In vitro and in silico analysis of anticancer and antioxidant potential of camphor derivatives

Abstract

Structural modification of natural products is a key strategy in drug discovery. In this work, a series of camphor derivatives were synthesized by incorporating heteroaromatic aldehydes, and their anticancer and antioxidant potential was evaluated through an integrated in vitro and in silico approach. The results highlighted the thiophene 5 and benzofuran 9 derivatives as the most potent agents, exhibiting high selective cytotoxicity against gastric, colon, and prostate cancer cell lines, with IC₅₀ values as low as 31.8 µM and a selectivity index of 7.0. The cytotoxic mechanism was found to be mediated by apoptosis induction via the mitochondrial pathway. Moreover, these active compounds displayed a favorable safety profile, showing low hemolytic activity and low predicted toxicological risk according to computational models. Overall, these findings demonstrate that camphor functionalization with specific heterocycles represents an effective strategy for developing novel anticancer drug candidates with high selectivity and a promising safety profile.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-27596-4.

Introduction

Cancer is a degenerative disease characterized by the uncontrolled proliferation of abnormal cells, posing one of the major threats to public health worldwide due to its high incidence and mortality. The World Health Organization (WHO) estimated approximately 9.7 million cancer-related deaths in 2022, a figure projected to increase to 15.3 million by 2040¹. This rise is associated with factors such as progressive population aging and chronic exposure to carcinogens^2,3. At the cellular level, malignant neoplasms frequently exhibit critical survival adaptations, including the Warburg effect (elevated glucose uptake) and the frequent dysregulation of key signaling pathways like PI3K/AKT, which is deeply involved in proliferation, migration, and metastasis^4–6.

The primary goal of oncological therapies is to selectively eliminate tumor cells without affecting the surrounding normal tissue. However, this selectivity remains a challenge. Conventional chemotherapeutic agents act via mechanisms like cell cycle interruption or the induction of apoptosis, a process regulated by caspases⁷. The complex etiology of cancer, which involves genetic alterations and the accumulation of reactive oxygen and nitrogen species (ROS/RNS) that cause oxidative DNA damage⁸, underscores the difficulty in designing effective, low-toxicity treatments. Consequently, the search for and characterization of new compounds with potential antineoplastic activity is a top priority in current biomedical research.

In this critical context, the strategy of chemoprevention and therapy using natural products has gained prominence. Historically, natural sources have been indispensable in drug discovery, with over 50% of current clinical drugs—including a significant number of anticancer agents—derived from them, validating their therapeutic potential^9,10. Among the bioactive phytochemicals, secondary metabolites have long been recognized as medicinal agents. Specifically, monoterpenes stand out due to their reported potential anti-tumor effects and low toxicity. The structural diversity of monoterpenes provides them with greater flexibility for cellular interaction, which significantly broadens their therapeutic potential in oncology.

One such monoterpene is camphor, a bicyclic monoterpenone found in various aromatic plants. It exists in two readily accessible enantiomers, R-(+)-camphor and S-(–)-camphor, and its rigid structure makes it an excellent scaffold for chemical modification. Although camphor is widely used in cosmetics and as a chemical intermediate¹¹, its potential for developing derivatives with diverse biological activities—including anticancer properties—has been scarcely explored^12–14. The Claisen-Schmidt condensation offers a straightforward method to synthesize camphor derivatives; however, the biological activity of products derived from heteroaromatic aldehydes has not been sufficiently assessed.

This study is based on the hypothesis that incorporating heteroaromatic aldehydes into the camphor scaffold via Claisen-Schmidt condensation will yield novel derivatives with enhanced biological activity. Specifically, we hypothesize that these new compounds will exhibit selective cytotoxicity against HT-29, PC-3, and AGS cancer cell lines while showing minimal toxicity to the normal GES-1 cell line. Furthermore, we postulate that their mechanism of action involves the induction of apoptosis through changes in mitochondrial membrane permeability and that they will possess a favorable safety profile, characterized by significant antioxidant capacity and low hemolytic activity.

Materials and methods

General

Melting points were determined using a Stuart Scientific SMP3 apparatus. Optical rotations were measured on an Atago PO-1 digital polarimeter at 20 ± 1 °C. FT-IR spectra were recorded on a Thermo Scientific Nicolet iS5 spectrophotometer.

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Avance 400 Digital NMR spectrometer. Detailed acquisition and processing parameters are provided in the Supplementary Information.

Gas chromatography–mass spectrometry (GC–MS) analyses were conducted on a Shimadzu GCMS-QP5050A system and an Agilent 7890B GC coupled to a 5977 A mass spectrometer (EI, 70 eV) fitted with an HP-5MS column (30 m × 0.25 mm × 0.25 μm). Detailed GC-MS operational parameters are provided in the Supplementary Information.

Chemicals

All chemicals were purchased from Sigma-Aldrich–Merck or AK Scientific and used as received without further purification. These included R-(+)-camphor (≥ 95%), S-(–)-camphor (≥ 98%), furfural (≥ 98%), 2-thiophenecarboxaldehyde (98%), 2-benzofurancarboxaldehyde (98%), 3-thiophenecarboxaldehyde (≥ 95%), 3-furancarboxaldehyde (98%), tert-butanol (99%), potassium tert-butoxide (99%), ethyl acetate (≥ 99%), sodium chloride (≥ 99%), and hydrochloric acid (37%).

General procedure for the synthesis of Camphor derivatives

The camphor derivatives were synthesized via a Claisen-Schmidt condensation. In a typical procedure, R-(+)- or S-(-)-camphor (4 mmol) and potassium tert-butoxide (4 mmol) were dissolved in tert-butanol (40 mL) and stirred under reflux for 30 min. The corresponding aromatic aldehyde (5 mmol) was then added, and the mixture was refluxed for an additional 4–6 h (Fig. 1). After cooling to room temperature, the reaction was neutralized with 1 N HCl and extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography using a hexane/ethyl acetate gradient of increasing polarity, up to a final eluting mixture of 8:2 (v/v).

Fig. 1.

Synthesis of camphor derivatives 3–12. Reagents and conditions: (a) K-OtBu, t-BuOH, reflux at 75 °C for 30 min; (b) Ar-CHO, reflux at 75 °C for 4–6 h.

