Synthesis, anticancer evaluation, and electrochemical investigation of new chiral pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides

Abstract

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides (MM-compounds) represent a novel class of heterocyclic compounds with promising anticancer potential. In this study, we report the synthesis and biological evaluation of two enantiomeric derivatives: the R-enantiomer (MM124) and the S-enantiomer (MM125). Both compounds exhibited potent and selective cytotoxicity against a panel of cancer cell lines derived from various tissue types, with a median IC₅₀ value of 0.35 µM. Mechanistic investigations in colorectal (HT-29) and prostate (PC-3) cancer cell lines demonstrated that the compounds induce apoptosis, oxidative stress, and DNA damage. Electrochemical assays and computational studies further suggested that MM124 and MM125 interact with DNA. Additionally, in silico pharmacokinetic and toxicological profiling indicated favorable drug-like properties. These findings support the potential of MM124 and MM125 as candidates for the development of new anticancer agents, warranting further structural optimization and preclinical evaluation.

Introduction

Triazine constitutes an important scaffold in drug discovery. Depending on the position of the nitrogen atom three isomers of triazine can be distinguished: 1,2,3-triazines, 1,2,4-triazines, and 1,3,5-triazines¹. 6-azauracil (2H-1,2,4-triazine-3,5-dione) was one of the first analogs of 1,2,4-triazine, which exhibited anticancer activity and was investigated in clinical trials. However, its clinical application was limited due to adverse side effects^2,3.

Tirapazamine (3-aminobenzo[e][1,2,4]triazine 1,4-dioxide) was another 1,2,4-triazine analog with cytotoxic activity associated with its conversion into toxic radicals, which induced single or double-strand DNA breaks (SSBs and DSBs) and apoptosis of treated cells^4,5. In 2009, Gucký et al. synthesized 3,7-diarylo-5-(3,4,5-trimetoksyphenylo)pirazolo[4,3-e][1,2,4]triazine derivatives that exhibited cytotoxic activity towards multiple hematological and lung cancer cell lines⁶. Based on these findings Mojzych et. al synthesized various derivatives of the pyrazolo[4,3-e][1,2,4]triazines and assessed their biological activity in multiple cancer cell lines⁷. Various pyrazolo-triazines exhibited inhibitory activity towards histone deacetylases⁸, metalloproteinases⁹, tubulin¹⁰, urease, and tyrosinase^11,12. Tricyclic pyrazolo[4,3-e] [1, 2, 3]riazines fused with a triazole or tetrazole ring have emerged as an important class of pyrazolo-triazine derivatives with profound anticancer activity ⁷. Furthermore, the incorporation of the sulfonamide group into the pyrazolo-triazine scaffold enabled the extension of the spectrum of potential molecular targets of the pyrazolo-triazne compounds¹³ including ABL kinase^14,15, carbonic anhydrases¹⁶, cyclin-dependent kinases (CDKs)^7,17, casein kinase 2 (CK2)^18,19, and glycogen synthase kinase 3 (GSK3)²⁰.

The pyrazolo[4,3-e][1,2,4]triazine derivatives are, so far, one of the least known groups of heterocycles. Nevertheless, the above literature data indicate that this structure has significant biological activity. Attachment of a tetrazole or triazole ring to the bicyclic structure of the pyrazolo[4,3-e][1,2,4]triazine and the addition of sulfonamide moiety can significantly improve the biological activity of compounds⁷. At present, numerous pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine derivatives undergo various phases of in vitro and in vivo studies (for the structural details of the compounds see Table 8 of the discussion section).

For example, the most pre-clinically advanced derivative MM129 was demonstrated to effectively limit cell viability via Bruton’s tyrosine kinase (BTK) inhibition resulting in apoptosis of colorectal cancer cells in vitro. The activity of the compound was also evaluated in the zebrafish embryo xenograft model, where MM129 showed synergistic anti-tumor activity with the chemotherapeutic agent – 5-fluorouracil (5-FU)²¹. Additionally, MM129 exhibited good pharmacokinetics parameters and safety profile in mice²². Bukowski et al. evaluated the cytotoxic activity of MM129 together with two other pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides MM130, and MM131 in four cancer cell lines (cervical cancer (HeLa), colorectal cancer (HCT-116), prostate cancer (PC-3), and pancreatic cancer (BxPC-3), where the cytotoxic 50% inhibitory concentration (IC₅₀) of compounds ranged from 0.17–1.15 μM²³. More recently, we have reported micromolar cytotoxicity of MM134, MM136, MM137, and MM139 compounds in BxPC-3, HCT-116, and PC-3 cells. Furthermore, the cytotoxic effect was selective for cancer cells and not normal human cells in vitro²⁴.

In this paper, we present the general method for the synthesis of two novel enantiomeric sulfonamide derivatives: the R-enantiomer (MM124) and the S-enantiomer (MM125) (Fig. 1) of the pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine ring system compound series incorporated with leucinol moiety and investigate their anticancer activity including cytotoxicity (for multiple cancer and normal cell lines of different tissue origin) as well as genotoxic, pro-apoptotic and pro-oxidative properties in HT-29 (colorectal adenocarcinoma) and PC3 (prostate cancer) cancer cell lines. Additionally, an electrochemical investigation was carried out to evaluate the DNA binding activity of the compounds. Molecular docking and molecular dynamics computation methods were employed to confirm the interaction between the compounds and DNA molecules.

Fig. 1

Chemical structure of two investigated enantiomeric sulfonamides MM124 (R-enantiomer) and MM125 (S-enantiomer) incorporated with leucinol moiety.

Materials and methods

Synthesis of MM124 and MM125

Melting points were determined on a Mel-Temp apparatus and were uncorrected. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Varian spectrometer (400 MHz for ¹H and 100 MHz for ¹³C). The chemical shift values were expressed in ppm (part per million) with tetramethylsilane (TMS) as an internal reference. The relative integrals of peak areas agreed with those expected for the assigned structures. The molecular weight of the final compounds was assessed by electrospray ionization mass spectrometry (ESI/MS) on Agilent Technologies 6538 UHD Accurate mass qadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC/MS) (Warsaw, Poland). The attenuated total reflectance infrared spectroscopy (ATR-IR) spectra were recorded over the range of 4000–400 cm⁻¹ on the Thermo Scientific Nicolet 6700 Fourier transform infrared (FTIR) spectrophotometer. Elemental compositions were within ± 0.4% of the calculated values. For preparation and spectroscopic data of compounds 1 see the literature²⁵.

The synthesis of the key tricyclic compounds with a sulfonamide moiety (MM124 and MM125) was performed by the standard procedure described in our previous articles^7,24,26 (Fig. 2). Briefly, the known chlorosulfonyl derivative 1 was reacted with enantiomeric leucinols to give the two corresponding chiral derivatives 2ab, which, when reacted with sodium azide in anhydrous ethanol under reflux, provided the final designed tricyclic sulfonamides MM124 and MM125. The structure and purity of the newly synthesized compounds were characterized by ¹H and ¹³C NMR and high-resolution mass spectrometry (HRMS) methods along with elemental and ATR-IR analysis (provided in the supplementary material – SM1).

Fig. 2

Reagents and conditions: (a) appropriate leucinol, MeCN, rt, 3 h; (b) NaN3, EtOH, reflux. For the details see the main text below.

Synthesis of sulfonamides (2ab)

Reaction (a): Derivative 1 (194 mg, 0.5 mmol) was dissolved in anhydrous acetonitrile (5 mL) and the appropriate amount of pure S or R enantiomer of leucinol (1.75 mmol) was added. The reaction was stirred overnight at room temperature, and then the reaction mixture was concentrated in vacuo to afford the crude sulfonamide, as a yellow solid. The residue was purified on silica gel using a mixture of CH₂Cl₂:EtOH (25:1) as eluent to give the titled compounds as a yellow solid.

Synthesis of N-(R)-(1-hydroxy-4-methyl-pent-2-yl)-4-(3-methyl-5-methylsulfonyl-1H-pyrazolo[4,3-e][1,2,4]triazyn-1-yl)benzenesulfonamide (2a)

Yield 98%. Melting point: 138–142 °C; IR cm^-1: 3503, 2933, 1596, 1505, 1456, 1410, 1313, 1147, 1094, 950, 826, 727, 612; ¹H NMR (DMSO) δ: 0.59 (d, 3H, J = 6.4 Hz), 0,75 (d, 3H, J = 6.8 Hz), 1.10–1.17 (m, 1H), 1.28–1.35 (m, 1H). 1.48–1.55 (m, 1H), 2.79 (s, 3H), 3.10–3.17 (m, 2H), 3.28 (t, 1H, J = 5.6 Hz), 3.62 (s, 3H), 4.66 (t, 1H, J = 5.2 Hz, exchanged with D₂O, OH), 7.65 (d, 1H, J = 7.2 Hz, exchanged with D₂O, NH), 8.10 (d, 2H, J = 8.8 Hz), 8.53 (d, 2H, J = 8.8 Hz); ¹³C NMR (DMSO) δ: 11.12, 21.46, 23.35, 23.68, 40.53, 40.83, 53.43, 64.12, 120.00, 128.39, 138.24, 140.12, 140.26, 146.08, 148.30, 160.96; HRMS (ESI, m/z) Calcd for C₁₈H₂₄N₆O₅S₂ [M⁺ + H] 469.13224. Found [M⁺ + H] 469.13293. Anal. Calcd for C₁₈H₂₄N₆O₅S₂: C, 46.14; H, 5.16; N, 17.94. Found: C, 46.25; H, 5.32; N, 17.72.

