New Caracasine Acid Derivatives with Anti-Leukemic Activity

1. Introduction

Medicinal plants constitute a crucial reservoir of bioactive compounds essential for developing novel therapeutics aimed at addressing various pathologies. The structural diversity of secondary metabolites derived from natural products is paramount in ongoing pharmacological research, highlighting the necessity of their isolation, structural elucidation, and mechanistic evaluation against clinically relevant diseases. Among these plants, the Croton genus (Euphorbiaceae) has become a significant source of pharmacologically active constituents. Scientific investigations have demonstrated a wide array of multifunctional bioactivities, including, but not limited to, antinociceptive, analgesic, antimicrobial, antidiabetic, anti-HIV, and antineoplastic effects. Systematic reviews and meta-analyses have extensively documented the therapeutic potential of its phytochemicals, underscoring their significance in the drug discovery process [1,2,3,4,5,6,7,8,9].

The medicinal importance of Croton species is increasingly acknowledged due to their remarkable diversity of secondary metabolites, particularly diterpenoids, which are critical to their therapeutic potential. Among these metabolites, clerodane, crotofolan, kaurane, and labdane-type diterpenoids are particularly noteworthy, as they are commonly found within the phytochemical composition of Croton plants [10]. Due to their structural diversity, diterpenoids hold significant potential in developing practical and selective anticancer pharmaceuticals. A prominent example of such a compound is paclitaxel; however, numerous other diterpenoids exhibit substantial bioactivities.

The ent-kauranes, classified as a group of diterpenoids, represent a significant category of secondary metabolites that exhibit various biological activities. These activities encompass anti-inflammatory, antiviral (specifically anti-HIV), antibacterial, leishmanicidal, hypotensive, insect antifeedant, and antitumor effects [11-12]. These compounds are found in several plant families but are especially abundant in genera such as Isodon (Asteraceae) and Croton (Euphorbiaceae). Due to their broad pharmacological potential, ent-kauranes have garnered considerable scientific interest, particularly for their antitumor properties as demonstrated by numerous studies [13-19]. Structurally, kauranes are tetracyclic compounds consisting of three six-membered rings and one five-membered ring (Fig. 1). However, under the ent-kaurane classification, significant structural diversity arises due to skeletal rearrangements and metabolic reactions, including oxidations, ring-opening, and the formation of new rings. Among ent-kauranes, a relatively small subgroup known as seco-ent-kauranes features structures with one of the four constitutive rings opened. These are distinguished by the carbon positions involved in the ring cleavage, such as 2,3-seco-ent-kaurane [20], 6,7-seco-ent-kaurane [21], 8,9-seco-ent-kaurane [22], and 8,15-seco-ent-kaurane [23]. To date, the least common are the 3,4-seco-ent-kauranes, which nevertheless exhibit notable pharmacological properties [24,25,26], and recently, a new 8,14-seco-ent-kaurane has been reported [27].

Figure 1. Ent-kaurane structure.

Figure 1. Ent-kaurane structure.

In our research, the 3,4-seco-ent-kauranes caracasine acid (1, Fig. 2) and its methyl ester, caracasine (2), were first isolated from Croton micans SW (initially misidentified as Croton caracasana [26]). Later, we reported a series of dimers derived from these compounds [28]. Both 1 and 2 display broad-spectrum antitumor activity against multiple human cancer cell lines [29]. We have also elucidated their mechanism of action in human leukemia cells [30-31]. As part of this investigation, a small group of caracasine acid derivatives were synthesized and evaluated for cytotoxicity, leishmanicidal, antibacterial, and anti-trypanosomal activity [32].

Building on our previous findings [32], which demonstrated that modifications to the α-β unsaturated system of caracasine acid (1) lead to a loss of activity, while esterification yields compounds with enhanced bioactivity, we explored the hemisynthesis of two new esters (3, 4) derived from the parent carboxylic acid. Additionally, we reduced the double bond between C-16 and C-17 to obtain ent-3,4-seco-15-oxo-kaur-4(19)-en-3-oic acid (5), and synthesized two carboxylic anhydrides (6, 7) from compounds 1 and 5. The results on different human cancer cell lines encourage us to obtain different derivatives from these seco-ent-kauranes. As a continuation of our ongoing research, herein we report the synthesis and structural characterization of five new derivatives (3-7) of the parent compound, caracasine acid (1), and the screening of anti-tumoral activities on twelve human cells, including eight cancer cell lines and four normal fibroblasts.