The structure of the synthesized compounds was elucidated and confirmed by spectroscopic analyses as follows:

(1S,3E)-3-[(furan-2-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (3): The compound was isolated as a red colored oil with a yield of 90%. [α]²⁰_D = -234.77 (0.33 mg/mL in ethanol). FT-IR: 2950–2850 cm⁻¹ (aliphatic C–H), ~ 1725 cm⁻¹ (conjugated C = O), 1600–1650 cm⁻¹ (C = C), 1500–1600 cm⁻¹ and 1260–1100 cm⁻¹ (furanyl ring). ¹H NMR (400 MHz, CDCl₃): δ = 7.51 (d, J = 1.5 Hz, 1H, H-13), 6.95 (s, 1H, H-11); 6.57 (d, J = 3.4 Hz, 1H, H-15), 6.46 (dd, J = 1.5 and 3.4 Hz, 1H, H-14), 3.34 (d, J = 4.2 Hz, 1H, H-3), 2.14–1.4 (m, 4 H, H-4 and H-5), 1.0 (s, 3 H, H-10), 0.99 (s, 3 H, H-9), 0.97 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.4, 152.0, 144.1, 139.5, 114.9, 114.4, 112.1, 57.3, 49.5, 46.4, 30.7, 26.2, 20.7, 18.3, 9.3. EI-MS (70 eV), m/z (%): 230 [M]+ (100), 215 (18), 202 (39), 187 (92), 147 (87), 91 (88), 55 (55), 81 (49), 65 (47), 77 (44), 119 (43), 159 (43). HR-MS (ESI): m/z 231.1381 [M + H]⁺ (calcd. For C₁₅H₁₈O₂, 231.1385).

(1R,3E)-3-[(furan-2-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (4): The compound was isolated as a red colored oil with a yield of 92%. [α]²⁰_D = + 234.83 (0.33 mg/mL in ethanol). FT-IR: 2948–2852 cm⁻¹ (aliphatic C–H), ~ 1720 cm⁻¹ (conjugated C = O), 1605–1647 cm⁻¹ (C = C), 1500–1600 cm⁻¹ and 1260–1100 cm⁻¹ (furanyl ring). ¹H NMR (400 MHz, CDCl₃): δ = 7.48 (d, J = 1.6 Hz, 1H, H-13), 6.92 (s, 1H, H-11); 6.55 (d, J = 3.4 Hz, 1H, H-15), 6.44 (dd, J = 1.8 and 3.4 Hz, 1H, H-14), 3.31 (d, J = 4.1 Hz, 1H, H-3), 2.35–1.7 (m, 4 H, H-4 and H-5), 1.0 (s, 3 H, H-10), 0.99 (s, 3 H, H-9), 0.97 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.2, 144.0, 139.4, 114.8, 114.3, 112.0, 57.4, 49.4, 46.5, 30.2, 26.5, 20.6, 18.6, 9.2. EI-MS (70 eV), m/z (%): 230 [M]⁺ (100), 215 (17), 202 (41), 187 (92), 147 (86), 91 (88), 55 (56), 81 (49), 65 (48), 77 (45), 119 (43), 159 (43). HR-MS (ESI): m/z 231.1379 [M + H]⁺ (calcd. For C₁₅H₁₈O₂, 231.1385).

(1S,3E)-1,7,7-trimethyl-3-[(thiophen-2-yl)methylidene]bicyclo[2.2.1]heptan-2-one (5): The compound was isolated as a white solid with a yield of 95%. Mp: 41–42 °C. [α]²⁰_D = -442.23 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2960, 2926 (aliphatic C–H), 1723 (conjugated C = O), 1673 (conjugated C = C), 1427, 1330). ¹H NMR (400 MHz, CDCl₃): δ = 7.42 (d, J = 5.0 Hz, 1 H, H-13), 7.25 (s, 1 H, H-11), 7.08 (d, J = 3.7, 1 H, H-15), 7.07 (dd, J = 3.6 and 5.0, 1 H, H-14), 3.18 (d, J = 4.0, 1 H, H-3), 2.20–1.10 (m, 4 H, H-4 and H-5), 1.02 (s, 3 H, H-10), 1.02 (s, 3 H, H-9), 0.89 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.0, 139.7, 139.5, 131.9, 128.4, 127.7, 120.4, 57.4, 49.7, 46.8, 31.0, 25.7, 20.6, 18.4, 9.3. EI-MS (70 eV), m/z (%): 246 [M]⁺ (100), 231 (15), 218 (24), 203 (69), 163 (56), 97 (54), 91 (52), 135 (46), 134 (39), 55 (35), 161 (32), 175 (32). HR-MS (ESI): m/z 247.1149 [M + H]⁺ (calcd. For C₁₅H₁₈OS, 247.1157).

(1R,3E)-1,7,7-trimethyl-3-[(thiophen-2-yl)methylidene]bicyclo[2.2.1]heptan-2-one (6): The compound was isolated as a white solid with a yield of 96%. Mp: 42–43 °C. [α]²⁰_D = + 442.78 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2962, 2929 (aliphatic C–H), 1725 (conjugated C = O), 1632 (conjugated C = C), 1427, 1330 (C–H bend, aromatic vibrations C–C). ¹H NMR (400 MHz, CDCl₃): δ = 7.39 (d, J = 5.0 Hz, 1 H, H-13), 7.23 (s, 1 H, H-11), 7.22 (d, J = 3.5, 1 H, H-15), 7.04 (dd, J = 3.4 and 5.0, 1 H, H-14), 3.15 (d, J = 4.1, 1 H, H-3), 2.3–1.2 (m, 4 H, H-4 and H-5), 0.99 (s, 3 H, H-10), 0.98 (s, 3 H, H-9), 0.89 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 207.6, 139.5, 139.2, 131.7, 128.3, 127.5, 120.2, 57.2, 49.5, 46.6, 30.8, 25.5, 20.5, 19.0, 9.2. EI-MS (70 eV), m/z (%): 246 [M]⁺ (100), 231 (15), 218 (27), 203 (69), 163 (56), 97 (57), 91 (55), 135 (47), 134 (39), 55 (39), 161 (32), 175 (32). HR-MS (ESI): m/z 247.1155 [M + H]⁺ (calcd. For C₁₅H₁₈OS, 247.1157).

(1S,3E)-3-[(furan-3-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (7): The compound was isolated as a light yellow solid with a yield of 87%. Mp: 56–57 °C. [α]²⁰_D = -88.25 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2963, 2930, 2872 (aliphatic C–H), 1744 (conjugated C = O), 1670 (conjugated C = C), 1571, 1518 (furan ring), 1459, 1388 (C–H bend). ¹H NMR (400 MHz, CDCl₃): δ = 7.65 (s, 1 H, H-13), 7.44 (t, J = 2.8, 1 H, H-14), 7.06 (s, 1 H, H-11), 6.6 (d, J = 1.6, 1 H, H-15), 2.95 (d, J = 4.0, 1 H, H-3), 2.38–1.32 (m, 4 H, H-4 and H-5), 1.0 (s, 3 H, H-10), 0.96 (s, 3 H, H-9), 0.95 (s, 3 H, H-8). ¹³C NMR: (100 MHz, CDCl₃) δ = 210.9, 144.5, 143.9, 141.0, 121.8, 117.6, 110.0, 57.7, 46.8, 43.3, 29.9, 27.0, 19.8, 19.1, 9.2. EI-MS (70 eV), m/z (%): 230 [M]⁺ (100), 215 (21), 202 (24), 147 (92), 91 (88), 187 (79), 55 (56),119 (55), 95 (46), 120 (45), 65 (44), 77 (44). HR-MS (ESI): m/z 231.1380 [M + H]⁺ (calcd. For C₁₅H₁₈O₂, 231.1385).