Synthesis of N-(S)-(1-hydroxy-4-methyl-pent-2-yl)-4-(3-methyl-5-methylsulfonyl-1H-pyrazolo[4,3-e][1,2,4]triazyn-1-yl)benzenesulfonamide (2b)

Yield 82%. Melting point: 141–145 °C; IR cm^-1: 3493, 2933, 1596, 1506, 1313, 1150, 968, 727, 612; ¹H NMR (DMSO) δ: 0.59 (d, 3H, J = 6.4 Hz), 0,76 (d, 3H, J = 6.4 Hz), 1.10–1.19 (m, 1H), 1.28–1.35 (m, 1H). 1.45–1.55 (m, 1H), 2.79 (s, 3H), 3.10–3.17 (m, 2H), 3.27 (m, 1H), 3.62 (s, 3H), 4.66 (t, 1H, J = 5.6 Hz, exchanged with D₂O, OH), 7.65 (d, 1H, J = 7.2 Hz, exchanged with D₂O, NH), 8.10 (d, 2H, J = 8.8 Hz), 8.53 (d, 2H, J = 8.8 Hz); ¹³C NMR (DMSO) δ: 11.83, 22.22, 23.99, 24.57, 41.13, 41.59, 54.13, 64.78, 121.27, 129.14, 138.90, 140.64, 141.03, 147.37, 148.89, 161.50; HRMS (ESI, m/z) Calcd for C₁₈H₂₄N₆O₅S₂ [M⁺ + H] 469.13224. Found [M⁺ + H] 469.13286. Anal. Calcd for C₁₈H₂₄N₆O₅S₂: C, 46.14; H, 5.16; N, 17.94. Found: C, 46.30; H, 5.28; N, 17.78.

Synthesis of tricyclic sulfonamides (3ab—MMs)

Reaction (b): A sulfonamide derivative with a methylsulfonyl group (0.33 mol) was dissolved in anhydrous ethanol (5 mL), and sodium azide (26 mg, 0.4 mol) was added. The reaction mixture was refluxed until the substrate disappeared (control thin-layer chromatography (TLC)). Then, the solvent was evaporated and the crude product was purified using column chromatography and CH₂Cl_2: MeOH (50:1) mixture as eluent to give the final compounds as a yellow solid.

N-(R)-(1-hydroxy-4-methyl-pent-2-yl)-4-[7-methyl-5H-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazin-5-yl)]benzenesulfonamide (3a: MM-124)

Yield 76%. Melting point: 157–160 °C; IR cm^-1: 3406, 3161, 2926, 1592, 1507, 1461, 1411, 1320, 1142, 1095, 969, 832, 643; ¹H NMR (methanol) δ: 0.66 (d, 3H, J = 6.4 Hz), 0,81 (d, 3H, J = 6.8 Hz), 1.18–1.26 (m, 1H), 1.32–1.38 (m, 1H), 1.48–1.55 (m, 1H), 2.85 (s, 3H), 3.24–3.30 (m, 1H), 3.30–3.35 (m, 1H), 3.42–3.45 (m, 1H), 4.57 (t, 1H, J = 5.6 Hz, OH), 8.08 (d, 2H, J = 8.8 Hz), 8.43 (d, 2H, J = 8.8 Hz); ¹³C NMR (methanol) δ: 11.19, 22.05, 23.62, 25.34, 42.02, 54.88, 65.95, 120.13, 129.68, 140.88, 141.86, 143.30, 148.33, 148.88, 149.30; HRMS (ESI, m/z) Calcd for C₁₇H₂₁N₉O₃S [M⁺ + H] 432,48,235. Found [M⁺ + H] 432,48,276. Anal. Calcd for C₁₇H₂₁N₉O₃S: C, 47.32; H, 4.91; N, 29.22. Found: C, 47.49; H, 5.08; N, 29.00.

N-(S)-(1-hydroxy-4-methyl-pent-2-yl)-4-[7-methyl-5H-pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazin-5-yl)]benzene-sulfonamide (3b: MM-125)

Yield 88%. Melting point: 159–161 °C; IR cm^-1: 3411, 3158, 2923, 1592, 1506, 1460, 1411, 1319, 1151, 1095, 970, 853, 832, 725, 643; ¹H NMR (methanol) δ: 0.66 (d, 3H, J = 6.4 Hz), 0,81 (d, 3H, J = 6.4 Hz), 1.20–1.26 (m, 1H), 1.32–1.38 (m, 1H), 1.48–1.55 (m, 1H), 2.85 (s, 3H), 3.25–3.35 (m, 2H), 3.42–3.46 (m, 1H), 4.57 (bs, 1H, OH), 8.08 (d, 2H, J = 8.8 Hz), 8.43 (d, 2H, J = 8.8 Hz); ¹³C NMR (methanol) δ: 11.18, 22.05, 23.62, 25.35, 42.03, 54.89, 65.96, 120.13, 129.70, 140.91, 141.86, 143.32, 148.33, 148.87, 149.31; HRMS (ESI, m/z) Calcd for C₁₇H₂₁N₉O₃S [M⁺ + H] 432,48,235. Found [M⁺ + H] 432,48,282. Anal. Calcd for C₁₇H₂₁N₉O₃S: C, 47.32; H, 4.91; N, 29.22. Found: C, 47.55; H, 4.88; N, 29.05.

Biological studies

Chemicals

Trypsin–EDTA and all culture media (RPMI-1640, DMEM-F12, MEM) were purchased from Biowest (CytoGen, Poland). EMEM medium was purchased from the American Type Culture Collection (ATCC, Rockville, USA). 4′,6-diamidino-2-phenylindole (DAPI), acridine orange/ethidium bromide (AO/BE), propidium iodide (PI), Hoechst 33,342 (HE), bleomycin, phosphate-buffered saline (PBS), dimethylformamide (DMF), penicillin–streptomycin solution stabilized, fetal bovine serum (FBS), histopaque1077, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide (MTT), normal melting point agarose (NMP), low melting point agarose (LMP), sodium dodecyl sulfate (SDS), Triton X-100 were supplied by Sigma Aldrich Chemical Co (USA). MitoTracker Red was purchased from Invitrogen (UK). 5-fluorouracil (5-FU) was obtained from MedChemExpress LLC (TriMen Chemicals, Lodz, Poland).

Cell culture

Cancer cell lines: BxPC-3 (pancreas adenocarcinoma, ATCC® CRL-1687™), DLD-1 (colorectal adenocarcinoma, ATCC CCL-221 ™), HCT-116 (colorectal carcinoma, ATCC® CCL-247™), HT-29 (colorectal adenocarcinoma, ATCC® HTB-38™) and PC3 (prostate cancer, ATCC® CRL-1435™) and normal cell lines: CCD 841 CoN (large intestine normal cells, CRL-1790 ™) and WI-38 (human lung fibroblasts, ATCC® CCL-75™) were obtained from American Type Culture Collection (ATCC). The MycoBlue™ Mycoplasma Detector kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used at least every month for the control of mycoplasma contamination in the cell cultures. The human peripheral blood lymphocytes (PBLs) were isolated from leucocyte buffy-coat obtained from blood collection at the Blood Bank in Lodz, Poland. Blood came from healthy, non-smoking donors (adults, aged 25–40) who had no signs of infection during collection. The use of a human leucocyte buffy-coat was approved by the Bioethics Committee for Scientific Investigation, University of Lodz (agreement no. KBBN-UŁ/I/8/2019). PBLs were isolated from blood by centrifugation in a density gradient of Histopaque®1077 (300 g for 25 min). The obtained pellet was resuspended in RPMI-1640 medium to the density of about 1–3 × 10⁵ cells per mL. Lymphocytes were grown in RPMI-1640 medium with 10% (v/v) FBS, 1% (v/v) penicillin–streptomycin, and phytohaemagglutinin (PHA). Adherent cells were cultured in dedicated cell media as shown in Table 1.

Table 1 Composition of culture media used to cultivate adherent cells.

MTT assay

MTT cytotoxicity assay is a common method for determining the metabolic activity in living cells. The assessment is based on the enzymatic conversion of a tetrazolium salt that has a light color to its purple-blue formazan product that can be quantified using spectrophotometry. The obtained absorbance value is directly proportional to the metabolically active cells in the sample^27,28.