Figure 2. Caracasine acid (1), a 3,4-seco-ent-kaurane.

Figure 2. Caracasine acid (1), a 3,4-seco-ent-kaurane.

The present study aimed to elucidate the cytotoxic potential of a select group of caracasine acid derivatives, with consideration for future structural modifications via click chemistry.

2. Materials and Methods

2.1. Biological activity

2.1.1. Cell lines

The cancer cell lines used in the cytotoxic assays were acquired from the American Tissue Culture Collection (ATCC) (Manassas, VA, USA) and maintained as described previously [33]. The MRC-5 and BJ human fibroblasts were used as non-tumoral control cells. The (MRC-5 LD and BJ LD) are also human fibroblast cell lines resistant to doxorubicin. The cell line CCRF-CEM is derived from T-lymphoblastic leukemia; they are cells with high chemosensitivity. Also, the cell line K562 represents cell samples from a patient with acute myeloid leukemia that presents Bcr-Abl translocation. The Raji and Ramos cell lines correspond to B-cell leukemia. Other cells like U2OS represent a child osteosarcoma, HCT116 is a colorectal tumor cell line, and (HCT116p53-/-, Horizon Discovery Ltd., Cambridge, UK) is a similar cell line with its p53 gene knocked down, a model of human cancer frequently associated with poor prognosis. The cells were maintained in Nunc/corning 80 cm2 plastic tissue culture flasks and cultured in a cell culture medium according to ATCC or Horizon recommendations (DMEM/RPMI 1640 with 5g/L glucose, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal calf serum, and NaHCO₃).

2.1.2. Lymphocytes from normal donors.

Leukocytes from 5 normal donors were obtained from the Transfusion Medical Department at the Olomouc University Hospital. The cells were separated by the standard Ficoll-Hypaque method, as described previously [30-31]. The cells were incubated for 72 hours, as described for the standard compound screening method performed at the Institute of Molecular and Translational Medicine.

2.1.3. Cytotoxic MTS assay

In vitro cytotoxicity of compounds (1-7) was ascertained using a standard 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(sulfophenyl)-2H-tetrazolium (MTS), and it was performed at the Institute of Molecular and Translational Medicine by a robotic platform (High-ResBiosolutions). Cell suspensions were prepared and diluted according to the particular cell type and the expected target cell density (25.000-35.000 cells/mL based on cell growth characteristics). An automatic pipettor (30 μL) added cells into 384-well microtiter plates. All tested compounds were dissolved in 100% DMSO, and four-fold dilutions of the intended test concentrations were added in 0.15 μL aliquots at time zero to the microtiter plate wells by the echo acoustic non-contact liquid handler Echo550 (Labcyte). The assays were accomplished in technical duplicates and at least three biological replicates. The cells were incubated with the tested compound for 72 hours at 37 ^oC, in a 5% CO₂ atmosphere at 100% humidity. At the end of the incubation period, cells were assayed using the MTS test. Aliquots (5 μL) of the MTS stock solution were pipetted into each well and incubated for 1-4 h. After this incubation period, the optical density (OD) was measured at 490 nm with an Envision reader (Perkin Elmer). Tumor cell survival (TCS) was calculated using the following equation: TCS = (ODdrug-exposed well/meanODcontrol wells) x 100%. The IC₅₀ value, the compound concentration lethal to 50% of the tumor cells, was calculated from the appropriate dose-response curves in Dotmatics software (Updated version 2022, London, UK) [33]. After three days of incubation on the cancer cell lines and normal fibroblasts, the minimum inhibitory concentrations (IC₅₀) were obtained.

As previously described, no significant effect was observed when primary leukocytes were treated with caracasine or caracasine acid [30-31]; the results were confirmed with the different structures.

