Synthesis and Preliminary Anticancer Activity Assessment of N-Glycosides of 2-Amino-1,3,4-thiadiazoles

The addition of 2-amino-1,3,4-thiadiazole derivatives with parallel iodination of differently protected glycals has been achieved using a double molar excess of molecular iodine under mild conditions. The corresponding thiadiazole derivatives of N-glycosides were obtained in good yields and anomeric selectivity. The usage of iodine as a catalyst makes this method easy, inexpensive, and successfully useable in reactions with sugars. Thiadiazole derivatives were tested in a panel of three tumor cell lines, MCF-7, HCT116, and HeLa. These compounds initiated biological response in investigated tumor models in a different rate. The MCF-7 is resistant to the tested compounds, and the cytometry assay indicated low increase in cell numbers in the sub- G1 phase. The most sensitive are HCT-116 and HeLa cells. The thiadiazole derivatives have a pro-apoptotic effect on HCT-116 cells. In the case of the HeLa cells, an increase in the number of cells in the sub-G1- phase and the induction of apoptosis was observed.

It is well known that derivatives of 1,3,4-thiadiazoles exhibit anti-tumor activity against human cancer cells (MCF-7 (breast cancer), SMMC-7721 (human hepatocarcinoma), HL-60 (human leukemia), A549 (non-small cell lung carcinoma), Hep-G2 (human liver hepatocellular carcinoma), PC-3 (human prostate adenocarcinoma), and HCT-116 (human Priebe et al. [30] reported the synthesis of new iodo-hexose compounds and their preliminary antitumor application for the treatment of glioblastoma and pancreatic cancer, which showed better anti-tumor activity in contrast to their deoxy-analogs. On the other hand, glycals are versatile building blocks in carbohydrate synthetic chemistry and have been extensively investigated for the synthesis of 2-deoxy-O-, S-, N-glycosides. The method for entry into 2-deoxy glycosides has been discussed in several reviews [31][32][33][34][35].
Continuing this study, we present the synthesis and biological activity of conjugates possessing different 2-deoxy-2-iodomonosaccharide scaffolds joined with thiadiazole derivatives.

