Glycoconjugates of Mucochloric Acid—Synthesis and Biological Activity

The pharmacological effects of the presence of a sugar moiety, 1,2,3-triazole ring and silyl groups in the structure of biologically active compounds have been extensively studied in drug design and medicinal chemistry. These components can be useful tools to tailoring the bioavailability of target molecules. Herein we present the study on the impact of the sugar substituent structure and triisopropylsilyl group presence on the anticancer activity of mucochloric acid (MCA) derivatives containing the furan-2(5H)-one or 2H-pyrrol-2-one core. The obtained results clearly indicated that tested compounds caused a significant decrease in cell viability of HCT116 and MCF-7 cell lines. MCF-7 cells indicate serious resistance toward investigated compounds in comparison with HCT116 cell line, it suggests that estrogen-dependent breast cancer cells are significantly less sensitive to the tested derivatives. Depending on the structure of the sugar, the type and site of connection with the furanone or 2H-pyrrol-2-one derivative and the presence of the silyl group, the selectivity of the compound towards cancer cells can be controlled. The obtained results may have an impact on the design of new furanone-based anticancer compounds.


Introduction
The furanone scaffold is found in many complex natural products and exhibits diverse biological properties, such as antibacterial [1][2][3][4], anticancer [5][6][7][8][9][10], antifungal [11,12], antiviral [13], anti-inflammatory [14][15][16][17] and antioxidant [15][16][17][18] (Figure 1). In recent years, several derivatives, such as 3,4-dihalogeno-furan-2(5H)-one derivatives, have been obtained, which exhibited various anticancer activities [19,20]. Molecular targets for these derivatives were such key enzymes as kinases, COX-1, topoisomerase I or MDM2-p53 interaction. As a result of the reductive amination reaction of 3,4-dihalogeno-furan-2(5H)-one derivatives in the presence of various amines, highly functionalized α,β-unsaturated αhalogeno-β-aryl-γ-butyrolactams were obtained [21]. These compounds have been shown to exhibit cytotoxic effects [9] and increase the sensitivity of tumor cells to oncogenic viruses [22]. Additionally, as we have shown in our previous work [19], the introduction of a silyl group into the structure of 3,4-dihalogeno-furan-2(5H)-one increases the cytotoxicity of the tested compound with reference to the initial structure. Carbohydrates and their glycoconjugates are involved in many biological processes and play an important role in various diseases. The structure of carbohydrates, including the presence of functional groups, allows numerous structural modifications leading to creation of new compounds with biological activity as well as enhancing the activity of existing drugs. A good example of this strategy are carbohydrate-based metallo-drugs such as platinum carbohydrate complexes [23]. Glycosylation of peptides can protect against proteolysis and improve aqueous solubility. Sugar residues are most often attached to biologically active compounds in order to increase their bioactivity, improve their physical and chemical properties or target their action [24]. Many of them are subjected to further modifications in order to obtain better activity and selectivity compared to the parent structure. Carbohydrate-based drugs have e.g., antibacterial [25], antidiabetic [26,27], anticancer and antiviral effects, and act as enzyme inhibitors [28][29][30][31].

Synthesis
Derivatives of 3,4-dichloro-furan-2(5H)-one and 2H-pyrrol-2-one were joined to appropriate sugar derivative via O-CH2-CH2-CH2-1,2,3-triazole linker or 1,2,3-triazole ring, which was built directly on anomeric carbon atom (Scheme 1). 1,2,3-Triazole is an important heterocycle in medicinal chemistry, often use as a convenient coupler joining two parts into final product. It is formed via 1,3-dipolar addition of appropriate azido derivatives (1,3-dipole) with corresponding propargyl fragment of 1,3-dipolarophile. It occurs in many compounds with anticancer activity, inhibiting various enzymes such as EGFR [33][34][35], VEGFR [36] and PARP [37]. Carbohydrates and their glycoconjugates are involved in many biological processes and play an important role in various diseases. The structure of carbohydrates, including the presence of functional groups, allows numerous structural modifications leading to creation of new compounds with biological activity as well as enhancing the activity of existing drugs. A good example of this strategy are carbohydrate-based metallo-drugs such as platinum carbohydrate complexes [23]. Glycosylation of peptides can protect against proteolysis and improve aqueous solubility. Sugar residues are most often attached to biologically active compounds in order to increase their bioactivity, improve their physical and chemical properties or target their action [24]. Many of them are subjected to further modifications in order to obtain better activity and selectivity compared to the parent structure. Carbohydrate-based drugs have e.g., antibacterial [25], antidiabetic [26,27], anticancer and antiviral effects, and act as enzyme inhibitors [28][29][30][31].