96-well plates were seeded at a density of approximately 8 × 10³ ( DLD-1, HCT-116, HT-29 cells), 1 × 10⁴ (CCD 841 CoN, BxPC-3, PC3, and WI-38), and 8 × 10⁴ (PBL_s) cells per 100 µL medium per well. After 24-h incubation in controlled conditions (37 °C; 5% CO₂), cells were exposed to tested MM-compounds in the range of 0.1–5 µM. These concentrations were obtained by diluting the compounds in DMSO (final concentration was < 0.5% v/v)²⁹ and in the culture medium. 5-FU was used in the concentration range of 2–500 μM.

The experimental design included non-treated controls and blanks (wells without cells).

After 72-h of incubation, 20 µl of MTT tetrazolium salt (5 mg/mL in PBS) was added to each well, and plates were incubated in a humidified atmosphere for 3 h (37 °C; 5% CO₂).

The solutions were removed and 100 µL of DMSO was added to dissolve formazan complexes in case of adherent cancer cells or by directly adding a 100 µL mixture of 20% SDS and 50% DMF to each well for 24 h PBL_s. A spectrophotometer (microplate reader Power Wave XS BioTek Instruments, Inc., USA) reading was performed at 570 nm.

A statistical program (GraphPad Prism 7) was used to analyze the obtained data. The dose–response analysis was performed to estimate the inhibitory concentration (IC₅₀) of tested compounds. The IC₅₀ value is defined as a concentration of tested compound that leads to a reduction of cell pool viability by 50% compared to the negative control (accepted as 100%).

$$\text{\% }cell\:viability= \frac{\left(Absorbance\:value\:of\:treated\:cells -Absorbance\:value\:of\:blank\right)}{(Absorbance\:value\:of\:untreated\:cells-Absorbance\:value\:of\:blank)}\times 100\text{\%}$$

The selectivity index (SI) was calculated as a ratio of median IC₅₀ values in normal cell lines and cancer cell lines.

$$\text{SI}= \frac{\left(Median\:IC50\:value\:calculated\:for\:normal\:cell\:lines \right)}{(Median\:IC50\:value\:calculated\:for\:cancer\:cell\:lines )}$$

MitoTracker Red – mitochondrial membrane potential changes (ΔѰm)

MitoTracker Red is a derivative of dihydro-X-rosamine. Reduced MitoTracker dyes undergo oxidation in the viable cells, and form conjugates with thiol groups of proteins which make them fluorescent³⁰. The intensity of fluorescence correlates with the fitness of the mitochondria and changes with alterations in mitochondrial membrane potential (MMP)³¹.

Tumor (HT-29 and PC-3) cells were seeded at a density of 2 × 10⁴ on 96-well black clear-bottom plates. Cells were incubated with 0.5, 1, and 2 µM concentrations of MM124 and MM125 for 24 h. After 24-h cells were incubated with MitoTracker Red (0.1 µM/200 µL PBS/well) for 40 min. After incubation time MitoTracker Red solution was discarded and PBS (200 µL/well) was added. Fluorescence was read at an absorbance/emission of 581/644 nm using the SpectraMax® i3x Multi-Mode Detection Platform.

Dual acridine orange/ethidium bromide (AO/EB) and propidium iodide/Hoechst 33,342 (PI/HE) double staining

Tumor HT-29 and PC-3 cells were seeded at a density of 1.5 × 10⁵ on 12-well plates Following 24 h, cells were exposed to MM124 and MM125 in 0.5, 1, and 2 µM concentrations for 24 h. Following the incubation period, cells were incubated with fluorochromes (AO/EB: 100 µM; 1:1, v/v) or (PI/HE: 1 μg/mL; 1:1, v/v) for 5 min at 37 °C in the dark. Cells were examined in a fluorescence microscope (Olympus BX60 F5 Olympus Optical Co.Ltd.) at 360 nm. The results were obtained in duplicates and presented as mean percentage of necrotic and apoptotic cells ± standard deviation (SD) values. The differences between the experimental samples and control samples were estimated by the ANOVA test followed by the post-hoc Dunnetts test (p < 0.05, N = 200).

Study of reactive oxygen species (ROS) formation

Assessment of intracellular levels of reactive oxygen species (ROS) was carried out using a fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). The DHCF-DA probe is hydrolyzed by intracellular esterases after penetrating the cell membrane. The resulting product is oxidized in the presence of oxidants such as H₂O₂, ·OH, ·NO_2, and others, to the highly fluorescent 2’,7'-dichlorofluorescein (DCF). This allows fluorescence emission in a population of cells to be measured or identified microscopically^32,33.

HT-29 and PC-3 cells were seeded on 96-well black microplates at density 12 × 10⁵/mL and cultured in standard conditions (37 °C; 5% CO₂) for about 48 h until they reached the exponential growth phase. After this time, the culture medium was removed and the cells were washed three times with PBS. A 20 µM DCFH-DA solution (in PBS) was then applied and the cells were incubated for 20 min. Subsequently, the fluorescent probe was removed and cells were washed with PBS again. Solutions of MM124 and MM125 were applied at concentrations equivalent to 0.5xIC₅₀, IC₅₀, and 2xIC₅₀ values obtained in the MTT assay. Negative control cells were incubated with PBS, and the positive control included cells treated with H₂O₂ at a concentration of 500 µM. Fluorescence was measured on reader SpectraMax i3 Molecular Devices using an excitation wavelength of 485 nm and emission of 535 nm following 120 min.

Genotoxicity studies

The comet assay is a commonly used method for measuring DNA strand breaks at a single-cell level. The method stems from the simple principle that DNA fragmented following treatment with a genotoxic compound migrates more quickly than undamaged DNA in agarose gel during electrophoresis. In summary, single-cell suspensions mixed with LMP are layered on microscope slides covered in NMP, then lysed with detergent to rupture cell membranes and remove histone proteins, and finally electrophoresed to separate the fragmented DNA from intact DNA. In the presence of strand breaks, DNA moves in the direction of the anode, creating a picture that, when stained with a fluorescent dye and viewed using a fluorescence microscope, resembles the tail of a comet. Alkaline comet assay allows easy estimation of SSBs and apurinic/apyrimidinic sites^34,35,36,37.

The alkaline version of the comet assay was used in the current study according to Singh et al. (1988)³⁸, with modifications. Cells were seeded at a density of 150 000/2 mL (HT-29), 250 000/2 mL (PC3), and 600 000/2 mL of PBL_s onto 6-well plates. Following 24 h, cells were subjected to MM124 and MM125 in 0.5 µM, 1 µM, and 2 µM concentrations. Untreated control and positive control (cells treated with 20 µM bleomycin) were used in the experiment. Cells were exposed to compounds for another 24 h (37 °C; 5% CO₂). Afterward, they were transferred to Eppendorf tubes and centrifuged at 1400 rpm for 10 min at 4 °C. The precipitate was diluted in PBS. The next steps were performed as it was previously described by our group^23,24. Following electrophoresis, slides were stained with DAPI (1 µg/ml) and coverslipped. DNA damage was evaluated with the use of fluorescence microscopy at 360 nm using CellSens (Olympus) software. A total number of approximately 100 cells per slide was chosen for further analysis.

CASP: Comet Assay Software Project Lab (http://casplab.com) software was used to establish the mean value of DNA (%) in comet tails. The data were presented with a standard error of the mean (± standard error of the mean; SEM) and analyzed with ANOVA followed by post-hoc Dunnett’s test (p < 0.05, N = 200).

Electrochemical studies

Chemicals and reagents

Stock solutions of MM124 and MM125 were prepared by dissolving the required amount of the analytes in DMSO. Lower concentrations were achieved by properly diluting the stock sulfonamide derivatives solutions. The voltammetric analysis utilized PBS as a supporting electrolyte with a pH of 7.4. Both PBS and double-stranded salmon sperm DNA (dsDNA) were supplied from Sigma Aldrich Chemical Co (USA). A standard stock solution of DNA was prepared by dissolving the requisite amount of DNA powder in PBS. Aqueous solutions were made using distilled and deionized water.

Apparatus and instrumentation

Electrochemical measurements were performed using a versatile potentiostat EmStat3 (PalmSens, the Netherlands), run by the PS Trace software (version no. 5.9). A traditional three-electrode system was used, with a boron-doped diamond electrode (Redoxme AB, Sweden, diameter: 2 mm) as a working electrode, a platinum wire (Mineral, Poland) used as an auxiliary electrode, and an Ag/AgCl electrode (Mineral, Poland) used as a reference electrode. During all experiments, a voltammetric cell of 10 mL was used. The pH of the supporting electrolytes was measured using a pH meter (Elmetron, Poland) equipped with a combination glass electrode.

