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Article

Semi-Synthesis, Anti-Leukemia Activity, and Docking Study of Derivatives from 3α,24-Dihydroxylup-20(29)-en-28-Oic Acid

by
Mario J. Noh-Burgos
1,2,
Sergio García-Sánchez
3,
Fernando J. Tun-Rosado
4,
Antonieta Chávez-González
3,
Sergio R. Peraza-Sánchez
1 and
Rosa E. Moo-Puc
5,*
1
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán (CICY), Mérida 97205, Mexico
2
Unidad Médica de Alta Especialidad, Centro Médico Ignacio García Téllez, Instituto Mexicano del Seguro Social (IMSS), Mérida 97200, Mexico
3
Unidad de Investigación Médica en Enfermedades Oncológicas, UMAE Hospital de Oncología, Centro Médico Nacional Siglo XXI, IMSS, Ciudad de México 06720, Mexico
4
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico
5
Secretaría de Ciencia, Humanidades, Tecnología e Innovación-Hospital Regional de Alta Especialidad de la Península de Yucatán, IMSS-BIENESTAR, Mérida 97130, Mexico
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(15), 3193; https://doi.org/10.3390/molecules30153193
Submission received: 23 June 2025 / Revised: 20 July 2025 / Accepted: 21 July 2025 / Published: 30 July 2025
(This article belongs to the Special Issue Synthesis and Derivatization of Heterocyclic Compounds)

Abstract

Current treatments against leukemia present several limitations, prompting the search for new therapeutic agents, particularly those derived from natural products. In this context, structural modifications were performed on the triterpene 3α,24-dihydroxylup-20(29)-en-28-oic acid (T1), isolated from Phoradendron wattii. Among the five derivatives obtained, 3α,24-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T1c) exhibited the highest activity, with an IC50 value of 12.90 ± 0.1 µM against THP-1 cells. T1c significantly reduced cell viability in both acute lymphoblastic leukemia (CCRF-CEM, REH, JURKAT, and MOLT-4) and acute myeloid leukemia (THP-1) cell lines, inducing apoptosis after 48 h of treatment, while showing minimal cytotoxicity toward normal mononuclear cells (MNCs). In silico molecular docking studies were conducted against three key protein targets: BCL-2 (B-cell lymphoma 2), EGFR (epidermal growth factor receptor, tyrosine kinase domain), and FLT3 (FMS-like tyrosine kinase 3). The lowest binding energies (kcal/mol) observed were as follows: T1–BCL-2: −10.12, EGFR: −12.75, FLT3: −14.05; T1c–BCL-2: −10.23, EGFR: −14.50, FLT3: −14.07; T2–BCL-2: −11.59, EGFR: −15.00, FLT3: −14.03. These findings highlight T1c as a promising candidate in the search for anti-leukemic drugs which deserves further study.

Graphical Abstract

1. Introduction

Globally, cancer is the second leading cause of death, with 20 million new cases and 9.7 million deaths reported in 2022 [1,2]. Leukemias rank thirteenth among the most common cancers and represent the tenth leading cause of cancer-related death worldwide [2]. Among the types of acute leukemia, acute lymphoblastic leukemia (ALL) is most frequently diagnosed in children and young adults, while acute myeloid leukemia (AML) is more prevalent in adults but is also common in children [3,4,5].
The treatment of choice for leukemia is allogeneic hematopoietic stem cell transplantation [6]. However, it presents major challenges such as the lack of compatible donors and its low efficacy in advanced stages of the disease [6]. Chemotherapy continues to be widely used but faces common challenges in oncology, such as toxicity, adverse side effects, development of cell drug resistance mechanisms, and disease recurrence [7,8,9]. Therefore, the search for new, more effective, and selective drugs against leukemic cells remains an ongoing challenge [8,9].
Natural products have been widely used in the discovery and development of new drugs. It is estimated that 40% of the drugs used in cancer treatment have been inspired by natural products. The isolation of bioactive compounds from different natural sources, together with the generation of structural analogues of these compounds by chemical derivatization, are the most important strategies in the search for anticancer drugs [10,11].
Pentacyclic lupane-type triterpenes (PLTTs) are compounds isolated from natural sources that exhibit notable chemopreventive and antineoplastic properties, but face limitations such as low solubility, bioavailability, and selectivity [12,13,14,15]. To address these challenges, researchers have synthesized derivatives and employed strategies like complexation with cyclodextrins, liposomes, carbon nanotubes, or gold nanoparticles [16,17,18]. Additionally, introducing delocalized lipophilic cationic groups (e.g., TPP, F16, and Rho123) has enhanced their stability, solubility, and selective targeting of tumor cells, improving their therapeutic potential in cancer treatment [19,20].
PLTTs serve as a valuable scaffold for developing compounds with enhanced anti-leukemic activity due to their modifiable positions (C-3, C-17, C-20, and C-28), which improve activity and selectivity against cancer cells [15,21]. For instance, introducing a 1,2,3-triazole at C-3 of betulinic acid enhances activity in acute myeloid leukemia (HL-60 and THP-1) cells [15]. Similarly, adding a propynoyl group at C-28 of betulin increases cytotoxicity in acute lymphoblastic leukemia (CCRF-CEM) and murine leukemia (P388) cells [22]. Additionally, modifying betulin at C-3 with a 4-fluorophenylhydrazone group significantly improves activity against MOLT-4 leukemia cells [23].
Recently, studies have highlighted the importance of structural modifications at the C-30 position of triterpenes to improve their bioactivity and reduce their cytotoxicity in normal cells [7]. For example, the introduction of an aldehyde group on C-30 of betulinic acid resulted in the formation of 30-oxobetulinic acid with cytotoxic activity on cancer and CCRF-CEM leukemia cell lines [24]. In this context, the presence of the formyl group, in the form of α,β-unsaturated aldehyde on C-30, contributed to obtaining compounds more active against leukemic cells compared to their non-functionalized analogues [25,26,27].
Previous studies evaluated the pentacyclic triterpenes 3α,24-dihydroxylup-20(29)-en-28-oic acid (T1) and 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T2) (Figure 1), isolated from Phoradendron wattii [28], in chronic (K562) and acute (HL60) myeloid leukemia cell lines. T2 induced cell cycle arrest at G2/M, likely due to its α,β-unsaturated aldehyde at C-30, which enhanced activity against leukemic cells compared to T1 [29]. Additionally, modifying T1 by introducing a methyl group at C-3 (3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid, T1a) improved its anti-leukemic effects, including apoptosis induction, ROS generation, and mitochondrial membrane disruption [30].
In this context, PLTTs, specifically those isolated from P. wattii, present favorable characteristics such as good yields, structural stability, biological activity, and selectivity [28,29,30]. The triterpene T1 represents an important scaffold due to the presence of promising sites, such as C-3 and C-30, making it a good candidate for simple structural modifications with the intention of generating new semi-synthetic derivatives with improved biological activity on acute lymphoblastic leukemia cell lines (CCRF-CEM, REH, JURKAT, and MOLT-4) and an acute myeloid leukemia cell line (THP-1).