(1R,3E)-3-[(furan-3-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (8): The compound was isolated as a light yellow solid with a yield of 87%. Mp: 58–59 °C. [α]²⁰_D = + 88.78 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2970, 2930, 2870 (aliphatic C–H), 1741 (conjugated C = O), 1670 (conjugated C = C), 1571, 1518 (furan ring), 1459, 1388 (C–H bend). ¹H NMR (400 MHz, CDCl₃): δ = 7.63 (s, 1 H, H-13), 7.42 (d, J = 2.8, 1 H, H-14), 7.03 (s, 1 H, H-11), 6.6 (d, J = 1.6, 1 H, H-15), 2.96 (d, J = 4.0, 1 H, H-3), 2.30–1.31 (m, 4 H, H-4 and H-5), 0.99 (s, 3 H, H-10), 0.95 (s, 3 H, H-9), 0.94 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 210.9, 144.5, 143.8, 141.0, 121.8, 117.6, 110.1, 57.6, 46.8, 43.3, 29.9, 27.0, 19.7, 19.1, 9.2. EI-MS (70 eV), m/z (%): 230 [M]⁺ (100), 215 (15), 202 (18), 147 (96), 91 (84), 187 (81), 75 (58, )55 (50),120 (45), 95 (43), 120 (45), 65 (42), 77 (43). HR-MS (ESI): m/z 231.1383 [M + H]⁺ (calcd. For C₁₅H₁₈O₂, 231.1385).

(1S,3E)-3-[(1-benzofuran-2-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (9): The compound was isolated as a white solid with a yield of 85%. Mp: 159–160 °C. [α]²⁰_D = -295.23 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2957, 2927, 2872 (aliphatic ν C–H); 1731 (conjugated C = O); 1637 (conjugated C = C); 1449, 1387, 1350 (ring vibrations δ C–H); 1256 (benzofuran ring ν C–O). ¹H NMR (400 MHz, CDCl₃): δ = 7.57 (d, J = 7.7, 1 H, H-15), 7.50 (d, J = 8.3, 1 H, H-19), 7.39 (m, 1 H, H-17), 7.24 (m, 1 H, H-16), 7.06 (s, 1 H, H-11), 6.91 (s, 1 H, H-13), 3.56 (d, J = 4.2, 1 H), 2.37–1.32 (m, 4 H, H-4 and H-5), 1.04 (s, 3 H, H-10), 1.03 (s, 3 H, H-9), 0.95 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.1, 155.5, 153.8, 142.5, 129.4, 125.6, 123.2, 121.3, 114.5, 111.3,111.2, 57.3, 49.9, 46.8, 30.6, 26.3, 20.9, 19.7, 9.3. EI-MS (70 eV): m/z 280 [M]+ (100), 265 (9), 252 (30), 237 (75), 131 (57), 197 (55), 115 (43), 209 (32), 55 (31), 252 (29), 170 (26), 141 (26). HR-MS (ESI): m/z 281.1540 [M + H]⁺ (calcd. For C₁₉H₂₀O₂, 281.1542).

(1R,3E)-3-[(1-benzofuran-2-yl)methylidene]-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (10): The compound was isolated as a white solid with a yield of 85%. Mp: 159–160 °C. [α]²⁰_D = + 295.23 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2963, 2931, 2872 (aliphatic ν C–H, sp³); 1747 (conjugated C = O); 1644 (conjugated ν C = C); 1449, 1387, 1350 (ring vibrations δ C–H); 1256 (benzofuran system ν C–O). ¹H NMR (400 MHz, CDCl₃): δ = 7.55 (d, J = 7.7, 1 H, H-15), 7.49 (d, J = 8.2, 1 H, H-19), 7.32 (m, 1 H, H-17), 7.25 (m, 1 H, H-16), 7.04 (s, 1 H, H-11), 6.89 (s, 1 H, H-13), 3.54 (d, J = 4.16, 1 H, H-3), 2.36–1.24 (m, 4 H, H-4 and H-5), 0.96 (s, 3 H, H-10), 0.93 (s, 3 H, H-9), 0.89 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.0, 155.5, 153.7, 142.5, 128.3, 125.6, 123.1, 121.3, 114.4, 111.3, 111.1, 57.2, 49.9, 46.7, 30.5, 26.3, 20.8, 19.7, 9.2. EI-MS (70 eV), m/z (%): 280 [M]+ (100), 265 (9), 252 (30), 237 (76), 131 (58), 197 (56), 115 (46), 209 (32), 55 (35), 252 (29), 170 (27), 141 (27). HR-MS (ESI): m/z 281.1539 [M + H]⁺ (calcd. For C₁₉H₂₀O₂, 281.1542).

(1S,3E)-1,7,7-trimethyl-3-[(thiophen-3-yl)methylidene]bicyclo[2.2.1]heptan-2-one (11): The compound was isolated as a light yellow solid with a yield of 93%. Mp: 46–47 °C. [α]²⁰_D = -416.23 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2962, 2927, 2869 (aliphatic ν C–H, sp³); 1727 (α,β-unsaturated ketone ν C = O); 1648 (conjugated C = C); 1454 (δ C–H ring); ~800–700 (thiophene ring, weak, ν C–S and δ C–H). ¹H NMR (400 MHz, CDCl₃): δ = 7.45 (d, J = 2.0, 1 H, H-13), 7.34 (dd, J = 3.0 and 4.9 1 H, H-14), 7.26 (d, J = 5.3, 1 H, H-15), 7.21 (s, 1 H, H-11), 3.05 (d, J = 4.0, 1 H, H-3), 2.36–1.31 (m, 4 H, H-4 and H-5), 1.01 (s, 3 H, H-10), 0.99 (s, 3 H, H-9), 0.85 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.3, 140.6, 137.4, 128.0, 127.4, 126.2, 121.2, 57.2, 49.4, 46.6, 30.7, 27.0, 20.5, 19.1, 9.3. EI-MS (70 eV), m/z (%): 246 [M]⁺ (100), 231 (15), 218 (30), 163 (99), 135 (66), 203 (64), 91 (56), 97 (44), 55 (42), 134 (40), 164 (36), 95 (34). HR-MS (ESI): m/z 247.1154 [M + H]⁺ (calcd. For C₁₅H₁₈OS, 247.1157).