Synthesis
In the present work, the synthesis of aminoglycosides in the reaction of glycals with 2-amino-1,3,4-thiadiazole derivatives has been studied. The preliminary antitumor activity of obtained conjugates was determined. The targeted conjugates were obtained by the addition of the 2-amino-1,3,4-thiadiazole derivative (2) to an unsaturated sugar (1) in the presence of iodine (Scheme 1). Scheme 1. Synthesis of glycoconjugates, derivatives of 1,2,4-thiadiazole.
The glycosylation of the glycal derivatives was performed in the dark at room temperature. The main products formed by the addition of iodine (Scheme 1) to the double bond of 3,4-di-O-acetyl-L-rhamnal (1a), 3,4-di-O-benzyl-L-rhamnal (1d), 3,4,6-tri-O-acetyl-D-glucal (1b), and 3,4,6-tri-O-acetyl-D-galactal (1c), followed by a substitution reaction, were isolated by column chromatography. The structures of the obtained compounds were established based on the analysis of their NMR spectra. The signals were identified using literature data [36,37]. Additionally, in the 1 H NMR spectra, the following were diagnostic: chemical shifts of acetyl groups, hydrogen at the anomeric position, hydrogens at C-6 position for rhamnopyranose derivatives, and hydrogen at C-2. In the 13 C NMR spectrum were the C-1, C-2, and S-C=N carbon chemical shifts.
To determine the reaction conditions, we performed preliminary experiments presented in: Table 1. compounds were established based on the analysis of their NMR spectra. The signals were identified using literature data [36,37]. Additionally, in the 1 H NMR spectra, the following were diagnostic: chemical shifts of acetyl groups, hydrogen at the anomeric position, hydrogens at C-6 position for rhamnopyranose derivatives, and hydrogen at C-2. In the 13 C NMR spectrum were the C-1, C-2, and S-C=N carbon chemical shifts.
To determine the reaction conditions, we performed preliminary experiments presented in Table 1. When the 3,4-di-O-acetyl-L-rhamnal (1a) was treated with 2-amino-5-(4-tert-butylphenyl)-1,3,4-thiadiazole (2a) in different solvents (Entry a-c) and in the absence of iodine, no substrate conversion was observed. In the second set of experiments, the reaction was carried out in the presence of 1 eq. of iodine in the mixture of solvents with different polarities (Entry d-f). Under applied conditions, only traces of the product were detected. The third set of experiments was carried out in THF in the presence of a different concentration of iodine, namely, 0.5, 1.0, and 2.0 eq (Entry g-i). These results indicated that the reaction occurred successfully in the presence of double molar excess of molecular iodine. To determine which of the iodoamination methods would be the most beneficial, an experiment with NIS was also performed (Entry j), this reactant is recommended as conventional in synthesis 2-deoxy-2-halogenosugars starting from glycals [36]. The presence of an additional product in a very high concentration prompted us to abandon this method. Other methods of addition using heavy metals as catalysts are also known [38]. Unfortunately, the residue of those metals in the tested sample could adversely affect the survival of cells and the results of biological tests, so we decided to give up these methods as well.
In a series of experiments, we carried out reactions of 3,4-di-O-acetyl-L-rhamnal (1a), 3,4,6-tri-O-acetyl-D-glucal (1b), 3,4,6-tri-O-acetyl-D-galactal (1c), and 3,4-di-O-benzyl-Lrhamnal (1d) with 2-amino-5-phenyl-1,3,4-thiadiazole derivatives (2a-2d) containing different substituents in the aromatic ring in anhydrous THF containing molecular iodine. As a result of these experiments, hitherto unknown derivatives 3a-3m were obtained. The results are presented in Table 2. When the 3,4-di-O-acetyl-L-rhamnal (1a) was treated with 2-amino-5-(4-tert-butylphenyl)-1,3,4-thiadiazole (2a) in different solvents (Entry a-c) and in the absence of iodine, no substrate conversion was observed. In the second set of experiments, the reaction was carried out in the presence of 1 eq. of iodine in the mixture of solvents with different polarities (Entry d-f). Under applied conditions, only traces of the product were detected. The third set of experiments was carried out in THF in the presence of a different concentration of iodine, namely, 0.5, 1.0, and 2.0 eq (Entry g-i). These results indicated that the reaction occurred successfully in the presence of double molar excess of molecular iodine. To determine which of the iodoamination methods would be the most beneficial, an experiment with NIS was also performed (Entry j), this reactant is recommended as conventional in synthesis 2-deoxy-2-halogenosugars starting from glycals [36]. The presence of an additional product in a very high concentration prompted us to abandon this method. Other methods of addition using heavy metals as catalysts are also known [38]. Unfortunately, the residue of those metals in the tested sample could adversely affect the survival of cells and the results of biological tests, so we decided to give up these methods as well.
The electrophilic addition of halogens to cyclic enol ethers has been investigated earlier by Lemieux and Fraser-Reid [39], who proposed a general mechanism involving the initial formation of carbenium ions which, upon nucleophilic attack by halide ion, give mainly the products of thermodynamic control. Later on, it has been shown that the product formation is under kinetic control and that the stereoselectivity depends on the solvent polarity [7,9,10], the structure of the enol ether, and the nature of the halogen [16,[40][41][42].
To improve the stereoselectivity of glycosylation reactions involving glycals, directing groups such as Cl, Br, and I are introduced. Most commonly, a directing group is temporarily introduced at C-2, and the acceptor is then incorporated at C-1. An example is NISor NBS-mediated glycosylation developed by Thiem et al. These protocols have found widespread use in the construction of complex amine-containing N-glycosides [39,43]. The addition of halogen to double bonds is expected to occur by a bimolecular process, with the approach of a halogen molecule perpendicularly to the system of the vinyl ether from both site of a molecule plane, and the compound formed may rearrange in the rate-limiting step to intermediate ions (ion pairs, open carbocation, or carboxonium ion) which, according to their relative reactivities, control the stereochemical course of the reaction (Scheme 2).

