In vitro anticancer studies of new derivatives based on the furanocoumarin scaffold

Abstract

Furanocoumarins, known for their diverse bioactivity, were chemically modified to develop new derivatives with potential anticancer properties. This study reports the synthesis and comprehensive biological evaluation of seven aminoalkyl furanocoumarin derivatives. In vitro cytotoxicity was assessed against four human cancer cell lines (HTB-140, A549, HeLa, SW620) and a normal keratinocyte line (HaCaT) using MTT and LDH assays. Compounds 4 and 6 demonstrated the strongest antiproliferative effects, particularly against SW620 and HTB-140 cells, with IC₅₀ values around 11–18 µM, indicating potent anticancer activity. Flow cytometry revealed that these effects were largely mediated through apoptosis, not nonspecific toxicity. Molecular docking studies identified interactions with EGFR and Bcl-2 family proteins, suggesting a pro-apoptotic mechanism, though additional pathways may contribute to their selectivity. Importantly, antimicrobial screening showed negligible activity against representative Gram-positive and Gram-negative strains, indicating a low risk of microbiota disruption - an important feature for cancer therapy. These findings position furanocoumarin derivatives, particularly compounds 4 and 6, as promising lead structures for the development of selective, microbiota-sparing anticancer agents.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-30998-z.

Introduction

Despite decades of research and numerous therapeutic advancements, cancer continues to pose a significant global health burden. Based on data from 185 countries, the number of cancer cases recorded in 2022 is projected to increase by 77% by 2050¹. Lung, colorectal, and breast cancers were identified as the three most prevalent cancer types in 2022, highlighting critical directions for future research.

According to a recent report published by the American Cancer Society, despite a declining mortality rate, the annual incidence of cancer continues to rise in the United States of America². Moreover, many cancer types have developed mechanisms of drug resistance, including altering the inhibition of metabolic activation, as well as alterations in apoptosis-related proteins^3–5. This highlights the urgent need for continued scientific efforts to discover and develop new anticancer agents.

To address this issue, our research team synthesized eight new furanocoumarin derivatives and evaluated their cytotoxic activity. The study design was inspired by the current scientific trend of extracting bioactive compounds from natural sources, particularly plants, in the search for effective anticancer agents^6–8.

Aydoğmuş-Öztürk et al. isolated the bioactive compound visnagin (Fig. 1) from Ammi visnaga L. and demonstrated its significant inhibitory effect on the proliferation of human melanoma cells through the induction of intracellular reactive oxygen species (ROS) production⁹. Such findings inspire scientists to apply structural modifications to biocompounds scaffolds to enhance the activity of naturally occurring substances.

Fig. 1.

Chemical structures of biologically active compounds (a) Khellin and (b) Visnagin.

Notably, several furocoumarin derivatives have been reported to exhibit significant antiproliferative activity against various cancer cell lines, further supporting the rationale for our study.

Sharma et al. assessed structural modifications on Khellin (Fig. 1) to enhance its inhibitory effect on CYP1A1, identifying two derivatives as potential cancer chemopreventive agents¹⁰. Ragab et al. synthesized several new natural compound derivatives of flavonoids, including furanocoumarin, and conducted extended anticancer screening¹¹. Among the synthesized compounds, ten demonstrated significant antiproliferative activity against many cancer cell lines. Notably, one of those compounds (25e) was successfully transferred to an in vivo antitumor activity test, resulting in a significant decrease in tumor size. Another study reported promising cytotoxic results of a new furanocoumarin derivative (IC₅₀ = 2 nM), exhibiting comparable activity to the reference drug sorafenib¹². Amin et al. obtained a series of new furanocoumarin and benzofuran compounds hybridized with various heterocyclic rings and evaluated their cytotoxic potential through in vitro and molecular docking studies. Most compounds demonstrated antiproliferative activity against breast cancer cell lines, comparable to the reference drug ciprofloxacin¹³. In the next very similar study, new furanocoumarin derivatives modified by featuring bioactive functional groups were tested for cytotoxic activity against human breast cancer cell lines. The best registered IC₅₀ concentrations were on the level of 1–3 µM¹⁴.

Research should focus not only on the efficacy of therapy but also on the patient’s overall well-being. Preservation of the gut microbiota has emerged as a critical consideration in the design and administration of cancer therapies. The intestinal microbiota plays a vital role in modulating immune responses, maintaining mucosal integrity, and influencing the efficacy and toxicity of anticancer agents. Disruption of this microbial balance, often caused by broad-spectrum antibiotics or off-target effects of therapeutic compounds, can lead to dysbiosis, which is associated with impaired treatment responses, increased side effects, and greater susceptibility to infections¹⁵. Recent studies have emphasized the importance of evaluating the microbiota safety profile of investigational drugs, particularly those considered as adjuncts or alternatives to traditional chemotherapeutics. In vitro antimicrobial assays using representative Gram-positive (e.g., Staphylococcus aureus, S. epidermidis) and Gram-negative (e.g., Escherichia coli, Pseudomonas aeruginosa) bacterial strains serve as early indicators of a compound’s potential to disturb gut flora. Compounds that show no activity against these model strains, evidenced by high minimum inhibitory concentrations (MICs) or absence of growth inhibition, are considered microbiota-safe¹⁶. Such selectivity allows for targeted anticancer or antimicrobial action without compromising the patient’s commensal microbiome. This property is especially valuable in immunocompromised cancer patients, where microbiome integrity supports systemic immunity and may enhance the response to immunotherapies and other treatments¹⁷.

In this research article, our team presents the synthesis of eight furanocoumarin derivatives, followed by an extensive cytotoxic activity evaluation against selected cancer cell lines. To gain deeper insight into their biological potential, we investigated their possible mechanism of action, focusing on molecular targets and pathways involved in cancer progression. These findings were further supported by modern in silico methods, including target predictions, molecular docking, and ADMET prediction, to assess their binding affinities, pharmacokinetic properties, and overall drug-likeness. Additionally, preliminary microbiological safety was assessed using a panel of bacterial strains.

Our study provides valuable insights into the potential of furanocoumarin derivatives as promising anticancer agents and highlights the significance of computational approaches in modern drug discovery.

Results and discussion

Chemistry

The objective of the synthetic work was to obtain a library of aminoalkyl derivatives of furanocoumarins (Fig. 2). The commercially available starting material, 7-ethyl-4,9-dihydroxy-5H-furo[3,2-g]¹benzopyran-5-one, was selectively mono-methylated using dimethyl sulphate to protect one of the hydroxyl groups. The remaining free hydroxyl group was then subjected to nucleophilic substitution via reaction with epichlorohydrin to afford the corresponding epoxide intermediate. This epoxide was subsequently reacted with various amines to yield the desired aminoalkyl derivatives^18,19.

Fig. 2.

Simplified synthetic pathway for furocoumarin derivatives.

All synthesized amino derivatives were purified by flash chromatography and subsequently converted to their hydrochloride salts. The structures of the final compounds were confirmed by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high-resolution mass spectrometry (HRMS).

In vitro cytotoxic activity

To evaluate cytotoxic activity of obtained compounds, we assessed MTT test²⁰, which determines the minimum concentration at which the tested compound inhibits the growth of a cancer cells culture by 50%, compared to the untreated control group. This concentration is expressed as the IC₅₀ value (in µM). To establish selectivity, the IC₅₀ value of each compound against a normal human cell line was divided by the corresponding value obtained for the cancer cell line, yielding the Selectivity Index (SI). The SI reflects the compound’s ability to selectively target cancer cells while sparing normal cells.

All synthesized furocoumarin derivatives were subjected to this assay, and the results are summarized in Table 1. The cancer cell lines included HTB-140 (human melanoma), A549 (human lung adenocarcinoma), HeLa (human cervical cancer), and SW620 (human colorectal cancer, derived from a metastatic site). The normal human cell line was represented by HaCaT (immortalized human keratinocytes).

Table 1.