(1R,3E)-1,7,7-trimethyl-3-[(thiophen-3-yl)methylidene]bicyclo[2.2.1]heptan-2-one (12): The compound was isolated as a light yellow solid with a yield of 93%. Mp: 46–47 °C. [α]²⁰_D = -416.23 (0.33 mg/mL in ethanol). FT-IR (ATR, cm⁻¹): 2958, 2928, 2872 (aliphatic ν C–H, sp³); 1722 (α,β-unsaturated ketone ν C = O); 1644 (conjugated C = C); 1454 (ring vibrations δ C–H ); ~800–700 (tiophene ring weak, ν C–S and δ C–H). ¹H NMR (400 MHz, CDCl₃): δ = 7.46 (d, J = 2.4, 1 H, H-13), 7.35 (dd, J = 3.0 and 5.0, 1 H, H-14), 7.28 (dd, J = 0.88 and 5.0, 1 H, H-15), 7.22 (s, 1 H, H-11), 3.06 (d, J = 4.1, 1 H, H-3), 2.37–1.25 (m, 4 H, H-4 and H-5), 1.2 (s, 3 H, H-10), 1.0 (s, 3 H, H-9), 0.83 (s, 3 H, H-8). ¹³C NMR (100 MHz, CDCl₃): δ = 208.2, 140.5, 137.3, 127.9, 127.4, 126.1, 121.1, 57.1, 49.3, 46.5, 30.7, 26.9, 20.5, 19.0, 9.2. EI-MS (70 eV), m/z (%): 246 [M]⁺ (100), 231 (15), 218 (27), 246 (99), 135 (66), 203 (65), 91 (59), 97 (45), 55 (46), 134 (42), 164 (36), 95 (35). HR-MS (ESI): m/z 247.1151 [M + H]⁺ (calcd. For C₁₅H₁₈OS, 247.1157).

Cell culture

Experimental cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA): HT-29 (human colorectal adenocarcinoma), PC-3 (human prostate cancer), AGS (human gastric adenocarcinoma), and the control group GES-1 (healthy gastric epithelial cells). All cancer cell lines were cultured in DMEM-F12 medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 1 mM glutamine. Cells were seeded in 96-well microplates at a density of 3 × 10³ cells per well in 100 µL of medium. After 24 h of incubation at 37 °C under a humidified atmosphere containing 5% CO₂ to allow cell attachment, cells were treated with different concentrations of the test compounds and incubated for 72 h under the same conditions. Stock solutions were prepared in ethanol, maintaining a constant concentration, with a final solvent concentration of 1%. Control cultures received 1% ethanol only. To ensure a consistent biological response, 21 passages were used for the GES-1 cell lines and 64, 22 and 34 for the PC-3, AGS and HT-29 cell lines, respectively.

Cytotoxicity assay

Cell viability was determined using the sulforhodamine B (SRB) assay, following the protocol previously described by Villena et al.¹⁵. Briefly, cells were seeded into 96-well plates (3 × 10³ cells/well) and incubated for 24 h. Subsequently, the cells were treated with the test compounds (at concentrations ranging from 0 to 100 µM) and incubated for an additional 72 h. After the treatment period, cells were fixed with trichloroacetic acid (10% final concentration) and stained with 0.1% sulforhodamine B. The protein-bound dye was then solubilized with a 10 mM Tris base solution, and the absorbance was measured at 540 nm using a microplate reader (BioTek EL808 microplate reader). The percentage of cell viability was calculated using the following Eq. (1):

Where “A sample” is the absorbance of cells treated with the test compound and “A control” is the absorbance of the control cells treated with the vehicle (1% ethanol). Daunorubicin and 5-fluorouracil were used as positive controls, and untreated cells served as the negative control. Ethanol was used as the vehicle solvent. IC₅₀ values were calculated by fitting dose-response curves to a four-parameter logistic model using SigmaPlot software, and 95% confidence intervals (CI) were determined for each value. Statistical significance of differences among treatments was assessed using the Kruskal–Wallis test followed by appropriate post hoc comparisons, as data did not follow a normal distribution. All experiments were performed in triplicate and repeated independently three times, with results expressed as mean ± standard deviation (SD).

Selectivity index

The selectivity index (SI) was calculated as the ratio between the IC₅₀ value determined in GES cells and the corresponding IC₅₀ value in the cancer cell line, according to Eq. (2):

A SI value > 2 is considered the threshold to classify a compound as selective toward cancer cell lines. Conversely, an SI < 2 indicates low selectivity, suggesting cytotoxicity toward both tumor and normal cells¹⁶.

DPPH radical scavenging assay

Antioxidant activity, evaluated as free radical scavenging capacity, was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, following a previously reported methodology¹⁷. For the assay, each test compound (0.1 mL, at concentrations ranging from 5 to 400 µg/mL) was mixed with a 50 µM ethanolic DPPH solution (2.9 mL). The mixture was incubated for 15 min at room temperature in the dark, and the resulting absorbance was measured at 517 nm. A blank sample containing solvent instead of the test compound was used as the control. The radical scavenging capacity (RSC) was then calculated using the following Eq. (3):

where “A control” is the absorbance of the control sample (without the test compound) and “A sample” is the absorbance in the presence of the test compound. The IC₅₀ values, corresponding to the concentration required to scavenge 50% of the radicals, were determined through linear regression analysis of the dose-response curves (percentage of radical scavenging vs. concentration). Data are presented as the mean ± standard deviation (SD) of three independent experiments, each performed in triplicate.

Antioxidant activity measurement by ABTS radical scavenging

The 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was determined according to a previously reported methodology¹⁸. Briefly, the ABTS⁺ radical cation was generated by reacting ABTS solution with potassium persulfate. Prior to the assay, this stock solution was diluted to an initial absorbance of approximately 0.700 at 732 nm. The scavenging capacity was then determined by mixing the test compounds at various concentrations with the ABTS⁺ solution and measuring the subsequent decrease in absorbance. Ascorbic acid was used as the positive control, and the antioxidant activity was expressed as the percentage of ABTS radicals scavenged, calculated using Eq. 3. Data are presented as the mean ± standard deviation (SD) of three independent experiments, each performed in triplicate.

Determination of mitochondrial membrane permeability by flow cytometry

Changes in mitochondrial membrane potential were evaluated using the cationic probe Rhodamine 123, following an established protocol¹⁹. Briefly, cells were treated with various concentrations of compounds 3, 5, or 9 for 48 h. After treatment, the cells were stained with Rhodamine 123 (1 µM), harvested by trypsinization, and immediately analyzed by flow cytometry. The results are expressed as the percentage of cells that retained the fluorescent probe, corresponding to the population with intact mitochondrial membrane potential.

