipinski's rule of five as is the case with T2e. These criteria are intended to aid in the development of orally bioavailable drugs with

high gastrointestinal absorption, a key goal in drug discovery.^35,46 The low solubility of T2c and T2d suggests a lower likelihood of passive absorption in the gastrointestinal tract. In addition, the results of pharmacokinetic analysis of T2c and T2d do not predict that they are inhibitors of CYP450 isoforms, which could reduce the risk of toxicity and minimize the occurrence of unwanted side effects, such as drug and byproduct interaction, and accumulation.³⁵ These findings underscore the need for future structural modifications to improve the physicochemical parameters of T2c and T2d, thereby enhancing their therapeutic potential.

These results indicate that T2c-T2e derivatives could be potential drug candidates for future studies in the treatment of leukemia, as there is currently no effective treatment for this disease. Among all the derivatives evaluated, compound T2e demonstrated the ability to induce cell death and generate apoptotic effects to varying degrees in leukemia cell lines, potentially as a possible inhibitor of BCL-2 and EGFR. However, it is important to deepen our understanding of the mode of action of T2e, in particular by exploring the expression of genes and proteins involved in the main mechanisms of resistance and survival of leukemic cells, the evaluation of the collateral population and stem cells, the determination of its bioavailability, and, finally, toxicity studies. These results highlight the importance of future research focused on key aspects to validate and advance in the development of T2e as a potentially effective therapy against leukemia.

Limitations of Study

This research allowed us to determine which structural modification enhances the biological activity, the effect on apoptosis, and the possible target observed in the molecular docking analysis. In this study we chose two proteins overexpressed in leukemia cell lines and involved in apoptosis, EFGR and BCL-2. Nevertheless, it is possible that in the future a larger quantity of the active compound (T2e) may be obtained through semi-synthesis, and its effects on cell viability at different times, apoptosis, and molecular bioassays (gene and protein expression) may be evaluated in active cell lines.

On the other hand, in the molecular docking analysis, the ligand as well as the protein were prepared following standard AutoDock Vina conditions, without an explicit assignment of protonation states at pH 7.4. However, the visualization in ChimeraX may not accurately reflect the actual protonation used in docking.

Conclusion

The triterpenes T2, T3, and betulin were isolated from Phoradendron wattii and characterized by ¹H- and ¹³C-NMR experiments. A series of derivatives from 3α,23-O-isopropylidenyl-3α,23-dihydroxy lup-20(29)-en-28-oic (T2) was generated, from which the 3α,23-dihydroxy-30-oxolup-20(29)-en-28-ol derivative (T2e) showed inhibition in leukemia cell lines (CCRF-CEM, REH, JURKAT, MOLT-4, and THP-1) and induction of death by apoptosis to different degrees. The T2e derivative is a promising candidate for future research, as it showed a minor cytotoxic effect on MNC, and it was found to interact with BCL-2 and EGFR in silico. Overall, the present work shows that simple modifications at promising sites in PT can generate derivatives with enhanced biological activity against leukemia cell lines.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251328289 - Supplemental material for Semi-Synthetic Derivatives from a Lupane-Type Triterpene Isolated from Phoradendron wattii with Anti-Leukemia Activity

Supplemental material, sj-docx-1-npx-10.1177_1934578X251328289 for Semi-Synthetic Derivatives from a Lupane-Type Triterpene Isolated from Phoradendron wattii with Anti-Leukemia Activity by Mario J. Noh-Burgos, Sergio García-Sánchez, Fernando J. Tun-Rosado, Antonieta Chávez-González, Rosa E. Moo-Puc, Sergio R. Peraza-Sánchez and Carlos J. Quintal-Novelo in Natural Product Communications

Footnotes

Acknowledgments

R.E.M.-P. thanks the IMSS Foundation, A.C., for the facilities used at Centro de Investigación en Salud Jesús Kumate Rodríguez. Additional thanks to María Antonieta Chávez González for the donation of leukemia cells (CCRF-CEM, JURKAT, MOLT-4, REH, and THP-1) and for the facilities used at Laboratorio de Células Troncales Tumorales, Unidad de Investigación Medica en Enfermedades Oncológicas, UMAE Hosptital de Oncología, CMN Siglo XXI, IMSS. Thanks also to M.Sc. Luis W. Torres-Tapia for his guidance in the Natural Products Chemistry Laboratory of the Biotechnology Unit at CICY.