Cytotoxic activity of the new furanocoumarin derivatives established by MTT^a assay.

Compound	Cancer cells								Normal cells
	HTB-140d		A549e		HeLaf		SW620g		HaCaTh
	IC50b	SIc	IC50b	SIc	IC50b	SIc	IC50b	SIc	IC50
1	30.4 ± 8.7	1.64	39.8 ± 7.9	1.25	41.5 ± 4.6	1.20	21.1 ± 4.4	2.36	49.8 ± 7.9
2	29.4 ± 3.4	1.58	36.5 ± 6.5	1.27	29.2 ± 3.9	1.59	17.4 ± 3.5	2.67	46.5 ± 8.5
3	30.1 ± 4.7	1.44	33.6 ± 4.1	1.29	34.2 ± 4.9	1.27	12.7 ± 5.1	3.41	43.3 ± 9.1
4	18.2 ± 3.6	2.32	32.3 ± 7.9	1.31	27.4 ± 6.5	1.54	11.5 ± 6.6	3.68	42.3 ± 7.9
5	29.4 ± 4.9	1.49	43.8 ± 8.3	1.00	32.6 ± 3.7	1.34	26.3 ± 3.3	1.67	43.8 ± 8.3
6	17.5 ± 3.4	2.48	26.3 ± 7.4	1.65	22.4 ± 4.9	1.94	11.4 ± 4.9	3.81	43.4 ± 7.4
7	32.5 ± 4.1	1.60	35.2 ± 6.3	1.47	32.3 ± 3.8	1.61	28.6 ± 5.5	1.81	51.9 ± 6.3
8	31.3 ± 2.6	1.47	38.4 ± 7.3	1.20	33.4 ± 4.6	1.38	26.3 ± 4.3	1.75	46.1 ± 7.3
9	23.4 ± 2.6	2.29	37.6 ± 7.3	1.43	29.5 ± 4.6	1.82	21.9 ± 4.3	2.45	53.6 ± 7.3
*Ref 1	1.4 ± 0.69	2.00	2.7 ± 1.1	1.04	1.8 ± 0.74	1.56	1.9 ± 0.9	1.47	2.8 ± 1.1
**Ref 2	0.5 ± 0.21	2.20	0.9 ± 0.19	1.22	0.6 ± 0.14	1.83	0.7 ± 0.16	1.57	1.1 ± 0.19

*Ref 1 = cisplatin, ** Ref 2 – doxorubicin.

^a The MTT assay is a colorimetric assay for measuring cell metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple color. Data are expressed as mean SD, ^b IC₅₀ (µM)—the concentration of the compound that corresponds to a 50% growth inhibition of the cell line (as compared to the control) after the cells were cultured for 72 h with the individual compound. ^c The SI (Selectivity Index) was calculated using the formula: SI = IC₅₀ for normal cell line/IC50 cancer cell line, ^d Human Melanoma (HTB-140), ^e Human Lung Adenocarcinoma (A549), ^f Human Cervical cancer (HeLa), ^g Human Colorectal Cancer (SW620), ^h Human immortal keratinocyte cell line from adult human skin (HaCaT).

The whole series of tested furanocoumarin derivatives exhibited significant cytotoxic activity, underscoring how targeted semi-synthetic modification of a natural scaffold can generate promising anticancer agents. Overall, the compounds were the most potent toward the human colorectal cancer (SW620) cells. Derivatives 4 and 6 produced half-maximal inhibitory concentrations IC₅₀ of 11.5 ± 6.6 and 11.4 ± 4.9 µM, respectively. Moderate, but still meaningful activity was recorded for derivatives 1 (21.1 ± 4.4 µM), 2 (IC₅₀ = 17.4 ± 3.5 µM), and 3 (IC₅₀ = 12.7 ± 5.1 µM).

A similar potency pattern emerged in the human melanoma line HTB-140, where compounds 4 and 6 again ranked as the top performers, showing IC₅₀ values of 18.2 ± 3.6 µM and 17.5 ± 3.4 µM, respectively. Using the criterion of sub- 17.4 µM potency, derivatives 2, 3, 4, and 6 were selected for deeper mechanistic profiling. Next steps of this research are focused on mapping their effects on apoptosis induction, key signaling cascades, and potential molecular targets to elucidate the mechanistic basis of their cytotoxic potency and selectivity. Collectively, these data position furanocoumarin derivatives 4 and 6, supported by derivatives 2 and 3, as the most compelling candidates for further preclinical evaluation against colorectal and melanoma malignancies.

Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) is a cytoplasmic enzyme involved in glucose metabolism, and it is released into the extracellular space following cell membrane damage. Elevated levels of LDH in the culture medium are therefore commonly used as an indicator of loss of membrane integrity and serve as a reliable marker of cell viability and cytotoxicity²¹.

To further evaluate the cytotoxic effects of the most promising furanocoumarin derivatives identified in the preliminary screening, compounds 2, 3, 4, and 6 were selected for LDH assays. Each compound was tested at three concentrations- 25, 50, and 100 µM — to assess their dose-dependent effects on LDH release from cancer cells. The results, presented in Fig. 3, demonstrate a clear correlation between compound concentration and the extent of LDH leakage, supporting the compounds’ cytotoxic potential and suggesting membrane-disruptive effects at higher doses.

Fig. 3.

The effect on LDH release of the new furanocoumarin derivatives assessed in three concentrations of 100, 50, and 25 µM (presented as the % of cytotoxicity) on HTB-140, A549, HeLa, and SW620 cancer cell lines, compared to the normal human cell line (HaCaT). *** p ≤ 0.001, ** p ≤ 0.01.

The release of LDH into the culture medium was noticeably higher in SW620 and HTB-140 cancer cells compared to HaCaT keratinocytes, particularly at concentrations of 100 µM and 50 µM for all four tested furocoumarin derivatives. These findings are consistent with the high selectivity indices observed in the MTT assay. At these concentrations, the compounds induced cytotoxicity in approximately 20–70% of cancer cells, with derivatives 4 and 6 showing the most significant effects (Fig. 3).

Interestingly, at the lowest tested concentration (25 µM), compound 2 produced the strongest cytotoxic response in HTB-140 cells, resulting in 30.4% LDH release, compared to 13.5% in HaCaT cells. This was followed by compound 6, which induced 24.6% LDH release in HTB-140 cells versus 6.8% in HaCaT (selectivity index, SI = 2.48). These data further support the selective cytotoxicity of the compounds toward cancer cells.

From a structural perspective, the most active derivatives shared common features. Comparing structural differences of compound 2 and the rest of the set indicates that the substitution of the epoxide group in the 7’ position of furanocoumarin heterocycle ring with different amine-derived substituents improved the motif activity. Moreover, the presence of N-alkyl substituents at the R position was the most favorable structural modification for enhancing derivative activity. Notably, for compound 6, the elongation of the alkyl chain (relative to derivatives 3, 4, and 8) did not significantly diminish either cytotoxicity or selectivity, suggesting a degree of tolerance in this position concerning biological activity.

In summary, the LDH release assay complemented the MTT results, reinforcing that the studied furocoumarin derivatives exhibit highly selective cytotoxic effects toward cancer cells. These findings provided a rationale to further explore their mechanism of action, which was addressed in the next phase of the study.

Apoptotic activity

Apoptosis is a genetically regulated process that leads to controlled cell death through a series of highly organized molecular events. It plays a crucial role in maintaining tissue homeostasis by eliminating damaged, dysfunctional, or potentially harmful cells²². In the context of cancer therapy, selectively triggering apoptosis in malignant cells is considered a safe and effective strategy, as it targets the tumor while sparing healthy tissue^23–25. This mechanism may also underlie the observed antiproliferative activity of the furanocoumarin derivatives investigated in this study.