Hemolysis assay

The hemocompatibility of the compounds was evaluated using a human red blood cell (RBC) hemolysis assay, adapted from a previously established protocol²⁰. First, human blood was collected from a healthy donor into EDTA-containing tubes. The RBCs were isolated from plasma by centrifugation (500 × g, 10 min). The resulting RBC pellet was then washed three times with phosphate-buffered saline (PBS) to remove any remaining plasma and buffy coat. For the assay, the washed RBCs were resuspended in PBS and incubated with the test compounds at various concentrations (10–300 µg/mL) for 30 min at 37 °C with gentle agitation. After incubation, the samples were centrifuged (1000 × g, 5 min) to pellet intact RBCs. The amount of hemoglobin released into the supernatant was quantified by measuring its absorbance at 540 nm. Deionized water and PBS were used as the positive (100% hemolysis) and negative (0% hemolysis) controls, respectively. The percentage of hemolysis was calculated using the following equation:

Where “A sample”: Absorbance of the supernatant with the sample; “A positive control”: Absorbance of the supernatant with the positive control; “A negative control”: Absorbance of the supernatant with the negative control. All experiments were performed in triplicate and repeated independently three times.

Physicochemical properties and in Silico toxicity prediction of Camphor-Derived compounds

The physicochemical properties of the camphor-derived compounds (3–12), including molecular weight (MW), logP, number of hydrogen bond acceptors (HBA), number of hydrogen bond donors (HBD), and topological polar surface area (TPSA), as well as gastrointestinal (GI) absorption and blood-brain barrier (BBB) permeability, were calculated using SwissADME (https://www.swissadme.ch). Lipinski’s rule of five and PAINS alerts were also obtained using this tool. Toxicity prediction (toxicological class and activity against hepatotoxicity [HT], immunotoxicity [IT], non-hepatotoxicity [NHT], reproductive toxicity [RT], and carcinogenicity [CT]) was performed with ProTox-II²¹ and pkCSM²². The results were summarized in a table to simultaneously evaluate physicochemical properties, absorption, and potential toxicity of camphor derivatives.

Statistical analysis

Data are expressed as mean ± standard deviation of three independent experiments performed in triplicate. A Kruskal–Wallis ANOVA test was applied with a 95% confidence level using STATISTICA 7.0, as the data did not follow a parametric distribution.

Results and discussion

Synthesis of target compounds

Ten camphor-derived Michael acceptors were synthesized using the Claisen-Schmidt condensation, a type of crossed aldol reaction ideal for forming α,β-unsaturated ketones. This reaction requires a strong, non-nucleophilic base, such as potassium tert-butoxide, to generate the necessary enolate intermediate from the camphor scaffold. The use of a strong base is critical because the enolate’s stability is limited by camphor’s rigid bicyclic structure, rendering milder bases ineffective. The condensation is completed by the subsequent addition of a respective aldehyde, which couples with the enolate to form the final product.

In this work, we investigated the previously unexplored Claisen-Schmidt condensation between camphor and heteroaromatic aldehydes. Our results demonstrate that this approach is highly effective, affording yields between 85% and 96%. These yields are not only comparable to but often superior to those reported in studies using traditional homonuclear aromatic aldehydes (80–90%). The introduction of a heteroaromatic ring introduces distinct electronic and steric properties, suggesting that these novel camphor derivatives may possess unique biological activities. Figure 2 shows the structures of the camphor isomers used as starting materials and the derivatives synthesized from them. These derivatives were subsequently isolated, purified, and characterized using FT-IR, NMR, and HR-MS.

Fig. 2.

Chemical structures of the starting materials, camphor isomers 1 and 2, and the synthesized derivatives 3–12.

In the IR spectrum confirmed the presence of bands corresponding to the carbonyl group (C = O), the unsaturated alpha-beta double bond (C = C), the typical bonds (= C-H) of the heterocyclic system, and the aliphatic-type bonds (C-H). showed singlet signals with chemical shifts in the range of 7.25–6.92 ppm for ¹H and 128.0–114.8 ppm for ¹³C. These signals, observed in the ¹H spectrum of synthetic compounds 3–12, were attributed to the presence of the typical trans-olefinic proton, which corresponds to the bond between camphor and the aromatic heterocyclic ring. These data were corroborated for all the molecules using the heteronuclear multiple-bond correlation (HMBC) spectra. In general, The H-11 proton of the camphor derivatives showed ²J heteronuclear couplings with carbon 2 and 12, and ³J couplings with carbon 1 and 3. For its part, mass spectrometry (EI-MS) revealed the molecular ion corresponding to all molecules, as well as their characteristic fragmentations.

Cytotoxicity and selectivity of compounds

The cytotoxic activity of the synthesized compounds was evaluated by determining the IC₅₀ values in three human cancer cell lines: AGS (gastric cancer), HT-29 (colorectal cancer), and PC-3 (prostate cancer). The results are summarized in Table 1, including the selectivity index (SI), calculated as the ratio between the cytotoxic concentration in cancer cells and that in normal cells.

Table 1.

Cytotoxic activity (IC₅₀, µM) of Camphor derivatives in human tumor cell lines and selectivity index (SI) values.

Cell lines IC50 (µM)a (SI)b
Compound	AGS	HT-29	PC-3	GES-1
1	> 100	> 100	> 100	201.5 ± 1.4
2	> 100	> 100	> 100	322.7 ± 1.7
3	99.9 ± 0.2 (1.85)	> 100	> 100	184.8 ± 0.33
4	> 100	> 100	> 100	154.8 ± 0.3
5	43.2 ± 0.3* (4.2)	87.5 ± 0.5* (2.1)	63.57 ± 0.6* (3.0)	181.44 ± 1.6
6	> 100	> 100	> 100	315.9 ± 1.4
7	> 100	> 100	> 100	238.2 ± 0.31
8	> 100	> 100	> 100	264.6 ± 0.5
9	31.8 ± 0.4* (7.0)	76.3 ± 0.2* (3.0)	97.0 ± 0.2* (2.3)	222.6 ± 0.6
10	> 100	> 100	> 100	278.5 ± 0.6
Daunorubicin	13.0 ± 0.3* (27.1)	15.1 ± 0.5* (23.3)	10.41 ± 0.4* (33.9)	352.89 ± 0.6
5-FU	56.1 ± 0.5* (2.5)	2.9 ± 0.7* (48.3)	16.4 ± 0.6* (3.4)	140.25 ± 0.31

ᵃIC₅₀ values (µM) represent the mean ± SD of three independent experiments performed in triplicate and were estimated by fitting dose-response curves to a four-parameter logistic model (95% confidence intervals). ᵇ The selectivity index (SI) was calculated as the ratio between IC₅₀ in non-tumor cells (GES-1) and tumor cells. Statistical differences versus ethanol-treated controls were evaluated using the Kruskal–Wallis test (p < 0.05), with significant differences indicated by an asterisk (*). Compounds with IC₅₀ > 100 µM were considered inactive. Daunorubicin and 5-fluorouracil (5-FU) were included as positive controls.