2. Results and Discussion

2.1. Chemical Synthesis of the Target Compounds

Due to the cytotoxic effects observed for compound T1, its derivative T1a, and compound T2 in leukemia cell lines [29,30], in the present study, a series of T1 analogues were developed by minor structural modifications to be evaluated in a panel of leukemia cell lines.
Compound T1 was isolated from the aerial parts of P. wattii according to a previous study [28]. Derivatives were obtained from T1 by a series of reactions targeting the C-3 and C-30 carbons. Initially, T1 was subjected to a C-3 methylation reaction employing CH3I/K2CO3 in anhydrous Me2CO, resulting in the derivative 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid (T1a) with a 68.4% yield (Scheme 1, step a). The structure of T1a was confirmed based on the 1H-NMR spectrum (Figure S1, Supplementary Materials); a new single signal at δ 3.66 characteristic of a methyl group attached to a heteroatom was observed. Furthermore, the 13C-NMR spectrum (Figure S2) showed a new signal at δ 51.4. These results confirmed that T1a is 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid. Subsequently, this compound (T1a) reacted with SeO2 in anhydrous EtOH, carrying out an allylic oxidation reaction on C-30, yielding the acid derivative 3α-methoxy-24-hydroxy-30-oxolup-20(29)-en-28-oic acid (T1b) with a 50% yield (Scheme 1, step b). In the 1H-NMR spectrum (Figure S3), the absence of the signal at δ 1.60–1.80, corresponding to the H-30 protons, is noticeable, and a new signal at δ 9.52, characteristic of aldehyde groups, can be observed. Furthermore, in the 13C-NMR spectrum (Figure S4) the signal at δ 194.1 was assigned to this aldehyde group at C-30. These results confirm that T1b is 3α-methoxy-24-hydroxy-30-oxolup-20(29)-en-28-oic acid.
On the other hand, under the same conditions as for T1b, an allylic oxidation reaction was carried out at the C-30 position of compound T1, yielding the acid derivative 3α,24-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T1c) with a 17% yield and the by-product 3α,24,30-trihydroxylup-20(29)-en-28-oic acid (T1d) with a 19% yield. In the 1H- and 13C-NMR spectra for T1c (Figures S5 and S6), similar signals were observed as for T1b, a new signal at δ 9.51 characteristic of aldehyde groups and a signal at δ 196.9, respectively. On the other hand, the structure of the by-product T1d was confirmed based on the 1H-NMR spectrum (Figure S7); a new signal was observed at δ 4.05 (JAB = 15.8 Hz, 2H) with three lines, the inner signal overlapped, and the exterior signals were very attenuated (roof effect). This pattern is characteristic of AB systems and corresponds to the isolated geminal nuclei in H-30 adjacent to the hydroxyl group. Furthermore, in the 13C-NMR spectrum (Figure S8), a new signal was observed at δ 65.2 that corresponds to the oxygen-bound methylene of C-30. These results confirm that T1d is 3α,24,30-trihydroxylup-20(29)-en-28-oic acid.
Compound T1d was obtained through a selective oxidation of the allylic system present in the precursor triterpene, using selenium dioxide (SeO2) as the oxidizing agent. This type of transformation is a well-known allylic oxidation that proceeds via a mechanism involving an Alder-ene-type cycloaddition, followed by a [2,3]-sigmatropic rearrangement, which generates an unstable seleninic intermediate. Hydrolysis of this intermediate and elimination of reduced selenium species (Se(IV)) lead to the formation of the allylic alcohol at the C-30 position, corresponding to compound T1d [31].
Finally, C-3 acetylation of T1 employing acetic anhydride yielded the derivative 3α-acetyl-24-hydroxylup-20(29)-en-28-oic acid (T1e) with a 62% yield (Scheme 1, step c). The structure of T1e was confirmed based on the 1H-NMR spectrum (Figure S9), a new single signal at δ 2.02 ppm, corresponding to the methyl group of the acetyl substituent. In addition, in the 13C-NMR spectrum, two new signals were observed at δ 171.5 and δ 21.4 ppm corresponding to the carbonyl and methyl groups of the acetyl substituent, respectively. Under mild acetylation conditions (Ac2O/pyridine, room temperature), only the C-3 hydroxyl group was acetylated, while the C-24 hydroxyl remained unchanged. This selectivity can be explained by the greater accessibility and nucleophilicity of the equatorial C-3 hydroxyl, whereas the C-24 hydroxyl is less accessible due to steric hindrance from the rigid lupane side chain. Consistently, NOESY data from lupane-type triterpenes isolated from P. wattii showed that H-24 and H-25 are spatially close, indicating that the C-24 hydroxyl resides in a hindered environment, which likely limits its reactivity [28].

