Synthesis and Cytotoxic Activity of New 1,3,4-Thiadiazole Thioglycosides and 1,2,3-Triazolyl-1,3,4-Thiadiazole N-glycosides

New 1,3,4-thiadiazole thioglycosides linked to substituted arylidine systems were synthesized via glycosylation of the prepared 1,3,4-thiadiazole thiol compounds. Click strategy was also used for the synthesis of new 1,3,4-thiadiazole and 1,2,3-triazole hybrid glycosides by reaction of the acetylenic derivatives with different glycosyl azids followed by deacetylation process. The cytotoxic activities of the prepared compounds were studied against HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. The results showed that the key thiadiazolethione compounds 2 and 3, the triazole glycosides linked to p-methoxyarylidine derivatives 14 and 15 in addition to the free hydroxyl glycoside 20 were found potent in activity comparable to the reference drug doxorubicin against MCF-7 human cancer cells. The acetylenic derivative 2 and glycoside 20 were also found highly active against HCT-116 cell lines.


Introduction
The enhanced global encumbrance of cancer in the last decades devoted the extensive research to the discovery of novel selective potent candidates with promising ability either to destroy cancer cells or limit their proliferation. Click chemistry is now considered as one of the most efficient strategies providing rapid and mild routes for novel bioorganic compounds such as anticancer candidates. The strategy involves the Cu (I)-catalyzed 1,3-dipolar cycloaddition of an azide and alkyne leading, with outstanding selectivity, to the formation of functionalized 1,4-disubstitued 1,2,3-triazole under mild conditions with high yields [1]. The formed products via click cycloaddition possess the 1,2,3-triazole core which is well known to be incorporated in a large number of compounds reported by their important pharmacological properties [2,3]. Triazole-modified analogs possessing significant anticancer activity have been synthesized by means of an azide-alkyne click chemistry strategy [4]. In addition to its importance as a basic motif of numerous nucleosides analogs featured with antiviral, as anti-HIV, or cytostatic activities [5][6][7], the triazole nucleus was also revealed with potential anticancer activity [8][9][10]. The interesting structural characteristics involving the superb plain immutability to metabolic transformation, aromaticity nature, as well as the high dipole moment and H-bonding ability of the triazole core, prompted its potential usefulness as a connecting group [11,12]. Consequently, such ring motif could be a useful linker for joining two different kinds of functionalities with biological Thiadiazole ring system, being featured as a structural bio-isostere of pyrimidine and oxadiazole, has shown numerous biological activities including anticancer, antifungal, antiviral, antidiabetic, analgesic, antieplileptic, antibacterial, and anti-inflammatory activities [16][17][18][19][20]. The enhanced liposolubility imparted by the sulfur atom of the thiadiazole core in addition to the mesoionic nature allowed compounds incorporating such motif to interact with biological targets with distinct affinities after penetration across cellular membranes. Recent studies displayed the ability of 1,3,4-thiadiazole compounds to inhibit a variety of molecular targets, including kinases which allowed their activity against different cancer cell lines presenting them as a promising scaffold for antitumor drug discovery [16][17][18][19][20][21][22][23][24][25].
On the other hand, nucleoside analogues have been recognized as active drug candidates for the treatment of different cancer forms. A number of nucleoside analogues such as Cytarabine, Azacitidine, Capecitabine, Floxuridine, and Decitabine are indeed ratified with antitumor activity [26]. Figure 1 displays a number of reported highly active 1,3,4-thiadiazole and triazole glycosides (I, III, and IV) and the triazole-thiadiazole hybrid II against different cancer cells [7][8][9]27,28]. Such significances and our interest in synthesizing new active glycosyl heterocycles [29][30][31][32][33], enhanced our foresight that the 1,3,4-thiazoles and triazoles motif hybrid compounds and their sugar linked products could be useful systems for designing novel cytotoxic agents. In the current study, new thioglycosides of arylamino-1,3,4-thiadiazole and glycosyl-1,3,4-thiadiazole-triazole conjugates were synthesized and studied for their cytotoxic activity against human colorectal carcinoma and breast adenocarcinoma cell lines. Thiadiazole ring system, being featured as a structural bio-isostere of pyrimidine and oxadiazole, has shown numerous biological activities including anticancer, antifungal, antiviral, antidiabetic, analgesic, antieplileptic, antibacterial, and anti-inflammatory activities [16][17][18][19][20]. The enhanced liposolubility imparted by the sulfur atom of the thiadiazole core in addition to the mesoionic nature allowed compounds incorporating such motif to interact with biological targets with distinct affinities after penetration across cellular membranes. Recent studies displayed the ability of 1,3,4-thiadiazole compounds to inhibit a variety of molecular targets, including kinases which allowed their activity against different cancer cell lines presenting them as a promising scaffold for antitumor drug discovery [16][17][18][19][20][21][22][23][24][25].

