Preparation and biological evaluation of coumarin amide derivatives bearing fluorine as potential fungicides and anticancer agents

2.1 General experimental

Thin-layer chromatography (TLC) was performed on silica gel plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Melting points were determined on The X-5A micro melting point tester (Gongyi Kerui instrument Co., Ltd). NMR spectra were performed on a Bruker Avance neo 400 MHz instrument (Bruker, Bremerhaven, Germany). HRMS was performed on Xevo G2-SQTOF instrument (Waters, Milford, MA, USA).

2.2 Antifungal assay

In vitro evaluations were conducted on compounds A1 ∼ 9, B1 ∼ 14, and C1 ∼ 11 to assess their impact on the growth of phytopathogenic fungi by inhibiting mycelial growth. Five phytopathogenic fungi, including Alternaria brassicae (AB), Alternaria alternata (AA), Colletotrichum gloeosporioides (CG), Pyricularia grisea (PG), Fusarium oxysporium f. sp.vasinfectum (FV) were used for the assays. The dissolved compounds in acetone were combined with the PDA at 50 μg/mL before being transferred to sterilized Petri dishes. Acetone served as blank controls, and kresoxim-methyl, a commercial fungicide, used as the positive control. Mycelia disks measuring 4 mm in diameter were inoculated onto PDA Petri dishes and incubated at 25 °C for a period of 4 days. Three replicates were performed for every treatment, measuring the radial growth of fungal colonies and analyzing the data statistically. Subsequently, the inhibition rates (%) versus seven concentrations of some compounds and kresoxim-methyl were obtained, and the median effective concentration (EC₅₀) values were calculated.

2.3 Molecular docking studies

ChemDraw2019 was used to create a 2D structure of compound C5, which was saved as a cdx file. Next, the 3D structure of C5 was optimized using the MM2 force field in Chem3D 2019. Chitinase's 3D configurations were obtained from the PDB repository (https://www.rcsb.org, PDB: 1W9U). Subsequently, the proteins of interest were brought into AutoDock Tools for the processes of adding hydrogen atoms, calculating charges, and combining non-polar hydrogens, with the final outcome being saved in PDBQT format. Ultimately, execute AutoDock Vina to perform molecular docking, then utilize Discovery studio for visualizing the outcomes.

2.4 Anticancer assay

Three human cancer cells, including HeLa, HePG-2, HCT116, and normal human cells (BEAS-2B) were used to determine the cytotoxic effects of the prepared compounds (A1 ∼ 9, B1 ∼ 14, C1 ∼ 11), and CCK-8 assay was used to assess the impact on growth. Cells were placed in 96-well plates and exposed to the compounds at a concentration of 100 μmol/L for 48 h in triplicate. Each well was treated with CCK-8 solution at a concentration of 20 %, followed by incubation for 1 h and measurement using spectrophotometry at 450 nm. As a positive control, 5-fluorouracil (5-FU) was also used in identical conditions. The additional IC₅₀ values for certain compounds was also tested. Each experiment was conducted thrice.

2.5 Cell cycle arrest assay

HeLa cells (2 × 10⁶/well) were seeded in 6-well plates and incubated overnight, and then exposed to A4 (0, 4, 8, 16 μM) for 48 h. Afterward, PBS was rinsed over the cells and trypsin was added. After centrifugation, the liquid above the sediment was discarded and the remaining material was soaked in 70 % ethanol overnight. The cells were rinsed with PBS two times and then placed in a staining buffer with 190 µL volume, which included propidium iodide (1 mg/mL, 4 µL) and RNaseA (ribonuclease, 10 mg/mL, 4 µL), and Tritonx-100 (0.2 µL). Following a 20-minute staining period in darkness, the distribution of the cell cycle was analyzed with flow cytometry.

2.6 Cell apoptosis assay

The incubated HeLa cells (2 × 10⁶/well) were exposed to A4 (0, 4, 8, 16 μM) for 48 h. Following the incubation period, PBS was rinsed over the cells and trypsin was added. Then the resulting cell was tmixed with Annexin binding buffer. Subsequently, annexin V/FITC (5 μL) and PI (10 μL) were introduced to the cell suspension and allowed to incubate at appropriate temperature for 15 min. Afterward, flow cytometry underwent analysis the stained cells.

2.7 Data analyses

The formula used to calculate the inhibitory effects on these fungi in vitro is Inhibition rate (%) = (C-T) × 100/(C-4 mm), with C representing the diameter in the blank control group and T representing the diameter in the treatments. The cell Inhibition rate (%) = (OD1-OD2)/(OD1-OD3) × 100 %. The cell viability rate (%) = (OD2-OD3) / (OD1-OD3) × 100 %. with OD1, OD2,OD3 representing the negative control, experiment and blank group. GraphPad Prism 8 software (GraphPad, Inc., San Diego, CA) was used to conduct statistical analysis.