To better understand how the tested furocoumarin derivatives exert their cytotoxic effects, we investigated whether they induce apoptosis in cancer cells. The same cell lines used in the MTT and LDH assays- HTB-140 (melanoma), A549 (lung carcinoma), HeLa (cervical cancer), SW620 (colorectal cancer) were treated with compounds 2, 3, 4, and 6 at their previously determined IC₅₀ concentrations. Apoptosis levels were measured using flow cytometry (Fig. 4), allowing us to distinguish between early and late apoptotic events, as well as necrosis.

Fig. 4.

Percent of cells led to early apoptosis or late apoptosis and necrosis by compounds 2, 3, 4, 6, and camptothecin (CPT), as positive control against HaCaT cells, A549, HeLa, and SW-620 cancer cells, detected with Annexin V-FITC/7-AAD by flow cytometry. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Among the tested compounds, compound 4 emerged as the most potent apoptosis inducer. It drove 68.8% of HTB-140 cells and 55.6% of A549 cells into late apoptosis or necrosis, indicating a strong and consistent pro-apoptotic effect across both cell types (Fig. 4E and D). Similarly, compound 2 showed marked activity in A549 cells, where 63.2% of cells underwent late apoptosis or necrosis. In HTB-140 cells, it triggered early apoptosis in 19.5% and late apoptosis in 20.8% of the population.

Compound 6 also demonstrated notable activity, although slightly weaker. It pushed 48.8% of HTB-140 and 44.9% of HeLa cells into late apoptosis or necrosis and affected 39.7% of A549 cells. Compound 3, while included in the assay, showed more moderate effects (data in Fig. 3), suggesting potential cell-line-dependent activity.

Importantly, the tested compounds exhibited minimal toxicity toward normal human keratinocytes (HaCaT line). Across all treatments, only 0.3–0.6% of HaCaT cells underwent late apoptosis or necrosis, supporting a high degree of selectivity for cancer cells.

Together, these results indicate that the cytotoxicity of our furanocoumarin derivatives is largely associated with the induction of apoptosis, rather than nonspecific cell damage. Later in this paper, we plan to apply in silico pathway analysis to further explore the molecular mechanisms involved, with a focus on apoptosis-related signaling, such as the EGFR and BCL-2 pathways.

The positive control was generated by incubation of HaCaT, SW620, HeLa, A549, HTB-140 cells with camptothecin (CPT, 4 µM) for 4 h. Cells were stained with Annexin V-FITC and 7-AAD, and the percentage of apoptotic cells was analyzed by flow cytometry. Positive control induced apoptosis in all cells (Fig. 5).

Fig. 5.

The effect of compounds 2, 3, 4, 6, and camptothecin (CPT), as positive control on early and late apoptosis or necrosis in HaCaT cells (A), SW-620 (B), HeLa (C), A549 (D), HTB-140 (E) cancer cells detected with Annexin V-FITC/7-AAD by flow cytometry (Q1-Alive, Q2- Early Apoptotic, Q3- Necrotic/Late Apoptosis, Q4- Necrotic).

Interleukin-6 study

Interleukin-6 (IL-6) is a cytokine that activates inflammatory and autoimmune responses in various diseases, such as pancreatic, prostate, and colon cancers. Elevated levels of IL-6 are observed in advanced and metastatic cancers, where it plays a role in tumor development and progression²⁶. All investigated human cell lines were treated with IC₅₀ doses of the most potent derivatives 2, 3, 4, and 6 (see Fig. 6).

Fig. 6.

Effects of obtained derivatives on IL-6 levels measured by ELISA test. Data are expressed as the mean ± SD from three independent experiments performed in triplicate. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 as compared to control.

All derivatives exhibited a significant effect on HTB-140 cells, reducing the release of interleukin-6 (IL-6) compared to the control. The strongest effect was found for compound 4. In SW620 cells, compounds 4 and 6 showed the strongest effect among the tested derivatives, reducing IL-6 secretion by around 50%. In HeLa cells, derivative 3 inhibited IL-6 release by an average of 48%. In the case of A549 cells, the best result was found for derivative 2, showing IL-6 release by an average of 38%.

In summary, all selected derivatives significantly reduced IL-6 levels in HTB-140, SW620, HeLa, and A549 cancer cells. In contrast, no sensitivity was observed in HaCaT cell lines.

Molecular docking studies

To explore the molecular basis of apoptosis induction, we performed molecular docking of all new furocoumarin derivatives against two classes of targets: anti-apoptotic Bcl-2 family proteins and the epidermal growth factor receptor (EGFR). The Bcl-2 family is the anti-apoptotic proteins recognized as a critical inhibitors of apoptosis²⁷. If a tested compound can bind to the hydrophobic groove of these proteins and block their function, their regulatory activity may be disrupted, leading to the activation of apoptotic pathways in cancer cells. EGFR is a transmembrane tyrosine kinase whose ligand-induced dimerization and autophosphorylation trigger downstream MAPK/ERK and PI3K/Akt pathways, driving proliferation and suppressing apoptosis. Small molecules that bind in the ATP-binding cleft of EGFR block its kinase activity, thereby shutting off these pro-survival signals and promoting apoptotic death in cancer cells^28,29. We chose EGFR and anti-apoptotic Bcl-2 family members as mechanistically complementary controllers of cancer cell survival: EGFR drives pro-survival kinase signaling, whereas Bcl-2 proteins block mitochondrial apoptosis via the BH3-binding groove. Docking across both classes was intended to distinguish plausible mechanisms consistent with our cytotoxicity data and to prioritize targets for subsequent validation.

Following in-silico study conventions, the co-crystallized ligands of the selected targets are referred to as native ligands (referent compounds). To gain deeper insight into the molecular interactions between the targets and both reference and tested ligands, we selected Bcl-2 family protein and EGFR structures that were co-crystallized with known inhibitors. Molecular docking was performed using these structures to evaluate the binding modes and affinities of both native ligands and the synthesized compounds. The selected crystallographic structures included: Apoptosis Regulator Bcl-2 (PDB IDs: 4LVT³⁰, 6O0P³⁰, 6O0O³⁰, Bcl-2-like protein 1 also called Bcl-xL (PDB IDs: 1YSG³¹, 2YXJ³², Myeloid Cell Leukemia 1 (Mcl-1) (PDB IDs: 8AV9³³, 6FS0³³, and five EGFR kinase-domain variants (PDB ID: 1M17³⁴, G719S (PDB IDs: 2ITO³⁵, L858R/T790M (PDB ID: 4I23³⁶, exon 20 insertion (PDB ID: 4LQM³⁷ and C797S triple mutant (PDB ID: 7AEM³⁸. Detailed information regarding the protein–native ligand complexes was obtained from the Protein Data Bank and DrugBank and is provided in the supplementary material (Table S.2).

Docking was carried out using Smina, an optimized fork of AutoDock Vina, first by redocking each native ligand to validate our protocol (RMSD ≤ 3,5 Å), then by placing each furanocoumarin derivative into the same binding sites. For every ligand–protein pair, the ten lowest-energy binding poses were retained. Binding affinities (minimum and maximum kcal/mol) and redocking RMSDs are summarized in Tables 2 and 3.

Table 2.

Molecular Docking results of newly synthesized furanocoumarin derivatives to selected Bcl-2 family protein crystal structures compared to the native ligands.