Chemistry
On the other hand, nucleoside analogues have been recognized as active drug candidates for the treatment of different cancer forms. A number of nucleoside analogues such as Cytarabine, Azacitidine, Capecitabine, Floxuridine, and Decitabine are indeed ratified with antitumor activity [26]. Figure 1 displays a number of reported highly active 1,3,4-thiadiazole and triazole glycosides (I, III, and IV) and the triazole-thiadiazole hybrid II against different cancer cells [7][8][9]27,28]. Such significances and our interest in synthesizing new active glycosyl heterocycles [29][30][31][32][33], enhanced our foresight that the 1,3,4-thiazoles and triazoles motif hybrid compounds and their sugar linked products could be useful systems for designing novel cytotoxic agents. In the current study, new thioglycosides of arylamino-1,3,4-thiadiazole and glycosyl-1,3,4-thiadiazole-triazole conjugates were synthesized and studied for their cytotoxic activity against human colorectal carcinoma and breast adenocarcinoma cell lines.

Chemistry
In the current study, two, structurally, related glycosyl heterocyclic systems incorporating different glycol-linkages were synthesized via synthetic strategies starting with available simple molecules. The functionalized 1,3,4-thiadiazole thione bases 2 and 3, which are linked to arylidine systems, were obtained via reaction of aromatic aldehydes; namely p-methoxybenzaldehyde or p-N-dimethylaminobenazaldehyde with 3-amino-1,3,4-thiadiazole-5-thiol in acidic ethanolic solution. The obtained products were reacted with the acetylated bromosugar, tetraacetyl-d-galactopyranosylor triacetyl-d-xylopyranosyl bromide to afford the corresponding thioglycosides 4-7, respectively. The particular 1 H-NMR spectra revealed the signal attributed to anomeric hydrogen of the glycosyl unit as doublet at δ 5.20-5.55 ppm, exhibiting coupling constants in the range 9.4-9.8 Hz indicating the β-type orientation of the thioglycosidic attachment. For the compounds possessing β-N-glycosidic part near to the C=S group, the anomeric proton has been reported to be exist [34][35][36] at higher chemical shifts (δ 6.5-7.2 ppm). Such relatively raised δ values in such type of structures are explained by the anisotropic deshielding influence of the C=S. The thio-linkage and β-conformation in the thioglycosides 4-7 were also confirmed by the assigned signal at δ 85.1-86.5 ppm for the anomeric C-1 in the 13 C-NMR spectra in addition to the absence of the well-characteristic signal corresponding to the C=S group which accounts for the attachment of the glycosyl part at the sulfur atom and the regioselectivity of the glycosylation reaction. The derived thioglycosides with unprotected OH groups 8-11 were obtained by deacetylation of the acetylated 4-7, respectively, by the treatment with methanolic ammonia solution (Scheme 1). The IR spectra of the deacetylated thio-glycoside 8-11 showed the absorption bands which were attributed to the hydroxyl groups in the sugar unit and disappearance of the characteristic C=O of the acetyl bands which was also confirmed by the corresponding 1 H-NMR spectra which did not possess the methyl proton signals and instead displayed the hydroxyl peaks. In the current study, two, structurally, related glycosyl heterocyclic systems incorporating different glycol-linkages were synthesized via synthetic strategies starting with available simple molecules. The functionalized 1,3,4-thiadiazole thione bases 2 and 3, which are linked to arylidine systems, were obtained via reaction of aromatic aldehydes; namely p-methoxybenzaldehyde or p-Ndimethylaminobenazaldehyde with 3-amino-1,3,4-thiadiazole-5-thiol in acidic ethanolic solution. The obtained products were reacted with the acetylated bromosugar, tetraacetyl-D-galactopyranosylor triacetyl-D-xylopyranosyl bromide to afford the corresponding thioglycosides 4-7, respectively. The particular 1 H-NMR spectra revealed the signal attributed to anomeric hydrogen of the