Cytotoxicity and Anticancer Activities
The 72 h in vitro MTT assay, performed on HCT116 and MCF-7 cancer cell lines, allowed the assessment of cell viability and IC 50 parameter for tested compounds. IC 50 for cell viability was described with a dose of the drug, with 50% reduction in the whole population when compared to the untreated controls. Obtained results allow for comparison of different compounds using the same or different targeted cell lines. The calculated IC 50 parameters for tested compounds are presented in Table 2. As positive control, MCA was used with IC 50 value determined on the same cell lines in previous research [20]. Cells survival fractions (SF) followed by 72 h MTT viability assay are presented in Supplementary File S1.  Figure S1 (Supplementary File), MCA-mucochloric acid, a IC 50 values from previous studies [20]. chloro-5-hydroxy-2H-pyrrol-2-one 8 reacted with triisopropylsilyl chloride in the presence of imidazole and trimethylamine to obtain product 9 in 71% yield [42,43].

Cytotoxicity and Anticancer Activities
The 72 h in vitro MTT assay, performed on HCT116 and MCF-7 cancer cell lines, allowed the assessment of cell viability and IC50 parameter for tested compounds. IC50 for cell viability was described with a dose of the drug, with 50% reduction in the whole population when compared to the untreated controls. Obtained results allow for comparison of different compounds using the same or different targeted cell lines. The calculated IC50 parameters for tested compounds are presented in Table 2. As positive control, MCA was used with IC50 value determined on the same cell lines in previous research [20]. Cells survival fractions (SF) followed by 72 h MTT viability assay are presented in Supplemen- Based on the obtained results of the cytotoxicity, chemical modifications did not always improve the drug activity against cancer cell lines. The compounds 10, 11, 15, 17, 19, 20 and 23 were not active at all, or reduced viability of only one of used cell lines, HCT116 or MCF-7, respectively. Such results may be explained by the modification in the compound structure, e.g., the presence of a sugar residue may facilitate the introduction of the compound into the cell, and the presence of a 1,2,3-triazole ring may increase cytotoxicity. Compound 12 showed high cytotoxicity against both colorectal and breast cancer cell lines, with the IC 50 9.1 ± 2.4 and 10.8 ± 0.7 µM, respectively. Other tested compounds exhibit moderate toxicity, but at doses over 10 µM.
Analysing the relationship between compound structure and activity against selected cancer cell lines, a significant improvement in the cytotoxicity was observed when the molecule contains 2,3-unsaturated sugar fragment (10, 11, 12, 25 and 26). The smallest effect is observed in the case of derivatives containing L-fucose in their structure (19, 20 and 21). Making a further comparison of the obtained derivatives, it can be concluded that an attachment of the sugar moiety through the nitrogen atom in the furan-2(5H)-one ring has a negligible effect on increasing cytotoxic properties. Moreover, the pattern of the sugar rest addition by the nitrogen/oxygen atom and the distance from the furan-2(5H)one ring were not significant for improving the cytotoxic activity of the tested derivatives. However, it is noticeable that the compounds containing a silyl substituent in their structure (12, 18, 21, 24 and 26) significantly inhibit the proliferation of cancer cells. More detailed cytotoxic effects are presented as particular HCT116 and MCF cells viability graphs in Supplementary File ( Figure S1).