Author Contributions

Conceptualization, R.E.M.-P. and A.C.-G.; methodology, M.J.N.-B., S.G.-S.; formal analysis, M.J.N.-B. and R.E.M.-P.; investigation, M.J.N.-B., C.Q-N., S.G.-S., F.J.T.-R., A.C.-G., S.R.P.-S., and R.E.M.-P.; resources, R.E.M.-P.; data curation, M.J.N.-B., S.G.-S., and F.J.T.-R.; writing: original draft preparation, M.J.N.-B.; writing: review and editing, M.J.N.-B., C.Q-N., S.G.-S., F.J.T.-R., A.C.-G., S.R.P.-S., and R.E.M.-P.; visualization, M.J.N.-B. and R.E.M.-P.; supervision, C.Q-N., S.G.-S., and A.C.-G.; project administration, R.E.M.-P.; funding acquisition, R.E.M.-P. All authors have read and agreed to the published version of the manuscript.

Availability of Data and Materials

The data used to support the findings of this study are included within the article and are available from the first author upon request.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

This article does not contain any studies with human or animal subjects. In the case of MNC, these were collected according to institutional guidelines, including written informed consent from each donor. All procedures were approved by Ethics and Scientific Committee of the Mexican Institute of Social Security (IMSS), ethics approval number CNIC R-2021-785-025.

Funding

This work was supported by the Consejo Nacional de Humanidades Ciencias y Tecnologías de Mexico (CONAHCYT) under grant A1-S-10616 CB 2017–2018; M.J.N.-B. received a scholarship from CONAHCYT (application number 774184); R.E.M.-P. thanks Fundación IMSS, A.C., for providing facilities at the Centro de Investigación en Salud Dr. Jesús Kumate Rodríguez.

ORCID iDs

Mario J. Noh-Burgos

Fernando J. Tun-Rosado

Antonieta Chávez-González

Rosa E. Moo-Puc

Sergio R. Peraza-Sánchez

Carlos J. Quintal-Novelo

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

Supplemental Material

Figures S1-S2. Nuclear magnetic resonance spectra of compound T2a [Figure S1. ¹H-NMR (400 MHz, CDCl₃), Figure S2. ¹³C-NMR (100 MHz, CDCl₃)]. Figures S3-S4. Nuclear magnetic resonance spectra of compound T2b [Figure S3. ¹H-NMR (400 MHz, CDCl₃), Figure S4. ¹³C-NMR (100 MHz, CDCl₃)]. Figures S5-S6. Nuclear magnetic resonance spectra of compound T2c [Figure S5. ¹H-NMR (400 MHz, CDCl₃), Figure S6. ¹³C-NMR (100 MHz, CDCl₃)]. Figures S7-S8. Nuclear magnetic resonance spectra of compound T2d [Figure S7. ¹H-NMR (400 MHz, CDCl₃), Figure S8. ¹³C-NMR (100 MHz, CDCl₃)]. Figures S9-S10. Nuclear magnetic resonance spectra of compound T2e [Figure S9. ¹H-NMR (400 MHz, CDCl₃), Figure S10.¹³C-NMR (100 MHz, CDCl₃)]. Figures S11-S12. Nuclear magnetic resonance spectra of compound T2f [Figure S11. ¹H-NMR (400 MHz, CDCl₃), Figure S12. ¹³C-NMR (100 MHz, CDCl₃)]. Supplemental material for this article is available online.

Trial Registration Number Data

Not applicable because this article does not contain any clinical trials.

References

Siegel

Miller

Fuchs

, et al. Cancer statistics, 2022. CA Cancer J Clin. 2022;72(1):7–33. doi:https://doi.org/10.3322/caac.21708

Bray

Laversanne

Sung

, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229–263. doi:https://doi.org/10.3322/caac.21834

Ahmad

Kifayatullah Shah

, et al. Prevalence of acute and chronic forms of leukemia in various regions of Khyber Pakhtunkhwa, Pakistan: needs much more to be done!. Bangladesh J Med Sci. 2019;18:222–227. doi:https://doi.org/10.3329/bjms.v18i2.40689

Machado

Jacques

Marceli

, et al. Anti-leukemic activity of semisynthetic derivatives of lupeol. Nat Prod Res. 2021;35(22):4494–4501. doi:https://doi.org/10.1080/14786419.2020.1737051

Moreno

Avilés

Sandoval

, et al. CDKIs p18INK4c and p57Kip2 are involved in quiescence of CML leukemic stem cells after treatment with TKI. Cell Cycle. 2016;15(9):1276–1287. doi:https://doi.org/10.1080/15384101.2016.1160976