Compound	Crystal PDB ID
	4LVT	6O0P	6O0O	1YSG	2YXJ	8AV9	6FS0
	Calculated Binding Affinity (min|max) [kcal/mol]
Native Molecule	−11.4	−12.3	−10.9	−7.9	−10.8	−16.2	−16.4
RMSD	1.54	1.25	0.67	0.70	1.40	0.04	0.17
1	−7.1 | −5.9	−7.2 | −5.8	−6.6 | −5.4	−5.6 | −4.8	−7.1 | −6.0	−7.8 | −6.5	−7.6 | −6.6
2	−6.8 | −5.7	−6.6 | −5.9	−6.4 | −5.3	−5.3 | −4.2	−6.4 | −5.5	−6.7 | −5.8	−6.9 | −6.3
3	−6.6 | −6.0	−6.6 | −5.8	−5.9 | −5.0	−5.4 | −4.1	−6.6 | −6.0	−7.3 | −5.8	−7.0 | −5.8
4	−6.5 | −6.0	−6.9 | −5.8	−6.2 | −5.3	−5.6 | −3.9	−6.4 | −5.7	−7.4 | −6.0	−6.9 | −6.0
5	−7.5 | −6.7	−7.7 | −5.9	−6.9 | −6.3	−6.4 | −4.6	−8.4 | −7.0	−8.0 | −7.1	−7.8 | −7.3
6	−6.3 | −6.0	−7.3 | −6.0	−5.7 | −5.5	−5.1 | −4.4	−7.3 | −6.6	−6.8 | −5.8	−7.4 | −6.6
7	−7.1 | −6.2	−8.0 | −6.4	−6.4 | −5.9	−6.1 | −4.8	−7.7 | −7.0	−8.3 | −6.5	−8.1 | −6.9
8	−7.0 | −6.2	−7.1 | −6.1	−6.2 | −5.4	−5.4 | −4.1	−7.1 | −6.0	−7.3 | −5.6	−7.3 | −6.1
9	−6.7 | −6.1	−7.3 | −6.2	−6.7 | −5.7	−5.2 | −4.1	−7.1 | −6.5	−7.1|−6.5	−7.5 | −6.6

Table 3.

Molecular Docking results of newly synthesized furanocoumarin derivatives to selected EGFR family protein crystal structures compared to the native ligands.

Compound	Crystal PDB ID
	1M17	2ITO	4I23	4LQM	7AEM
	Calculated Binding Affinity (min|max) [kcal/mol]
Native Molecule	−7.0	−8.2	−8.1	−8.0	−8.2
RMSD	2.4	3.02	0.24	2.08	1.75
1	−8.2 | −6.9	−7.7 | −6.8	−8.1 | −6.7	−7.3 | −6.7	−8.1 | −7.6
2	−7.4 | −6.5	−7.5 | −6.5	−7.3 | −6.5	−7.0 | −6.4	−7.9 | −6.9
3	−7.3 | −6.6	−7.0 | −6.5	N\A	−7.2 | −6.5	−7.4 | −6.3
4	−7.4 | −6.6	−7.4 | −6.9	−7.0 | −6.2	−7.1 | −6.5	−7.6 −6.7
5	−7.9 | −7.0	−8.1 | −7.1	−7.8 | −7.3	−8.2 | −7.2	−8.2 | −7.1
6	−7.3 | −6.4	−6.9 | −6.4	−6.4 | −6.0	−6.7 | −6.1	−7.3 | −6.7
7	−8.2 | −7.3	N/A	−7.9 | −7.1	−7.8 | −7.0	−8.3 | −7.5
8	−7.7 | −7.0	N/A	−7.5 | −6.5	−7.5 | −6.6	−7.4 | −8.1
9	−8.0 | −7.0	−7.7 | −6.7	−7.7 | −6.7	−7.5 | −6.8	−7.8 | 7.0

The performance of the docking protocol was validated through redocking of native ligands, with results showing binding affinities greater than − 7 kcal/mol and RMSD values below 3.5 Å. These findings confirm the reliability of the docking affinities obtained for the test compounds. Overall, the binding affinities of the newly designed furanocoumarin derivatives toward the Bcl-2 protein family were lower (i.e., less negative) than those of the native ligands, suggesting weaker predicted interactions within the binding pockets. The most notable result was observed for the complex of compound 3 with the 1YSG receptor, exhibiting predicted binding affinities of − 6.4 and − 4.6 kcal/mol, respectively (Table 2).

Despite acceptable RMSD values following the redocking protocol for the crystal structures of 2ITO and 4I23 (3.02 Å and 0.24 Å, respectively), a few docking attempts resulted in N/A outcomes (Table 3). These cases indicate that certain compounds failed to dock within the 4 Å vicinity of the binding pocket, which was defined using the autobox grid centered on the native ligand position.

Overall, the molecular docking analysis indicates that the EGFR signaling pathway may serve as a biologically relevant mechanism responsible for the observed induction of apoptosis in cancer cells upon treatment with the tested compounds. Notably, the predicted binding affinities of the new furanocoumarin derivatives were consistently comparable to those of native ligands across all evaluated EGFR crystal structures, with differences not exceeding 2.0 kcal/mol — a threshold commonly considered indicative of similar binding potential³⁹.

Among the EGFR structures analyzed, the 1M17 conformation revealed particularly promising results, with all docked tested compounds biding energies very similar to the native ligand (−7.0 kcal/mol) (Table 3). Compounds 5, 7, 8, and 9 displayed superior binding affinities with both min and max values from 10 docking poses better in relative to the redocked native ligand, strongly suggesting enhanced stabilization within the ATP-binding pocket. Specifically, compounds 7 and 9 emerged as the most potent binders, exhibiting docking scores of − 8.2 | −7.3 kcal/mol and − 8.0 | −7.0 kcal/mol, respectively, compared to − 7.0 kcal/mol for the native ligand. These results underscore their potential as lead candidates for further optimization (see Figs. 7 and 8).

Fig. 7.

Molecular docking visualization of compound 5 bound to the 1M17 crystal structure, with predicted binding affinities of − 8.2 and − 7.3 kcal/mol. Dashed lines indicate specific interactions between the ligand and protein residues: red for hydrogen bonds and yellow for hydrophobic contacts.

Fig. 8.

Molecular docking visualization of compound 8 bound to the 1M17 crystal structure, with predicted binding affinities of − 8.0 and − 7.0 kcal/mol. Dashed lines indicate specific interactions between the ligand and protein residues: red for hydrogen bonds and yellow for hydrophobic contacts.

Detailed interaction analysis (Fig. 9) demonstrated that the enhanced binding affinities of the tested compounds were primarily supported by a diverse network of hydrogen bonds, alkyl interactions, and van der Waals contacts within the EGFR binding cleft. Notably, the lipophilic contacts (π–alkyl/alkyl) act in concert with the hydrogen-bond network, jointly contributing to stabilization within the EGFR cleft.

Fig. 9.

Two-dimensional interaction diagrams illustrating key intermolecular contacts between the 1M17 EGFR protein and: (a) compound 5, (b) compound 8. Hydrogen bonds, van der Waals interactions, alkyl contacts, and other non-covalent interactions are shown between ligand atoms and amino acid residues within the binding pocket.

Both compounds 7 and 9 shared structurally related features at the R position, with compound 7 bearing a piperidine ring and compound 9 having a morpholine moiety. These heterocyclic substituents appear to expand the available chemical space and facilitate the formation of additional π-alkyl interactions, which may further stabilize the ligand–receptor complex.

This multi-layered interaction profile likely contributes to both the increased binding strength and improved specificity of these compounds toward the EGFR active site. Such properties are particularly desirable in the context of anticancer drug design, as they may translate into greater biological efficacy and reduced off-target effects. Nonetheless, these in-silico results are promising but require mechanistic confirmation in vitro. On the other hand, those results do not explain enhanced cytotoxicity and selectivity of the best candidates among the tested furanocoumarins. Compounds 3, 4, 6 bearing various N-alkyl derived substituents in R positions were enabled to create an additional π-alkyl interaction with heterocycle ring stabilizing them in the biding cleft. Thus, distinctive results of those compounds must be explained by different mechanism than creating stable complex with ATP-biding pocket of EGFR.

Primary antimicrobial evaluation

All obtained derivatives were transferred to antimicrobial testing against a panel of representative bacteria (see Table 4). These include both Gram-positive and Gram-negative strains common in the human microbiome:

Table 4.

Activity of synthesized furanocoumarin derivatives against standard bacteria strains, expressed by minimal inhibitory concentrations (µg/mL).