Electrochemical measurements

In the present study square-wave (SWV) and cyclic voltammetry (CV) were applied for the general characterization of the chosen sulfonamide derivatives (MM124, MM125). Whereas, interaction studies were performed using SW voltammetry. Interaction studies were performed as follows: measurements of MM124, and MM125 (at their fixed concentrations) were performed in the presence and absence of dsDNA in PBS at pH 7.4. 10 ml of a chosen supporting electrolyte was introduced in an electrochemical cell and then purged with argon for 5 min. After recording the voltammogram of the pure PBS, precise amounts of MM124 or MM125 and dsDNA were added using a micropipette. Following each addition, the solution underwent another 15-s deoxygenation, and a voltammogram was again recorded. The working electrode surface (boron-doped diamond electrode; BDDE) was polished with alumina slurry suspension on a polishing cloth before each voltammetric measurement. After that, the electrode surface was rinsed with distilled water. All experiments were performed in triplicate. The SWV conditions used during the experiments were as follows: amplitude of 25 mV, step potential of 5 mV, and frequency of 25 Hz. Cyclic voltammograms were obtained using a scan rate of 50–500 mV s⁻¹.

Computational analysis

Drug likeness and ADMET

The oral drug-likeness of compounds (here MM124 and MM125) can be predicted using SwissADME (http://www.swissadme.ch/). Based on several assumptions (parameters) referred to as Lipinski’s rule of five: (a) octanol/water partition coefficient (log P) of the molecule not be greater than five, (b) molecular weight less than 500 Da, (c) not more than five hydrogen bond donors, (d) and no more than 10 hydrogen bond acceptors a drug-likeness of the compound can be estimated. In contrast, bioavailability can be predicted based on several features of compounds, including flexibility (no more than 9 rotatable bonds), lipophilicity (XLOGP3 parameter between − 0.7 and + 5.0), size: molecular weight (between 150 and 500 g/mol), polarity (topological polar surface area (TPSA) between 20 and 130 Å2), solubility (LogS not higher than 6), and saturation (fraction of carbons in the sp3 hybridization (Csp3) not less than 0.25).

Moreover, key parameters including gastrointestinal absorption (GI), blood–brain barrier (BBB) permeability, cytochrome P450 3A4 (CYP3A4) and cytochrome P450 2D6 (CYP2D6) inhibition potential and toxicity can be predicted using pkCSM (http://biosig.unimelb.edu.au/pkcsm/)³⁹, SwissADME⁴⁰ and PreADMET online servers (https://preadmet.qsarhub.com/). Additionally, multiple toxicological features including hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity can be predicted using Protox-II (http://tox.charite.de/protox_II).

Molecular docking

A crystallized structural model of the 6-BP DNA molecule complexed with ellipticine was revealed by X-ray crystallography technique at a resolution of 1.50 Å (PDB code: 1Z3F)⁴¹.

The structure model comprises a dsDNA molecule with the intercalating agent. The DNA molecule was prepared for docking analysis by the addition of polar hydrogen, Gasteiger charge, and assigning autodock atom type, while the complex ligand was prepared by providing the flexibility followed by saving both in the default PDBQT format of Autodock software. Grid-box was prepared by covering the extended conformations of the reference ligand ellipticine as well as the interacting residues of the DNA and the grid parameters used in the current docking analysis are tabulated in Table 2.

Table 2 The coordinates of the grid box used in the MM124 and MM125 in silico DNA interactions studies.

The docking analysis was conducted using the Lamarckian Genetic Algorithm (LGA), employing a population size of 150, 2.5 million energy evaluations, and 27,000 generations across 30 independent runs for each ligand. The docking protocol and parameters were validated by re-docking the reference ligand to the target DNA. Validation criteria included the comparison of binding scores, structural alignment, and chemical interactions to confirm that the docking simulation accurately reproduced the binding mode observed in the reference bioactive complex. The validated docking parameters were further utilized for the docking of the MM124 and MM125 compounds with the intent to identify their DNA interacting properties that could explain the genotoxic potential of the compounds.

Molecular dynamics simulation

The obtained docking results were further validated for the stability of the macromolecular drug-receptor complex concerning time by performing molecular dynamics (MD) simulations. MD simulation was executed for each of the above-mentioned macromolecular complexes for 100 ns by using the Desmond module of Schrodinger’s Maestro software^34,35,36,37. Addition of explicit solvent molecules followed by their neutralization by adding the respective ions. The steepest-descent algorithm was used to relax the system and eliminate any steric clashes or poor contacts within atoms to minimize the system’s energy. Using a short series having low temperature with constant pressure (NPT) simulations, the system was brought to equilibrium. Positional constraints were applied to the system in addition to a progressive increase in temperature which usually makes it more likely that the system will be in a stable, balanced state before the simulation. To get the appropriate outcomes, the simulation was performed for 100 ns while taking into account the system’s energies, atom positions, and deviation values^42,43,44.

Results

Biological studies

MTT assay

MTT assay used to determine the cytotoxicity of compounds showed that both MM124 and MM125 possess cytotoxic activity towards tested cell lines (Table 3). MM124 compound exhibited the highest cytotoxic activity towards HT-29 colorectal cancer cells (IC₅₀ = 0.22 ± 0.02 µM) and PC-3 cells (IC₅₀ = 0.3 µM), while showing lower cytotoxicity in normal human colon tissue cells (CCD 841 CoN) (IC₅₀ = 1.12 ± 0.04 µM), human fibroblasts (WI-38 cells) (IC₅₀ = 0.98 ± 0.13 µM) and PBLs (IC₅₀ = 0.87 ± 0.07 µM). In contrast, MM125 exerted the highest activity in HT-29 cells (IC₅₀ = 0.27 ± 0.02 µM) and pancreatic cancer (BxPC-3) cells (IC₅₀ = 0.21 ± 0.02 µM). Similarly, to the other investigated derivative, MM125 showed preferential activity towards cancer cells, compared to normal human colon tissue cells (CCD 841 CoN cells) (IC₅₀ = 1.15 ± 0.02 µM), human fibroblasts (IC₅₀ = 1.21 ± 0.03) and PBLs (IC₅₀ = 1.109 ± 0.13). In summary, MM-compounds exhibited higher activity in cancer cell lines (median IC₅₀ = 0.35 µM) compared to normal cell lines (median IC₅₀ = 1.11 µM). We have decided to focus on HT-29 and PC-3 cells in further investigations.

Table 3 Determination of relative cell viability (IC₅₀ values) of cancer cell lines (colorectal cancer (DLD-1, HT-29, HCT-116), prostate cancer (PC-3) and pancreatic cancer (BxPC-3)) or normal cells: normal human colon tissue cells (CCD 841 CoN), human lung fibroblasts (WI-38 cells) and peripheral blood lymphocytes (PBLs), treated with MM124 and MM125 using 72-h MTT assay. The values were provided with standard deviation (± SD) [µM]. Additionally, median IC₅₀ values obtained for MM-compounds in cancer and normal cells were summarized to calculate the selectivity index (SI) (ratio of median IC₅₀ values in normal cell lines and cancer cell lines).

MM-compounds showed higher cytotoxic activity in cancer cell lines than 5-FU used as a reference chemotherapeutic drug. SI calculated as the ratio of median IC₅₀ values in normal cell lines and cancer cell lines for MM-derivatives equaled 3.17, indicating potential preference of compound’s activity in cancer cells.

MitoTracker Red – mitochondrial membrane potential changes (ΔѰm)

The change in the MMP results in the deposition of Mitotracker Red reagent in the mitochondrial matrix. The fluorescence intensity reflects the health of the mitochondria and varies with changes in MMP⁴⁵.

The fitness of the mitochondria of HT-29 and PC-3 tumor cells was assessed following 24-h treatment of cells with MM124 and MM125 in concentrations of 0.5, 1, and 2 µM. A decrease in the fluorescence intensity was observed with an increase in the compounds’ concentration (Fig. 3). In the highest concentration of the compound, MM125 induced the most pronounced decrease in MMP potential in HT-29 (% of control = 78.8 ± 3.32) and PC-3 cell line (% of control = 58 ± 0.58).

Fig. 3

Changes in the mitochondria membrane potential (MMP) after 24-h incubation with MM124 and MM125 in HT-29, and PC-3 cells. Data were presented as a percentage [%] of control cells’ fluorescence intensity ± SD value.

Dual acridine orange/ethidium bromide (AO/EB) fluorescent staining

AO/EB fluorescent staining was used to detect apoptosis and necrosis in HT-29 and PC-3 lines exposed to MM124 and MM125 compounds (Fig. 4). These cancer cell lines were chosen based on the results of MTT and Mitotracker Red assay. AO/EB staining combines the use of two fluorescent dies that allow distinguishing alive cells (acridine orange stained—green nucleus with red–orange cytoplasm); apoptotic (green irregular nuclei with chromatin condensation or fragmentation) and necrotic cells (ethidium bromide stained—orange cell nuclei) upon differential uptake of fluorescent dyes and morphology of cell and chromatin. Apoptosis was observed under a fluorescent microscope and a total of 200 cells were counted ⁴⁶.