2.2. In Vitro Anti-Leukemia Activity

The viability effect of T1, T2, and T1 derivatives using different concentrations was evaluated on five leukemia cell lines: CCRF-CEM, REH, JURKAT, MOLT-4, and THP-1, the results being compared with dasatinib (Table 1). Of particular interest, the derivatives T1b and T1c showed significant activity in all cell lines evaluated, with IC50 values lower than that of the parent compound T1, which showed no activity, while T2 showed greater selectivity against the CCRF-CEM and REH cell lines compared to that against the JURKAT cell line. On the other hand, the T1a derivative showed moderate activity on the REH cell line (IC50 = 64.22 µM), while T1d and T1e showed no activity.
Among all the derivatives, compounds T1b and T1c exhibited the highest activity across the tested cell lines. Notably, both contain an α,β-unsaturated aldehyde group at C-30, a key structural feature associated with increased cytotoxicity. The methylation of the hydroxyl group at C-3 in T1b further enhanced its activity. α,β-Unsaturated carbonyl groups, such as aldehydes and ketones, exert cytotoxic effects by forming Michael-like adducts, as the electrophilic β-carbon readily reacts with thiol groups in proteins and peptides involved in cancer progression and drug resistance. Beyond their interaction with cellular thiols, these groups can also trigger apoptosis via the mitochondrial pathway, a crucial mechanism of cell death and a promising target for cancer therapy [32,33].
For example, oxidation at C-30 of lupeol, which leads to the formation of an α,β-unsaturated aldehyde in the isopropylidene fragment, has produced derivatives with cytotoxic activity against leukemia cell lines. The compound 3β-hydroxylup-20(29)-en-30-al showed IC50 values of 11.72 ± 1.06 µM in JURKAT cells and 19.52 ± 0.47 µM in K562 cells [7]. However, this compound also reduced the viability of normal peripheral blood lymphocytes (PBLs) by 32.17% compared to untreated controls, suggesting a potential cytotoxic effect on non-tumor cells [7]. In a previous study, another 30-oxo-lupane compound (T2) was reported to reduce K562 cell viability to 88.6 ± 0.40% at 102 µM and to 24.3 ± 1.95% at 204 µM, while showing a moderate effect on HL-60 cells (88.35 ± 3.85% and 85.15 ± 3.05%, respectively) [29].
In comparison, compounds T1b and T1c exhibited potent cytotoxic activity across all leukemia cell lines tested, with IC50 values ranging from 4.05 to 14.97 µM for T1b and from 12.90 to 24.92 µM for T1c. These results highlight the significance of introducing an α,β-unsaturated carbonyl group at C-30 as a key structural modification to enhance cytotoxic potential in leukemia models, while also emphasizing the importance of assessing selectivity toward cancer cells. In this context, previous studies have demonstrated that incorporating a formyl group at C-30 is a critical modification, yielding derivatives with markedly higher activity against leukemic cells compared to the precursor molecule [7,24,32].
On the other hand, compounds T1c and T2 are epimers, differing in the orientation of the hydroxymethyl group at the C-4 position, either forward (C-24) or backward (C-23), respectively. Compound T1c showed a significant effect on all cell lines tested, whereas T2 generated less effect on CCRF-CEM, REH, and JURKAT cell lines. Based on these differences in activities and stereochemistry, interaction with enzymes or receptors is possibly the main mechanism of action involved [29].

2.3. Effect of Compounds on the Viability of Peripheral Blood Mononuclear Cells (MNCs)

The safety of the compounds was measured by assessing the viability effect on MNCs (Table 2). When the T1b derivative was evaluated at the maximum concentration of 200 µM, it showed a potent cytotoxic effect on normal cells (IC50 = 10.78 µM), while T1c showed a non-significant effect on MNCs, implying a high selectivity (SI > 10) of this derivative against leukemia cell lines. On the other hand, T1a, T1d, and T1e showed no activity at the maximum concentration evaluated.
The T1b derivative was cytotoxic in normal MNC cells. Therefore, simple modifications such as the introduction of methyl groups together with the presence of the formyl group in this type of triterpenes may confer cytotoxic effects on normal MNC cells. The presence of the Michael-type acceptor on C-30 could facilitate their covalent binding to proteins, which contributes to their cytotoxicity [33]. Therefore, structural modifications can be made by replacing the formyl group with hydrazones or azines, which can interact in a similar manner [24]. One study showed that the introduction of asymmetric azines into 30-oxobetulinic acid generated derivatives selective for CCRF-CEM leukemia cells and proved to be inactive in non-malignant human fibroblasts [24]. Therefore, the design and structural modification of T1b could be oriented towards the introduction of azine groups to improve its selectivity.

2.4. Apoptosis Assay

Since compound T1c was the only derivative that decreased the cell viability of leukemic cells (IC50 ≤ 20 µΜ), without affecting viability in normal MNC cells, its mode of action to exert cytotoxic effects was evaluated by flow cytometry techniques, determining the apoptotic effect at 48 h, using ½ IC50 and IC50 concentrations obtained in the viability assay in the different leukemia cell lines. Dasatinib at different concentrations was included as a positive control. Cell death was quantified by dual staining with annexin V-FITC and DAPI. Annexin V binds to externalized phosphatidylserine residues during early and late apoptosis, whereas DAPI penetrates membrane-compromised cells, distinguishing between late apoptotic and necrotic cells [34,35]. The total percentage of apoptotic cells was calculated as the sum of early and late apoptotic populations. The results were normalized to 100% with respect to the negative control.
To better understand the type of cell death induced by T1c, it is essential to distinguish the mechanistic differences between early and late apoptotic processes. Early apoptosis involves the activation of initiator caspases (caspase-8 or -9) and mitochondrial outer membrane permeabilization (MOMP), mediated by Bax/Bak oligomerization. This leads to cytochrome c release and apoptosome formation. At this stage, phosphatidylserine is externalized to the outer leaflet of the plasma membrane, without loss of membrane integrity. In contrast, late apoptosis is characterized by the activation of executioner caspases (caspase-3, -6 and -7), which degrade structural proteins and activate caspase-activated DNase (CAD), resulting in nuclear condensation, internucleosomal DNA fragmentation, formation of apoptotic bodies, and irreversible cell disintegration. These events ensure the efficient and controlled elimination of apoptotic cells, minimizing the risk of triggering inflammatory responses [36,37,38,39].
In the present results, it was observed that T1c induces apoptotic death in leukemic cells to varying degrees. For example, for the MOLT-4 cell line (Figure 2), the apoptosis rate was 96.13 ± 0.71% (early apoptotic cells: 1.40%; late apoptotic cells: 94.74%) for the positive control, while T1c at ½ IC50 exhibited apoptosis rates of 69.91 ± 2.17% (early apoptotic cells: 41.15%; late apoptotic cells: 28.76%). When the IC50 was evaluated, the apoptosis rate changed to 94.58 ± 0.45% (early apoptotic cells: 30.12%; late apoptotic cells: 64.46%), similar to the positive control. These results highlight the apoptotic effect of T1c on the MOLT-4 cell line.
Under the same conditions, for the CCRF-CEM cell line at 48 h of treatment, the apoptosis rate was 94.84 ± 1.07% (early apoptotic cells: 1.66%; late apoptotic cells: 92.90%) in the positive control (Figure 3), while for T1c at ½ IC50 concentration it was 62.40 ± 1.57% (early apoptotic cells: 16.98%; late apoptotic cells 45.42%). However, when evaluated at the IC50 concentration, an increase in apoptosis of 97.13 ± 0.36% (early apoptotic cells: 16.92%; late apoptotic cells: 80.21%) similar to the positive control was observed.
On the other hand, when assayed against the JURKAT cell line (Figure 4), the positive control showed 97.19 ± 0.11% apoptosis (early apoptotic cells: 0.59%; late apoptotic cells: 96.60%). The T1c derivative showed apoptosis rates of 67.14 ± 2.18% (early apoptotic cells: 30.60%; late apoptotic cells: 36.55%) at the concentration of ½ IC50. When evaluated at IC50, the apoptosis rate changed to 96.37 ± 0.49% (early apoptotic cells: 47.61%; late apoptotic cells: 48.72), similar to that of the positive control.
For the acute myeloid leukemia cell line THP-1 (Figure 5), the positive control showed 32.14 ± 3.15% apoptosis (early apoptotic cells: 7.21%; late apoptotic cells: 24.93%). The T1c derivative showed an effect at ½ IC50 of 29.40 ± 2.66% (early apoptotic cells: 14.50%; late apoptotic cells: 14.90%). When IC50 values were used, death by apoptosis was clearly higher at 52.23 ± 1.69% (early apoptotic cells: 22.84%; late apoptotic cells: 29.39%) than the positive control. Thus, the THP-1 line is clearly less sensitive to this compound than the rest of the cell lines. The T1c derivative induces apoptotic death in leukemic cells to varying degrees.