Compound	S. aureus NCTC 4163	S. aureus ATCC 25,923	S. aureus ATCC 6538	S. aureus ATCC 29,213	S. epidermidis ATCC 12,228	S. epidermidis ATCC 35,984	E. coli ATCC 25,922	P. aeruginosa ATCC 15,442
2	128	64	64	128	64	64	> 256	> 256
2a	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
3	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
4	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
5	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
6	256	256	256	256	> 256	128	> 256	> 256
7	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
8	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
9	> 256	> 256	> 256	> 256	> 256	> 256	> 256	> 256
Ciprofloxacin	0.25	0.5	0.25	0.5	0.25	0.25	0.003	125

• Gram-Positive: Staphylococcus aureus (NCTC 4163) and Staphylococcus epidermidis (ATCC 12228) were tested as representative Gram-positive bacteria. These species are typical members or surrogates of the commensal skin/gut flora and opportunistic pathogens.

• Gram-Negative: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442) were tested as representative Gram-negative bacteria. E. coli is a common gut commensal, while P. aeruginosa represents an opportunistic pathogen; both are relevant when considering gut microbiome safety. Additionally, potency against Mycobacterium tuberculosis was also assessed (see Supplementary file, Table S1).

Neither S. aureus nor S. epidermidis cultures were inhibited by the compounds. The minimum inhibitory concentrations (MICs) for all compounds against these Gram-positive strains were above the top of the tested range (e.g. on the order of > 128–256 µg/mL, meaning no growth inhibition even at the highest concentration). In practical terms, no clear zones of inhibition or low MIC values were observed for any compound with these bacteria, signifying a lack of antibacterial potency. Similarly, no activity was observed against E. coli or P. aeruginosa. All compounds had MIC values exceeding the highest tested dose for these Gram-negative strains (e.g., MIC > 128 µg/mL), again indicating that the compounds did not suppress or kill these bacteria within the test limits. Even P. aeruginosa – a notoriously hardy Gram-negative – showed no sensitivity to the compounds at maximum concentrations. Results of antitubercular screening (see Supplementary file, Table S1) showed similar outcomes to those presented for Gram-positive and Gram-negative bacteria.

Preserving the gut microbiome is particularly important for cancer patients. Research has shown that antibiotic-induced dysbiosis (disruption of the normal microbiota) can have negative consequences for cancer therapy outcomes. For instance, broad-spectrum antibiotics may eliminate beneficial bacteria and induce gut dysbiosis⁴⁰, which has been associated with unfavorable responses to chemotherapy and immunotherapy. Loss of commensal bacteria can lead to side effects (like diarrhea or infections) and can impair immune system modulation. In contrast, a treatment that does not act as an antibiotic will avoid these issues. By not eradicating gut bacteria, the compounds allow patients to retain their protective and immunomodulatory microbial species. This can translate into better tolerance of treatment and potentially improved efficacy, since a stable microbiome supports the immune system’s ability to fight cancer and reduces the likelihood of opportunistic infections.

The absence of antibacterial activity across both Gram-positive and Gram-negative bacteria supports the thesis that these compounds are “microbiome-safe.” In a cancer patient, this means that administering these compounds is unlikely to disrupt the normal gut microbial community. The gut microbiome is composed of many Gram-positive (e.g. Lactobacillus, Bifidobacterium) and Gram-negative (e.g. Bacteroides) commensals. A compound that does not inhibit model Gram-positive or Gram-negative bacteria (serving as surrogates for gut bacteria strains) in vitro is unlikely to kill off beneficial gut microbes in vivo. Thus, the tested compounds should spare the gut flora, helping to maintain microbial balance.

Conclusions

All synthesized compounds demonstrated notable cytotoxic effects on cancer cell lines (HTB-140, A549, HeLa, SW620), with the most potent activity observed against SW620 (colorectal cancer) cells. Compounds 4 and 6 stood out with the lowest IC₅₀ values (~ 11.4–11.5 µM), suggesting strong anticancer potential. Flow cytometry analysis confirmed that cytotoxic effects were primarily due to apoptosis, not necrosis. Compound 4 was the most effective in inducing late apoptosis/necrosis, with up to 68.8% apoptosis in melanoma cells. Minimal apoptosis was observed in normal HaCaT cells across all compounds, reinforcing tumor-specific selectivity. LDH release correlated with MTT results, confirming dose-dependent membrane damage in cancer cells. Compounds 2, 4, and 6 induced the strongest LDH release, particularly in SW620 and HTB-140 cells. Again, HaCaT cells showed limited LDH release, aligning with their lower observed cytotoxicity. Docking to EGFR and Bcl-2 family proteins suggested that these compounds may exert pro-apoptotic effects via EGFR inhibition, especially compounds 5, 7, 8, and 9. However, the most cytotoxic compounds (2, 3, 4, 6) did not show the strongest docking affinity, implying additional mechanisms beyond EGFR binding might be involved. The structural modifications, particularly N-alkyl substitutions, influenced activity and selectivity. None of the tested compounds exhibited significant antibacterial activity against Gram-positive or Gram-negative bacteria at clinically relevant concentrations. This suggests they are unlikely to disrupt the gut microbiome, which is beneficial for cancer patients’ immune support and therapy outcomes.

Compounds 4 and 6 are the most promising anticancer candidates, demonstrating potent cytotoxicity, strong apoptosis induction, minimal impact on healthy cells, and favorable microbiota-sparing profiles. Compounds 2 and 3 also merit further investigation due to moderate activity. Overall, the study supports the development of furanocoumarin derivatives as microbiota-safe anticancer agents, with compound 4 being the leading candidate for future preclinical evaluation.

Materials and methods

Chemistry

All solvents, chemicals, and commercially available reagents were obtained from Aldrich Chemical Company (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA, USA) and used without further purification. Melting points were determined using an Electrothermal 9100 capillary melting point apparatus and are reported uncorrected.

The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker BioSpin GmbH spectrometer (Bruker, Billerica, MA, USA) operating at 300 MHz for ¹H and 75.49 MHz for ¹³C. Spectra were recorded in DMSO-d₆ or CDCl₃, with chemical shifts (δ) reported in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are given in hertz (Hz), and signal multiplicities are abbreviated as follows: s (singlet), d (doublet), t (triplet), and m (multiplet). The spectra of the compounds were provided in the Supplementary file (Figures S1-S9).

High-resolution mass spectrometry (HRMS) was performed using an LCT Micromass time-of-flight (TOF) instrument in positive ion mode. Reaction progress was monitored by thin-layer chromatography (TLC) on silica gel plates (Kieselgel 60 F₂₅₄, Merck) with a 0.2 mm layer thickness and UV detection at 254 nm. Chloroform–methanol mixtures (98:2 or 95:5, v/v) were used as eluent systems.

Column chromatography was carried out using Merck silica gel 60 (particle size 0.05–0.2 mm, 70–325 mesh ASTM), with chloroform or chloroform–methanol (100:5, v/v) as the eluent.

Synthesis of 7-ethyl-4-hydroxy-9-methoxy-5 H-furo[3,2-g][1]benzopyran-5-one (2)

7-ethyl-4,9-dihydroxy-5 H-furo[3,2-g][1]benzopyran-5-one (1) (0.02 mol), K₂CO₃ (0.02 mol), and (CH₃O)₂SO₂ (0.02 mol) in acetone (50 mL) was refluxed for 24 h. When the reaction was complete, the mixture was filtered, and the solvent was evaporated. The residue was purified by column chromatography on silica gel using chloroform as the eluent. Yield: 70% (yellow solid); m.p. 153–154 °C; ¹H-NMR (300 MHz, CDCl₃ δ/ppm): 13,16 (s, 1H, -OH), 7.61 (d, J = 2.1 Hz, 1H, Har,), 7.00 (d, J = 2.1 Hz, 1H, Har), 6.08 (s, 1H, Har), 4.10 (s, 3 H, -OCH₃), 2.72 (q, J = 7.6 Hz, 2 H, -CH₂-ethyl), 1.35 (t, J = 7.6 Hz, 3 H, ethyl-CH₃); ¹³C NMR (75 MHz, CDCl₃), δ (ppm): 184.37, 172.02, 151.05, 149.92, 145.95, 144.88, 125.89, 113.67, 106.15, 105.77, 104.67, 61.82, 27.55, 10.90.