Scheme 2.
Addition of iodine to the double bond of glycals followed glycosylation.
Unsaturated pyranoid rings are conformationally more flexible than saturated ones. Therefore, even insignificant changes to the spatial disposition of substituents attached to the ring may give rise to changes in the 4 H 5 , 5 H 4 conformational equilibrium of glycals. The factor affecting the conformational equilibrium of glycals is the 1,3-diaxial interactions destabilizes the 5 H 4 form of D-glycals with the C-3 and C-5 substituents in the cis orientation [44].
Compounds 1a exist mainly in the 5 H 4 conformation (Scheme 2), where all of the substituents have quasiequatorial orientations. In the 4 H 5 , the acetoxyl group at C-4 is in the quasiaxial disposition, and this destabilizes this structure.
The stereoselectivity of reaction depends on the structure of protecting groups. In the reaction of 3,4-di-O-benzyl-L-rhamnal (1d) a mixture of 1,2-trans L-gluco and L-manno glycosides was formed, suggesting that the addition of iodine proceeds through the stabilized carbo-oxonium ion (Scheme 3). It is proposed that the stereoselectivity of the reaction is based on a Curtin-Hammett kinetic scheme involving the in situ generation of the more reactive glycosyl halide [58].
The 1,2-trans diiodie is formed by preferential attack of Ion the oxocarbenium ion and the next activation in reaction with iodine takes place. The isomerization of iodides is a fast reaction, and an equilibrium mixture of α,β-iodides is formed [59].
The 1,2-cis glycoside is formed by attack of the nucleophile on the more reactive 1,2-trans-di-iodide via an SN2-like substitution (Scheme 3) and gives 1,2-cis glycoside as the main product of the reaction. The 1,2-trans isomer can alternatively be received by the competitive formation of 2-iodo-α-glycosyl iodide, which could then give a N-glycoside in direct SN2 substitution with the amine. On the other hand, the 1,2-trans glycoside may be obtained in the reaction of amines with carbooxonium ion.  [59], was compound having the α-Dmanno configuration. This result may be explained by the formation of bridged iodonium ions that induce the attack of iodide from the opposite side (trans opening), affording the production of the trans configuration (β-gluco iodide). The fast equilibration leads to the mixture of α/β iodides, and the nucleophilic substitution of the anomeric carbon atom by attack of 4-chloro or 5-tertbutyl substituted derivatives is controlled by steric effects. The preferred reaction by equatorial attack leads to cis β-manno glycoside formation [60].
The results of galactal 1c are opposite to the results presented above. The main products are 1,2-cis glycosides. The overall results were very close to those for the bromination of tri-O-acetyl-D-galactal, indicating that electronic effects rather than purely steric effects of the C-6 substituent are responsible for the product distribution during the bromination of glycals [15][16][17], where galactals with electron-withdrawing substituents at C-6 gavẽ 90% of the trans addition products [16]. The approach of iodine from above the molecular plane leads to the β-galacto isomer and is energetically more favorable than when the attack is from below the plane of the molecule. Due to steric reasons, the S N 2 substitution of I 3 led to the α-D-galacto isomer.     The results from the MTT assay for MCF-7 cells did not allow the IC 50 calculation. However, in the case of two compounds (3c and 3j), the SF of the cells decreased significantly at the highest doses ( Figure 2).
Based on the results obtained for the HCT116 cell line, we concluded that this cell line was more sensitive to the tested compounds than the MCF-7 cell line, as can be seen in Figure 3.
In the case of HeLa cells, the effect of the compounds on the SF value was visible for most of the compounds, but it was not as strong as in the case of HCT116 cells (Figure 4).