2.2. Spheroid formation

Spheroid culture aims to analyze the drug's effect on complex structures resembling solid tumors. The spheroids were prepared using the specific nonadherent plates for spheroid formation (Nunc Sphera Thermo Fisher) using the simple protocol outlined by the manufacturer. The cell lines HCT116 parental, HCT116KOTp53, A549, and U2OS were cultured at a density of 2500 cells per well using the medium with 10% fetal calf serum and antibiotics: McCoy for HCT116, F-12K (Kaighn's Modified) for A549, and Dulbecco Modified Eagle Media (DMEM). The cells were grown in culture for 48 hours as recommended by the manufacturer and as described by Das et al. [34] and Muñoz-Garcia et al [35].

Mixed spheroids were made by mixing MRC-5 fibroblasts with HCT116 or HCT116 p53 KO cells at a ratio of 1:1 (1000 cells/well); the cells were added immediately one after the other, and the formation of spheroids was monitored for 48 hrs. The mixed spheroids of both cell types had similar morphological features.

2.2.1. Spheroid viability assay

The cell viability of the treated spheroids was assessed using the kit CellTiter-Glo 3D (Promega Corporation). The kit is designed to measure ATP as an indicator of viability and generates a luminescent readout that is much more sensitive than colorimetric or fluorescence-based methods. The spheroids were cultivated with compounds ranging from 100 pM to 50 μM concentrations in triplicate for 72 hrs, and the luminescence was read with the Enspire apparatus (Perkin Elmer). A standard curve was performed for each analysis as recommended by the manufacturer.

Morphological assessment of spheroids was performed under the inverted microscope using grids every 12 hrs. No changes in morphology were observed during the process.

3.1.1. Isolation of caracasine acid (1) 

The naturally occurring diterpene caracasine acid (1) was used as the starting material for the chemical transformations presented in this report. Compound 1 was isolated by water decoction of dry leaves, partitioned with chloroform, and purified by column chromatography on silica gel. Spectroscopic methods characterized compound 1, and the data were compared with those previously reported by us [26].

3.1.2. Synthesis of derivatives 

The transformations made from 1 included the hemi-synthesis of five 3,4-seco-ent-kaurane diterpenes (3-7) whose structures are depicted in Scheme 1. Ester derivatives 2 and 3 were synthesized by a Fischer-type esterification reaction using methanol and cyclohexanol, with p-TsOH as catalyst, under reflux conditions and anhydrous MgSO₄ as desiccant. The eugenol ester was obtained by Steglich esterification using DIC and DMAP in dichloromethane.

Compound 5 was prepared by catalytic hydrogenation on 10% Pd/C of the double bond between carbons 16 and 17 of 1. This reaction occurs selectively in one of the olefins of the structure, and the compound 5 was obtained in high yields (85.2 %). It is known that the selective hydrogenation of carbon–carbon double bonds of conjugated carbonyl compounds in the presence of other isolated double bonds is a complex reaction, and it is greatly influenced by local structure. In this case, unpredictably, the conjugated olefin was the only one hydrogenated. It is possible that due to the structural arrangement of compound 1, the reduction occurs more easily and exclusively at the double bond of the α,β-unsaturated system, leaving the other double bond present in the molecule unaffected.

Compound 7 was synthesized by reacting acid 1 with acetyl chloride in the presence of pyridine, and, in a similar procedure, acid 5 was treated with acetyl chloride to obtain anhydride 6. The structures of these compounds were confirmed using HREIMS, ¹H NMR, ¹³C NMR, DEPT, HMQC, and HMBC.

3.1.3. Plant material 

Plant material of Croton micans to obtain caracasine acid 1 was collected in Ocumare de la Costa, Estado Aragua, Venezuela, and identified by the Botanist Giovannina Orsini. A voucher specimen (MYF26071) has been deposited at the Herbario Víctor Manuel Ovalles, Facultad de Farmacia, Universidad Central de Venezuela.

3.1.4. Reagents and equipment 

All reagents and solvents were from standard commercial sources and of analytical grade. TLC was used routinely to check all reactions using Merck Kieselgel 60 F254 aluminum plates; spots were examined under UV light at 254 nm and further visualized by sulphuric acid and p-anisaldehyde spray. Column chromatography was performed on silica gel (200–400 mm, 60 Å).