Synthesis of 7-ethyl-9-methoxy-4-[(oxiran-2-yl)methoxy]−5 H-furo[3,2-g][1]benzopyran-5-one (2a)

A mixture of 7-ethyl-4-hydroxy-9-methoxy-5H-furo[3,2-g][1]benzopyran-5-one (2) (0.02 mol) and 1-chloro-2,3-epoxypropane (100 mL) was refluxed in the presence of anhydrous potassium carbonate (K₂CO₃) for 72 h. Upon completion of the reaction, the inorganic salt was removed by filtration, and the excess 1-chloro-2,3-epoxypropane was evaporated under reduced pressure. The resulting residue was purified by column chromatography on silica gel using chloroform as the eluent. Yield: 57% (oil);

¹H-NMR (300 MHz, CDCl₃ δ/ppm): 7.63 (d, J = 2.1 Hz, 1H, Har,), 7.07 (d, J = 2.1 Hz, 1H, Har), 6.06 (s, 1H, Har), 4.50 (dd, J₁ = 8,7 Hz, J₂ = 2,7 Hz, -CH-epox), 4.20 (s, 3 H, -OCH₃), 4.07 (m, 1H, -CH₂-epox), 3.48 (m, 1H, -CH₂-epox), 2.88 (m, 1H, -CH₂-epox), 2.76 (m, 1H, -CH₂-epox), 2.71 (q, J = 7.6 Hz, 2 H, -CH₂-ethyl), 1.33 (t, J = 7.6 Hz, 3 H, -CH₃ ethyl). ¹³C NMR (75 MHz, CDCl₃), δ (ppm): 178.35, 168.85, 148.47, 146.99, 145.76, 145.33, 130.56, 120.89, 114.26, 108.77, 105.04, 76.20, 61.48, 50.69, 44.48, 26.99, 10.85.

General synthesis of amino alkyl derivatives and its hydrochlorides (3–9)

7-Ethyl-9-methoxy-4-[(oxiran-2-yl)methoxy]−5H-furo[3,2-g][1]benzopyran-5-one (0.02 mol) was dissolved in a methanol–water mixture (50:1, v/v). The appropriate alkylamine (0.02 mol) was then added, and the reaction mixture was refluxed for 72 h. After completion of the reaction, the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel using chloroform or a chloroform-methanol mixture (90:0.1–90:0.5 v/v) as the eluent. An appropriate amino alkyl derivative (3–9) was dissolved in anhydrous methanol, and the solution was acidified using methanol saturated with hydrochloric acid. Finally, the corresponding hydrochloride was precipitated by diethyl ether.

Hydrochloride of 7-ethyl-9-methoxy-4-[3-(diethylamino)−2-hydroxypropoxy]−5 H-furo[3,2-g][1] benzopyran-5-one (3)

Yield: 50% (white solid), m.p. 194–195 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 9.80 (br. s, 1H, NH⁺), 8.17 (d, J = 2.1 Hz, 1H, Har,), 7.32 (d, J = 2.1 Hz, 1H, Har), 6.12 (m, 2 H, Har, -OH), 4.37 (m, 1H, -CH-epox), 4.25 (m, 2 H, -CH₂-epox), 4.11 (s, 3 H, -OCH₃), 3.47 (m, 1H, -CH₂-epox), 3.34 (m, 1H, -CH₂-epox), 3.26 (m, 4 H, 2 x -CH₂-amine), 2.72 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.28 (m, 9 H, 3 x -CH₃, ethyl, amine). ¹³C NMR (75 MHz, DMSO), δ (ppm): 177.75, 169.24, 148.54, 147.30, 145.44, 145.25, 129.57, 118.74, 112.27, 108.38, 105.36, 76.40, 64.35, 61.40, 53.79, 47.80, 47.08, 26.23, 10.73, 8.51, 8.37; HRMS (m/z): calculated value for C₂₁H₂₇NO₆ [M+⁺]: 390.1911; found: 390.1917.

Hydrochloride of 7-ethyl-9-methoxy-4-{2-hydroxy-3-[(propan-2-yl)amino]propoxy}−5 H-furo[3,2-g][1]benzopyran-5-one (4)

Yield: 70% (white solid), m.p. 163–164 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 8.96 (br. s, 1H, -NH₂⁺), 8.76 (br. s, 1H, NH₂⁺), 8.17 (d, J = 2.4 Hz, 1H, Har), 7.34 (d, J = 2.4 Hz, 1H, Har), 6.14 (s, 1H, Har) 6.04 (m, 1H, -OH), 4.26 (m, 3 H, -CH-epox, -CH₂-epox), 4.11 (s, 3 H, -OCH₃), 3.34 (m, 2 H, -CH₂-epox), 3.15 (m, 1H, -CH-), 2.73 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.28 (m, 9 H, 3 x -CH₃ ethyl, amine); ¹³C NMR (75 MHz, DMSO), δ (ppm): 177.93, 169.42, 148.63, 147.31, 146.46, 145.23, 129.52, 118.63, 112.64, 108.39, 105.44, 76.54, 65.25, 61.42, 49.95, 46.81, 26.25, 18.52, 18.28, 10.72; HRMS (m/z): calculated value for C₂₀H₂₅NO₆ [M + H⁺]: 376.1754; found: 376.1760.

Hydrochloride of 7-ethyl-9-methoxy-4-[2-hydroxy-3-(4-methylpiperidin-1-yl)propoxy]−5 H-furo[3,2-g][1]benzopyran-5-one (5)

Yield: 60% (white solid), m.p. 195–196 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 10.08 (br. s, 1H, -NH⁺), 8.16 (d, J = 2.4 Hz, 1H, Har), 7.32 (d, J = 2.4 Hz, 1H, Har), 6.11 (s, 1H, Har) 6.08 (m, 1H, -OH), 4.43 (m, 1H, -CH-epox), 4.17 (m, 2 H, -CH₂-epox), 4.10 (s, 3 H, -OCH₃), 3.59 (m, 2 H, -CH₂-epox), 3.46 (m, 1H, -CH-piper), 3.26 (m, 2 H, -CH₂-piper), 3.02 (m, 2 H, -CH₂-piper), 2.71 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.79 (m, 2 H, -CH₂-piper), 1.55 (m, 2 H, -CH₂-piper), 1.24 (t, J = 7.5 Hz, 3 H, ethyl -CH₃), 0.92 (m, 3 H, piper-CH₃); ¹³C NMR (75 MHz, DMSO), δ (ppm): 177.67, 169.25, 148.42, 147.35, 146.39, 145.10, 129.72, 119.22, 113.01, 108.36, 105.26, 76.65, 64,23, 61.38, 59.23, 53.40, 52.44, 30.71, 30.60, 28.03, 26.25, 21.12, 10.73; HRMS (m/z): calculated value for C₂₃H₂₉NO₆ [M + H⁺]: 416.2068; found: 416.2072.