Fig. 4

Determination of apoptosis and necrosis in HT-29 (A-B) and PC-3 (C-D) cancer cell lines treated with 0.5, 1, and 2 µM concentrations of MM124 and MM125 with AO/EB double staining following 24-h incubation of cells with tested compounds. Data were presented as mean percentage of necrotic and apoptotic cells [%] ± SD values. The differences between the experimental samples and control samples were estimated by the ANOVA test followed by post-hoc Dunnetts’s test (p < 0.05, N = 200).

MM124 and MM125 used in all three tested concentrations (0.5, 1, and 2 µM) induced statistically significant (p < 0.05) increase in the % of apoptotic cells in the HT-29 cell line. The number of apoptotic cells increased with an increase in MM124 and MM125 concentrations. MM124 exhibited stronger pro-apoptotic activity in HT-29 cells than MM125 when used in 1 and 2 µM concentrations. MM124 induced apoptosis of 27.5 ± 4.24 (p = 0.0002) and 38.5 ± 2.12 (p < 0.0001) cells after their incubation with 1 and 2 µM concentrations of compound respectively, while the slightly lower pro-apoptotic potential was observed for MM125 compound (% of apoptotic cells equaled to 23 ± 2.82 (p = 0.0007) and 32.35 ± 3.75 (p < 0.0001) for incubation of cells with 1 and 2 µM concentrations of the compound respectively) (Fig. 4A). A statistically significant (p < 0.05) increase in the necrosis prevalence was observed following 24-h incubation of HT-29 with MM124 in 2 µM (mean necrosis % = 19 ± 1.41; p = 0.0013) concentration and 0.5 (mean necrosis % = 21 ± 1.41; p = 0.0003) and 1 µM (mean necrosis % = 16.5 ± 2.12; p = 0.0133) concentration of MM125. An increase in necrotic cells was observed with the increase in MM124 concentration. No such effect was observed following incubation with MM125 (Fig. 4B).

In contrast, in the PC-3 cell line, MM124 and MM125 used in the 2 µM concentration induced a statistically significant increase in the number of apoptotic cells (the % of apoptotic cells equaled 13 ± 1.41 (p = 0.002) and 14 ± 1.41 (p = 0.0012) for MM124 and MM125 respectively) (Fig. 4C). Necrosis was the prevalent form of cell death. A statistically significant increase in the number of necrotic cells was observed following 24-h incubation of cells with tested compounds (p < 0.05) except from 0.5 µM concentration of MM125. MM124 used in the highest concentrations induced necrosis of 82 ± 2.82 cells (p < 0.0001), while MM125 exhibited lower necrotic potential (% of necrotic cells equalled to 42 ± 2.83; p < 0.0001) (Fig. 4D).

Dual propidium iodide/Hoechst 33,342(PI/HE) staining

The rationale for dual labeling with PI/HE is to exploit the contrasting staining characteristics of these dyes to differentiate between viable and dead cells (apoptotic and necrotic) and to observe cell nuclei simultaneously. Functional cells with intact membranes will internalize HE, resulting in a blue fluorescence in their nuclei. However, they will prevent the entry of PI, thus avoiding the emission of red fluorescence from their nuclei. Cells that are dead or have permeabilized membranes will absorb both HE and PI leading to the production of pink fluorescence. Through the analysis of the fluorescence released by the labeled cells using suitable excitation wavelengths, it is feasible to differentiate between living apoptotic and necrotic cells based on their distinct fluorescence characteristics⁴⁷.

In the HT-29 cell line MM124 and MM125 compounds used in all tested concentrations induced a statistically significant increase in apoptotic cell fraction following 24-h incubation time. MM124 used in 0.5, 1, and 2 µM concentrations induced apoptosis of 21.5 ± 2.12 (p = 0.0012), 32 ± 4.24 (p < 0.0001), and 39 ± 5.7% (p < 0.0001) of cells respectively, while MM125 induced apoptosis of 23.5 ± 2.12 (p = 0.0007) 25.5 ± 2.12 (p = 0.0004) and 37 ± 1.4% (p < 0.0001) of cells compared to negative control cells where apoptotic cell fraction was estimated as 0.9 ± 0.28% (Fig. 5A). At the same, MM124 used in 2 µM (mean necrosis % = 24 ± 1.4; p = 0.005) and MM125 used in 1 µM (mean necrosis % = 21 ± 1.4; p = 0.0074) and 2 µM (mean necrosis % = 26.5 ± 0.7; p < 0.0001) concentrations induced a statistically significant (p < 0.05) increase in necrosis prevalence compared to negative control (mean necrosis % = 16 ± 1.4) (Fig. 5B).

Fig. 5

Determination of apoptosis and necrosis in HT-29 (A-B) and PC-3 (C-D) cancer cell lines treated with 0.5, 1, and 2 µM concentrations of MM124 and MM125 with PI/HE double staining following 24-h incubation of cells with tested compounds. Data were presented as mean percentage of necrotic and apoptotic cells ± SD values. The differences between the experimental samples and control samples were estimated by the ANOVA test followed by post-hoc Dunnetts’s test (p < 0.05, N = 200).

In PC-3 cells, MM124 used in 0.5 µM (mean apoptosis % = 8.5 ± 2.12; p = 0,0249), 1 µM (mean apoptosis % = 9.5 ± 0.7; p = 0.0098) or 2 µM (mean apoptosis % = 13 ± 1.4; p = 0.0007) concentration and MM125 employed in 1 µM (mean apoptosis % = 9 ± 1.4; p = 0.0155) or 2 µM (mean apoptosis % = 17 ± 1.4; p < 0.0001) concentrations induced statistically significant (p < 0.05) increase in apoptotic cell fraction (Fig. 5C). Similarly to AO/EB staining, the response of prostate cancer cells was directed towards necrosis where MM124 used in 0.5 µM (mean necrosis % = 21 ± 4.24; p = 0.0148), 1 µM (mean necrosis % = 42.5 ± 3.53; p < 0.0001) and 2 µM (mean necrosis % = 64.5 ± 6.36; p < 0.0001) and MM125 used in 1 µM (mean necrosis % = 21.5 ± 3.56; p = 0.0126) and 2 µM (mean necrosis % = 51 ± 1.4; p < 0.0001) induced statistically significant increase in necrotic cell fraction compared to negative control (mean necrosis % = 5.25 ± 0.35) (Fig. 5D).

Study of reactive oxygen species (ROS) formation

Time-dependent change in total cellular ROS levels was analyzed in HT-29 and PC-3 cancer cells by using a DCFH-DA fluorescent probe (Fig. 6)^32,33. In HT-29 cells (A), the MM124 compound used in IC₅₀ and 2xIC₅₀ concentrations induced a statistically significant (p < 0.05) increase in ROS production following 2-h treatment. ROS generation increased with the rising concentration of MM-compound and exceeded levels produced in cells in response to 500 μM H₂O₂. In contrast, the MM125 compound induced an increase in ROS production when used in the 0.5xIC₅₀ and IC₅₀ concentrations. However, it has not surpassed the ROS-inducing properties of H₂O₂. In PC-3 cells (B), MM124 used only in 2xIC₅₀ concentration and MM125 used in IC₅₀ concentration induced a statistically significant increase in ROS production. Nevertheless, ROS levels generated in response to MM124 used in 2xIC₅₀ concentration exceeded the ROS formation following H₂O₂ treatment.

Fig. 6

Reactive oxygen species (ROS) generation in HT-29 (A) and PC-3 (B) cells after 2-h treatment with MM124 and MM125 at concentrations of 0.5xIC₅₀, IC₅₀, and 2xIC₅₀ concentrations and 500 μM H₂O₂ used in the experiment as a positive control. Data were presented as median relative fluorescence intensity units (RFU) with interquartile range and minimal and maximal values. The Kruskal–Wallis test was employed to show a statistically significant difference between groups. Dunn’s multiple comparisons test was used to compare mean rank differences between control and treated groups. In all groups, N = 15. Significant changes: * p < 0.05; ** p < 0.01, ***; p < 0.001; p < 0.0001.

