Abstract
Background/Aim: As part of our continuing investigation in coumarin derivatives as potential anticancer substances, a series of alkylpsoralens were synthesized, and their antiproliferative activity was evaluated in leukemic HL60 cells. Materials and Methods: Alkylpsoralens were systematically synthesized from the combination of several chloroketones and 7-hydroxycoumarin derivatives. Results: Among the compounds synthesized, 4,4’,8-trimethylpsoralen demonstrated the most potent activity (IC50=6.6 μM). Conclusion: The correlation between the alkylation pattern and antiproliferative activity showed the importance of the C4-methyl and C8-methyl moieties in the psoralen nucleus as well as the importance of lipophilicity for their antiproliferative activity.
Coumarins are a large group of naturally occurring compounds with a wide range of biological properties. They represent a privileged scaffold for medicinal chemists because of their peculiar physicochemical properties, and versatile synthetic transformation into a large variety of functionalized coumarins, including the 7-substituted coumarin (1), the coumarin-dimer (2) and 3-arylcoumarin derivatives (3).
Furocoumarins can be divided into the linear type, where the furan ring is attached at the C6-C7 position of the coumarin nucleus, and the angular type carrying the furan ring at the C7-C8 position. The linear furocoumarins are mainly distributed in Apiaceae, Moraceae, Rutaceae, and Leguminosae, and the angular furocoumarins are found among Apiaceae and Leguminosae (4, 5). They have been used medicinally in eastern countries for ages; the Ayruveda, the Indian hallowed book, portrays Psoralea carylifolia poultice, and old Egyptians utilized Ammi majus for leukoderma.
Psoralens, the most abundant linear furocoumarin, have been shown to possess a wide spectrum of biological activities including photosensitizing, insecticidal, antifungal, and antibacterial activities (4). Furthermore, psoralens have been isolated from natural resources as potential therapeutic agents for several types of cancer (6–9). Because of the growing body of evidence regarding their anticancer potential, more attention is being paid to the elucidation of the possibility of using them thereof in clinical practice (10–12). Although several efforts have been made to obtain new alkylated furocoumarins and their biological activity has been discussed (13, 14), little is known about the effect of alkylation of psoralens on their antiproliferative activity.
In the course of our investigation on the structure-activity relationship of coumarins (15–17), the effect of alkylation of psoralen drew our attention. To obtain a better understanding of the biological activity of alkylpsoralen, we synthesized 28 alkylpsoralens and evaluated their biological activity. This research could provide useful guidance to the design and optimization of potent coumarin-type antiproliferative agents for anticancer therapy.
Materials and Methods
General procedures. Chemicals and solvents from commercial sources were used without further purification unless specified. Reactions were carried out under argon and monitored by thin-layer chromatography on silica gel (mesh size 60, F254) with visualization under UV light. Standard and flash column chromatography procedures were not optimized. Nuclear magnetic resonance (NMR) spectra were recorded on a 400-MHz JEOL ECP-400 spectrometer (JEOL, Tokyo, Japan), and chemical shift values are expressed in ppm (δ) relative to the residual 1H signal of the solvents. Unless otherwise specified, compounds were dissolved in 2HCCl3.
Synthesis of 4,4’-dimethylpsoralen (6b) (Figure 1). The general procedure for the synthesis of alkylpsoralen is shown in Figure 1 (6a-6y). Various alkylated psoralens were systematically synthesized from the combination of 7-hydroxy-4-methylcoumarin with the appropriate chloroketone. To an ice-cold solution of resorcinol (1a, 1.24 g, 11.2 mmol) in dioxane (ca. 0.5 ml), conc. H2SO4 (10 ml) was added dropwise at 10°C. After the addition of conc. H2SO4, ethyl 3-oxobutanoate (2a, 1.73 g, 13.3 mmol) was added dropwise over 30 min, and the mixture was stirred for 21 h at room temperature. After the reaction, ice water was added to the mixture and stirred for 0.5 h, and the precipitate was filtered and dried under reduced pressure. The resulting mixture was recrystallized from ethanol to afford 7-hydroxy-4-methylcoumarin (3a; yield 76%). For 3a: 1H-NMR (CDCl3) δ 2.40 (s, 3H), 6.14 (s, 1H), 6.83 (dd, J=2.6, 8.2 Hz), 6.92, (d, J=2.6 Hz, 1H), 7.48 (d, J=8.8 Hz), 9.2 (br. s, 1H). To a solution of 1-chloropropan-2-one (4a, 0.33 g, 3.4 mmol), K2CO3 (0.58 g, 4.3 mmol) and KI (0.12 g, 0.72 mmol) in acetone (30 ml) 3b (0.51 g, 2.9 mmol) were added, and the reaction mixture was refluxed for 15 h. After being cooled to room temperature, the organic solvent was removed under reduced pressure to provide 4-methyl-7-(2-oxopropyloxy)coumarin (5b, 0.41 g, 1.8 mmol, yield from 3b: 63%). The whole amount of 5b was dissolved in 0.1 M NaOH (50 ml) and ethanol (50 ml), and the reaction mixture was refluxed for 2 h. After being cooled to room temperature, the reaction mixture was neutralized by the addition of 0.6 M HCl, and the resulting precipitate was filtered and dried under reduced pressure. The residue was added to an appropriate volume of deionized water and partitioned between ethyl acetate and water. The organic phase was dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. The residue was chromatographed over a silica gel column (hexane/ethyl acetate; 8:2) to obtain the desired product (6b, 0.19 g, 0.89 mmol, yield from 5b; 49%). For 6b: 1H-NMR (CDCl3) δ 2.28 (d, 3H, J=1.1 Hz), 2.52 (s, 3H), 6.26 (s, 1H), 7.40 (s, 1H), 7.46 (q, 1H, J=1.1 Hz), 7.67 (s, 1H).