O’Reilly

Zeinabad

Szegezdi

. Hematopoietic versus leukemic stem cell quiescence: challenges and therapeutic opportunities. Blood Rev. 2021;50:1–17. doi:https://doi.org/10.1016/j.blre.2021.100850

Newman

Cragg

. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J Nat Prod. 2020;83(3):770–803. doi:https://doi.org/10.1021/acs.jnatprod.9b01285

Barreto

Gotardi

Baggio

, et al. Natural and semisynthetic pentacyclic triterpenes for chronic myeloid leukemia therapy: reality, challenges and perspectives. ChemMedChem. 2021;16(12):1835–1860. doi:https://doi.org/10.1002/cmdc.202100038

Bednarczyk

Wachowiak

Szulc

, et al. Strong and long-lasting antinociceptive and anti-inflammatory conjugate of naturally occurring oleanolic acid and aspirin. Front Pharmacol. 2016;7:202. doi:https://doi.org/10.3389/fphar.2016.00202

10.

Palomares

Pérez

García

, et al. Nutraceutical role of polyphenols and triterpenes present in the extracts of fruits and leaves of Olea europaea as antioxidants, anti-infectives and anticancer agents on healthy growth. Molecules. 2022;27(7):2341. doi:https://doi.org/10.3390/molecules27072341

11.

Kaur

Sharma

Gupta

, et al. Structure-activity-relationship and mechanistic insights for anti-HIV natural products. Molecules. 2020;25(9):2070. doi:https://doi.org/10.3390/molecules25092070

12.

Ahmed

Sajid

Javeed

, et al. Antioxidant, antifungal, and aphicidal activity of the triterpenoids spinasterol and 22,23-dihydrospinasterol from leaves of Citrullus colocynthis L. Sci Rep. 2022;12:4910. doi:https://doi.org/10.1038/s41598-022-08999-z

13.

Majeed

Sangwan

Chinthakindi

, et al. Synthesis of 3-O-propargylated betulinic acid and its 1,2,3-triazoles as potential apoptotic agents. Eur J Med Chem. 2013;63:782–792. doi:https://doi.org/10.1016/j.ejmech.2013.03.028

14.

Bębenek

Chrobak

Marciniec

, et al. Biological activity and in silico study of 3-modified derivatives of betulin and betulinic aldehyde. Int J Mol Sci. 2019;20(6):1–17. doi:https://doi.org/10.3390/ijms20061372

15.

Dzhemileva

Tuktarova

Dzhemilev

, et al. Pentacyclic triterpenoids-based ionic compounds: synthesis, study of structure–antitumor activity relationship, effects on mitochondria and activation of signaling pathways of proliferation, genome reparation and early apoptosis. Cancers (Basel). 2023;15(3):756. doi:https://doi.org/10.3390/cancers15030756

16.

Chodurek

Orchel

Gwiazdoń

, et al. Antiproliferative and cytotoxic properties of propynoyl betulin derivatives against human ovarian cancer cells: in vitro studies. Int J Mol Sci. 2023;24(22):16487. doi:https://doi.org/10.3390/ijms242216487

17.

Chrobak

Kadela

Bębenek

, et al. New phosphate derivatives of betulin as anticancer agents: synthesis, crystal structure, and molecular docking study. Bioorg Chem. 2019;87:613–628. doi:https://doi.org/10.1016/j.bioorg.2019.03.060

18.

Nistor

Trandafirescu

Prodea

, et al. Semisynthetic derivatives of pentacyclic triterpenes bearing heterocyclic moieties with therapeutic potential. Molecules. 2022;27(19):6552. doi:https://doi.org/10.3390/molecules27196552

19.

Bębenek

Kadela

Chrobak

, et al. New acetylenic derivatives of betulin and betulone, synthesis and cytotoxic activity. Med Chem Res. 2017;26:1–8. doi:https://doi.org/10.1007/s00044-016-1713-9

20.

Rajendran

Jaggi

Singh

, et al. Pharmacological evaluation of C-3 modified betulinic acid derivatives with potent anticancer activity. Invest New Drugs. 2008;26(1):25–34. doi:https://doi.org/10.1007/s10637-007-9081-4

21.

Urban

Sarek

Klinot

, et al. Synthesis of A-seco derivatives of betulinic acid with cytotoxic activity. J Nat Prod. 2004;67(7):1100–1105. doi:https://doi.org/10.1021/np049938m

22.