Genotoxicity study

DNA damage following 24-h incubation of cells with tested MM-compounds was assessed using OpenComet software⁴⁸ (Fig. 7). Data were presented as % DNA in the comet tail. Mean tail DNA % results recorded for three concentrations (0.5 µM, 1 µM, and 2 µM) of MM124 and MM125 were compared to positive and negative controls. MM-compounds used in the lowest concentration (0.5 µM) did not induce a statistically significant increase in DNA damage following treatment of both tumor and normal cell lines. In the HT-29 cell line (Fig. 7A), MM124 used in 1 µM (mean = 28.43 ± 4.77; p = 0.0004) and 2 µM (mean = 29.51 ± 2.44; p = 0.0001) concentrations and MM125 used in 2 µM (mean = 24.14 ± 2.83; p = 0,0166) concentration induced significantly higher increase in DNA damage compared to negative control. However, DNA damage has not exceeded the one induced by positive control (20 µM bleomycin; mean = 33.58 ± 3.62; p < 0.0001). DNA damage induced by MM124 and MM125 in all tested concentrations was substantially higher in the PC-3 cancer cell line (Fig. 7B) compared to PBL_S (Fig. 7C). A statistically significant (p < 0.05) increase in DNA damage was observed for the 1 and 2 µM concentrations of MM124 (mean_1µM = 49.52 ± 4.28; mean_2µM = 56.5 ± 6.65; p < 0.0001) and MM125 (mean_1µM = 38.8 ± 4.36; mean_2µM = 57.99 ± 6.96; p < 0.0001) in PC3 cells compared to the negative control (Fig. 7B). The DNA damage induced by the compounds used in the highest concentration has exceeded the levels induced by 20 µM bleomycin (mean = 44.99 ± 2.37; p < 0.0001). In PBLs (Fig. 7C), MM124 used in 1 µM (mean = 13.08 ± 0.86; p < 0,0001) concentration, and MM125 used in 1 µM (mean = 15.4 ± 1.8; p = 0.0319) and 2 µM (mean = 27.76 ± 3.12; p < 0.0001) concentrations induced statistically significant increase in DNA compared to negative control. The levels of DNA damage have not exceeded the ones induced by positive control (mean = 41.33 ± 1.95; p < 0.0001). DNA damage induced by MM124 and MM125 increased with the increase in their concentration – a dose–response relationship was observed in all cell lines. Examples of comets obtained in the experiment in the HT-29 and PC-3 cell lines were shown in Figs. 8 and 9 respectively.

Fig. 7

DNA damage induced by 0.5 µM,1 µM, and 2 µM concentrations of MM124 and MM125 in cancer cell lines (HT-29 (B), PC-3 (C)) and PBLs (D). Data were represented as mean tail DNA % with ± SEM values. The differences between the experimental samples and control samples were estimated by the ANOVA test followed by post-hoc Dunnett’s test (p < 0.05, N = 200). * significant difference compared to the negative control (untreated cells).

Fig. 8

Images of comets obtained in an alkaline comet assay. Control samples (A and B), 2 µM MM125 (C and D), and 20 µM bleomycin (E and F) in the HT-29 cell line. Upper images are shown in magnification of 200X. Lower images are shown in magnification 400X.

Fig. 9

Increase in DNA damage following treatment of PC3 cells with three tested concentrations (0.5, 1, and 2 µM) of MM124 (A-C) and MM125 (D-F). Images were shown in magnification of 400X.

Electrochemical studies

The electrochemical characteristics of MM124 and MM125 were examined under physiological pH conditions, for a better understanding of the occurring DNA interaction with the chosen sulfonamide derivatives. In the preliminary experiments performed using SW voltammetry, both, oxidation and reduction of the sulfonamides were observed. Figure 10A shows the SW voltammograms of MM124 and MM125 recorded within the potential window from 0 to − 0.5 V, for the same concentrations of the compounds. As can be seen, MM124 and MM125 exhibited one reduction and one oxidation peak, both ca. − 0.265 V vs. Ag/AgCl, in the applied potential range. For both compounds, the observed reduction signals were significantly higher than those caused by compound oxidation. Sequential studies were conducted using cyclic voltammetry. Interestingly, for both compounds, only signals originating from analytes reduction were observed (see Fig. 10B) despite changes in the direction of the potential sweep. Based on the obtained results, it can be clearly stated how significantly the scan rate affects the kinetics of the electrode processes.

Fig. 10

(A) SW voltammograms of MM124 (blue lines) and MM125 (red lines) for anodic (solid lines) and cathodic (dotted lines) direction of the potential sweep, c(_MM125) = c(_MM124) = 1.0 × 10⁻⁴ mol L⁻¹; supporting electrolyte: PBS pH 7.4; (B) Cyclic voltammogram of MM124 (main plot) and MM125 (inset) recorded at various scan rates of 50 (1), 75 (2), 100 (3), 200 (4), 300 (5), 400 (6), 500 (7) mV s⁻¹, c(_MM125) = c(_MM124) = 1.0 × 10⁻⁴ mol L⁻¹; supporting electrolyte: PBS pH 7.4.

To evaluate whether the mass transfer of MM124 and MM125 toward the BDD electrode surface was controlled by adsorption or diffusion, the effect of various scan rates on chosen sulfonamide derivatives signals was investigated. The CV curves of MM124 and MM125 recorded within the potential window ranging from − 0.7 to 0.2 V at various scan rates are presented in Fig. 10B. It is worth noting that the surface of the BDD electrode, used in the present study, exhibits relatively low adsorption properties, suggesting that diffusion-controlled processes were expected⁴⁹. The linear dependence between the peak current (I_p) of both MM124 and MM125 and the square root of the scan rate (v^1/2) were determined, demonstrating that diffusion played a crucial role in the electrode reaction of chosen sulfonamides. Additionally, the plots of log I_p vs. log v yielded straight lines with slopes of 0.47 for MM124 and 0.45 for MM125, which are close to the theoretical value of 0.5⁵⁰, typically observed for diffusion characteristics of the registered currents.

The interaction of MM124 and MM125 with dsDNA was investigated using the SWV technique. The addition of dsDNA to the sulfonamides solutions significantly decreased the peak currents of both analytes (See Fig. 11). It is worth mentioning, that dsDNA was also analyzed separately in the same experimental conditions and neither anodic nor cathodic peaks were observed (data not shown). Therefore, it can be concluded that the observed decreases in the peak currents of MM124 and MM125 in the presence of dsDNA are likely due to the binding of the analytes to the large, slowly diffusing dsDNA, resulting in a significant decrease in the diffusion coefficient value.

Fig. 11

(A) SW voltammograms of MM124 (blue line) recorded with progressively increasing amounts of dsDNA (gray lines), c(_MM124) = 1.0 × 10⁻⁵ mol L⁻¹, c(_dsDNA) = 10 − 50 mg L^-1; (B) SW voltammograms of MM125 (red line) recorded with progressively increasing amounts of dsDNA (gray lines), c(_MM125) = 1.0 × 10⁻⁵ mol L⁻¹, c(_dsDNA) = 10 − 50 mg L^-1;

During the evaluation of DNA interaction with the selected sulfonamides, no notable shifts in peak potentials were detected. Since a shift in peak potential is typically associated with intercalative interactions, we can likely exclude this type of interaction between MM124 /MM125 and dsDNA. Additionally, during the voltammetric analyses, interaction studies were conducted by incubation of the analyzed compounds with dsDNA for a specified period. However, no significant differences in the obtained signals were observed as a result of the incubation of these compounds.

In summary, considering the observed significant decrease in current values of MM124 and MM125 in the presence of increasing amounts of dsDNA, it can be concluded that interactions occur between dsDNA and the selected sulfonamides. However, these studies indicate the DNA interaction other than the intercalative type typical for hydrophobic and planar compounds like the tested sulfonamides.

Computational analysis

Drug likeness and ADMET

ADMET properties and drug-likeness prediction for the designed compounds—MM124 and MM125 have revealed that both compounds follow Lipinski’s rule of five with one common violation related to the number of hydrogen bond acceptors. The obtained physicochemical properties for both the designed compounds as per Lipinski’s rule of five were tabulated in Table 4.

Table 4 Predicted molecular properties describing Lipinski’s rule of five.

The ADMET properties of both the designed compounds MM124 and MM125 related to their BBB permeability, GI absorption, hepatotoxicity, cardiotoxicity, and CYP inhibition predicted by pkCSM³⁹, SwissADME⁴⁰, and PreADMET online server were summarized in Table 5.

Table 5 Predicted ADMET properties of MM124 and MM125.

The presence of other major toxic effects like immunotoxicity, mutagenicity, etc. for both the designed molecules MM124 and MM125 were additionally predicted by using the Protox-II webserver was tabulated in Table 6.

Table 6 Toxicity profiles of MM124 and MM125 estimated using Protox-II (http://tox.charite.de/protox_II).

MM124 and MM125 compounds may exhibit low oral bioavailability due to the low GI absorption. Also, their activity in the brain compartment may be limited because of no BBB permeability and the fact that both compounds may act as P-glycoprotein substrate (P-gp) substrates. The BBB is composed of endothelial cells of capillaries that protect the brain cells from potentially toxic compounds. P-gp is an ATP-dependent drug transport protein located mostly in the apical membranes of several epithelial cell types that actively transport agents out of the cell⁵¹.

Human cytochrome CYP P450 enzymes are extensively involved in drug detoxification, cellular metabolism, and homeostasis. Members of the cytochrome CYP P450 family are responsible for almost 80% of oxidative metabolism and roughly 50% of the overall elimination of major pharmaceutical agents in humans. CYPs can modify drug responses in many ways, including their influence on drug elimination, action, safety, bioavailability, and drug resistance⁵². MM124 compound may not inhibit the activity of major drug-metabolizing CYP enzyme isoforms CYP2D6 and CYP3A4, while the effect of MM125 on CYP3A4 inhibition seems to be ambiguous.