2.5. Molecular Docking

The ability of the T1c derivative to induce apoptosis in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cell lines highlighted the importance of performing molecular docking analysis with key targets in the search for new compounds with anti-leukemic activity. For this purpose, the Molecular Operating Environment (MOE) software was used [40], focusing on key leukemia-associated targets: the first one was BCL-2, which is related to the control and regulation of apoptosis; this protein blocks apoptosis through the intrinsic pathway [38,39,41]. The second one was the epidermal vascular endothelial growth factor receptor (EGFR), which plays a key role in cell survival and proliferation; in addition, it has downstream signaling pathways associated with the BCL-2 family [42,43,44]. Finally, the third one was the FMS-like tyrosine kinase 3 (FLT3) gene, which is mainly expressed in the bone marrow, especially in CD34+ hematopoietic stem cells and early progenitor cells of the myeloid and lymphoid lineages; besides, it has downstream signaling pathways associated with PI3K/AKT, RAS/MAPK, and STAT5 that promote proliferation and suppress apoptosis [45,46,47]. The docking scores of compounds T1, T2, and T1c were compared with betulinic acid and a co-crystallized inhibitor (Table 3).

2.5.1. Molecular Docking with the BCL-2 Protein

Molecular docking analysis with the BCL-2 protein revealed that the T1c derivative presented an affinity energy similar to that of its molecular precursor (T1) and a lower affinity than its T2 epimer. Relative to betulinic acid, T1c showed better affinity (−10.23 kcal/mol). However, the co-crystallized inhibitor presented the best affinity score (−17.72 kcal/mol). Taken together, these results indicate that the effects observed for compounds T1c and T2 are not directly related to BCL-2 inhibition. The crystal structures of BCL-2 in complex with a BH3 domain inhibitor were used. Figure 6 shows the different binding modes of the most active compound (T1c) with this key molecular target.

2.5.2. Molecular Docking with the EGFR Tyrosine Kinase

The crystal structures of EGFR with erlotinib inhibitors were used. In the present docking results with the EGFR kinase domain, the T1c derivative (−14.50 kcal/mol) showed higher affinity than its precursor molecule, T1 (−12.75 kcal/mol), betulinic acid (−12.73 kcal/mol), and even presented better affinity energy than the co-crystallized inhibitor (−13.97 kcal/mol), and, furthermore, it presented similar affinity energy to its epimer compound, T2 (−15.00 kcal/mol). On the other hand, the T1c derivative showed two favorable interactions at the binding site through hydrogen bridge interactions with residues GLY833 and LYS836 (Figure 7). These results suggest that the apoptotic effect observed during the experimental analysis could be due to the interaction with the EGFR molecular target, which in turn can trigger cell death in the different cell lines evaluated.

2.5.3. Molecular Docking with FLT3 Protein

Finally, for the FLT3 kinase domain, the binding energies of compounds T1, T2, and the T1c derivative were found to be slightly more affine than betulinic acid (−13.64 kcal/mol) and showed a lower affinity than the co-crystallized inhibitor (−18.17 kcal/mol). Based on the docking scores for interaction with FLT-3, none of the exerted effects of the T1c derivative were found to be related to this target. The crystal structures of FLT3 with quizartinib inhibitors were used. Figure 8 shows the different binding modes of T1c with this key molecular target.

2.5.4. Similarity to Drugs

As a complementary study, we submitted the molecular structures of the four compounds to in silico analysis via the SwissADME platform to predict their physicochemical and pharmacokinetic properties (Table 3).
Lipinski’s rule of six was applied for betulinic acid, T1, T2, and the T1c derivative. Of all the compounds, the T1c derivative and its epimer compound T2 complied with all the rules, with acceptable values for molecular weight (≤500), hydrogen bond donors (nHBA ≤ 5), hydrogen bond acceptors (nHBD ≤ 10), lipophilicity (expressed as cLogP ≤ 5), molar refractivity (TPSA ≤ 140 Å), and rotatable bonds (nROTB ≤ 10) [48,49]. On the other hand, results from the SwissADME platform showed that the T1c derivative and T2 have a higher probability of passive absorption in the gastrointestinal tract than T1 and betulinic acid. In addition, T1c and T2 are predicted not to inhibit with some of the key CYP450 isoforms (Table 4) [48]. The results obtained suggest the T1c derivative as a good candidate for further studies.
The results suggest that the T1c derivative is a good candidate for future studies on its role in the treatment of leukemia, as it is able to induce apoptosis in ALL and AML cells, as well as potentially act as an EGFR inhibitor. Notably, the SwissADME platform showed promising results for the T1c derivative, as it complies with all Lipinski’s rules, with good water solubility (Log P: 4.66), and it has a higher probability of passive absorption in the gastrointestinal tract. In addition, T1c is predicted to be a non-substrate of P-glycoprotein, which is a positive feature, since this drug efflux pump has been linked to anticancer drug resistance due to low drug accumulation in multidrug-resistant cells. Putting all this together, it can be stated that T1c is a promising molecule. Therefore, gaining a thorough understanding of its mode of action, exploring gene and protein expression, evaluating the effects of T1c on leukemia stem cells, and conducting in vivo bioavailability and toxicity studies are crucial.

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded in CDCl3 and CD3OD on a Bruker Avance 400 spectrometer (Billerica, MA, USA). All chemical shifts (δ) were referenced to the residual signals of the deuterated solvent, and coupling constants are reported in hertz (Hz).
Thin-layer chromatography (TLC) was performed on an aluminum sheet precoated with silica gel 60 F254 (Sigma-Aldrich, Saint Louis, MO, USA), using a layer of 0.25 mm for analytical purposes and 0.5 mm for preparative application. Spots were detected under UV light at 254 and 366 nm or after spraying with a mixture of H2SO4-AcOH-H2O (1:20:4). Column chromatography (CC) was carried out, employing silica gel 60 with particle sizes ranging from 63 to 200, 40 to 63, or 2 to 25 µm, as well as Sephadex LH-20 (Sigma-Aldrich), depending on the purification needs.
All bioassays were conducted under sterile conditions inside a Class II, Type A2 laminar flow hood (NuAire, Plymouth, MN, USA). Cell cultures were maintained in a NuAire CO2 incubator equipped with a water-jacketed chamber and an HEPA filtration system.