glycosyl unit as doublet at δ 5.20-5.55 ppm, exhibiting coupling constants in the range 9.4-9.8 Hz indicating the β-type orientation of the thioglycosidic attachment. For the compounds possessing β-N-glycosidic part near to the C=S group, the anomeric proton has been reported to be exist [34][35][36] at higher chemical shifts (δ 6.5-7.2 ppm). Such relatively raised δ values in such type of structures are explained by the anisotropic deshielding influence of the C=S. The thio-linkage and β-conformation in the thioglycosides 4-7 were also confirmed by the assigned signal at δ 85.1-86.5 ppm for the anomeric C-1 in the 13 C-NMR spectra in addition to the absence of the well-characteristic signal corresponding to the C=S group which accounts for the attachment of the glycosyl part at the sulfur atom and the regioselectivity of the glycosylation reaction. The derived thioglycosides with unprotected OH groups 8-11 were obtained by deacetylation of the acetylated 4-7, respectively, by the treatment with methanolic ammonia solution (Scheme 1). The IR spectra of the deacetylated thio-glycoside 8-11 showed the absorption bands which were attributed to the hydroxyl groups in the sugar unit and disappearance of the characteristic C=O of the acetyl bands which was also confirmed by the corresponding 1 H-NMR spectra which did not possess the methyl proton signals and instead displayed the hydroxyl peaks. In order to add a 1,2,3-trizole core as an additional ring system to the previous arylidine thiadiazole compounds, click 1,3-dipolar cycloaddition strategy was applied for synthesizing the target compounds. The necessary terminal acetylenic active center was formed by reaction of the thioles 2 and 3 with propargyl bromide in a basic medium. The produced terminal acetylenic Scheme 1. Synthesis of thiadiazole glycosides based arylidine system.
In order to add a 1,2,3-trizole core as an additional ring system to the previous arylidine thiadiazole compounds, click 1,3-dipolar cycloaddition strategy was applied for synthesizing the target compounds. The necessary terminal acetylenic active center was formed by reaction of the thioles 2 and 3 with propargyl bromide in a basic medium. The produced terminal acetylenic compounds 12 and 13 were reacted with glycopyranosyl azides namely; 2,3,4,6-tetra-O-acetyl-d-gluco-or xylopyranosyl azides by means of Cu(I) catalysis in 1,3-dipolar cycloaddition reactions by applying click conditions, to give the targeted 1,2,3-triazole-N-glycoides, in 73-76% yields. In the latter reactions, sodium ascorbate and copper sulfate pentahydrate coupled system was utilized for generation of necessary Cu(I) species by the in-situ reduction of copper (II) salt. THF/water (2:1) mixed solvent system was found to be the most efficient solvent system resulting, regioselectively, in highest yields of the desired click products 14-17. The 1 H-NMR spectra of the N 1 -glycosyl-1,2,3-triazolyl linked to thiadiazole system showed the corresponding signals assigned for the glycopyranosyl protons including the protons of the CH 3 C=O and signals of the remaining protons in the sugar part. The revealed J values corresponding to the anomeric hydrogen (H-1) at 9.6-10.2 Hz also confirmed the β-N-glycosidic linkage mode of the glycopyranosyl ring to the triazole motif. The afforded acetylated 1,2,3-triazole glycosides 14-17 were deacetylated by using a saturated ammonia/methanol solution which led to the formation of the derived 1,2,3-triazole-N-glycosides 18-21, respectively (Scheme 2). The absence of the carbonyl band of CH 3 C=O in the assigned infra-red spectra and appearance of the particular absorptions corresponding to -OH groups evidenced the deacetylation process. Furthermore, their 1 H-NMR spectra revealed the signals assigned to the hydroxyl protons and, in addition, disappearance of the signals of the acetyl groups of the protected precursors which is in complete stratification with the structures of the resulted free hydroxy glycosides.