Spectroscopic methods elucidated the structures, including 1D and 2D NMR and HREIMS techniques. NMR spectra were recorded in the specified deuterated solvent at 500 MHz on a Brucker DRX-500 Avance spectrometer. Chemical shifts are expressed in ppm (δ) relative to the residual solvent signals, and the coupling constants J are given in Hertz (Hz). HREIMS were recorded with a Finnigan Trace mass spectrometer. IR spectra were performed on a FT-IR Thermo Nicolet Nexus 470 spectrophotometer. Melting points (mp) were uncorrected on an Electrothermal apparatus.

4. Synthesis and characterization

4.1.1. Caracasine acid (1) 

Compound 1 was isolated from Croton micans Sw. by decoction from dried leaves, liquid-liquid extraction with CHCl₃, and purification by column chromatography on silica gel using a mixture of Hex/ EtOAc 70:30. White solid, 220–221 °C. IR mmax: 3305, 2933, 2863, 1735, 1720, 1698, 1638, 1120, 936 cm1. ¹H NMR (500 MHz, CDCl₃) δ ppm: 5.95 (s, 1H, H-17b), 5.24 (s, 1H, H-17a), 4.86 (s, 1H, H-19a), 4.60 (s, 1H, H-19b), 3.04 (bs, 1H, H-13), 2.35 (d, J = 12.1 Hz, 2H, H-14), 2.06 (m, 2H, H-2), 2.05 (m, 1H, H-5), 1.88 (m, 2H, H-12), 1.72 (s, 3H, H-18), 1.68 (m, 2H, H-1), 1.63 (m, 2H, H-11), 1.50 (m, 2H, H-6), 1.36 (m, 1H, H-9), 1.27 (m, 2H, H-7), 1.06 (s, 3H, H-20). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 210.3(C-15), 179.4 (C-3), 149.3 (C-16), 146.8 (C-4), 114.7 (C-17), 114.1 (C-19), 51.9 (C-8), 49.9 (C-5), 43.4 (C-9), 41.5 (C-10), 38.1 (C-13), 36.4 (C-14), 33.5 (C-1), 32.2 (C-12), 32.1 (C-7), 28.4 (C-2), 24.7 (C-6), 23.4 (C-18), 21.6 (C-20), 18.3 (C-11). EIMS: m/z 316.2034 [M]⁺ (Calcd for C₂₀H₂₈O₃, 316.2038).

4.1.2. Caracasine (2) 

This compound was isolated together with 1 from plant material, but with the purpose of obtaining it in larger amounts, it was obtained by a Fisher esterification. To a solution of the acid 1 (101.8 mg, 0.3217 mmol) in methanol (5 mL), p-TsOH (0.16 mmol) and anhydrous MgSO₄ were added. The reaction mixture was stirred at reflux for 6 h and then filtered. The solvent was removed under reduced pressure and the residue purified by column chromatography on silica (Hex/EtOAc 80:20) to afford 2 as a white solid, 76%, 73-75oC. IR mmax: 2931 m, 2857, 1738, 1723, 1700, 1638, 1406, 1117, 932 cm1. ¹H NMR (500 MHz, CDCl₃) δ ppm: 5.93 (s, 1H, H-17a), 5.24 (s, 1H, H-17b), 4.87 (s, 1H, H-19a), 4.65 (s, 1H, H-19b), 3.61 (s, 3H, H-10 ), 3.05 (bs, 1H, H-13), 2.35 (d, J = 11.9 Hz, 2H, H-14), 2.06 (m, 2H, H-2), 2.03 (m, 1H, H-5), 1.87 (m, 2H, H-12), 1.73 (s, 3H, H-18), 1.68 (m, 2H, H- 1), 1.63 (m, 2H, H-11), 1.53 (m, 2H, H-6), 1.36 (m, 1H, H-9), 1.28 290 (m, 2H, H-7), 1.05 (s, 3H, H-20). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 210.0 (C-15), 173.8 (C-3), 149.4 (C-16), 146.9 (C-4), 114.5 (C- 17), 114.0 (C-19), 51.9 (C-8), 51.5 (C-10 ), 50.0 (C-5), 43.5 (C-9), 41.5 (C-10), 38.2 (C-13), 36.5 (C-14), 33.8 (C-1), 32.3 (C-12), 32.1 (C-7), 28.4 (C-2), 24.8 (C-6), 23.4 (C-18), 21.6 (C-20), 18.4 (C-11). EIMS: m/z 330.2193 [M]⁺ (Calcd for C₂₁H₃₀O₃, 330.2195).