3.2. Extraction and Isolation of Compounds

In a previous study, the compounds 3α,24-dihydroxylup-20(29)-en-28-oic acid (T1) and 3α,23-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T2) were isolated from the aerial parts of Phoradendron wattii [28]. Briefly, the dried aerial material of P. wattii was macerated at room temperature with methanol. The methanolic extract obtained was concentrated under reduced pressure and suspended in a hydroalcoholic solution of methanol and water (1:3). Subsequently, the mixture was successively partitioned with hexane, dichloromethane, and ethyl acetate to obtain the different organic fractions. The hexane and dichloromethane fractions were purified by silica gel column chromatography, using different solvent systems, to isolate compounds T1 and T2. Structural elucidation was performed by NMR spectroscopy as well as high-resolution mass spectrometry (HRMS) [28].

3.3. Synthesis of Compound T1a from T1

A mixture of T1 (100 mg, 0.21 mmol) and K2CO3 (540 mg, 3.90 mmol) was solubilized in 2.5 mL of CH3I and 5 mL of anhydrous acetone. The reaction was stirred at room temperature for 24 h. After completion, the mixture was subjected to liquid–liquid extraction with H2O and EtOAc (1:2 × 2, followed by 1:1). The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure [30]. The crude product was purified by column chromatography (hexane-acetone, 80:20) to yield 78 mg (75.7%) of 3α-metoxy-24-hydroxylup-20(29)-en-28-oic acid (T1a).
3α-Metoxy-24-hydroxylup-20(29)-en-28-oic acid (T1a): white, amorphous powder (CHCl3), Rf = 0.36 (hexane-Me2CO, 80:20); 1H-NMR (CDCl3, 400 MHz) δ 4.73 (d, J = 2.1 Hz, 1H), 4.60 (dd, J = 2.3, 1.4 Hz, 1H), 3.66 (s, 4H), 3.52 (dd, J = 10.6, 5.1 Hz, 1H), 3.38 (d, J = 11.3 Hz, 1H), 3.08 (br s, 1H), 2.99 (ddd, 1H), 2.25–2.16 (m, 1H), 2.17 (br s, 1H), 1.93–1.85 (m, 3H), 1.68 (s, 3H), 1.66–1.54 (m, 3H), 1.54–1.30 (m, 13H), 1.29–1.10 (m, 3H), 1.04 (dd, J = 13.0, 4.6 Hz, 1H), 0.99 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.67 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ 176.9, 150.8, 109.7, 71.5, 56.7, 51.4, 50.5, 49.6, 47.2, 43.1, 42.6, 41.0, 40.6, 38.4, 37.2, 37.1, 34.0, 33.1, 32.3, 30.8, 30.8, 29.8, 26.6, 25.5, 21.0, 19.5, 18.2, 17.9, 16.3, 16.1, 15.0; see Figures S1 and S2.

3.4. Synthesis of Compound T1b from T1a

Compound T1b was obtained by reacting T1a (45.1 mg, 0.09 mmol) with SeO2 (51.4 mg, 0.46 mmol) in 10 mL of anhydrous ethanol under reflux for 48 h. After completion, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting crude product was treated with 20 mL of water and extracted with dichloromethane (3 × 30 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography using a 1:1 mixture of hexane and ethyl acetate, affording 23 mg (49.5%) of 3α-metoxy-24-hydroxy-30-oxolup-20(29)-en-28-oic acid (T1b) [50,51,52].
3α-Metoxy-24-hydroxy-30-oxolup-20(29)-en-28-oic acid (T1b): white, amorphous powder (CHCl3), Rf = 0.38 (hexane-EtOAc, 50:50); 1H-NMR (CDCl3, 400 MHz) δ 9.52 (s, 1H), 6.28 (s, 1H), 5.90 (s, 1H), 3.67 (s, 3H), 3.65 (pt, 1H), 3.51 (d, J = 11.3 Hz, 1H), 3.38 (d, J = 11.3 Hz, 1H), 3.32 (ddd, 1H), 2.97 (br s, 1H), 2.29–2.25 (m, 1H), 2.24 (br s, 1H), 2.13–2.04 (m, 1H), 1.98–1.86 (m, 4H), 1.70–1.56 (m, 3H), 1.54–1.24 (m, 11H), 1.26–1.14 (m, 3H), 0.94 (dd, J = 13.0, 4.1 Hz, 1H), 0.96 (s, 3H), 0.90 (s, 3H), 0.84 (s, 3H), 0.67 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ 194.1, 175.7, 155.6, 133.0, 70.5, 55.8, 50.5, 50.5, 49.9, 49.3, 42.1, 42.1, 41.6, 39.9. 39.6, 37.2, 36.2, 36.0, 33.0, 32.1, 31.3, 31.1, 28.7, 26.4, 25.6, 19.9, 17.2, 16.9, 15.3, 15.0, 13.9; see Figures S3 and S4.

3.5. Synthesis of Compounds T1c and T1d from T1

A solution of compound T1 (50 mg, 0.11 mmol) and SeO2 (58.7 mg, 0.53 mmol) in 10 mL of anhydrous ethanol was prepared and subjected to the same procedure described for the synthesis of T1b. The resulting residue was purified by column chromatography (hexane-EtOAc, 1:1), yielding 5.4 mg (17%) of 3α,24-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T1c) and 6.0 mg (19%) of the subproduct 3α,24,30-trihydroxylup-20(29)-en-28-oic acid (T1d).
3α,24-Dihydroxy-30-oxolup-20(29)-en-28-oic acid (T1c): white, amorphous powder (MeOH), Rf = 0.40 (hexane-EtOAc, 50:50); 1H-NMR (CD3OD, 400 MHz) δ 9.51 (s, 1H), 6.39 (s, 1H), 6.02 (s, 1H), 3.59 (pt, 1H), 3.51 (d, J = 10.9 Hz, 1H), 3.39 (ddd, 1H), 3.33 (d, J = 10.7 Hz, 1H), 2.38–2.24 (m, 1H), 2.26 (br s, 1H), 2.14–2.02 (m, 1H), 1.98–1.86 (m, 4H), 1.68–1.56 (m, 3H), 1.54–1.30 (m, 13H), 1.26–1.14 (m, 3H), 0.94 (dd, J = 12.7, 4.3 Hz, 1H), 1.01 (s, 3H), 0.98 (s, 3H), 0.88 (s, 3H), 0.70 (s, 3H). 13C-NMR (CD3OD, 100 MHz) δ 196.9, 180.6, 158.8, 135.0, 76.5, 71.5, 57.9, 52.4, 51.6, 44.5, 43.6, 41.9, 41.3, 39.5, 38.2, 38.1, 35.1, 34.2, 33.7, 33.3, 30.8, 30.5, 28.7, 26.7, 22.0, 19.0, 17.8, 16.9, 16.7, 15.0; see Figures S5 and S6.
3α,24,30-Trihydroxylup-20(29)-en-28-oic acid (T1d): white, amorphous powder (MeOH), Rf = 0.22 (hexane-EtOAc, 50:50); 1H-NMR (CD3OD, 400 MHz) δ 4.96 (s, 1H), 4.88 (s, 1H), 4.05 (JAB = 15.8, 2H), 3.59 (pt, 1H), 3.51 (d, J = 11.0 Hz, 1H), 3.32 (d, J = 11.0 Hz, 1H), 2.91 (ddd, 1H), 2.38–2.24 (m, 1H), 2.26 (br s, 1H), 2.10–1.99 (m, 1H), 1.93–1.83 (m, 2H), 1.78–1.68 (t, 1H), 1.64–1.24 (m, 18H), 1.22–1.11 (m, 3H), 0.94 (dd, J = 12.7, 4.3 Hz, 1H), 1.05 (s, 3H), 0.98 (s, 3H), 0.89 (s, 3H), 0.71 (s, 3H). 13C-NMR (CD3OD, 100 MHz)δ180.7, 156.5, 106.8, 76.5, 71.5, 65.2, 57.7, 51.8, 51.1, 44.5, 44.1, 43.6, 42.0, 41.3, 39.5, 38.2, 38.1, 35.1, 34.3, 33.5, 33.4, 30.9, 28.0, 26.7, 22.1, 19.1, 17.9, 16.9, 16.7, 15.2; see Figures S7 and S8.