Molecules 2019, 24, 3738 4 of 14 compounds 12 and 13 were reacted with glycopyranosyl azides namely; 2,3,4,6-tetra-O-acetyl-Dgluco-or xylopyranosyl azides by means of Cu(I) catalysis in 1,3-dipolar cycloaddition reactions by applying click conditions, to give the targeted 1,2,3-triazole-N-glycoides, in 73-76% yields. In the latter reactions, sodium ascorbate and copper sulfate pentahydrate coupled system was utilized for generation of necessary Cu(I) species by the in-situ reduction of copper (II) salt. THF/water (2:1) mixed solvent system was found to be the most efficient solvent system resulting, regioselectively, in highest yields of the desired click products 14-17. The 1 H-NMR spectra of the N 1 -glycosyl-1,2,3triazolyl linked to thiadiazole system showed the corresponding signals assigned for the glycopyranosyl protons including the protons of the CH3C=O and signals of the remaining protons in the sugar part. The revealed J values corresponding to the anomeric hydrogen (H-1) at 9.6-10.2 Hz also confirmed the β-N-glycosidic linkage mode of the glycopyranosyl ring to the triazole motif. The afforded acetylated 1,2,3-triazole glycosides 14-17 were deacetylated by using a saturated ammonia/methanol solution which led to the formation of the derived 1,2,3-triazole-N-glycosides 18-21, respectively (Scheme 2). The absence of the carbonyl band of CH3C=O in the assigned infra-red spectra and appearance of the particular absorptions corresponding to -OH groups evidenced the deacetylation process. Furthermore, their 1 H-NMR spectra revealed the signals assigned to the hydroxyl protons and, in addition, disappearance of the signals of the acetyl groups of the protected precursors which is in complete stratification with the structures of the resulted free hydroxy glycosides.

Cytotoxic Activity
The synthesized compounds were examined in vitro for their activities on HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. The percentages of intact cells were calculated and compared to those of the control. Activities of tested Scheme 2. Synthesis of thiadiazolyl-1,2,3-triazole glycosides.

Cytotoxic Activity
The synthesized compounds were examined in vitro for their activities on HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. The percentages of intact cells were calculated and compared to those of the control. Activities of tested compounds against the two cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the two human cells in an insignificant dose-dependent manner as shown in Figures 2 and 3. In order to study the efficacy of the synthesized compounds, a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follow. In case of HCT-116 human colorectal carcinoma cells, Figure 2, Table 1, shows that compounds 2 and 20 had equipotent activities. Furthermore, the tested compounds 10, 14, 3, 15, and 12, respectively, had slightly lower cytotoxic activities than that of doxorubicin. The rest of the tested compounds had significantly less cytotoxic activities when compared to that of the reference drug. compounds against the two cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the two human cells in an insignificant dose-dependent manner as shown in Figures 2 and 3. In order to study the efficacy of the synthesized compounds, a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follow.
In case of HCT-116 human colorectal carcinoma cells, Figure 2, Table 1, shows that compounds 2 and 20 had equipotent activities. Furthermore, the tested compounds 10, 14, 3, 15, and 12, respectively, had slightly lower cytotoxic activities than that of doxorubicin. The rest of the tested compounds had significantly less cytotoxic activities when compared to that of the reference drug.    compounds against the two cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the two human cells in an insignificant dose-dependent manner as shown in Figures 2 and 3. In order to study the efficacy of the synthesized compounds, a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follow.
In case of HCT-116 human colorectal carcinoma cells, Figure 2, Table 1, shows that compounds 2 and 20 had equipotent activities. Furthermore, the tested compounds 10, 14, 3, 15, and 12, respectively, had slightly lower cytotoxic activities than that of doxorubicin. The rest of the tested compounds had significantly less cytotoxic activities when compared to that of the reference drug.     The results of cytotoxic activities investigation against MCF-7 human breast cancer cells showed that compounds 3, 2, 20, and 14, respectively, had significantly more potent cytotoxic activities. Furthermore, compounds 15, 16, 21, and 10 had comparable activities to that of doxorubicin, whereas compounds 17, 6, 18, 7, 5, 13, and 12 had insignificantly less cytotoxic activities than the latter derivatives. The two other compounds, 4 and 11, in the current investigation were significantly less active compared to that of doxorubicin Figure 3, Table 1.