4.1.3. Caracasine acid cyclohexyl ester (3) 

To a solution of 1 (0.1002 g, 0.3167 mmol) in benzene (4 mL), cyclohexanol (0.033 mL, 0.3167 mmol), p-TsOH (0.16 mmol), and anhydrous MgSO₄ were added. The reaction mixture was stirred at reflux for 6 h and then filtered. The solvent was removed under reduced pressure, and the residue purified by column chromatography on silica (Hex/EtOAc 90:10) to afford 7 as a white solid, 72.5 %, m.p. 86-91°C. ¹H NMR (500 MHz, CDCl₃) δ ppm: 5.93 (s, 1H, H-17a), 5.24 (s, 1H, H-17b), 4.88 (s, 1H, H-19a), 4.67 (s, 1H, H-19b), 4.70 (m, 2H, H-1’), 3.05 (bs, 1H, H-13), 2.35 (m, 2H, H-14), 2.25 (m, 2H, H-2), 2.03 (m, 1H, H-5), 1.91 (m, 2H, H-12), 1.80 (m, 4H, H-2’, H-6’), 1.73 (s, 3H, H-18), 1.68 (m, 2H, H-1), 1.61 (m, 2H, H-11), 1.53 (m, 2H, H-6), 1.50 (m, 4H, H-3’, H-5’), 1.37 (m, 1H, H-9), 1.27 (m, 2H, H-7), 1.04 (s, 3H, H-20), 0.89 (m, 2H, H-4’). NMR ¹³C (125 MHz, CDCl₃) δ ppm: 210.2 (C-15), 173.2 (C-3), 149.4 (C-16), 146.9 (C-4), 114.4 (C-17), 113.9 (C-19), 72.4 (C-1’), 51.9 (C-8), 49.8 (C-5), 43.4 (C-9), 41.5 (C-10), 38.1 (C-13), 36.4 (C-14), 33.8 (C-1), 32.3 (C-7), 32.1 (C-12), 31.7 (C-2’), 31.7 (C6’), 29.1 (C-2), 25.4 (C-6), 24.8 (C4’), 23.8 (C-3’), 23.8 (C5’), 23.7 (C-18), 21.5 (C-20), 18.4 (C-11). EI:MS: m/z = 398.4 [M]⁺ for C₂₆H₃₈O₃.

4.1.4. Caracasine acid eugenol ester (4) 

To a stirred solution of 1 (100.3 mg, 0.32 mmol) in 10 ml of DCM, DIC(43 μL. 0.32 mmol) was added followed by addition of DMAP (15mg, 0.01 mmol) and after 10 min of stirring, eugenol (52.74 mg, 0.32 mmol) was added and allowed to stir the reaction mixture at rt for 24h. Work-up was done with water and the mixture extracted with CHCl3 (3x10 ml), washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude compound was purified by column chromatography using 40% EtOAc: Hexane as the solvent system. The compound was obtained as a pink oil, 79% yield; ¹H NMR: CDCl₃ (500 MHz): 6.90 (s, 1H, H-3'), 6.87 (m, 2H, H-5'), 6.69 (m, 1H, H-6'), 5.96 (m, 1H, H-8'), 5.92 (s, 1H, H-17b), 5.09 (m, 2H, H-9'), 5.24 (s, 1H, H-17a), 4.85 (brs, 1H, H-19b), 4.65 (brs, 1H, 19a), 3.82 (s, 3H, H-10'), 3.62 (s, 3H, H-21), 3.35 (d, 2H, H-7̛ ), 3.04 (brs, 1H, H-13), 2.35 (d, J = 12.3 Hz, 2H, H-14), 2.10 (m, 2H, H-2), 2.05 (dd, J = 9.4, 3.5 Hz, 1H, H-5), 1.85 (m, 2H, H-12), 1.72 (s, 3H, H-18), 1.65 (m, 2H, H-11), 1.63 (dd, J = 6.7, 3.10 Hz, H-1), 1.50 (m, 2H, H-6), 1.34 (d, J = 8.35 Hz, H-9), 1.27 (m, 2H, H-7), 1.06 (s, 3H, H-20). NMR ¹³C (100 MHz, CDCl₃) δ ppm: 210.2 (C-15), 173.2 (C-3), 149.4 (C-16), 148.0 (C-2'), 146.9 (C-4), 144.0 (C-1'), 137.9 (C-8'), 132.3 (C-4'), 121.3 (C-5'), 117.3 (C-6'), 114.5 (C-3'), 114.4 (C-17), 113.9 (C-19), 111.3 (C-9’), 55.9 (C-10'), 51.9 (C-8), 49.8 (C-5), 43.4 (C-9), 41.5 (C-10), 39.8 (C-7'), 38.1 (C-13), 36.4 (C-14), 33.8 (C-1), 32.3 (C-7), 32.1 (C-12), 31.7 (C-2’), 31.7 (C6’), 29.1 (C-2), 25.4 (C-6), 24.8 (C4’), 23.8 (C-3’), 23.8 (C5’), 23.7 (C-18), 21.5 (C-20), 18.4 (C-11). EIMS: m/z = 462.62 [M]⁺ for C₃₀H₃₈O₄.