3.6. Synthesis of Compound T1e from T1

Compound T1 (20.0 mg, 0.04 mmol) was dissolved in 10 mL of acetic anhydride (Ac2O) and 0.5 mL of pyridine (C5H5N), and the mixture was stirred at room temperature for 72 h. Upon completion, the reaction was quenched with water and extracted by liquid–liquid extraction using H2O-EtOAc (1:2 × 2 and 1:1). The organic phase was washed with 5% HCl and brine [53], dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (hexane-Me2CO, 95:5) to give 12.3 mg (61.5%) of 3α-acetyl-24-hydroxylup-20(29)-en-28-oic acid (T1e).
3α-Acetyl-24-hydroxylup-20(29)-en-28-oic acid (T1e): white, amorphous powder (CHCl3), Rf = 0.46 (hexane-Me2CO, 80:20); 1H-NMR (CDCl3, 400 MHz) δ 4.82 (t, J = 2.5 Hz, 1H), 4.74 (d, J = 2.3 Hz, 1H), 4.60 (s, 1H), 4.02 (d, J = 10.4 Hz, 1H), 3.80 (d, J = 10.4 Hz, 1H), 3.00 (td, J = 10.7, 4.7 Hz, 1H), 2.28 (dt, J = 12.7, 3.0 Hz, 1H), 2.19 (td, J = 12.3, 3.6 Hz, 1H), 2.02 (s, 3H), 1.97 (d, J = 7.4 Hz, 1H). 1.88–1.77 (m, 1H), 1.69 (s, 3H), 1.67–1.60 (m, 2H), 1.58–1.15 (m, 18H), 1.14–1.04 (m, 2H), 1.02 (s, 3H), 0.97 (s, 3H), 0.94 (s, 3H), 0.88 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ 171.5, 170.5, 150.5, 109.9, 73.2, 70.7, 56.5, 50.5, 49.4, 47.7, 47.0, 42.7, 40.8, 39.7, 38.5, 37.2, 37.2, 33.9, 33.6, 32.3, 30.7, 29.8, 25.6, 22.3, 21.4, 21.0, 20.8, 19.5, 18.7, 17.3, 16.2, 15.0; see Figures S9 and S10.

3.7. Leukemia Cell Lines

The acute myeloid leukemia cell line (THP-1, ATCC TIB-202) and the 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) were obtained from the American Type Culture Collection (ATCC) and kindly provided by María Antonieta Chávez González from Unidad de Investigación Médica en Enfermedades Oncológicas, UMAE Hospital de Oncología, Centro Médico Nacional Siglo XXI, IMSS. All experimental procedures were approved by the Ethics and Scientific Committee at IMSS.
Cell lines were cultured in RPMI 1640 medium (Roswell Park Memorial Institute medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cultures were maintained at 37 °C in a humidified incubator with 5% CO2. All biological evaluations were performed using cell between passages 3 and 4.

3.8. Compounds and Controls

All compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL. In all assays, culture medium containing 0.1% DMSO was used as the negative control, while dasatinib was included as the positive control. Normal mononuclear cells (MNCs) were included as the normal cell control. All experiments were performed in triplicate.

3.9. Cell Viability Assay in Leukemic Cell Lines and Normal Mononuclear Cells

Leukemic cell lines and normal mononuclear cells (MNCs) were used to evaluate the cytotoxicity of the compounds. Leukemic cells (1 × 105 cells/well) were cultured in 96-well plates and treated with increasing concentrations of compounds (0.02, 0.2, 2.0, and 20 µM) for 48 h.
Normal mononuclear cells (MNCs) were isolated from peripheral blood mononuclear cells obtained from healthy human donors. Cell collection was conducted in accordance with institutional guidelines, including written informed consent from each participant. All procedures were approved by Ethics and Scientific Committee of the Mexican Institute of Social Security (IMSS, ethical approval number: CNIC R-2021-785-025).
The MNCs were purified using FicollPaque Plus (Pharmacia Biotech, Uppsala, Sweden) by centrifugation at 400× g for 30 min at room temperature, following the manufacturer’s protocol [54,55]. The resulting cells were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and counted using a hemocytometer after trypan blue staining, confirming a viability of 95% [56]. A total of 1 × 105 cells/well were cultured in 96-well plates and treated with various concentrations of compounds (25, 50, 100, and 200 µM) for 48 h.
After incubation, both leukemic cells and MNCs were collected, washed with phosphate-buffered saline (PBS), and stained with DAPI (500 ng/mL) for 15 min in darkness, following the manufacturer’s instructions [57]. Fluorescence was analyzed using a FACSVerse flow cytometer (BD Bioscience, San Jose, CA, USA).

3.10. Apoptosis Assay

Apoptosis induction was evaluated by flow cytometry. Cells (1 × 105 cells/well) were cultured in 96-well plates and exposed to the compounds at two concentrations (½ and 1 × IC50). After 48 h of treatment, the cells were washed with phosphate-buffered saline (PBS) and stained with annexin V-FITC (BD Bioscience) for 15 min in the dark. Following a second PBS wash, the cells were stained with DAPI (500 ng/mL) for 15 min in darkness. The stained samples were subsequently analyzed on a FACSVerse cytometer (BD Bioscience, San Jose, CA, USA) [58].