Hata

Hori

Ogasawara

, et al. Anti-leukemia activities of lup-28-al-20(29)-en-3-one, a lupane triterpene. Toxicol Lett. 2003;143(1):1–7. doi:https://doi.org/10.1016/s0378-4274(03)00092-4

23.

Hata

Hori

Takahashi

. Differentiation- and apoptosis-inducing activities by pentacyclic triterpenes on a mouse melanoma cell line. J Nat Prod. 2002;65(5):645–648. doi:https://doi.org/10.1021/np0104673

24.

Khwaza

Mlala

Oyedeji

, et al. Pentacyclic triterpenoids with nitrogen-containing heterocyclic moiety, privileged hybrids in anticancer drug discovery. Molecules. 2021;26(9):2401. doi:https://doi.org/10.3390/molecules26092401

25.

Hata

Ogawa

Makino

, et al. Lupane triterpenes with a carbonyl group at C-20 induce cancer cell apoptosis. J Nat Med. 2008;62:332–335. doi:https://doi.org/10.1007/s11418-008-0236-1

26.

Mutai

Abatis

Vagias

, et al. Lupane triterpenoids from Acacia mellifera with cytotoxic activity. Molecules. 2007;12(5):1035–1044. doi:https://doi.org/10.3390/12051035

27.

Mutai

Abatis

Vagias

, et al. Cytotoxic lupane-type triterpenoids from Acacia mellifera. Phytochemistry. 2004;65(8):1159–1164. doi:https://doi.org/10.1016/j.phytochem.2004.03.002

28.

Cragg

Pezzuto

. Natural products as a vital source for the discovery of cancer chemotherapeutic and chemopreventive agents. Med Princ Pract. 2016;25(2):41–59. doi:https://doi.org/10.1159/000443404

29.

Valencia

García

Torres

, et al. Lupane-type triterpenes of Phoradendron vernicosum. J Nat Prod. 2017;80(11):3038–3042. doi:https://doi.org/10.1021/acs.jnatprod.7b00177

30.

Valencia

Moreno

Ceballos

, et al. Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on leukemia cell lines. Molecules. 2022;27(17):5616. doi:https://doi.org/10.3390/molecules27175616

31.

Valencia

Estrada

Ceballos

, et al. Lupane triterpene derivatives improve antiproliferative effect on leukemia cells through apoptosis induction. Molecules. 2022;27(23):8263. doi:https://doi.org/10.3390/molecules27238263

32.

Castro

Careaga

Sacca

, et al. Lupane triterpenoids and new derivatives as antiproliferative agents against prostate cancer cells. Anticancer Res. 2019;39(7):3835–3845. doi:https://doi.org/10.21873/anticanres.13533

33.

Callies

Bedoya

Beltrán

, et al. Isolation, structural modification, and HIV inhibition of pentacyclic lupane-type triterpenoids from Cassine xylocarpa and Maytenus cuzcoina. J Nat Prod. 2015;78(5):1045–1055. doi:https://doi.org/10.1021/np501025r

34.

Soumya

Lakshmipriya

Klika

, et al. Anticancer potential of rhizome extract and a labdane diterpenoid from Curcuma mutabilis plant endemic to Western Ghats of India. Sci Rep. 2021;11:552. doi:https://doi.org/10.1038/s41598-020-79414-8

35.

Daina

Michielin

Zoete

. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017;7:42717. doi:https://doi.org/10.1038/srep42717

36.

Berman

Westbrook

Feng

, et al. The protein data bank. Nucleic Acids Res. 2000;28(1):235–242. doi:https://doi.org/10.1093/nar/28.1.235

37.

Morris

Huey

Lindstrom

, et al. Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30(16):2785–2791. doi:https://doi.org/10.1002/jcc.21256

38.

Meng

Goddard

Pettersen

, et al. UCSF Chimerax: tools for structure building and analysis. Prot Sci. 2023;32(11):1–13. doi:https://doi.org/10.1002/pro.4792

39.

Pettersen

Goddard

Huang

, et al. UCSF Chimerax: structure visualization for researchers, educators, and developers. Prot Sci. 2021;30(1):70–82. doi:https://doi.org/10.1002/pro.3943

40.

Litaudon

Jolly

, et al. Cytotoxic pentacyclic triterpenoids from Combretum sundaicum and Lantana camara as inhibitors of Bcl-xL/BakBH3 domain peptide interaction. J Nat Prod. 2009;72(7):1314–1320. doi:https://doi.org/10.1021/np900192r

41.

Debatin

. Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother. 2004;53:153–159. doi:https://doi.org/10.1007/s00262-003-0474-8

42.