4.1.5. Ent-3,4-seco-15-oxo-kaur-4(19)-en-3-oic acid (5) 

Compound 1 (0.202 g, 0.639 mmol) was dissolved in dry THF (10.0 mL), and then Pd/C 10% (35.6 mg, 31.6 μmol) was added. It was stirred under an H2 atmosphere for 4 h, and the mixture was filtered through celite, and the solvent was evaporated under a vacuum. The residue was purified by column chromatography on silica gel eluted with Hex/EtOAc 80:20, to afford 5 as a white solid, 86.6%, m.p. 169- 171 °C. ¹H NMR (500 MHz, CDCl₃) δ ppm: 4.87 (s, 1H, H-19a), 4.65 (s, 1H, H-19b), 2.42 (bs, 1H, H-13), 2.39; 1.37 (m, 2H, H-14), 2.29 (m, 2H, H-2), 2.23 (m, 1H, H-16), 2.20 (CH₃O), 2.05 (m, 1H, H-5), 1.78; 1.26 (m, 2H, H-7), 1.72 (s, 3H, H-18), 1.67 (m, 2H, H-6), 1.66 (m, 2H, H-1), 1.56 (m, 2H, H-12), 1.52; 1.23 (m, 2H, H-11), 1.20 (m, 1H, H-9), 1.09 (d, J = 6.92 Hz, 3H, H-17), 1.03 (s, 3H, H-20). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 224.6 (C-15), 179.8 (C-3), 146.7 (C-4), 113.9 (C-19), 52.0 (C-8), 49.6 (C-5), 47.7 (C-16), 43.2 (C-9), 41.0 (C-10), 37.1 (C-14), 34.8 (C-13), 33.3 (C-1), 32.7 (C-7), 28.3 (C-2), 24.9 (C-12), 24.5 (C-6), 23.3 (C-18), 21.6 (C-20), 18.1 (C-11), 10.0 (C-17). EIMS: m/z 318.2193 [M]⁺ (Calcd for C₂₀H₃₀O₃, 318.2195).

4.1.6. Acetic-ent-3,4-seco-15-oxo-kaur-4(19)-en-anhydride (6) 

To a round-bottom flask containing compound 5 (60 mg, 0.189 mmol) and pyridine (15.26μ μL, 0.189 mmol), under a nitrogen atmosphere, acetyl chloride (13.48μL, 0.189 mmol) was added dropwise. The resulting mixture was stirred for 3h. After complete reaction, workup was done adding 10 mL of water, followed by extraction with CH3Cl (3 × 5 5mL), dried with anhydrous sodium sulphate, and evaporated under vacuum. The crude product was purified by silica gel column chromatography eluted with n-hexane/CH₂Cl₂ (1:1), affording compound 6 as a white amorphous solid with 85% yield.