3.1 Chemistry

As shown in Scheme 1, initially, primary coumarin-3-formyl chlorides 5a ∼ c were synthesized through a four-step process beginning with readily available salicylaldehydes. Salicylaldehydes 1a ∼ c were subjected to Knoevenagle condensation with diethyl malonate (2) in ethanol by a catalytic amount of piperidine, forming coumarin-3-carboxylates 3a ∼ c. Next, the ethyl esters underwent hydrolysis using a NaOH solution to produce coumarin-3-carboxylic acids 4a ∼ c, which reacted with oxalyl chloride to give the intermediates 5a ∼ c. At last, a series of coumarin amide derivatives bearing fluorine (A1 ∼ 9, B1 ∼ 14, C1 ∼ 11) were prepared in 55 ∼ 89 % yields by reaction of 5a ∼ c with different amines in the presence of triethylamine at 50 °C. Fig. 2 summarizes all the structures. The chemical structures of these derivatives were verified using ¹H NMR, ¹³C NMR and HRMS spectrometry (see Supporting Information). Moreover, X-ray diffraction was used to identify the stereo structures of A6, B11, C2 and C7, as shown in Fig. 3. The crystallographic data of these compounds were submitted to the CCDC under no. 2337042, 2337478, 2,337,043 and 2337044, respectively (see Supporting Information).

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Scheme 1

Synthetic route for coumarin amide derivatives bearing fluorine.

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Fig. 2

Chemical Structures of compounds A1-9, B1-14, C1-11.

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Fig. 3

X-ray crystallographic structures of A6 (A), B11 (B), C2 (C) and C7 (D).

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3.2 Antifungal activities

All the target compounds were screened their antifungal activities in vitro against five phytopathogenic fungi at 50 μg/mL using the mycelium linear growth rate method (Liu et al., 2022). Kresoxim-methyl (Kre) was used as positive control. As illustrated in Table 1, several compounds displayed significant inhibitory effects on specific fungi, which was consistent with the previous research findings that coumarin-3-carboxamide derivatives have good antibacterial activity (Yu et al., 2018). Among them, six compounds A4, A7, B2, B5, B6 and C5 inhibited AB with inhibition rates higher than 50 %. Compounds A4, A7, B2, and C5 exhibited better effectiveness with inhibition rates exceeding 67 % against the AA strain, surpassing Kre (64.3 %). For CG strain, compounds B2 (65.3 %), C2 (65.3 %), C3 (66.7 %), C5 (86.1 %), C6 (75.0 %) and C8 (59.7 %) displayed higher antifungal activities than Kre (53 %). Furthermore, three compounds A4 (58.3 %), B13 (56.35) and C5 (59.2 %) demonstrated slightly greater or comparable antifungal efficacy compared to Kre (53 %) for PG strain. Regrettably, nearly all of the desired substances showed weaker antifungal properties against the FV strain. In general, compound C5 demonstrated a more favorable and wide-ranging antifungal impact compared to the agricultural fungicides Kre.

After analyzing the bioassay results mentioned earlier, certain structure–activity relationships (SARs) were examined as follow: (1) generally, introducing fluorine atom at C-6 and C-8 of coumarin was more beneficial for the improvement of the fungicigal activities (A1 ∼ 9, C1 ∼ 11 vs. B1 ∼ 14). (2) For compounds A1 ∼ 9, the introduction of 1-ethylproyl group at R position led to a sharp increase in bioactivity, such as A4. (3) For compounds B1 ∼ 14, adding n-propyl (B2) at the R position typically enhanced the antifungal effectiveness compared to other alkyl groups. (4) When the fluorine atom at the C-8 of coumarin, introduction of 1-ethylproyl group at R position could also obtain the best compound (C5). (5) Unfortunately, it was discovered that adding aromatic groups to the R position resulted in decreased potency for compounds A8 ∼ 9, B9 ∼ 14 and C9 ∼ 11. The findings of the aforementioned structure–activity relationship will serve as a theoretical guide for future optimization of the structure.

To further investigate the antifungal capabilities, the EC₅₀ values of compound C5 were examined against four phytopathogenic fungi (AB, AA, CG and PG) for more reliable results. As summarized in Table 2, this compound showed strong antifungal effects, with EC₅₀ values ranging from 11.5 to 36.5 μg/mL. Particularly, compound C5 exhibited superior fungicidal activity against AA, CG, and PG strains, with EC₅₀ values of 11.5, 18.0, and 33.8 μg/mL, significantly lower than Kre (AA: 15.5 μg/mL, BC: 21.5 μg/mL, PG: 53.7 μg/mL).

3.3 Molecular docking studies

Chitinase, a key enzyme in chitin production, plays a vital part in breaking down external chitin, as well as in degrading and restructuring fungal cell walls (Hartl et al., 2012). Literature studies have shown that amidocoumarins exhibit notable chitinase inhibitory effects and possess strong binding affinity to the receptor (Sharma et al., 2020). At present, molecular docking are commonly utilized in the study of natural products (López-López et al., 2021). Therefore, we employed molecular docking to investigate the binding interactions between compound C5 and chitinase models in order to explore potential antifungal mechanisms.