One of the leading reasons for new drug withdrawal from a clinical setting is drug-induced hepatotoxicity which can lead to liver failure⁵³. According to the in silico predictions performed for MM124 and MM125 the toxic effect of compounds on the liver cells is unclear. According to the Swiss-ADME webserver, both compounds may exhibit hepatotoxic activity. However, the Protox-II server classifies compounds as inactive for hepatotoxicity endpoint. Nonetheless, the probability of this prediction is low (p = 0.55).

The ability of laboratories to test compounds for mutagenicity was substantially improved after Bruce Ames created and published a bacterial strain and a mutagenicity assay in 1973. The now-famous “Ames Test” required little training, could be completed in two days, and demanded little equipment could be quickly and reliably replicated in different labs. The Ames test is still widely used in commercial chemical testing, despite the discovery of other methods to evaluate the mutational properties of compounds. Investigated in this study MM-compounds were found to lack the mutagenic properties by all the softwares/servers used in the in silico predictions⁵⁴.

Cardiotoxicity constitutes an important side-effect associated with the used of many commonly used drugs. In the majority of cases, it results from the interaction with the potassium ion channel of the human ether-a-go-go-related gene (hERG). The inhibition of hERG causes long QT syndrome (LQTS), a potentially fatal heart condition. The elimination of potentially harmful drug candidates through the use of a virtual screening method to anticipate drug-induced hERG-related cardiotoxicity is important for the drug discovery process⁵⁵.

The investigation revealed the effect of MM124 and MM125 compounds on hERG inhibition is ambiguous. Among other toxicological endpoints, it is important to evaluate the toxic effect of compounds on the immune system. Immunotoxicity of the drugs is a leading cause of morbidity and mortality in patients undergoing treatments. The most common immunotoxic consequence of medicines include hypersensitivity (allergic and anaphylactic) reactions⁵⁶. According to the in silico predictions MM124 and MM125 with high probability may not exert immunotoxic effects.

Molecular docking

The map files for various atom types of the macromolecular target as well as ligands generated by the Autogrid utility were used in the Autodock software to perform molecular docking simulations. The docking protocols for the concerned macromolecular targets used in the current study were successfully validated by considering the binding energy, overlay conformation, and chemical resemblance based on the observed chemical interactions of the docked conformation of the reference ligand ellipticine with reference to its bioactive conformation. The overlaid conformation (a) and chemical interactions (b) of the docked conformation of the reference ligand ellipitiicne was represented in Fig. 12.

Fig. 12

Overlaid conformation (a) and chemical interactions (b) of the docked conformation of the reference ligand ellipiticine.

After validation of molecular docking, we have employed the above-stated parameters to run the simulation studies of MM124 and MM125 chiral sulfonamides, and the observed docking results were provided in Table 7.

Table 7 Binding energy (kcal/mol) obtained after performing a docking analysis of both the designed molecules (MM124, MM125) and the standard inhibitor ellipticine complexed in the studied PDB structure (PDBid:1Z3F).

The two-dimensional binding interactions and three-dimensional binding conformation of MM124 and MM125 with DNA were represented in Fig. 13 and Fig. 14 respectively.

Fig. 13

Two-dimensional binding interactions and three-dimensional binding conformation of MM124 with DNA molecule.

Fig. 14

Two-dimensional binding interactions and three-dimensional binding conformation of MM125 with DNA molecule.

Based on the observed binding energy and type of the interacting residues it can be concluded that compounds exhibit a similar binding pattern as ellipticine used in our study as a reference compound.

Molecular dynamics simulation

The molecular dynamics (MD) simulation of the ligand MM125 in complex with DNA revealed several key insights into its flexibility, stability, and dynamic behavior. MM125 was found to possess eight flexible bonds involving 30 heavy atoms out of a total of 54 atoms. The flexibility of individual atoms or residues in the ligand or macromolecular structure was assessed through the root-mean-square fluctuation (RMSF). RMSF is a critical parameter that provides insights into the dynamic behavior and relative flexibility of the macromolecular complex. The RMSF values for the ligand during the simulation ranged from 0.5 to 3.5 Å, indicating relative flexibility within the binding site. The structural stability of the ligand within the DNA binding cavity was analyzed using the root-mean-square deviation (RMSD). During the simulation period, the ligand exhibited RMSD values within the range of 1.5–2.4 Å, demonstrating minimal fluctuations. The overall RMSD analysis confirmed that the MM125-DNA complex maintained a high degree of thermodynamic stability with only minor deviations from the initial position throughout the simulation. The radius of gyration (rGyr) was calculated to evaluate the compactness and conformational stability of the complexed ligand. The rGyr values for MM125 complexed with DNA were observed within the range of 4.0–5.0 Å, further confirming good conformational stability during the simulation. Overall, the MD simulation of the MM125-DNA complex over 100 ns concluded that the ligand displayed stable binding within the target DNA’s active site, with limited fluctuations and consistent conformational behavior. The RMSD (1.5–2.4 Å), RMSF (0.5–3.5 Å), and rGyr (4.0–5.0 Å) collectively highlight the robust interaction and stability of the complex. The RMSD (a), rGyr (b), and RMSF (c) for MM125 complexed with DNA throughout the 100 ns MD simulation was represented in Fig. 15.

Fig. 15

The root-mean-square deviation (RMSD) (a), radius of gyration (rGyr) (b) and root-mean-square fluctuation (RMSF) (c) for MM125 complexed with DNA throughout the 100 ns MD simulation.

In summary, docking analysis of MM124, and MM125 compounds against 6-BP DNA molecule to predict their intercalation property has revealed that both the compounds MM125 and MM124 are showing good binding affinity against the DNA similar to that of the reported standard intercalating ligand ellipticine. Although both the concerned compounds MM125 and MM124 are showing worse binding interactions with DNA when compared with the reference compound ellipticine. MD simulation of the compound MM125 has revealed that the ligand is sufficiently stabilized within the macromolecular cavity of DNA to impart the biological effect.

Discussion

Common methods of cancer treatment include radiotherapy, gene therapy, hormone therapy, immunotherapy, and surgery. Until now, the most popular therapeutic option is chemotherapy, based on the use of cytostatic drugs that cause damage to the genetic material of cancer cells. Due to the mechanism of action, cytostatics are divided into (a) alkylating agents that inhibit the ability of cells to replicate DNA (cyclophosphamide, cisplatin, carboplatin); (b) antimetabolites which disrupt the biosynthesis of nucleic acids (5-FU), gemcitabine, 6-mercaptopurine); (c) plant alkaloids, including topoisomerase inhibitors (topotecan, irinotecan) and taxols, causing abnormal mitotic spindle formation (docetaxel, paclitaxel), (d) anti-cancer antibiotics that disrupt the double-helix sugar-phosphate backbone (mitomycin C and other bleomycin)^57,58. The treatment process is selected individually and may involve both the combination of various therapeutic methods and various cytostatic agents. The effectiveness of multi-drug programs is based mainly on the synergistic effect of substances that surpass the effects of monotherapy⁵⁹.

The most common cause of chemotherapy failure is the emergence and development of multidrug resistance, defined as increased insensitivity of cancer cells to the therapeutic agents. This phenomenon may have a genetic basis or be associated with an increased outflow of chemotherapeutic agents, intensification of repair processes in cancer cells, deregulation of apoptotic pathways, or reduced affinity for drugs^58,60. Attempts to overcome drug resistance focus on understanding the biological basis of multidrug resistance development and the use of combination therapies with low-toxicity pharmaceuticals targeting imperfections in cellular mechanisms. The complicated nature and heterogeneity of neoplastic diseases are the main reasons for the growing need to search for new oncotherapeutic agents with selective action and safety profiles. Identification of substances that increase the effectiveness of existing drugs, or allow to bypass of the multidrug resistance phenomenon, brings hope to patients with the worst prognosis⁵⁸.

Heterocyclic compounds, especially those with nitrogen atoms are the cornerstone of pharmaceutical agents, antibiotics, and agrochemicals. Approximately 75% of FDA-approved drugs contain a nitrogen heterocycle, underscoring their importance in medicinal chemistry. Among these, 1,2,4-triazine derivatives are particularly noteworthy given their anticancer potential. For instance, avapritinib, a kinase inhibitor of platelet-derived growth factor receptor α (PDGFRα), was approved in 2020 for treating gastrointestinal stromal tumors. Its efficacy has prompted further clinical trials for treating other advanced solid tumors, such as lung, breast, melanoma, and sarcoma⁶¹.

A promising strategy in chemotherapeutic development involves combining fragments of known drugs into a single molecule to enhance biological activity. The natural pyrazolo[4,3-e][1,2,4]triazine system, when combined with pharmacophore groups, offers opportunities to design new compounds with potential anticancer properties^24,62. Studies have shown that pyrazolo[4,3-e][1,2,4]triazine derivatives exhibit moderate antitumor activity. For instance, aza-sildenafil analogs and aniline-substituted pyrazolo[4,3-e][1,2,4]triazine sulfonamides have demonstrated effectiveness against cancer cells. Modifying the pyrazolo[4,3-e][1,2,4]triazine structure by incorporating tetrazole or triazole rings has also been shown to increase cytotoxic activity against cancer cell lines. These modifications highlight the potential of structural adjustments in enhancing the efficacy of pyrazolo[4,3-e][1,2,4]triazine derivatives as recently reviewed⁷.