Compounds 1, 2, 4, 6, 7, 8, and 10 showed IC₅₀ values above 100 µM in the three evaluated cell lines, indicating low or no cytotoxic activity under the tested conditions. Both camphor enantiomers showed IC₅₀ activity over 100 µM under the conditions tested in all cell lines evaluated. Compound 3 exhibited moderate cytotoxicity against AGS (IC₅₀ = 99.9 ± 0.2 µM; SI = 1.85), while no significant activity was observed in HT-29 and PC-3 (IC₅₀ > 100 µM). Compounds 5 and 9 demonstrated the highest cytotoxic activity among the tested compounds. Compound 5 presented IC₅₀ values of 43.2 ± 0.3 µM (SI = 4.2), 87.5 ± 0.5 µM (SI = 2.1), and 63.57 ± 0.6 µM (SI = 3.0) in AGS, HT-29, and PC-3, respectively. In turn, compound 9 reached IC₅₀ values of 31.8 ± 0.4 µM (SI = 7.0), 76.3 ± 0.2 µM (SI = 3.0), and 97.0 ± 0.2 µM (SI = 2.3) in the same cell lines. The compounds were not active under the conditions tested on the GES-1 (healthy cells) cell line; all showed an IC₅₀ greater than 100 µM.

As positive controls, daunorubicin displayed strong cytotoxic activity, particularly in PC-3 (IC₅₀ = 10.41 ± 0.4 µM; SI = 33.9), while 5-fluorouracil (5-FU) exhibited high activity against HT-29 (IC₅₀ = 2.9 ± 0.7 µM; SI = 48.3). It is worth noting that, except for compound 3, all compounds that showed activity against cancer cell lines presented a selectivity index greater than 2 and therefore can be considered selective toward tumor cells^23–25.

Several studies have reported that camphor and its derivatives possess significant cytotoxic potential against different cancer cell lines. In a study with various monoterpenes, camphor showed moderate cytotoxicity in HCT-116 colon cancer cells with IC₅₀ values of 4.5 Mm²⁶. Likewise, essential oils with high camphor content exhibited significant cell growth inhibition in A375 melanoma cells, T98G glioblastoma cells, and MDA-MB-231 breast cancer cells. IC₅₀ values ranged from 47 to 97 µg/mL²⁷. Interestingly, the combination of camphor and menthol showed synergistic effects against breast cancer-associated cell lines, MCF-7, MDA-MB-231 and MDA-MB-436, reaching IC₅₀ values of 27.6, 31.2 and 36.5 µg/mL, respectively. It is worth mentioning that the use of this mixture of camphor and menthol showed lower toxicity in non-tumor cells²⁸.

Furthermore, heterocyclic camphor derivatives conjugated to pyrimidine rings showed marked activity against MDA-MB-231 (breast cancer), RPMI-8226 (multiple myeloma), and A549 (lung adenocarcinoma) cell lines, with reduced cytotoxicity in normal cells (GES-1), an effect also observed for the compounds described in this study. These findings suggest a good selectivity of this class of compounds. Likewise, quaternary camphor derivatives tested on lymphoid lines such as CEM-13, U-937, and MT-4 revealed potencies in the micromolar range, highlighting once again their potential as a structural scaffold for the development of selective anticancer agents^29,30. A more in-depth analysis of the structure-activity relationship (SAR) offers an explanation for these differences in potency. It is particularly noteworthy that compound 5 (a thiophene derivative) and compound 9 (a benzofuran derivative) exhibit greater cytotoxicity compared to their furan analog, compound 3. The higher activity of compound 5 suggests that the presence of the sulfur heteroatom may be a key factor for its biological activity, as the sulfur atom in the thiophene ring is known to enhance drug-receptor interactions, possibly through additional hydrogen bonding³¹. Similarly, the high potency of compound 9, which incorporates a larger and more rigid benzofuran ring system, indicates that a greater aromatic surface and structural rigidity could favor a more effective binding to its site of action. Fused heterocycles like benzofuran are known to provide better hydrophobic and aromatic (e.g., π-π) interactions, acting as excellent cores for drug design³². These structural features likely contribute to the enhanced cytotoxic profile observed in our study.

Antioxidant capacity

The antioxidant capacity of the synthesized compounds was evaluated using DPPH and ABTS radical scavenging assays, with ascorbic acid (AA) as a positive control. The resulting IC₅₀ values are summarized in Table 2; Fig. 3.

Table 2.

Antioxidant activity of camphor-derived compounds determined by DPPH and ABTS assays (IC₅₀, µM).

Compound	DPPH (µM)	ABTS (µM)
1	> 2627.7	> 2627.7
2	> 2627.7	> 2627.7
3	430.0 ± 3.8	953.0 ± 1.5
4	452.4 ± 2.7	1003.6 ± 2.0
5	586.4 ± 2.4	1301.2 ± 2.4
6	488.2 ± 1.8	1082.2 ± 1.7
7	471.5 ± 2.1	1046.0 ± 3.3
8	606.0 ± 2.4	1344.4 ± 3.4
9	428.9 ± 1.8	954.7 ± 2.7
10	516.1 ± 2.7	1145.5 ± 3.7
11	432.3 ± 3.8	1396.0 ± 3.8
12	462.7 ± 2.8	1024.6 ± 3.5
AAa	59.6 ± 0.6	141.2 ± 3.5

The IC₅₀ values correspond to the concentration required to inhibit 50% of free radicals in each assay. Results are expressed as mean ± standard deviation of three independent determinations. Values higher than 2627 µM were considered inactive. ^aAscorbic acid (AA) was used as a positive control (p < 0.05).

Fig. 3.

Percentage of DPPH (A) and ABTS (B) radical inhibition as a function of the logarithm of concentration (µM) for compounds 3–12 and ascorbic acid (AA, reference antioxidant). Each point represents the mean ± standard deviation (SD) of three independent experiments performed in triplicate. Error bars indicate the experimental variability among replicates.