Inhibition of Cell Cycle and Pro-Apoptotic Action
The mechanism of action of anticancer drugs may be based on cell cycle inhibition (cytostatic action), inducing apoptosis, cellular programmed death or rapid necrosis. To confirm the mechanism of action of the tested derivatives in cancer cells, a cycle using flow cytometry techniques was applied. The amount of DNA in a cell is variable and dependent on single stage of the cycle. In eukaryotic cells, there are two main phases, i.e., interphase and mitosis, in which the amount of nuclear DNA content is single (in human diploid cells 2n, which means a set of 46 chromosomes), or doubled after replication (S phase), in which mitosis begins (tetraploid cells 4n, with 46 chromosomes doubled into 92 sister chromatids) [44]. DNA staining with specific nuclear dyes such as propidium iodide (PI) allowed an easily counting and distinguishing between cells with a different DNA-nucleus contents. Using flow cytometry and appropriate analysis gating, the cell cycle phases can be matched: subG1 (apoptotic and necrotic cells); G0/G1 phase (2n diploid cells, also called mononuclear cells); S phase (replication with DNA synthesis; DNA content more than 2n); G2/M phase (4n tetraploids cells, with doubled DNA content in nucleus). The cells with disturbed and uncontrolled replication and damaged mitosis present polyploid fraction (DNA contend above 4n). Figure 2 shows histograms of typical DNA content in HCT116 and MCF-7 control cells, after 72 h of incubation.
The obtained results of the cell cycle analysis confirm the cytotoxic effects for most of the tested derivatives (Figures 3 and 4), while their divergence for both cell lines may indicate tissue-dependent effects presented on HCT116 or MCF-7 cells, respectively. Based on the graphs, it can be seen that for both HCT116 and MCF-7 cell lines, an impact on subG1 phase elevation can be observed compared to untreated control (Figures 3 and 4). An increase in the subG1 phase may indicate activation of programmed cell death, i.e., apoptosis and/or uncontrolled and rapid necrosis. Such aim of action confirms potentially pro-apoptotic effect of the tested compounds against HCT116 cancer cell line after addition followed by slight increase only for a few compounds, as confirmed by the cytotoxicity assay ( Figure 4). The subG1 fraction increased after the addition of 11, 12, 13, 15, 17, 18, 20, 21, 23, 25 and 26 ( Figure 4); however, the effect on MCF-7 cell line was not so spectacular as on HCT116 cell line. Decreased G0/G1 fraction in most of the treatments on MCF-7 cells was the result of apoptotic fraction formation rather than cytostatic cell cycle blockade.
interphase and mitosis, in which the amount of nuclear DNA content is single (in h diploid cells 2n, which means a set of 46 chromosomes), or doubled after replicat phase), in which mitosis begins (tetraploid cells 4n, with 46 chromosomes doubled i sister chromatids) [44]. DNA staining with specific nuclear dyes such as propidium i (PI) allowed an easily counting and distinguishing between cells with a different D nucleus contents. Using flow cytometry and appropriate analysis gating, the cell phases can be matched: subG1 (apoptotic and necrotic cells); G0/G1 phase (2n d cells, also called mononuclear cells); S phase (replication with DNA synthesis; DNA tent more than 2n); G2/M phase (4n tetraploids cells, with doubled DNA content cleus). The cells with disturbed and uncontrolled replication and damaged mitosis p polyploid fraction (DNA contend above 4n). Figure 2 shows histograms of typical DNA content in HCT116 and MCF-7 c cells, after 72 h of incubation. The obtained results of the cell cycle analysis confirm the cytotoxic effects for m the tested derivatives (Figures 3 and 4), while their divergence for both cell lines m dicate tissue-dependent effects presented on HCT116 or MCF-7 cells, respectively. on the graphs, it can be seen that for both HCT116 and MCF-7 cell lines, an imp subG1 phase elevation can be observed compared to untreated control (           Table 2. The subG1 fraction represents apoptotic and dead cells. Results presented as mean of 3 experiments ± SD. Statistical significance indicated by star; evaluated by T-test, where p < 0.05.

Conclusions
Glycoconjugate derivatives of 3,4-dichloro-furan-2(5H)-one and 2H-pyrrol-2-one were obtained and their in vitro biological activity was tested. The click chemistry approach in a simple, easy and inexpensive way leads to products with high yield and purity, which does not deteriorate the enantiomeric purity of the starting compounds.
The obtained results clearly indicated that tested compounds caused a significant decrease in cell viability of HCT116 and MCF-7 cell lines. When comparing the results for selected cell lines, it can be seen that the IC 50 values on the MCF-7 cell line are much higher or show not activity at all in comparison to the HCT116 cell line, indicating that estrogendependent breast cancer cells are significantly less sensitive to the tested derivatives.
Analysing the structures of the tested compounds in terms of the structure of the sugar molecule, among the 3,4-dichloro-furan-2(5H)-one derivatives, the glucose derivative was the most cytotoxic compound, while among the 2H-pyrrole-2-one derivatives, the 4-O-acetyl-2,3,6-trideoxy-L-erythro-hex-2-eno pyranoside proved to be the most cytotoxic. These differences may be due to interactions with other molecular targets in the cell. It was also observed that increasing the distance of the sugar unit from the 1,2,3-triazole ring decreases the IC 50 value of the tested compounds. The best IC 50 results were obtained for derivatives 12, 21 and 26 containing a silyl substituent in their structure, which may result from an increase in the lipophilicity of the compounds, and thus improve their bioavailability.
Performed investigation delivers a preliminary assessment of the anticancer effect of furanone derivatives and are a start point for further research focused on optimization of the structure and determination of the molecular target.