Reagents and conditions for synthesis of 6b and 12b: i) conc. H2SO4, rt, 21 h; ii) 1-chloropropan-2-one (4a), K2CO3, KI, acetone, reflux, 15 h; iii) 0.1 M NaOH (50 ml)-EtOH (50 ml), reflux, 2 h; iv) separation on silica gel column chromatography; v) 3-chloroprop-1-ene (8), K2CO3, KI, acetone, reflux, 15 h; vi) N,N’-dimethylaniline, reflux, 5 h.
By combination of the resorcinol derivatives, namely resorcinol (1a), 2-methylbenzene-1,3-diol (1b), 5-methylbenzene-1,3-diol (1c), the β-ketoester derivatives, namely ethyl 3-oxobutanoate (2a), ethyl 3-oxopentanoate (2b), ethyl 3-oxo-3-phenylpropanoate (2c), ethyl 2-methyl-3-oxobutanoate (2d), ethyl 2-ethyl-3-oxobutanoate (2e), ethyl 2-oxocyclopentane-1-carboxylate (2f), ethyl 2-oxocyclohexane-1-carboxylate (2g), ethyl 2-fluoro-3-oxobutanoate (2h), ethyl 2-chloro-3-oxobutanoate (2i) and ethyl 4,4,4-trifluoro-3-oxobutanoate (2j), and the α-chloroketone derivatives, namely 1-chloropropan-2-one (4a), 3-chlorobutan-2-one (4b) and 1-chloro-3,3-dimethylbutan-2-one (4c), 28 alkylpsoralens were synthesized. Spectral data for 6b are shown in Table I along with data for other synthetic alkylpsoralens.
Analytical data of synthesized compounds.
Synthesis of 8-allyl-4,4’-dimethylpsoralen (12b). General procedure for synthesis of 8-allylated alkylpsoralen is shown in Figure 1 (12a-12c). A mixture of 7-hydroxy-4-methylcoumarin (3a, 0.48 g, 2.7 mmol) 3-bromoprop-2-ene (8, 0.41 g, 3.4 mmol), K2CO3 (0.60 g, 4.3 mmol), and KI (0.11 g, 0.66 mmol) in acetone (30 ml) was refluxed for 15 h. After being cooled to room temperature, the organic solvent was removed under reduced pressure and the residue was partitioned between dichloromethane and water. Concentration of the organic phase produced 4-methyl-7-allyloxycoumarin (9b, 0.56 g, 2.6 mmol, yield from 3a: 96%). The whole amount of 9b was dissolved in N,N’-dimethylaniline (20 ml), and the reaction mixture was refluxed for 5 h. After being cooled to room temperature, the reaction mixture was neutralized by 5% HCl, portioned between ethyl acetate and water, and the organic phase was concentrated. The resulting residue was chromatographed over a silica gel column (dichloromethane/ethyl acetate; 95:5) to obtain 8-allyl-7-hydroxy-4-methylcoumarin (10b, 0.21 g, 0.97 mmol, yield from 9b: 38%) as the rearranged product.
Introduction of furan ring to 10b was similarly done as in the case of 3b. The whole amount of 10b was etherified by 4a to give 8-allyl-4-methyl-7-(2-oxopropyloxy)coumarin (11b), followed by the furan ring cyclization to afford 8-allyl-4,4’-dimethylpsoralen (12b, 0.11 g, 0.43 mmol, yield from 10b: 44%) as the desired product. For 12b: 1H-NMR (CDCl3) δ 2.28 (d, 3H, J=1.4 Hz), 2.51 (s, 3H), 3.48 (d, 2H, J=6.2 Hz), 5.03 (dd, 1H, J=10.2, 1.4 Hz), 5.12 (dd, 1H, J=17.0, 1.4 Hz), 6.07 (m, 1H), 6.25 (s, 1H), 7.47 (d, 1H, J=1.4 Hz), 7.57 (s, 1H).
Cell proliferation assay. HL60 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. The level of cellular proliferation of HL60 cells grown in a 96-well microplate was measured by using Alamar Blue (Life Technologies Ltd., Tokyo, Japan). To each well, 100 μl of HL60 cell suspension (1.0×104 cells/100 μl) was inoculated and 100 μl of medium containing serial dilution of the samples to be assayed was added. After three days of incubation, 20 μl of alamar blue was aseptically added to each well, and cells were further incubated for approximately 20 h. Cellular proliferation (as a percentage of the untreated control) was calculated with the following equation:
where A570 and A595 are the absorbance at 570 nm and 595 nm, respectively.