Cytostatic Effects on Cancer Cell Lines followed by Cell Cycle
Although the MTT assay did not deliver clear results for the cytotoxicity of the tested compounds, the effect of lowered viabilities may have been caused by the cell cycle arrest. For a better interpretation of the possible effects, we decided to perform flow cytometry analysis for the highest doses of the compounds (100 µM). The typical histograms of DNA content in cells are presented in Supplementary materials ( Figure S1).
In the case of MCF-7 cells, some of the tested compounds (3a and 3c) influenced the cell cycle distribution and increased the number of cells in the sub-G1 phase, which corresponds to the dead cells ( Figure 5). Furthermore, we observed a visible cell cycle arrest in the G0/G1 phase after the exposition of the cells on the compounds 3f, 3g, and 3h. Figure 5. Cell cycle in MCF-7 cells after a 72h incubation with compounds with the concentration of 100 µM. The results were presented as mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant in comparison to the untreated controls (indicated with a star).
Cells responded with more visible cytostatic and cytotoxic effects (Figure 6). For a panel of 3a, 3b, 3c, and 3m compounds, the number of cells in the sub-G1 phase increased. Additionally, the lowering of the number of cells in the S phase and cell cycle arrest in the G2/M phase indicated the DNA damage with inhibition of the replication process.
In the case of the HeLa cell line, we observed a significant increase in the number of cells in the sub-G1 phase, which suggests lethal effects of the tested compounds on this cell line (Figure 7).
We decided to further investigate the type of cell death for each cell line with Annexin V with PI assay, which allows to distinguish necrosis from apoptosis. The results were presented as mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant in comparison to the untreated controls Table 3 (indicated with a star).  of 3a-3c, 3f-3h, 3j, 3l and 3m    The results were presented as mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant in comparison to the untreated controls (indicated with a star).

Death Mechanism Induced in Cancer Lines
To conclude the cell death mechanism, we performed Annexin V with PI assay after a 72 h incubation of all the cell lines with the analyzed compounds in the concentration of 100 µM. The typical dot plots from Annexin-V apoptosis assay from cells are presented in Supplementary material ( Figure S2).
Based on the results, for the MCF-7 cell line (Figure 8), we concluded that the tested compounds did not exhibit any pro-apoptotic effects. The results were presented as mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant in comparison to the untreated controls (indicated with a star).
In contrast to the MCF-7, the HCT116 cells ( Figure 9)responded with apoptosis. It indicated that the analyzed compounds exhibit pro-apoptotic activity against this cell line. Similarly to the HCT116 cells, the tested compounds have exhibited a pro-apoptotic effect on the HeLa cell line.
However, in most of the cases, the effect was much stronger (Figure 10). Figure 10. Apoptosis in HeLa after a 72h incubation with compounds with the concentration of 100 µM. The results were presented as mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant in comparison to the untreated controls (indicated with a star).
The efficient formation of glycosidic linkages has played an important role in the development of modern synthetic carbohydrate chemistry. Among the various types of donors that can be employed for the construction of glycosidic bonds, glycosyl iodides should now be viewed as versatile glycosylation reagents [61,62].
The research found the ineffectiveness of the conventional methods for the preparation of N-glycosides of 2-deoxy monosaccharides in the nucleophilic substitution reaction with the participation of very weakly nucleophilic amine thiadiazole derivatives. The publication presents an efficient method for glycosylation starting from glycals, with the activation leading to the glycosyl donor taking place with 2eq of iodine, preferably in a polar solvent such as THF. It has been shown that by the appropriate optimization of conditions, it is possible to obtain highly stereoselective products of rhamnal and glucal. The advantage of this method is the use of an easily available, cheap, and environmentally friendly reagent, molecular iodine.
The biological activity of the tested compounds was determined based on the MTT assay complemented with flow cytometry analysis. The combination of these methods allows for the detection of cytotoxic, cytostatic, and lethal effects of the compounds on the analyzed cell lines.
The performed MTT assay indicated that the effect of the compounds depended significantly on the tested cell line (Table 3). Among the tested cell lines, MCF-7 cells were the most resistant and HCT116 cells were the most sensitive to the tested compounds. Unfortunately, the compounds exhibited low biological activity and hence neither the IC 50 parameter nor the selectiveness of the compounds against the tested cell lines could be evaluated for the examined doses. Nevertheless, based on the SF values, we observed that the highest concentrations of the compounds (100 µM) inhibited the cells proliferation. Because of it, we decided to test the effect of the compounds on the cell cycle only for the highest concentrations.
According to the biological assays, in the case of MCF-7 cells, despite the low cytotoxic effect of the compounds, we did not observe any cytostatic or pro-apoptotic effect. During the cell cycle analysis, we detected a slight increase in the number of cells present in the sub-G1 phase or an increase in cells arrested in the G0/G1 phase. Furthermore, there was no increase in apoptotic nor necrotic cells. Nevertheless, the detected effect was not significant when compared to the untreated control.
More promising effects were detected in the case of other cell lines. The cell line, which was the most sensitive in the MTT assay (HCT116) responded with both cytostatic and cytotoxic effects. During the cell cycle analysis, we observed an increase in the number of cells in the sub-G1 fractions, however, the strongest effect was visible in the inhibition of the S phase and the arrest of the cells in the G2/M phase. Additionally, we detected an increase in cells in the early apoptosis, which suggests that the compounds have pro-apoptotic activity against the HCT116 cell line.
The most auspicious effects were observed for the HeLa cells. Similarly to the HCT116 cell line, the compounds caused cytotoxic and cytostatic effects on this cell line. Moreover, in the case of the HeLa cells, we detected an increase in the number of cells in the sub-G1 phase. Furthermore, Annexin V staining proved that the cells underwent both apoptotic cell deaths.
Based on the carried out experiments we noticed a tissue-dependent effect of the tested compounds and we concluded that they exhibit a cytotoxic effect on the MCF-7, HCT116, and HeLa cell lines, a cytostatic effect on the HCT116 and HeLa cell lines, and a lethal effect on the HeLa cell line.
Our results indicate that the tested compounds in their current form cannot be exploited as drugs, however, they stand as promising models for further modification. In order to improve the antitumor activity of the conjugates, structural modifications are necessary. Especially interesting is applying in biological tests deprotected conjugates, it should affect the solubility of these derivatives in the applied medium. Another approach is the synthesis of the derivatives where a sugar moiety is linked to a thiadiazole derivative via an aliphatic, aromatic-aliphatic, or aromatic linker.