¹H NMR (500 MHz, CDCl₃) δ ppm: 4.87 (s, 1H, H-19a), 4.65 (s, 1H, H-19b), 3.45 (m, 2H, H-17), 3.45 (m, 2H, H-1’), 2.53 (brs, 1H, H-13), 2.25 (CH₃CO), 2.32 (m, 2H, H-14), 2.25 (brs, 2H, H-16), 2.10 (m, 2H, H-2), 2.03 (m, 1H, H-5), 1.72 (s, 3H, H-18), 1.68 (m, 2H, H-1), 1.64 (m, 2H, H-12), 1.51 (m, 1H, H-6), 1.49 (m, 2H, H-11), 1.25 (m, 1H, H-7), 1.23 (bs, 1H, H-9), 1.14 (t, J = 10.3 Hz, 3H, H-2’), 1.05 (s, 3H, H-20). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 221.8 (C-15), 165.7 (C-3), 165.4 (COCH3), 146.6 (C-4), 113.9 (C-19), 69.9 (C-17), 66.7 (C-1’), 53.4 (C-16), 52.8 (C-8), 50.3 (C-5), 43.3 (C-9), 41.8 (C-10), 36.5 (C-14), 34.1 (C-13), 34.0 (C-1), 32.9 (C-7), 30.9 (C-12), 28.6 (C-2), 25.5 (C-6), 23.8 (C-18), 22.0 (C-20), 20.5 (CH3CO), 18.9 (C-11), 15.6 (C-2’). EIMS: m/z = 360.49 [M]⁺ for C₂₂H₃₁O₄.

4.1.7. Acetic/caracasine anhydride (7) 

With a similar procedure to that described for compound 6, the acetyl anhydride of 1 was obtained. To (0.100 mg, 0.32 mmol) of 1 dissolved in 5 mL of THF, 25.8 μL (0.32 mmol) of pyridine was added, the mixture was stirred under N₂ for 15 min, and the acetyl chloride (22.8 μL, 0.32 mmol) was added dropwise. The solution was kept under stirring for 3h and then quenched with 10 mL of water, extracted with dichloromethane, washed with brine, and the organic phase dried over Na₂SO₄. Evaporation under vacuum gave 7 as a white powder. The product was further purified by silica gel chromatography eluted with a mixture of n-hexane/EtOAc (8:2), affording compound (7) with 87% yield, m.p. 123- 125 °C.

¹H NMR (500 MHz, CDCl₃) δ ppm: 1.08 (s, 3H, H-20), 1.27 (m, 2H, H-7), 1.36 (m, 1H, H-9), 1.50 (m, 2H, H-6), 1.64 (dd, 2H, J = 6.7, 1.10 Hz, H-1), 1.73 (s, 3H, H-18), 1.86 (m, 2H, H-12), 2.05 (dd, J = 9.4, 3.5 Hz, 1H, H-5), 2.12 (m, 2H, H-2), 2.37 (d, J =12 Hz, 2H, H-14), 2.22 (s, 3H, CH₃CO), 3.05 (brs, 1H, H-13), 3.60 (s, 3H, H-21), 4.65 (brs, 1H, H-19a), 4.89 (brs, 1H, H-19b), 5.24 (s, 1H, H-17a), 5.94(s, 1H, H-17b). ¹³C NMR (125.5 MHz, CDCl₃) δ ppm: 210.2 (C-15), 165.5 (C-3), 165.4 (CO-CH₃), 149.1 (C-16), 146.3 (C-4), 114.7 (C-17), 114.1 (C-19), 51.9 (C-8), 49.5 (C-5), 43.3 (C-9), 43.5 (C-9), 41.4 (C-10), 37.9 (C-13), 36.4 (C-14), 33.3 (C-1), 32.2 (C-12), 32.0 (C-7), 28.3 (C-2), 24.5 (C-6), 23.4 (C-8), 21.6 (C-20), 20.6 (CH3CO), 18.2 (C-11). ESI-MS: m/z = 358.472 [M]⁺ for C₂₂H₃₀O4.

3. Results

The compounds were not cytotoxic against normal human leukocytes, with viability > 80 % at concentrations up to 50 μM. The results are similar to those described previously for caracasine and caracasine acid [30-31].

Table 1 presents the effects of the compounds under investigation on two fibroblast cell lines. Compounds 7 and 3 had a similar impact to caracasine and caracasine acid against all fibroblast cell lines. Compounds 4, 5, and 6 did not affect the cells.

Table 2 shows that the compounds were highly effective against leukemic cells of different origins. Compounds 3 and 7 retained the cytotoxic activity against the cell lines, and the other structures had no effect.