MTT Cytotoxicity Assay
Collected after trypsinization, HCT116 and MCF-7 cells were counted under a Bürker chamber, and 2000 or 5000 cells were seeded, respectively, on each well of a sterile 96-well format plate (Sarstedt, Nümbrecht, Germany), in 100 µL of complete DMEM-F12 medium. For 24 h before compound addition, the cells were monitored directly on plates, and only well-attached cells were used for treatments with background controls (DMSO with a final maximum concentration of 1%) and untreated controls. Different proliferation rate of both cell lines discriminates the number of cells seeded into wells, where doubling time for HCT116 cells is 14-16 h, and for MCF-7 cells it is about 24-26 h (in-house experimental observations); such a procedure allowed for prolonged, up to 72 h lifetime observations, eliminating the contact inhibition and false cytostatic effects in vitro. To treated wells, 100 µL of tested compounds in fresh medium were added at a concentration range: 100, 50, 25, 12.5, 6.25, 3.13 µM (0 µM for control, untreated cells in DMEM-F12 without addition of tested compounds). After 72 h of incubation, the viability assay was performed according to the producer protocol (Promega), where the medium over the compounds was removed and cells were washed with PBS solution. A yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (0.5 mg/mL) in serum and phenol red free DMEM (Merck, Darmstadt, Germany) in volume of 50 µL was added to each well on plates for 2-3 h in dark, 37 • C incubations. The purple formazan crystals were produced only by the viable cells, containing NAD(P)H-dependent oxidoreductase enzymes that reduce MTT. The crystals were dissolved in acidic (0.04 M HCl) 2-propanolol solution (Merck, Darmstadt, Germany) after addition of 75 µL of dissolving mixture to each well on plates, for 5-10 min in darkness, at room temperature (r.t.) incubation. The absorption at 570 nm was measured directly using multiplate reader (Epoch, BioTek, Janki, Poland). Each time the biological experiments were repeated three times.

Cell Cycle and Cytometry Analysis of Apoptosis
For apoptotic dead cell and cell cycle estimation, cell cultures were plated in 6-well plates at a confluence of 3 × 10 5 cells in 2 mL of complete fresh DMEM-F12 medium. After 24 h, the medium was replaced with prepared sample solutions, at doses of calculated from MTT assay IC 50 values, respectively, for further long-term 72 h incubations. After incubation, the collected cells were centrifuged, and the pellet was dissolved in 250 µL of hypotonic buffer (hypotonic buffer comprised: PI 100 µg/mL in PBS (BD Biosciences, San Jose, CA, USA); 5 mg/L of citric acid; 1:9 Triton-X solution; RNase 100 µg/mL in PBS (Merck, Darmstadt, Germany)). The samples were incubated for 15 min at room temperature and in darkness. The cellular DNA contents were determined by fluorescence measurements using BD FACS Aria™ III sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) using a PE configuration (547 nm excitation laser line; emission: 585 nm).
At least 1 × 10 4 cells were analyzed for each sample, recording the DNA content as a differentiating parameter for mononuclear cells (G0/G1 phase); S (DNA replication phase); G2/M (binucleate and mitotic fraction) or dead, necrotic and apoptotic cells (sub-G1 fraction), respectively. The results were analyzed using the free software FlowingSoftware 2.5.1 (Perttu Terho, Turku Centre for Biotechnology, University of Turku, Finland) and are presented as mean fluorescence.

Statistical Analysis
Obtained absorbance was analysed to assess proliferation and survival fraction (SF), where tested samples were compared with the untreated controls (100%). Using Excel (Microsoft Office 365, A3 for faculty, access date: 2020), an IC 50 index was calculated for tested compounds/drugs, where used concentration reduced viability of treated populations by 50% in comparison to the untreated controls. The results from MTT and cell cycle were presented as mean from three experiments, and ± SD was added. The statistical significance was calculated with T-test and indicated by a star on the charts (p value < 0.05).