From the above results, it can be revealed that compounds 2 and 20 are nonspecifically active candidate drugs on both colon and breast human cancer types. It has also been shown that although compounds 3, 14, and 15 are active against both two cancer cell lines, they showed more specificity against human breast cancer lines. On the other hand, compounds 16 and 21 are specifically active candidate drugs against human breast cancer but not active on human colon cancer types.
In correlation of the afforded activity results with the structural characteristics of the highly active compounds, it was revealed that two candidates comprising two types of synthesized compounds, glycosyl-1,2,3-triazoles and 1,3,4-thiadiazole-thiol, were found to be of high activities against both cell lines. The results generally showed the importance of the 1,2,3-triazole ring system, introduced by click reactions, in highly active triazole glycosides 14-16, 20, and 21, in compounds with N-dimethylaminoarylidine part against either MCF-7 or HCT-116 cancer cells. It was well observed that the activity was lost in structurally related analogs, which lacks such ring system in compounds 4-6 and 11. It is noteworthy to mention that the obtained inhibition results also indicated that the attachment of the glucopyranosyl part with free unprotected hydroxyl groups to the 1,2,3-triazole ring system afforded N-glycosides with high cytotoxic activity more than its xylopyranosyl analog and also more than its acetylated precursors (the triazole glycosides 21 and 16). However, this was not the case when the phenyl ring in the arylidine part was substituted with p-methoxy substituent since the protected N-glycosides were found to be more active against both cells than their derived deacetylated compounds. The 1,3,4-thiadiazole glycosides incorporating thio-linkage attachment of the thiadiazole ring to the glycosyl part did not show high activities. The latter finding is, indeed, much more useful to show the importance of the free thiol-thione group at C-5 in the thiadiazole ring since both thiadiazolethione compounds 2 and 3 were highly active and such activities were lost in their derived glycosides in which such group was blocked. This conclusion was also supported by the afforded activity results of the acetylenic compounds 12 and 13, which showed much lower cytotoxic activities than their starting free thiol compound. On the other hand, the activity of the deprotected glycoside 10 against HCT-116 cell was improved compared to that of the glycoside 6, but the activity of the deprotected 11 was significantly declined compared to that of 7, especially against MCF-7 cancer cell which may refer to the influence of nature of sugar unit. The acetylenic derivative 12 with the para-methoxy substituent in the phenyl ring showed better activity, against HCT-116 cells, than its para-N-dimethylamino analog 13 which was also observed for the thiadiazole compounds 2 and 3.

General Procedures
Melting points were determined on a Böetius PHMK apparatus (Veb Analytik, Dresden, Germany). TLC was performed using aluminum plates, pre-coated with silica gel 60 or 60 F254 (Merck, Munchen, Germany.) and visualized by iodine or UV light (254 nm, Merck, Darmstadt, Germany). The NMR spectra were recorded on a Varian Gemini 300 and Bruker DRX 400 spectrometer (Varian, Palo Alto, CA, USA) at 25 • C. 1 H-and 13 C-NMR signals were referenced to TMS and the solvent shift ((CD 3 ) 2 SO δ H 2.50 and δ C 39.5). Coupling constants are given in Hz and without sign. The IR-spectra were recorded (KBr) on a Jasco FT/IR-410 instrument (Jasco, Easton, PA, USA). Microanalyses were operated using Perkin Elmer 240 instrument (Perkin Elmer 240, Waltham, MA, USA) and satisfactory results within the accepted range (±0.40) of the calculated values were obtained. All reagents and solvents were of commercial grade. The cytotoxic activity of the synthesized compounds was studied at National Research Center, Cairo, Egypt. The glycosyl azides were prepared as previously reported [37,38].

Conclusions
New 1,3,4-thiadiazole thioglycosides and 1,2,3-triazole glycosides based substituted arylidine systems were prepared via glycosylation and click chemistry cycloaddition, respectively. In addition to the free thiadiazolethione compounds, the target glycosides possessing the 1,2,3-triazole motif via click strategy base revealed more potent activity against HCT-116 and MCF-7 human cancer cell lines, comparable to doxorubicin, than their 1,3,4-thiadiazole analogs. These results showed the importance of the triazole core in the formed glycoside compounds which have characterized with higher cytotoxic activities.