Table 1. Effect of the different compounds on different cell types and cultures.

HUMAN FIBROBLASTS
CODE	BJ	BJ LD	MRC-5	MRC-5 LD
7	7.68	7.63	6.45	5.53
3	7.38	6.5	5.45	2.69
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	6.88	7.04	6.48	6.69
Caracasine (2)	7.28	7.42	6.84	6.36

Table 1. Effect of the different compounds on different cell types and cultures.

HUMAN FIBROBLASTS
CODE	BJ	BJ LD	MRC-5	MRC-5 LD
7	7.68	7.63	6.45	5.53
3	7.38	6.5	5.45	2.69
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	6.88	7.04	6.48	6.69
Caracasine (2)	7.28	7.42	6.84	6.36

Table 2. Effect of the compounds on leukemic cells.

HUMAN LEUKEMIA CELL LINES
CODE	CCRF-CEM	K562	RAJI	RAMOS
7	0.45	4.6	2.21	0.96
3	0.38	2.21	1.86	0.78
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	0.38	4.77	0.85	0.62
Caracasine (2)	0.86	3.29	1.45	0.96

Table 2. Effect of the compounds on leukemic cells.

HUMAN LEUKEMIA CELL LINES
CODE	CCRF-CEM	K562	RAJI	RAMOS
7	0.45	4.6	2.21	0.96
3	0.38	2.21	1.86	0.78
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	0.38	4.77	0.85	0.62
Caracasine (2)	0.86	3.29	1.45	0.96

The compounds also affected the growth of colon, lung, and osteosarcoma with values lower than 5 μM when the cells were grown in the standard 2D condition; however, the effect is lost when the spheroids are challenged with the compounds (Table 3). Mixed spheroids of different cell types, fibroblasts, and tumor cells that resemble the tumor microenvironment were also resistant to the effect of the compounds, as shown with the monogenic spheroids. There were also no changes in morphology in the spheroids upon treatment.

Table 3. Effects on other cell types.

HUMAN TUMORS 2D
CODE	HCT116 par	HCT116KO53	A549	U2OS
7	2.12	1.91	3.72	2.21
3	1.59	1.63	2.69	1.66
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	1.73	1.52	5.98	1.79
Caracasine (2)	2.02	1.91	8.39	3.19
HUMAN TUMORS SPHEROIDS
CODE	HCT116 par	HCT116KO53	A549	U2OS
7	>50	>50	>50	>50
3	>50	>50	>50	>50
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	>50	>50	>50	>50
Caracasine (2)	>50	>50	>50	>50

Table 3. Effects on other cell types.

HUMAN TUMORS 2D
CODE	HCT116 par	HCT116KO53	A549	U2OS
7	2.12	1.91	3.72	2.21
3	1.59	1.63	2.69	1.66
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	1.73	1.52	5.98	1.79
Caracasine (2)	2.02	1.91	8.39	3.19
HUMAN TUMORS SPHEROIDS
CODE	HCT116 par	HCT116KO53	A549	U2OS
7	>50	>50	>50	>50
3	>50	>50	>50	>50
4	>50	>50	>50	>50
5	>50	>50	>50	>50
6	>50	>50	>50	>50
Caracasine acid (1)	>50	>50	>50	>50
Caracasine (2)	>50	>50	>50	>50

5. Conclusions

The newly synthesized compounds numbered 3 to 7, which are derived from structural modifications of the parent acid (1), provide further confirmation that the α, β-unsaturated system is a critical pharmacophore in these bioactive compounds. Additionally, the findings indicate that substantial structural modifications should prioritize the carboxylic functional group. These results lay the groundwork for developing novel derivatives with improved anticancer activity and optimizing the active compounds presented in this study. Future research endeavors will concentrate on synthesizing innovative hybrid derivatives that link the ent-kaurane scaffold with other bioactive natural compounds through triazole rings employing click chemistry. Moreover, the data presented indicate that compounds 1, 2, 3, and 7 stand out as promising therapeutic candidates for advancing leukemia treatment. This study provides compelling evidence regarding the anticancer potential of caracasine acid (1) and its derivatives, suggesting that 3,4-seco-ent-kauranes may serve as promising lead candidates for further exploration in structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) studies.