Chemistry
General. All reagents were purchased from Merck (Darmstadt, Germany), Acros Organics (Geel, Belgium), Alpha Aesar (Haverhill, USA), and Sigma-Aldrich (Taufkirchen, Germany) and used without further purification. A column chromatography was per-formed on a silica gel packed column (Kieselgel 60 0.040-0.063 mm, 230-400 mesh, Merck, Darmstadt, Germany). Solvents for chromatography (n-hexane, ethyl acetate, methanol, dichloromethane) were distilled before use. Thin layer chromatography was performed on plates coated with silica gel 60 F 254 (Merck, Darmstadt, Germany); detection was carried out with either ultraviolet light (254 nm) or spraying with a solution of phosphomolybdic acid, a basic potassium permanganate solution, or ethanolic solution of concentrated H 2 SO 4 , with subsequent heating. Optical rotations were measured with a JASCO P-2000 polarimeter using a sodium lamp (589.3 nm) at room temperature. Melting points were measured on a Stuart SMP3 melting point apparatus. The 1 H NMR and 13 C NMR spectra were recorded on an Agilent 400 MHz spectrometer and Varian 600 MHz spectrometer in DMSO-d 6 or CDCl 3 using tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported as δ values (ppm). FT-IR spectra were recorded between 4000 and 650 cm −1 using an FT-IR Nicolet 6700 apparatus (IET, Mundelein, USA) with a Smart iTR accessory. HRMS spectra were recorded on a Waters ACQUITY UPLC/Xevo G2QT instrument.
General procedure for iodination of glycals: To a solution of glycal (1a-d: 1.0 eq) in THF (2 mL), 2-amino-1,3,4-thiadiazole derivative (2a-d: 2.0 eq) and I 2 (2.0 eq) were added. The reaction was stirred at room temperature for 1.5h. The TLC (Toluene:EtOAc, 2:1 v/v) indicated total consumption of glycal. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (gradient from toluene to toluene:EtOAc, 10:1 v/v) to give products as a pale yellow oil being a mixture of anomers. medium. After centrifugation (1500 rpm, 5 min.), the cells were resuspended in a fresh culture medium and counted using an automatic cell counter-Juli Stage (NanoEntek, Waltham, MA, USA).