Jan

Chaudhry

. Understanding apoptosis and apoptotic pathways targeted cancer therapeutics. Adv Pharm Bull. 2019;9(2):205–218. doi:https://doi.org/10.15171/apb.2019.024

43.

Liu

Yang

, et al. Inhibition of K562 leukemia angiogenesis and growth by expression of antisense vascular endothelial growth factor (VEGF) sequence. Cancer Gene Ther. 2003;10:879–886. doi:https://doi.org/10.1038/sj.cgt.7700645

44.

Song

Jiang

. Role of VEGF/VEGFR in the pathogenesis of leukemias and as treatment targets (Review). Oncol Rep. 2012;28(6):1935–1944. doi:https://doi.org/10.3892/or.2012.2045

45.

Alam

Shamsi

, et al. Bax/Bcl-2 cascade is regulated by the EGFR pathway: therapeutic targeting of non-small cell lung cancer. Front Oncol. 2022;12:1–23. doi:https://doi.org/10.3389/fonc.2022.869672

46.

Lipinski

Lombardo

Dominy

, et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46(1-3):3–26. doi:https://doi.org/10.1016/s0169-409x(00)00129-0

47.

Santos

Salvador

Marín

, et al. Synthesis and structure–activity relationship study of novel cytotoxic carbamate and N-acylheterocyclic bearing derivatives of betulin and betulinic acid. Bioorg Med Chem. 2010;18(12):4385–4396. doi:https://doi.org/10.1016/j.bmc.2010.04.085

48.

Hossain

Das

Dimmock

. Recent advances in α,β-unsaturated carbonyl compounds as mitochondrial toxins. Eur J Med Chem. 2019;183:111687. doi:https://doi.org/10.1016/j.ejmech.2019.111687

49.

Fulda

. Betulinic acid for cancer treatment and prevention. Int J Mol Sci. 2008;9(6):1096–1107. doi:https://doi.org/10.3390/ijms9061096

50.

Kaps

Chodurek

Orchel

, et al. Influence of 28-O-propynoylbetulin on proliferation and apoptosis of melanotic and amelanotic human melanoma cells. Postepy Hig Med Dosw (Online). 2016;70:1404–1408. doi:https://doi.org/10.5604/17322693.122767851

51.

Kaps

Gwiazdoń

Chodurek

. Nanoformulations for delivery of pentacyclic triterpenoids in anticancer therapies. Molecules. 2021;26(6):1764. doi:https://doi.org/10.3390/molecules26061764

52.

Lombrea

Scurtu

Avram

, et al. Anticancer potential of betulonic acid derivatives. Int J Mol Sci. 2021;22(7):3676. doi:https://doi.org/10.3390/ijms22073676

53.

Nistor

Rugina

Diaconeasa

, et al. Pentacyclic triterpenoid phytochemicals with anticancer activity: updated studies on mechanisms and targeted delivery. Int J Mol Sci. 2023;24(16):1–23. doi:https://doi.org/10.3390/ijms241612923

54.

Ehrhardt

Fulda

Führer

, et al. Betulinic acid-induced apoptosis in leukemia cells. Leukemia. 2004;18:1406–1412. doi:https://doi.org/10.1038/sj.leu.2403406

55.

Orchel

Chodurek

Jaworska

, et al. Anticancer activity of the acetylenic derivative of betulin phosphate involves induction of necrotic-like death in breast cancer cells in vitro. Molecules. 2021;26(3):1–23. doi:https://doi.org/10.3390/molecules26030615

56.

Dash

Chattopadhyay

Dash

, et al. Self assembled nano fibers of betulinic acid: a selective inducer for ROS/TNF-alpha pathway mediated leukemic cell death. Bioorg Chem. 2015;63:85–100. doi:https://doi.org/10.1016/j.bioorg.2015.09.006

57.

Lee

Meng

Flatten

, et al. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ. 2013;20:64–76. doi:https://doi.org/10.1038/cdd.2012.93

58.

Mariño

Kroemer

. Mechanisms of apoptotic phosphatidylserine exposure. Cell Res. 2013;23:1247–1248. doi:https://doi.org/10.1038/cr.2013.115

59.

Pokorny

Krajcovicova

Hajduch

, et al. Triterpenic azines, a new class of compounds with selective cytotoxicity to leukemia cells CCRF-CEM. Future Med Chem. 2018;10(5):483–491. doi:https://doi.org/10.4155/fmc-2017-0171

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.74 MB