3.11. In Silico Studies

Pharmacokinetic and physicochemical properties related to ADME (absorption, distribution, metabolism, and excretion) were predicted for the studied compounds using SwissADME [48]. Parameters analyzed included molecular weight (MW), numbers of hydrogen bond acceptors (nHBA) and donors (nHBD), lipophilicity (cLogP), number of rotatable bonds (nROTB), topological polar surface area (TPSA), and additional pharmacokinetic parameter descriptors.
For molecular docking analysis, three target protein structures were retrieved from the RCSB Protein Data Bank [59]: (1) the anti-apoptotic protein BCL-2 bound to a BH3 domain-specific inhibitor (PDB-ID: 4IEH), (2) the tyrosine kinase domain of epidermal growth factor receptor (EGFR) complexed with erlotinib (PDB-ID: 1M17), and (3) FMS-like tyrosine kinase 3 (FLT3) in complex the inhibitor quizartinib (PDB ID: 4RT7).
Molecular Operating Environment (MOE) software was used to perform molecular docking analysis and visualization [40]. First, MOE’s SiteFinder tool was used to identify the best binding site in the three-dimensional structure of the receptor. This tool evaluates possible binding sites and provides a ranking of them, allowing the selection of the area of highest affinity for the ligand.
Once the binding site was identified, molecular docking was performed using the Triangle Matcher, an MOE docking algorithm that specializes in the search and evaluation of ligand docking positions at the receptor binding site, considering geometric and steric interactions. The London dG scoring method was used both for the calculation of the energy during ligand docking at the binding site and in the refinement of conformations. During the docking process, a total of 300 ligand conformations were generated at the selected binding site. To improve the quality of the obtained poses and reduce the computational complexity, a set of 25 conformations showing the best energy scores was refined [40].

3.12. Analysis of Results

Flow cytometry data were analyzed using FlowJoTM software (version 10.9). Cell viability result IC50 values—defined as the compound concentrations that inhibit 50% of cell proliferation—were determined from dose–response curves using GraphPad Prism software (version 9). All experiments were performed in triplicate, and results were expressed as means ± standard errors of the means (SEMs). Statical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test to compare treated groups against the control. A p-value < 0.05 was considered statistically significant.

4. Conclusions

The triterpene 3α,24-dihydroxylup-20(29)-en-28-oic acid (T1), isolated from Phoradendron wattii, was modified and characterized by 1H- and 13C-NMR experiments. From T1, five derivatives were generated; among them, 3α,24-dihydroxy-30-oxolup-20(29)-en-28-oic acid (T1c) induced inhibition of cell proliferation and cell death by apoptosis in ALL and AML cell lines. The T1c derivative is a promising candidate because it exhibited minimal effects on MNC cells. Molecular docking analysis showed that T1c has a higher affinity for EGFR protein kinase than its precursor molecule (T1). These findings suggest that the introduction of an α,β-unsaturated aldehyde group at C-30 in pentacyclic triterpenes significantly enhances the biological activity on leukemia cell lines, highlighting the potential of T1c in future research for leukemia treatment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules30153193/s1. Figures S1 and S2. Nuclear magnetic resonance spectra of compound T1a [Figure S1. 1H-NMR (CDCl3, 400 MHz), Figure S2. 13C-NMR (CDCl3, 100 MHz)]. Figures S3 and S4. Nuclear magnetic resonance spectra of compound T1b [Figure S3. 1H-NMR (CDCl3, 400 MHz), Figure S4. 13C-NMR (CDCl3, 100 MHz)]. Figures S5 and S6. Nuclear magnetic resonance spectra of compound T1c [Figure S5. 1H-NMR (CD3OD, 400 MHz), Figure S6. 13C-NMR (CD3OD, 100 MHz)]. Figures S7 and S8. Nuclear magnetic resonance spectra of compound T1d [Figure S7. 1H-NMR (CD3OD, 400 MHz), Figure S8. 13C-NMR (CD3OD, 100 MHz)]. Figures S9 and S10. Nuclear magnetic resonance spectra of compound T1e [Figure S9. 1H-NMR (CDCl3, 400 MHz), Figure S10.13C-NMR (CDCl3, 100 MHz)].

Author Contributions

Conceptualization, R.E.M.-P. and A.C.-G.; methodology, M.J.N.-B. and S.G.-S.; formal analysis, M.J.N.-B. and R.E.M.-P.; investigation, M.J.N.-B., 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., 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, R.E.M.-P., 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.

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 Jesús Kumate Rodríguez.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