Hydrochloride of 7-ethyl-9-methoxy-4-[3-(dibuthylamino)−2-hydroxypropoxy]−5 H-furo[3,2-g][1] benzopyran-5-one (6)

Yield: 50% (white solid), m.p. 126–127 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 10.13 (br. s, 1H, -NH⁺), 8.17 (d, J = 2.4 Hz, 1H, Har), 7.32 (d, J = 2.1 Hz, 1H, Har), 6.12 (m, 2 H, Har, -OH), 4.41 (m, 1H, -CH-epox), 4.23 (m, 2 H, -CH₂-epox), 4.11 (s, 3 H, -OCH₃), 3.48 (m, 1H, -CH₂-epox), 3.32 (m, 1H, -CH₂-epox), 3.19 (m, 4 H, -(CH₂)₂-), 2.71 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.73 (m, 4 H, -(CH₂)₂-), 1.37 (m, 2 H, 2 X -CH₂-), 1.25 (t, J = 7.5 Hz, 3 H, ethyl -CH₃), 0.92 (t, 6 H, J = 7.35 Hz, 2 X-CH₃); ¹³C NMR (75 MHz, DMSO), δ (ppm): 177.65, 169.14, 148.49, 147.25, 146.41, 145.26, 129.54, 118.79, 112.80, 108.37, 105.36, 76.42, 64.45, 61.38, 54.92, 53.01, 52.71, 26.23, 24.74, 19.44, 13.53, 10.73; HRMS (m/z): calculated value for C₂₅H₃₅NO₆ [M + H⁺]: 446.2537; found: 446.2544.

Hydrochloride of 7-ethyl-9-methoxy-4-[2-hydroxy-3-(piperidin-1-yl)propoxy]−5 H-furo[3,2-g][1] benzopyran-5-one (7)

Yield: 40% (white solid), m.p. 212–213 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 10.13 (br.s, 1H,-NH⁺), 8.17 (d, J = 2.1 Hz, 1H, Har), 7.33 (d, J = 2.4 Hz, 1H, Har), 6.12 (m, 2 H, Har, -OH), 4.44 (m, 1H, -CH-epox), 4.19 (m, 2 H, -CH₂-epox), 4.11 (s, 3 H, -OCH₃), 3.50 (m, 2 H, -CH₂-epox), 3.03 (m, 2 H, -CH₂-piperidine), 3.02 (m, 2 H, -CH₂-piperidine), 2.72 (q, 2 H, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.82 (m, 6 H, -CH₂-piperidine), 1.25 (t, J = 7.5 Hz, 3 H, ethyl-CH₃); ¹³C NMR (75 MHz, DMSO), δ (ppm): 179.39, 169.16, 149.21, 147.06, 146.71, 145.58, 129.94, 119.25, 113.47, 108.98, 105.48, 78.40, 77.25, 67.06, 61.66, 61.04, 55.13, 27.06, 26.05, 24.26, 10.90; HRMS (m/z): calculated value for C₂₂H₂₇NO₆ [M + H⁺]: 402.1911; found: 402.1917.

Hydrochloride of 7-ethyl-9-methoxy-4-[3-(tert-butylamino)−2-hydroxypropoxy]−5 H-furo[3,2-g][1] benzopyran-5-one (8)

Yield: 70% (white solid) m.p. 176–177 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 9.04 (br. s, 1H, -NH₂⁺), 8.72 (br. s, 1H, -NH₂⁺), 8.17 (d, J = 2.1 Hz, 1H, Har), 7.34 (d, J = 2.4 Hz, 1H, Har), 6.14(m, 1H, Har), 6.04 (b.s, 1H, -OH), 4.27 (m, 3 H, -CH₂-CH-epox), 4.11 (s, 3 H, -OCH₃), 3.31 (m, 1H, -CH₂-epox), 3.09 (m, 1H, -CH₂-epox), 2.72 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.35 (s, 9 H, -C(CH₃)₃), 1.25 (t, J = 7.5 Hz, 3 H, ethyl -CH₃); ¹³C NMR (75 MHz, DMSO), δ (ppm): 177.86, 169.37, 148.60, 147.28, 146.45, 145.26, 129.50, 118.65, 112.64, 108.41, 105.44, 76.46, 65.53, 61.41, 56.38, 44.29, 39.51, 26.25, 24.98, 10.72; HRMS (m/z): calculated value for C₂₁H₂₇NO₆ [M + H⁺]: 390.1911; found: 390.1917.

Hydrochloride of 7-ethyl-9-methoxy-4-[2-hydroxy-3-(morpholin-4-yl)propoxy]−5 H-furo[3,2-g][1] benzopyran-5-one (9)

Yield: 60% (white solid) m.p. 222–223 °C; ¹H-NMR (300 MHz, DMSO, δ/ppm): 10.54 (br. s, 1H, -NH⁺), 8,17 (d, J = 2.4 Hz, 1H, Har), 7.31 (d, J = 2.4 Hz, 1H, Har), 6.13(s, 1H, Har), 4.36 (br.s, 1H, -OH), 4.18 (m, 3 H, -CH-CH₂-epox), 4.12 (s, 3 H, -OCH₃), 3.81(m, 4 H, -CH₂-morpholin, -CH₂-epox), 3.33 (m, 6 H, -CH₂-morpholin), 2.57 (q, J = 7.5 Hz, 2 H, -CH₂-ethyl), 1.26 (t, J = 7.5 Hz, 3 H, ethyl -CH₃); ¹³C NMR (75 MHz, DMSO), δ (ppm): 179.53, 169.33, 149.24, 147.04, 146.59, 145.70, 130.01, 119.09, 113.39, 109.00, 105.33, 78.27, 76.63, 67.04, 61.67, 60.79, 54.28, 27.07, 10.90; HRMS (m/z): calculated value for C₂₁H₂₅NO₇ [M + H⁺]: 404.1704; found: 404.1710.

Cytotoxic activity

Cell cultures

Cell lines HBT-140 (human melanoma), A549 (human lung adenocarcinoma), HeLa (cervical cancer), and SW620 (colorectal cancer), and the normal cell line HaCaT (immortalized human keratinocytes) were purchased from American Type Culture Collection (Rockville, USA), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% antibiotics (penicillin and streptomycin) and 10% heat-inactivated FBS-fetal bovine serum (Gibco Life Technologies, USA) at 37 °C and 5% CO₂ atmosphere. Cells were passaged using trypsin-EDTA (Gibco Life Technologies, USA) and cultured in 24-well or 96-well plates (1 × 10⁴ cells per well). Experiments were conducted in DMEM with 2% FBS.

MTT assay

Cell viability was evaluated using the MTT assay, which measures the reduction of 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase in viable cells. Cells were seeded in 96-well plates and incubated for 72 h with various concentrations of the tested compounds. Subsequently, 0.5 mg/mL of MTT solution was added to each well, and the plates were incubated for an additional 4 h. In metabolically active cells, MTT was converted into insoluble formazan crystals. These were then solubilized using 0.04 M HCl in absolute isopropanol.

The absorbance of the resulting solution was measured at 570 nm using an Epoch microplate reader (BioTek Inc., USA) with Gen5 software (BioTech Instruments, Inc., Biokom). Cell viability was expressed as the percentage of MTT reduction in treated samples relative to untreated control cells (cultured in serum-free DMEM). The relative MTT level (%) was calculated as: [A]/[B] × 100, where [A] is the absorbance of the treated sample, and [B] is the absorbance of the control. A lower relative MTT level corresponds to reduced cell viability.

LDH assay

As a marker of cell death, the release of lactate dehydrogenase (LDH) from the cytosol to the culture medium (cellular membrane integrity assessment) was used. The assay was performed after 72 h incubation of cells in 96-well plates with investigated compounds as described before. The activity of lactate dehydrogenase (LDH) released from the cytosol of damaged cells to the supernatant was measured according to the protocol of the cytotoxicity detection kit LDH test described by the manufacturer (Roche Diagnostics, Germany). An absorbance was measured at 490 nm using a microplate reader (using Epoch microplate reader, BioTek Inc., USA) equipped with Gen5 software (BioTech Instruments, Inc., Biokom). Compounds-mediated cytotoxicity expressed as the LDH release (%) was determined by the following equation: [(A test sample − A low control)/(A high control − A low control)] × 100% (A-absorbance); where “low control” were cells in DMEM with 2% FBS without tested compounds and “high control” were cells incubated in DMEM with 2% FBS with 1% Triton X-100 (100% LDH release).