Docking studies were conducted with AutoDock Tools (ADT) to explain the inhibitory activity of chitinase, utilizing a well-classified chitinase (PDB code: 1W9U) as the reference model. The docking result displayed that C5 had good binding affinity to chitinase with the docking binding energies of −7.42 kcal/mol. As shown in Fig. 4, C5 displayed hydrogen bonds and van der Waals attractions with polar residues within the binding cavity (Fig. 4, A), and shared similar binding modes with argadin, a natural chitinase inhibitor (Fig. 4, B) (Arai et al., 2000). The two carbonyl groups of C5 formed hydrogen bonds with residues GLY221 and LYS224, respectively. The coumarin ring of C5 had pi-sulfur interaction with MET243, pi-pi T-shaped interaction with TYR178, pi-alkyl interaction with PRO219. The fluorine atom formed halogen bond with MET243 and SER218 with bond length of 3.49 and 3.45 Å, respectively. The outcome suggested that the specific compound C5 perfectly matched the active site and had a positive interaction with chitinase.

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Fig. 4

Molecular models of C5 (A) and argadin (B) binding to chitinase.

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3.4 Anticancer activity

The synthesized compounds were evaluated for their anticancer properties at a concentration of 100 µmol/L on three human cancer cell lines: HeLa, HePG-2, and HCT116, and the results are collected in Table 3. Among these compounds, A4 demonstrated the highest effectiveness in inhibiting the growth of the three cancer cells, with rates of 90.28 %, 73.58 %, and 76.01 %, respectively. Additionally, several other compounds showed satisfactory cytotoxic effects on other cancer cells. For example, A2, A9, B2, B3 and C11 caused Hela cells growth inhibition rates exceeding 50 %. Compounds A2 and A7 showed moderate effectiveness in inhibiting cell growth in HePG-2 cells, with inhibition rates of 53.02 % and 50.14 %, respectively. On the other hand, all the target compounds exhibited poorer anticancer activities against HCT116 cells except A4. SARs indicated that introducing fluorine atom at C-6 of coumarin was more beneficial for the improvement of the anticancer activities. When the fluorine atom at the C-6 of coumarin, introducing of pentan-3-yl group at R position had positive influence on the anticancer activities.

In addition, as shown in Fig. 5, the compound A4 with the best anticancer activities were further examined to determine its IC₅₀ against the three cancer cells, and the result delivered that it exhibited intriguing and remarkable profiles of inhibiting cell growth with IC₅₀ values of 8.13 μM, 13.41 μM and 14.92 μM against Hela, HePG-2 and HCT116 cells, respectively, Approaching or exceeding the IC₅₀ values of 5-FU (14.69 μM, 33.35 μM and 4.45 μM). Moreover, A4 was further evaluated for its cytotoxic activity against normal cells BEAS-2B. As depicted in Fig. 6, A4 showed relative low cytotoxicity to BEAS-2B cells, and the IC₅₀ values was over 45 μM. This result indicated that A4 showed greater preference for cancer cells over human normal cells.

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Fig. 5

IC₅₀ values of compound A4 and 5-FU against three cancer cells. (** p < 0.01).

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Fig. 6

Cell viability of BEAS-2B treated with compound A4.

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3.5 Cell cycle analysis

Building on the promising results from the initial in vitro antiproliferative tests, we delved deeper into the effects of A4 on the cell cycle advancement of Hela cells through flow cytometry analysis. During the study, Hela cells were exposed to different amounts of A4 (0, 4, 8, 16 μM) for 48 h. As shown in Fig. 7, In the presence of A4, the proportion of cancer cells in the S phase was 26 %, 29.45 %, and 36.21 %, compared to 22.21 % in the control group. The results showed that A4 caused a significant S-phase arrest in a concentration-dependent manner.

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Fig. 7

Effect of compound A4 on cell cycle in Hela cells. Flow cytometry of Hela cells treated with A4 for 48 h. (** p < 0.01, *** p < 0.001).

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3.6 Cell apoptosis analysis

Additionally, Hela cells were subjected to increasing doses of A4 (0, 4, 8, 16 μM) for cell apoptosis assessment. As shown in Fig. 8, after incubating with A4 for 48 h, the combined proportion of Q2 and Q4 apoptotic cells was 6.9 %, 9.7 %, and 16.5 %, respectively. On the other hand, the group that was not treated showed only 3.7 % of cells undergoing apoptosis. As a result, it was shown that compound A4 had the ability to inhibit cell growth by causing cellular apoptosis.

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Fig. 8

Effect of compound A4 on cell apoptosis in Hela cells. Flow cytometric analysis of apoptotic cells after treatment of Hela cells with A4 for 48 h. (** p < 0.01, *** p < 0.001).

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