Cytotoxicity Test by MTT Assay
For the cytotoxicity determination of the tested derivatives, the cells in the exponential growth phase were trypsinized and seeded on 96-well plates (Nunc, ThermoFisher, Waltham, MA, USA) at concentration 5 × 10 4 cells/well/200 µL in completed grown medium. After approx. 24 h the culture medium was replaced with a fresh culture medium containing serial dilutions of the tested drugs. After 72 h, a solution of the compound was removed. Next, cell viability was determined by the MTT assay based on mitochondrial dehydrogenase enzyme activity. The basis of the method is the reaction of the reduction of tetrazolium salts to colored formazan carried out by dehydrogenase in living cells. Therefore, the amount of formazan formed is proportional to the number of functional cells. MTT assay was carried out according to the protocol (MTT, Sigma-Adrich, Taufkirchen, Germany) [63,64]. To each well on the plate, 50 µL of MTT solution (0.05 mg/mL in phenol red and FBS free DMEM-F12; PAA) was added. The plates were then placed in a CO 2 incubator for 1 to 2 h. After this time, MTT solution was removed, and formazan crystals were dissolved in acidic isopropanol. Next, spectrophotometrically absorbance measuring at 570 nm, using a multi-well plate reader SYNERGY4 (BioTek Instruments, Winooski, VT, USA) was performed. The fraction of viable cells was determined with concern to not treat with any factor control cells, according to the following formula: (1)

SF-survival fraction;
A-the absorbance value; A K -the absorbance value for control cells.

Cell Cycle and Apoptosis Analysis
The exponentially growing cells were harvested by trypsinization and seeded on a 6-well plate (Nunc, ThermoFisher, Waltham, MA, USA) in DMEM/F12 medium at concentrations of 1 × 10 5 cells/well/2 mL. After 72 h, the growth medium was exchanged for a fresh medium in control groups or medium containing the tested drugs at chosen concentrations. The cells were incubated for 72 h. After this time, the medium was collected and the cells were trypsinized with 1 mL trypsin/EDTA (Sigma-Aldrich, Taufkirchen, Germany). Trypsin was then neutralized by adding 2 mL of culture medium. The cells were centrifuged at 1300 rpm for 5 min at 4 • C, washed twice with 1 mL PBS, and finally fixed with 1 mL of ice-cold 70% ethanol. The samples were stored at −20 • C until analysis. Directly before the analysis by flow cytometer (Becton Dickinson Aria III, BD Company, San Diego, CA, USA), cells were centrifuged at 1300 rpm for 3 min, washed with 500 µL PBS, and centrifuged again. Then, cells were resuspended in 50 µL PBS and 50 µL of RNAse solution (Sigma-Aldrich, Taufkirchen, Germany) at a concentration of 100 µg/mL (in PBS). After 15 min of incubation at 37 • C, cells were stained with 250 µL propidium iodide solution (PI, Sigma-Aldrich, Taufkirchen, Germany) at a concentration of 100 µg/mL. Fluorescence was measured using a flow cytometer with the PE configuration (547 nm excitation laser line; emission: 585 nm). The samples were vortexed before analysis [65,66].
For apoptosis assay, the cells after collection were stained directly using FITC conjugated primary Annexin-V antibody (BioLegend, San Diego, USA) in binding buffer for 20 min in darkness at 37 • C and Annexin with PI solution 100 µg/mL (Sigma-Aldrich, Taufkirchen, Germany). Fluorescence was measured using a flow cytometer with the PE configuration (547 nm excitation laser line; emission: 585 nm) for necrotic and late apoptotic cell counting. For apoptotic cells, the FITC channel configuration was used (488 nm excitation laser line; emission: LP mirror 503, BP filter 530/30). The samples were vortexed before analysis [65,66].

Statistical Analysis
At least three replicates were performed for every kind of experiment. The results were presented as the mean value ± SD. The statistical analysis was based on a t-test, and a p-value less than 0.05 was considered statistically significant.
Supplementary Materials: The Supporting Information is available online. 1 H and 13 C NMR spectra of all obtained compounds, Figure S1: Typical histograms of PI stained DNA content during the cell cycle of MCF-7, HCT116, and HeLa cells, after 72 h of incubation with compounds at a dose of 100 µM; Figure S2:Typical dot plots of normal, early apoptotic, late apoptotic and necrotic MCF-7, HCT116, and HeLa cells after 72 h of incubation with compounds at a dose of 100 µM; General procedure for the synthesis of 2-benzoylhydrazinecarbothioamide derivatives (6).