Results and Discussion
Chemistry. To explore the effects of different property groups and their position on psoralen on HL60 antiproliferative activity, a series of alkylpsoralen derivatives were prepared from the combination of resorcinol (1a-1c), β-ketoester (2a-2j) and α-chloroketone (4a-4c) derivatives. The synthesis of 4,4’-dimethylpsoralen (6b) is illustrated in Figure 1 as a general procedure for alkylpsoralen synthesis. Synthesis of alkylpsoralens was largely divided into two steps; the first step was the construction of coumarin nucleus by the Pechmann reaction between the resorcinol derivative and the β-ketoester derivative (18), and the second was the fusion of furan-ring to the coumarin nucleus by the cyclization of 7-(2-oxoalkyloxy)coumarin under basic conditions.
In this study, the introduction of furan ring to the coumarin nucleus was achieved by the cyclization of the 7-(2-oxoalkyloxy)coumarin derivative prepared from the Williamson’s etherification of the appropriate combination of 7-hydroxycoumarin derivatives with several α-chloroketones. To suppress the production of angelicin-type structural isomer by the intermolecular nucleophilic addition of the carbonyl carbon by the C8 aromatic carbon, the cyclization was carried out under basic conditions. This process produced an alkylpsoralen as a main product.
Antiproliferative activity. These synthesized compounds were evaluated for their antiproliferative activity in vitro toward HL60 leukemic cells using serial dilutions in a 96-well microplate, and psoralen (13) was used as the positive control. The obtained results are summarized in Table II. Among the compounds tested, compound 6x demonstrated the most potent activity (IC50=6.6 μM, clog P=3.469), followed by 6b (IC50=7.1 μM, clog P=2.97), 6c (IC50=7.5 μM, clog P=3.499), 6w (IC50=21 μM, clog P=2.97) and 6a (IC50=30 μM, clog P=2.471). These compounds proved to be more potent than psoralen (13, IC50=133 μM, clog P=1.972).
Structures, IC50 (μM) and clog P of alkylpsoralens.
Structure and activity relationship. The biological data of the synthesized compounds led to a series of considerations that permitted a more defined structure-activity relationship to be delineated for the alkylpsoralen derivatives. To examine substituent effects at the C4 position in detail, the derivatives of methyl, ethyl, phenyl, and trifluoromethyl were synthesized. Phenyl and trifluoromethyl were selected for their hydrophobic and electron-withdrawing properties. Results showed that the maximal antiproliferative activity was obtained for 6b (IC50=7.1 μM) with 4-methyl followed by 6c (IC50=7.5 μM) with 4-ethyl. Introduction of phenyl at the C4 position (6d, IC50 >200 μM, clog P=4.359) led to the loss of the antiproliferative activity, probably because of the excessively elevated hydrophobicity. The replacement of C4-methyl (6b, clog P=2.97) with C4-trifluoromethyl (6v, clog P=3.354), which is a same-sized substituent compared with the methyl group, had a clear effect on lipophilicity. It is interesting to note that, despite the elevated lipophilicity, the biological activity of the C4-trifluoromethyl derivative (6v, IC50 >200 μM) completely diminished. These results suggested the importance of the electron-donating property at the C4 position for its biological activity.
None of the modifications at the C3 position of 6b (IC50=7.1 μM) proved successful in improving the biological properties of the compound. The introduction of either the methyl group 6e (IC50 >200 μM), the ethyl group 6f (IC50 >200 μM), the fluoro group 6t (IC50 >200 μM), or chloro group 6u (IC50 >200 μM), yielded compounds that were devoid of antiproliferative activity, suggesting the crucial role of the hydrogen atom at the C3 position. The necessity of the hydrogen atom at the C5 position for the biological activity was also indicated by the comparison of 6a (IC50=30 μM) with 6i (IC50 >200 μM), 6b (IC50=7.1 μM) with 6j (IC50 >200 μM), and 6c (IC50=7.5 μM) with 6k (IC50 >200 μM).
Compounds 6w (IC50=21 μM) and 6x (IC50=6.6 μM), which bear a methyl group at the C8 position, are slightly more active than their respective counterparts, namely 6a and 6b. However, this did not happen in the case of 6y (IC50 >200 μM, clog P=3.499); the C8 methylated derivative showed decreased activity when compared with 6c (IC50=7.5 μM, clog P=3.998); the difference could partly by attributed to its elevated lipophilicity.
In summary, we designed and synthesized a series of alkylpsoralens as anticancer agents and evaluated their antiproliferative activity against HL60 cells. It can be concluded that psoralen is a promising scaffold in anticancer drug design. Furthermore, methylation at the C4 and C8 positions induced their antiproliferative activity.
Footnotes
Authors’ Contributions
Conceptualization and Study Design: SK, YY; Execution of Experiments: SK, YM, HN, TS; Data Analysis: SK, YY; Manuscript writing and editing: SK, YY.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this article.
- Received January 26, 2022.
- Revision received February 15, 2022.
- Accepted February 16, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