Apoptotic activity

Apoptosis Detection Kit I; BD Biosciences Pharmingen) was used. Cells were preincubated for 72 h with IC₅₀ concentrations of tested compounds. The effect of the exposure of HBT-140 (human melanoma), A549 (human lung adenocarcinoma), HeLa (cervical cancer), SW620 (colorectal cancer), and HaCaT (immortalized human keratinocytes) cells to tested compounds was determined by dual staining with Annexin V: FITC and 7-AAD. Annexin V: FITC and 7-AAD were added to the cellular suspension as described in the manufacturer’s instructions and a fluorescence sample of 10,000 cells was analyzed by flow cytometry (Becton Dickinson). Cells that were Annexin V: FITC positive and 7-AAD negative were identified as early apoptotic. Cells that were Annexin V: FITC positive and 7-AAD positive were identified as late apoptotic or necrotic. Camptothecin was acquired from Sigma-Aldrich (C-9911) – incubation was done with 4 µM CPT for 4 h to perform a positive control study.

Interleukin-6 analysis

The Interleukin IL-6 ELISA kit was purchased from Diaclon SAS (Besancon Cedex, France). The HBT-140 (human melanoma), A549 (human lung adenocarcinoma), HeLa (cervical cancer), and SW620 (colorectal cancer), and the HaCaT (immortalized human keratinocytes) cells were treated with IC₅₀ concentration of selected tetrazole derivatives, and with IC₅₀ cytostatic drugs for 72 h. IL-6 level in cell culture supernatant was measured using enzyme-linked immunosorbent assay following the manufacturer’s protocol.

Molecular Docking studies

Protein structures were retrieved from the Protein Data Bank (PDB)⁴¹, identified by their PDB IDs (8AV9, 7AEM, 6O0P, 6O0O, 6FS0, 4LVT, 4LQM, 4I23, 2YXJ, 2ITO, 1YSG, 1M17), stored locally and preprocessed to remove alternate conformations, water molecules, ions, and crystallization additives. Hydrogen atoms were added, and protonation states were assigned using pdbfixer⁴². Cleaned receptor structures were converted to PDBQT format using AutoDockTools⁴³. Ligand structures, provided as SMILES representations, were transformed into 3D conformations using Open Babel⁴⁴ and converted to PDBQT format. The docking grids were automatically generated based on native ligand coordinates. The grid box was defined using the ligand center of mass with a padding of 4 Å to ensure the binding site was fully encompassed⁴⁵. Docking simulations were conducted using Smina, an optimized fork of AutoDock Vina⁴⁶. Parameters were set to default values, including exhaustiveness of 8 and 10 docking poses per ligand-receptor pair. Native ligand re-docking served as validation, with the root mean square deviation (RMSD) calculated to assess docking accuracy. An RMSD ≤ 3.0 Å was considered successful docking⁴⁷. Docking poses were evaluated based on binding affinities (kcal/mol). Ligands demonstrating binding affinities superior to native ligands were selected as hits. Interaction profiles of the best docking poses were analyzed using the Protein-Ligand Interaction Profiler (PLIP)⁴⁸, generating detailed reports of interaction types such as hydrogen bonds, hydrophobic interactions, and π-stacking. Complex structures (receptor-ligand) of the top hits were visualized using py3Dmol⁴⁹ within Jupyter Notebooks and 2D interaction diagrams were created using Discovery Studio Visualizer (BIOVIA, Dassault Systèmes)⁵⁰. Visualizations included highlighting binding sites, ligand conformations, and receptor-ligand interactions. Additionally, PLIP-generated interaction profiles were incorporated to illustrate specific molecular interactions. All steps were automated in a Python-based workflow. Configuration settings were managed using YAML files, facilitating easy modifications of parameters and file paths. Logging and error handling were implemented to ensure reproducibility and ease of debugging.

The complete docking pipeline project is publicly available on GitHub via https://github.com/TomaszSzostek/Molecular_docking.git.

Statistical analysis

The results are expressed as the mean ± SD from the indicated number of experiments. Comparisons were made using the student’s t-test. Experimental group differences were considered statistically significant at p ≤ 005.

Antimicrobial evaluation

Evaluation using Gram-positive and Gram-negative strains

The antimicrobial assays were conducted using reference strains of bacteria derived from international microbe collections: American Type Culture Collection (ATCC) and National Collection of Type Culture (NCTC). The following standard strains of bacteria were used: Gram-positive - Staphylococcus aureus NCTC 4163, Staphylococcus aureus ATCC 25,923, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29,213, Staphylococcus epidermidis ATCC 12,228, Staphylococcus epidermidis ATCC 35,984, Gram-negative: Escherichia coli ATCC 25,922, and Pseudomonas aeruginosa ATCC 15,442. Antimicrobial activity was examined by the Minimal Inhibitory Concentration (MIC) method under standard procedures provided by CLSI, with some modifications. MIC was determined by the two-fold serial broth microdilution method in 96-well microtitration plates using Mueller–Hinton II broth medium (Becton Dickinson, Franklin Lakes, NJ, USA). The final inoculum of all studied bacteria was 10⁶ CFU/mL (colony-forming unit per millilitre). The stock solution of tested compounds was prepared in dimethyl sulfoxide (DMSO) and diluted to a maximum of 1% of solvent content with a sterile medium. The MIC value recorded is defined as the lowest concentration of the tested antimicrobial agents (expressed in µg/mL) that inhibits the visible growth of the microorganism after 19 h of incubation at 35 °C.

Description related to conducted biological studies, including cell culture, suitable conditions, and methodology, was presented in our previous paper⁵¹.

In vitro tuberculostatic activity

The synthesized compounds were examined in vitro for their tuberculostatic activity using the broth microdilution method according to CLSI M24, 3rd ed⁵². Investigations were performed in 96-well microtiter plates by the twofold serial microdilution using Middlebrook 7H9 Broth medium (Beckton Dickinson) containing 10% of OADC (Beckton Dickinson). The inoculum was prepared from fresh LJ culture in Middlebrook 7H9 Broth medium with OADC, adjusted to a no. 0.5 McFarland tube, and diluted 1:100. The stock solution of the tested agent was prepared in DMSO. Each test compound stock solutions were diluted in Middlebrook 7H9 Broth medium with OADC by a four-fold the final highest concentration to be tested. Compounds were diluted serially in sterile 96-well microtiter plates using 100 µL Middlebrook 7H9 Broth medium with OADC. Concentrations of tested agents ranged from 512 to 0.0625 µg/ml.

A growth control containing no antibiotic and a sterile control without inoculation were also prepared on each plate. The plates were incubated at 37 °C for 21 days. After the incubation period, 30 µL of Alamar blue solution was added to each well, and the plate was re-incubated for 24 h. Growth is indicated by a color change from blue to pink, and the lowest concentration of compound that prevented the color change was noted as its MIC⁵³. Ciprofloxacin, Isoniazid, Linezolid, Rifampicin, Streptomycin, and Ethambutol were used for comparison as reference drugs.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

W.O.: Manuscript review and editing, Anticancer studies, Supervision. E.A.K.: Antitubercular studies, Supervision. A.G.: Antitubercular studies, Data interpretation. M.N.: Synthesis, Data interpretation, Manuscript review and editing: T.S.: In silico studies, Molecular docking. A.F.: Anticancer studies, Data collection. D.S.: Conceptualization, Data collection and interpretation, Original manuscript writing, Supervision.

Funding

No external grant supports this work.

Data availability

Part of the datasets generated and analyzed during the current study are not publicly available due to requests from co-authors (for possible patent submission), but are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Authors contribution

W.O.: Manuscript review and editing, Anticancer studies, Supervision. E.A.K.: Antitubercular studies, Supervision. A.G.: Antitubercular studies, Data interpretation. M.N.: Synthesis, Data interpretation, Manuscript review and editing: T.S.: In silico studies, Molecular docking. A.F.: Anticancer studies, Data collection. D.S.: Conceptualization, Data collection and interpretation, Original manuscript writing, Supervision.

Supplementary Information

Supplementary data for this article can be found online.