The combination of pyrazolo[4,3-e][1,2,4]triazine with tetrazole ring, sulfonamide moiety, and leucinol represents a promising chemical structure in cancer treatment. Incorporating leucinol in the chemical structure of the compound can offer several advantages for the development of potential drug-like molecules. First of all, the lipophilic side chain of leucinol can affect membrane permeability, helping the compound enter cells more efficiently. Leucinol can also mimic leucine residues in biological systems and can be used in peptidomimetic design. This allows interaction with amino acid-binding sites on enzymes, transporters, or receptors. Furthermore, cancer cells often overexpress L-type amino acid transporters (LAT1, etc.) to support rapid growth. Compounds incorporating amino acid-like moieties (like leucinol) may exploit these transporters for selective uptake by cancer cells. Adding leucinol can also impact water solubility, reduce metabolic degradation, or enhance plasma stability depending on the context making it useful for successful drug design^{63,64,65,66,67,68}.

The chirality of drug molecules can significantly influence their pharmacological activity. Research has demonstrated that different enantiomers of a compound can exhibit varying degrees of therapeutic efficacy and side effects. In studies on pyrazolo[4,3-e][1,2,4]triazine derivatives, it was observed that R-enantiomers generally exhibit slightly higher cytotoxic activity compared to their S-isomers⁶¹. Therefore, we aimed to investigated two enantiomeric pyrazolo[4,3-e][1,2,4]triazine leucinol derivatives MM124 (R-enantiomer) and MM125 (S-enantiomer).

The present study confirmed high, micromolar cytotoxicity (median IC₅₀ = 0.35 µM) of the MM124 and MM125 compounds towards cancer cell lines of different origins including colorectal (DLD-1, HCT-116, HT-29), pancreatic (BxPC-3) and prostate (PC-3) cancer with minor effect on normal human colon tissue cells (CCD 841 CoN), fibroblasts (WI-38) and peripheral blood lymphocytes (PBLs) in vitro. Although MM124 (R-enantiomer) exhibited slightly greater cytotoxic potency compared to MM125 (S-enantiomer), the difference was not sufficient to establish a clear structure–activity relationship. Importantly, both MM-compounds outperformed the standard chemotherapeutic agent 5-fluorouracil (5-FU) in terms of overall cytotoxic efficacy. However, 5-FU displayed superior selectivity toward malignant cells over normal cells.

It was also found that tested compounds induced oxidative stress and apoptosis of colorectal (HT-29) and prostate (PC-3) cancer cells as evidenced by the loss of MMP and features characteristic for apoptosis evidenced by dual fluorescent staining techniques. Nevertheless, the response of PC-3 cells seemed directed more to the necrotic type of cell death, which may lead to undesirable side effects. This is consistent with our previous studies where other pyrazolo[4,3-e][1,2,4]triazine sulfonamide compounds fused with tetrazole ring also induced necrotic response in PC-3 cells rather than apoptotic response compared to pancreatic BxPC-3. This may indicate the complex response of cancer cells to MM-compounds²⁴.

We have also found that the genotoxic effect exhibited by the compounds may be the direct consequence of the ongoing cell death process or may be the underlying cause of the high cytotoxicity exhibited by the compounds. This observation is also consistent with our previous findings for other compounds of the same class^23,69 and is common for other anti-cancer agents used in clinical practice like bleomycin used in our genotoxicity study as a positive control^70,71,72. Furthermore, MM124 and MM125 used in the highest concentrations (2 μM) induced similar levels of DNA damage compared to bleomycin used at 10 times higher concentration (20 μM). However, only MM124 used in 1 μM concentration induced a statistically significant increase in DNA damage in both cancer cell lines (HT-29 and PC-3) but not normal cells (PBLs).

For the first time, we have evaluated the DNA interacting potential of pyrazolo[4,3-e][1,2,4]triazine which could potentially indicate the underlying basis of their genotoxic properties. Evaluating the type of occurring interaction using electrochemical techniques should be generally supplemented with other methods, as these techniques only allow for the formulation of a hypothesis based on the observed shift in peak potential. Here, the absence of shifts in the peak potentials suggests that intercalative type of interactions likely did not occur. Therefore, groove binding may be suggested as more prevalent.

We have also performed in silico analysis of the compounds consisting of the ADMET properties prediction, molecular docking, and dynamics with DNA molecule to explore the binding mode of the compounds and the stability of the formed complexes. We have found that the compounds exhibit drug-likeness properties, do not show CYP inhibitory properties, and have favorable toxicity features, however, their use may be restricted by the low GI absorption. Docking and dynamics studies suggest the binding mode of the compounds as intercalative type. This could be attributed to the highly planar and hydrophobic nature of pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine core. However, distinct observations from the electrochemical and in silico studies need to be supplemented with other experimental approaches as described by other authors^73,74,75.

Our investigation complements the exploration of anti-cancer properties of pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides (MM-compounds series) designed and synthesized by Mojzych et al. that were extensively evaluated by several groups as recently described in our previous works^7,76. The existing data on the cytotoxicity of the compounds was provided in Table 8. All the compounds in the series are characterized by the common pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine scaffold connected to specific amide heterocyclic moiety (R; see Table 8) by the phenylsulfonyl linker^24,69.

Table 8 Results of the MTT assay for MM-compounds documented in the literature. The cytotoxic properties of compounds were documented across various cancer cell lines, including BxPC-3 (pancreatic adenocarcinoma), PC-3 (prostate cancer), HeLa (cervical cancer), and DLD1/HCT116/HT29 (colorectal adenocarcinoma), as well as normal cells such as human lung fibroblasts (WI-38) and human peripheral blood mononuclear cells (PBMCs). The data were acquired following a 72-hour incubation of the cells with investigated sulfonamides (or 24 hours as denoted by the (#) symbol). N/D – not-determined; * - not-published data. Based on⁷⁶

The data presented in Table 8 indicate that the incorporation of specific amide heterocyclic moieties does not appear to significantly influence the cytotoxic properties of the compounds in vitro. However, this structural modification profoundly impacts the physicochemical characteristics of the compounds, including ADMET profiles. These properties are challenging to fully evaluate through in vitro assays alone, necessitating further investigation through in vivo methods. The comparative evaluation of compound activity in cancerous versus normal cells is important for the assessment of the potential selectivity of chemical entities. Although the compounds exhibit only moderate selectivity in vitro, this differentiation is a critical starting point for therapeutic development. Notably, preclinical studies on the advanced derivative MM129 provided promising evidence that this class of heterocycles may possess anticancer efficacy in vivo, with an acceptable tolerability profile²².

Recent studies have highlighted the potential for MM-derivatives to function synergistically with established anticancer agents. For example, combinatorial regimens involving MM- compounds with 5-FU⁷⁷ or the kynurenine pathway inhibitor—indoximod⁷⁸ have shown potential to enhance their therapeutic efficacy. This suggests that these compounds could be integrated into multidrug strategies to overcome resistance mechanisms and improve clinical outcomes. Future work should focus on further delineating the pharmacodynamics and pharmacokinetics of MM-derivatives, exploring their effects on tumor microenvironment, and validating their efficacy in diverse cancer models. Such studies are crucial to advancing these compounds toward clinical translation and understanding their full therapeutic potential.

Conclusions

In summary, the field of heterocyclic chemistry continues to evolve, offering numerous opportunities for novel drug discovery. Advances in synthesis methods and modern organic chemistry techniques have accelerated the development of new heterocyclic compounds with diverse biological activities^79,80,81,82. Recent research has identified various pyrazolo-triazine derivatives with strong anticancer activity, particularly tricyclic pyrazolo[4,3-e][1,2,4]triazines fused with triazole or tetrazole rings^{7,12,14,16,22,23,24,26,61,69}. Investigated here enantiomeric derivatives of pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine incorporating leucinol (MM124 and MM125) exhibited comparable anticancer activity and similar cytotoxic properties to previously reported series of compounds. This indicates that incorporation of specific moieties with the phenylsulfonyl linker does not impact the biological activity of the pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine core. Nevertheless, both MM-compounds exhibited potent and selective cytotoxic activity against a panel of cancer cell lines derived from various tissue origins, with a median IC₅₀ value of 0.35 µM. Mechanistic studies demonstrated that MM124 and MM125 induce apoptosis, promote oxidative stress, and cause DNA damage of prostate and colorectal cancer cells. Electrochemical analyses and molecular simulations further supported the ability of these compounds to interact with DNA. Additionally, in silico pharmacokinetic and toxicological evaluations indicated favorable drug-like properties, highlighting their potential for further development as anticancer agents.