The parent camphor stereoisomers (compounds 1 and 2) were inactive in both assays. In contrast, most of the camphor derivatives exhibited moderate dose-dependent antioxidant activity. In the DPPH assay, the IC₅₀ values ranged from 428.9 to 606.0 µM. A similar trend, though with generally lower potency (higher IC₅₀ values), was observed in the ABTS assay, with IC₅₀ values ranging from 953.0 to 1396.0 µM. Among the derivatives, compound 3 consistently emerged as the most promising candidate, displaying the lowest IC₅₀ value in both the DPPH assay (430.0 ± 3.8 µM) and the ABTS assay (953.0 ± 1.5 µM). Nonetheless, all derivatives were significantly less potent than the ascorbic acid control (DPPH IC₅₀ = 59.6 ± 0.6 µM; ABTS IC₅₀ = 141.2 ± 3.5 µM).

The inactivity of the parent camphor molecule as a direct radical scavenger is consistent with existing literature. Several studies suggest that camphor’s protective effects in vivo are not due to direct scavenging but rather to its ability to modulate the endogenous antioxidant system. For example, in diabetic rat models, camphor treatment has been shown to increase the activity of enzymes like SOD, CAT, and GPx and elevate glutathione levels, thus counteracting oxidative stress³³. However, its effects can be complex, as other studies report that camphor may induce oxidative stress under different conditions, indicating a dose- and model-dependent response³⁴.

The fact that structural modification of the camphor nucleus in our study led to derivatives with direct radical-scavenging capacity is noteworthy. This aligns with reports on other camphor-derived heterocycles containing hydrazone, Schiff base, or thiazole moieties, which have demonstrated significant in vitro antioxidant activity, sometimes comparable to standards like Trolox^35,36. Therefore, these findings confirm that the camphor scaffold is a viable starting point for developing new chemical entities with an enhanced antioxidant profile.

Changes in membrane permeability induced by compounds 3, 5 and 9

To gain initial insight into the mechanism of action behind the observed cytotoxicity, we investigated whether the compounds induced apoptosis via the mitochondrial pathway, a central mechanism in cancer cell death³⁷. Our flow cytometry results (Fig. 4) conclusively demonstrate that treatment with compounds 3, 5, and 9 leads to a loss of the mitochondrial membrane potential.

Fig. 4.

Effect of the compounds 3, 5 and 9 on the mitochondrial membrane permeability of cancer cell line. Cells were exposed to different compound concentrations (µM) for 48 h as it is indicated in the graphic. C- are control cells (1% ethanol), C + are cells treated with FCCP (5 µM). Data represent the means ± S.D., (n = 3, *p < 0.05 vs. negative control).

This event is a hallmark of the opening of the mitochondrial permeability transition pore (mPTP), a multi-protein channel whose sustained activation is a critical event in initiating cell death³⁸. The opening of the mPTP leads to the collapse of the membrane potential and the release of pro-apoptotic factors, such as cytochrome c, from the intermembrane space, which in turn triggers the caspase cascade and executes apoptosis³⁹. Interestingly, while compound 3 also triggers the loss of mitochondrial potential, its overall cytotoxicity was significantly lower than that of compounds 5 and 9. This suggests that while mPTP opening is the common mechanism, the ultimate cytotoxic potency is modulated by other structural and functional properties. For instance, the potent antioxidant capacity of compound 3 might partially counteract the pro-apoptotic signals that lead to cell death. Alternatively, its furan structure may be less effective at inducing a sustained or complete pore opening compared to the thiophene and benzofuran moieties of the more potent compounds 5 and 9. Nevertheless, the ability of these camphor derivatives to induce mPTP-mediated events is significant, as many cancer cells develop resistance by suppressing this pathway. Therefore, small molecules that can bypass these defenses represent a highly promising therapeutic strategy^37,38.

Haemocompatibility of camphor-derived compounds

The hemolytic activity of the selected compounds was evaluated in human erythrocytes at different concentrations (10–300 µg/mL), and the percentages of hemolysis obtained are summarized in Table 3.

Table 3.

Hemolytic activity of camphor-derived compounds in human erythrocytes at different concentrations (µg/mL).

Compounds	Concentrations (µg/mL)
	10	25	50	100	200	300
	Hemolysis (%)
1	0	0.06	3.11	4.80	9.67	10.12
2	5.25	6.55	9.61	11.29	16.16	16.62
3	6.75	8.31	17.85	23.63	28.18	35.32
4	15.71	18.18	20.06	31.81	41.49	49.74
5	0	0	1.10	3.11	7.40	13.83
6	6.36	7.07	16.75	21.55	26.68	28.96
7	0	0	0	1.81	3.12	5.97
8	0	2.53	6.29	8.50	9.74	12.20
9	2.01	3.31	6.36	8.05	12.92	13.37
10	0	0.38	3.12	5.84	6.16	8.05
11	0	0.77	5.25	7.20	11.16	13.24
12	5.90	6.81	7.92	9.41	10.25	12.46
Triton X-100 1%	100	100	100	100	100	100

The values represent the percentage of hemolysis obtained as a function of the concentration of each compound. Results correspond to the mean of three independent assays (p < 0.05). Hemolysis values below 10% are generally considered indicative of low cytotoxicity in erythrocytes⁴⁰.

Compounds 1, 5, 7, 8, 10, and 11 exhibited low hemolysis across the entire concentration range tested, with maximum values below 14%, indicating good compatibility with cell membranes. In contrast, compounds 2, 3, 4, 6, and 12 displayed a concentration dependent increase in hemolysis, reaching maximum percentages ranging from 13% to 50%, with compound 4 inducing the highest hemolytic damage (49.74% at 300 µg/mL).

These results suggest that most of the compounds exhibit low hemolytic toxicity at moderate concentrations, although some, particularly compound 4, may exert adverse effects at higher doses. The low hemolysis observed for the majority of compounds supports their potential for biomedical applications; however, further studies are required to assess their safety under physiological conditions. This finding is particularly relevant since, to date, no studies have reported the hemolytic activity of camphor or its derivatives, suggesting that the potential interaction of these molecules with erythrocyte membranes remains unexplored.

Considering the hemolysis and cytotoxicity results together, a preliminary estimation of the therapeutic window of these compounds can be inferred. Derivatives 5 and 9, which exhibited cytotoxic effects against tumor cell lines at low micromolar concentrations while maintaining minimal hemolysis (< 15%) even at the highest tested doses, demonstrate a favorable safety margin. In contrast, compound 4 caused significant hemolysis at comparable concentrations, suggesting a narrower therapeutic window. These findings indicate that the structural features governing both cytotoxicity and membrane compatibility should be optimized to enhance selectivity toward tumor cells while minimizing adverse effects on erythrocytes and other normal cell types.