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 Médica en Enfermedades Oncológicas, UMAE Hospital 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.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Figure 1. Molecular structures of T1, T1a, and T2.
Figure 1. Molecular structures of T1, T1a, and T2.
Molecules 30 03193 g001
Scheme 1. Semi-synthesis of 3α,24-dihydroxylup-20(29)-en-28-oic acid derivatives (T1aT1e). Reagents and conditions: (a) CH3I, K2CO3, Me2CO anh., room temperature, 24 h; (b) SeO2, EtOH anh., reflux 48 h; (c) AcO2, pyridine, room temperature, 72 h.
Scheme 1. Semi-synthesis of 3α,24-dihydroxylup-20(29)-en-28-oic acid derivatives (T1aT1e). Reagents and conditions: (a) CH3I, K2CO3, Me2CO anh., room temperature, 24 h; (b) SeO2, EtOH anh., reflux 48 h; (c) AcO2, pyridine, room temperature, 72 h.
Molecules 30 03193 sch001
Figure 2. (A) T1c induced apoptosis in the MOLT-4 cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. * p < 0.05, ** 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 MOLT-4 cultures, treated with vehicle control (DMSO), positive control (dasatinib), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
Figure 2. (A) T1c induced apoptosis in the MOLT-4 cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. * p < 0.05, ** 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 MOLT-4 cultures, treated with vehicle control (DMSO), positive control (dasatinib), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
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Figure 3. (A) T1c induced apoptosis in the CCRF-CEM cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. ** 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), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
Figure 3. (A) T1c induced apoptosis in the CCRF-CEM cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. ** 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), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
Molecules 30 03193 g003
Figure 4. (A) T1c induced apoptosis in the JURKAT cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. ** 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 JURKAT cultures, treated with vehicle control (DMSO), positive control (dasatinib), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
Figure 4. (A) T1c induced apoptosis in the JURKAT cell line. T1c was evaluated at ½ IC50 and IC50 exposed for 48 h. ** 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 JURKAT cultures, treated with vehicle control (DMSO), positive control (dasatinib), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
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Figure 5. (A) T1c was 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), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
Figure 5. (A) T1c was 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), and T1c for 48 h. Cluster Q1: Necrotic or other death processes; Cluster Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Living cells. Apoptotic cell population in red box.
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Figure 6. Binding modes of T1c with BCL-2 protein. (A) Compound T1c docked to BCL-2 binding site. (B) Two-dimensional interactions between T1c and BCL-2 protein.
Figure 6. Binding modes of T1c with BCL-2 protein. (A) Compound T1c docked to BCL-2 binding site. (B) Two-dimensional interactions between T1c and BCL-2 protein.
Molecules 30 03193 g006
Figure 7. Binding modes of T1c with EGFR tyrosine kinase. (A) T1c docked to EGFRTyrKin binding site. (B) Two-dimensional interactions between T1c and EGFRTyrKin.
Figure 7. Binding modes of T1c with EGFR tyrosine kinase. (A) T1c docked to EGFRTyrKin binding site. (B) Two-dimensional interactions between T1c and EGFRTyrKin.
Molecules 30 03193 g007
Figure 8. Binding modes of T1c with FLT3 protein. (A) Compound T1c docked to FTL-3 binding site. (B) Two-dimensional interactions between T1c and FLT-3.
Figure 8. Binding modes of T1c with FLT3 protein. (A) Compound T1c docked to FTL-3 binding site. (B) Two-dimensional interactions between T1c and FLT-3.
Molecules 30 03193 g008
Table 1. The mean inhibitory concentration of triterpenes and derivatives evaluated against leukemia cell lines.
Table 1. The mean inhibitory concentration of triterpenes and derivatives evaluated against leukemia cell lines.
Compound IC50 µM (SI)
CCRF-CEM REH JURKAT MOLT-4 THP-1
T1N/AN/AN/AN/AN/A
T1aN/A64.22 ± 4.7N/AN/AN/A
T1b6.87 ± 0.6
(1.59)
9.46 ± 3.2
(1.13)
14.97 ± 1.6
(0.72)
4.05 ± 0.1
(2.66)
11.04 ± 0.1
(0.97)
T1c17.79 ± 1.0
(75.02)
24.92 ± 0.4
(53.55)
18.67 ± 1.4
(71.46)
13.86 ± 0.7
(96.30)
12.90 ± 0.1
(103.46)
T1dN/AN/AN/AN/AN/A
T1eN/AN/AN/AN/AN/A
T227.36 ± 0.9
(19.48)
46.74 ± 1.6
(11.40)
62.27 ± 2.3
(8.56)
N/AN/A
Dasatinib10.76 ± 1.25.43 ± 0.35.26 ± 0.52.63 ± 0.14.66 ± 0.3
SI: selectivity index (ratio of IC50 in the MNC line over IC50 in the leukemia cell line); N/A: not active.
Table 2. The mean inhibitory concentrations of triterpenes and derivatives evaluated against peripheral blood mononuclear cells.
Table 2. The mean inhibitory concentrations of triterpenes and derivatives evaluated against peripheral blood mononuclear cells.
IC50 µM
CompoundMNCsCompoundMNCs
T1275.01 ± 3.1T1dN/A
T1aN/AT1eN/A
T1b10.78 ± 1.3T2532.98 ± 6.1
T1c1334.73 ± 4.3DasatinibN/A
MNCs: peripheral blood mononuclear cells; N/A: not active; ≤200 µM.
Table 3. Docking score for compounds T1, T1c, T2, and betulinic acid.
Table 3. Docking score for compounds T1, T1c, T2, and betulinic acid.
CompoundScore (kcal/mol)
BCL-2EGFR (TK Domain)FLT3
T1−10.12−12.75−14.05
T1c−10.23−14.50−14.07
T2−11.59−15.00−14.03
Betulinic acid−9.74−12.73−13.64
Co-crystalized inhibitor−17.72−13.97−18.17
Table 4. In silico predicted physicochemical and pharmacokinetic parameters of T1, T1c, T2, and betulinic acid.
Table 4. In silico predicted physicochemical and pharmacokinetic parameters of T1, T1c, T2, and betulinic acid.
Compounds
T1T1cT2BA
Molecular Weight (g/mol) 472.70486.68486.68456.70
Physicochemical ParametersnHBA4553
nHBD3332
cLogP5.524.664.696.14
nROTB3442
TPSA (Å2)77.7694.8394.8357.53
Pharmacokinetic ParametersGI absorptionHighHighHighLow
CYP1A2 inhibitorNoNoNoNo
CYP2C19 inhibitorNoNoNoNo
CYP2C9 inhibitorYesNoNoYes
CYP2D6 inhibitorNoNoNoNo
CYP3A4 inhibitorNoNoNoNo
nHBA: num. of H-bond acceptors; nHBD: num. of H-bond donors; cLogP: lipophilicity (consensus Log Po/w); nROTB: num. of rotatable bonds; 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; BA: betulinic acid.
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Noh-Burgos, M.J.; García-Sánchez, S.; Tun-Rosado, F.J.; Chávez-González, A.; Peraza-Sánchez, S.R.; Moo-Puc, R.E. Semi-Synthesis, Anti-Leukemia Activity, and Docking Study of Derivatives from 3α,24-Dihydroxylup-20(29)-en-28-Oic Acid. Molecules 2025, 30, 3193. https://doi.org/10.3390/molecules30153193

AMA Style

Noh-Burgos MJ, García-Sánchez S, Tun-Rosado FJ, Chávez-González A, Peraza-Sánchez SR, Moo-Puc RE. Semi-Synthesis, Anti-Leukemia Activity, and Docking Study of Derivatives from 3α,24-Dihydroxylup-20(29)-en-28-Oic Acid. Molecules. 2025; 30(15):3193. https://doi.org/10.3390/molecules30153193

Chicago/Turabian Style

Noh-Burgos, Mario J., Sergio García-Sánchez, Fernando J. Tun-Rosado, Antonieta Chávez-González, Sergio R. Peraza-Sánchez, and Rosa E. Moo-Puc. 2025. "Semi-Synthesis, Anti-Leukemia Activity, and Docking Study of Derivatives from 3α,24-Dihydroxylup-20(29)-en-28-Oic Acid" Molecules 30, no. 15: 3193. https://doi.org/10.3390/molecules30153193

APA Style

Noh-Burgos, M. J., García-Sánchez, S., Tun-Rosado, F. J., Chávez-González, A., Peraza-Sánchez, S. R., & Moo-Puc, R. E. (2025). Semi-Synthesis, Anti-Leukemia Activity, and Docking Study of Derivatives from 3α,24-Dihydroxylup-20(29)-en-28-Oic Acid. Molecules, 30(15), 3193. https://doi.org/10.3390/molecules30153193

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