In Silico assessment of toxicological risk

As shown in Table 4, camphor-derived compounds (3–12) meet the drug-likeness criteria according to Lipinski’s rule of five. The number of hydrogen bond acceptors (HBA) ranges from 1 to 2, while all compounds lack hydrogen bond donors (HBD = 0), suggesting low polarity. LogP values range from 3.69 to 4.84, indicating moderate lipophilicity, and the total polar surface area (TPSA) varies between 30 and 45 Ų, compatible with good cellular permeability. According to the BOILED-Egg model, all compounds exhibit high gastrointestinal (GI) absorption and potential to cross the blood-brain barrier (BBB).

Table 4.

Physicochemical properties and in Silico toxicity predictions of camphor-derived compounds.

Criteria	3	4	5	6	7	8	9	10	11	12
MW	230.30	230.30	246.37	246.37	230.30	230.30	280.36	280.36	246.37	246.37
HBA	2	2	1	1	2	2	2	2	1	1
HBD	0	0	0	0	0	0	0	0	0	0
LogP	3.69	3.69	4.16	4.16	3.69	3.69	4.84	4.84	4.16	4.16
TPSA	30.21	30.21	45.31	45.31	30.21	30.21	30.21	30.21	45.31	45.31
GI	High	High	High	High	High	High	High	High	High	High
BBB	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
LP	0	0	0	0	0	0	0	0	0	0
TC	4	4	5	5	5	5	5	5	5	5
HT	I	I	I	I	I	I	I	I	I	I
NT	I	I	I	I	I	I	A	A	I	I
NPHT	I	I	I	I	I	I	I	I	I	I
RT	I	I	I	I	I	I	A	A	I	I
CT	I	I	I	I	I	I	I	I	I	I

The calculated values of molecular weight (MW), hydrogen bond acceptors and donors (HBA/HBD), logP, total polar surface area (TPSA), gastrointestinal absorption (GI), and blood-brain barrier permeability (BBB) are presented. Lipinski and PAINS (LP) alerts are indicated as the number of violations. Toxicity was predicted using in silico models, indicating the general toxicological class (TC) and activity (I = Inactive, A = Active) against hepatotoxicity (HT), neurotoxicity (NT), nephrotoxicity (NPHT), respiratory toxicity (RT), and cardiotoxicity (CT).

Regarding toxicity predictions, most compounds were classified as class 4–5, corresponding to low to moderate toxicity. In specific toxicity profiles, all compounds were predicted as inactive toward hepatotoxicity (HT), nephrotoxicity (NPHT), respiratory toxicity (RT), and cardiotoxicity (CT). Only compounds 9 and 10 showed potential activity against neurotoxicity (NT) and respiratory toxicity (RT), indicated as active. Despite the potential toxicity of these compounds, the results suggest that camphor derivatives possess a favorable pharmacokinetic profile, with good absorption and permeability, which meets the classic drug-likeness criteria. Their low polarity (HBD = 0 and TPSA < 50 Ų) and moderate lipophilicity (logP < 5) probably contribute to both the efficient cell penetration and the low hemolysis observed in previous experimental assays^41,42.

From a safety perspective, in silico predictions indicate that most compounds present a low toxicological risk, except for compounds 9 and 10, which may require further investigation regarding neurotoxicity and respiratory toxicity. These findings complement experimental data on antioxidant and hemolytic activity, suggesting that compounds 3–8 and 11–12 combine active biological profiles with low potential toxicity, making them the most promising candidates for further optimization and study.

Although all synthesized derivatives satisfy Lipinski’s rule of five and exhibit favorable gastrointestinal absorption according to the BOILED-Egg model, their experimental activities varied considerably. This discrepancy indicates that, while in silico predicted physicochemical properties support drug-likeness, specific molecular interactions such as the nature of heteroaromatic substituents, likely govern target engagement and biological response. Thus, in silico predictions align with the feasibility of oral bioavailability but cannot fully anticipate the variations in cytotoxic and antioxidant activity observed experimentally. Notably, the higher predicted toxicity of compounds 9 and 10 is consistent with experimental observations, where these compounds exhibited increased hemolysis relative to the rest of the series. This correlation reinforces the reliability of the in silico models, highlighting their value as a complementary tool for the early identification of potentially harmful derivatives. However, it is crucial to acknowledge their inherent limitations. These predictions depend on the quality of the algorithms and the data they were trained on, which may not accurately capture the complex biological interactions of novel scaffolds like camphor-based derivatives. Furthermore, factors such as metabolic activation or off-target interactions are typically not considered, underscoring the necessity of experimental validation.

In summary, the evaluated camphor derivatives exhibited a balanced biological profile, combining selective cytotoxicity against tumor cells (compounds 5 and 9) with notable antioxidant activity (compound 3). Low hemolysis and in silico toxicity predictions suggest limited risk to normal cells, although compounds 9 and 10 may require further evaluation for potential neurotoxic and respiratory effects.

Differences in biological activity among the isomers can be attributed primarily to stereochemistry, with derivatives from S-(-)-camphor and R-(+)-camphor showing variations in cytotoxicity, antioxidant activity, and tumor selectivity. Additionally, the electronic and steric properties of heteroaromatic substituents modulate activity: electron-donating groups enhance antioxidant capacity, whereas electron-withdrawing groups increase cytotoxic selectivity. These results indicate that subtle structural and stereochemical variations critically influence the balance between efficacy and safety^43,44, highlighting the potential of these derivatives as candidates for anticancer and antioxidant agents.

Conclusions

The thiophene (compound 5) and benzofuran (compound 9) derivatives are the most potent and selective agents against the tested cancer cells, acting through apoptosis induction. These active compounds also exhibit a favorable safety profile, with low toxicity toward red blood cells, suggesting a promising therapeutic window. Taken together, these findings confirm that these camphor derivatives are viable candidates for the development of future anticancer therapies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Evelyn Muñoz-Núñez: Conceptualization, Methodology, Writing, Validation, Funding. Susana Flores: Methodology, Formal analysis. Valentina Silva: Investigation, Writing, Validation, Formal analysis. Joan Villena: Methodology, Validation, Formal analysis. Rut Vergara: Conceptualization, Methodology. Iván Montenegro: Visualization, Formal analysis. Luis Fernandez Hernandez: Software, Methodology. Alejandro Madrid: Conceptualization, Visualization, Writing, Validation, Formal analysis. All authors reviewed the manuscript.

Funding

This work was supported by FONDECYT Postdoctoral Project N° 3230296, funded by ANID, Chile and Programa de Apoyo para el pago de Procesamiento de Artículos InES Género 2025, Dirección General de Investigación, Universidad de Playa Ancha, Chile, D.E. 0282/2025.

Data availability

All data that were analyzed during this study are included in this article, and further inquiries can be directed to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.