Synthesis, Anticancer and Antibacterial Activity of Mannose-based bis-1,2,3-Triazole Derivatives

: In the current work, aromatic amines and alkyl halides have been converted to the corresponding azides 2a‒d and 4a-d by the reaction with sodium nitrite and sodium azide respectively for amines and sodium azide for halides. Then, dipropargyl ether derivative of D-mannose 8 has been synthesized from diacetone mannose that has been obtained by the treatment of D-mannose ( 5 ) with dry acetone in the presence of sulfuric acid. Then, aldol condensation has been used to prepare diol 7 from the mannose diacetonide 6 . The reaction of compound 7 with propargyl bromide in alkaline media has been afforded dipropargyl derivative 8 . In a parallel step, both dialkyne with aromatic and aliphatic azide have been coupled to produce 1,2,3-triazole derivatives 9a ‒ d in the presence of Cu(I) salts. All synthesized compounds have been characterized by 1D and 2D NMR spectra alongside with HRMS data. The antibacterial activity against both gram-positive and gram-negative has been tested. Moreover, the anticancer activity has also been evaluated against AMJ13 cell line.


Introduction:
Cancer has life serious threats to global health that causes one out of six life losses in the world. 1 The triazoles derivatives revealed extensively antiproliferative potency against human prostate, 2 breast, 3 liver, 4 lung, 5 bladder 6 and other cancer cells, in drug discovery. Mimicking glycosides, Nerella and co-workers 7 built new1,2,3-triazoles based on carbohydrate molecule that have notable anticancer activity against breast and prostate cancer cell lines. Recently, Oubella et al. 8 synthesized series of novel (R)-Carvone-based 1,4-disubistituted-1,2,3-triazoles via regioselective cupper (I)-catalyzed alkyne-azide click methodology. The hybrids Carvone triazoles in vitro evaluated the anticancer activity against breast adenocarcinoma (MCF-7 and MDA-MB-231), (HT-1080) and fibro sarcoma (A-549) lung carcinoma, cell line. On the other hand, Covid-19 is the most life-threatening disease to global health till now. 9 The widely fast spread and aggressive symptoms caused by the crown virus results in tremendous economic loss and mortality during the pandemic. The significant heterocyclic azole compounds have a potential role as antimicrobial 10,11 and enzyme inhibitors motivated many researchers to improve pharmaceutically active 1,2,3-triazoles scaffolds. 12 The recent in silico studies were confirmed by Holanda et al., 13 that the phthalimide-based 1,2,3-triazoles derivatives were prepared via click are promising drug for COVID-19 treatment as the ability to disrupt virus spike, nucleocapsid or protease proteins. Also, many 1,2,3-triazole derivatives as FDA-approved drugs have exerted their pharmacological activities such as the antibacterial; tazobactam, 94 antibiotic; cefatrizine, 95 anticancer; Carboxyamido-triazole (CAI), 96 anti-HIV; TSAO, 97 and anti-Alzheimer; MTSMDL treatment. 18 1,2,3triazole employed superior antimicrobial, 19 antifungal, 20 antioxidant 21 and cytotoxic activities. 22 Collections of glycoconjugated 1,2,3-triazoles derivatives from different sugars functionalized with alkyne or azide moieties have been synthesized via CuAA-click reaction and their antibacterial were evaluated. [23][24][25] Many factors improve the 1,2,3triazoles biological value such as physical and chemical properties, high stability against oxidant agent, hydrolysis resistant in acid/base conditions, the hydrogen bonds forming and amide bioisoesters. 26 As a result of the special physicochemical properties, 1,2,3-triazole derivatives played a versatile role in the material sciences, 27 Anti-corrosions, 28 polymers, 29 dyes, 30 catalysts, 31 ligands, 32 surfactants, 33 chemo sensors for different species. 34 Click strategies is the concept introduced by Sharpless 35 and Meldal 36 in 2002 to design and synthesize wide scope of 1,2,3triazoles scaffold molecules. The 1,3-dipolar cycloaddition reaction of terminal alkyne and azide catalyzed by Cu(I) CuAAC afforded regioselective1,4-disubstituted -1,2,3-triazoles in high yield, stereoselective, easily removable byproducts, simplicity and green of click protocol. 37 In this work, novel bis-1,2,3-triazole derivatives have been synthesized starting from D-mannose and their structural properties were studied. They also have been examined against bacteria and breast cancer.

Material and Methods: General Information
Chemicals were gained from Sigma-Aldrich and Alfa Aesar Chemicals. Infrared spectra were obtained using SHIMADZU 2001 FT-IR. NMR spectra were verified using 600 MHz, Bruker DPX spectrometers, NMR assignments of the synthesized compounds supported by COSY and HSQC. Orbit rap LTQ XL ion trap MS in positive ion mode using electrospray ionization (ESI) source was employed to assemble HRMS. Silica TLC plates were used with an aluminum backing (0.2 mm, 60 F254). The reactions were monitored by TLC and envisioned by development of the TLC plates with an alkaline potassium permanganate solution dip. AMJ13 Cells have been provided by Iraqi Center for Genetics and Cancer Research ICGCR/ Mustansiriyah University -Baghdad / Iraq.

Biological Activities: Anticancer Activity Maintenance of Cell Cultures
AMJ13 Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 80% confluence twice a week, and incubated at 37 °C. 44

Cytotoxicity Assays
To determine the cytotoxic effect of compounds 9a and 9b, the MTT assay was done using 96-well plates. 45 Cell line was seeded at 1 × 96 cells/well. After 24 hours or a confluent monolayer was achieved, cells were treated with compounds 9a and 9b different concentrations (6.25 μg/mL-400 μg/mL). Cell viability was measured after 24, 48, and 72 hours of treatment by removing the medium, adding 28 µL of 2 mg/mL solution of MTT and incubating the cells for 2.5 hours at 37 °C. After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 µL of DMSO (Dimethyl Sulphoxide) followed by 37 °C incubation for 15 minutes with shaking . 46 The absorbency was determined on a micro plate reader at 492 nm; the assay was performed in triplicate. The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following equation: 47 The rate of Inhibition = -× 100 Where A is the optical density of control, and B is the optical density of the samples. 48 To visualize the shape of the cells under an inverted microscope, the cells were seeded into 24well micro-titration plates at a density of 1×105 cells mL −1 and incubated for 24 hours at 37 °C. Then, cells were exposed to 9a and 9b for 24 hours. After the exposure time, the plates were stained with crystal violet stain and incubated at 37 °C for 10-15 minutes. The stain was washed off gently with tap water until the dye was completely removed. The cells were observed under an inverted microscope at 100× magnification and the images were captured with a digital camera attached to the microscope. 49

Statistical Analysis:
The obtained data were statically analyzed using an unpaired t-test with GraphPad Prism 6. 50 The values were presented as the mean ± SD of triplicate measurements. 51

Antibacterial Activity
For assessing the antibacterial activity of the prepared compounds 9a-d, bacterial suspension was prepared by transferring 2-3 colonies with the same phenotypic characteristics growing on the Nutrient Agar medium to tubes containing the crystalline saline solution. The tubes were compared with a 0.5 McFarland standard, which gives an approximate number of cells to 1.5 × 10 8 colony /mL. The inhibitory efficacy of the prepared compounds with concentrations (50,100,200,400) μg/mL were tested against Gram positive bacteria S. aureus and Gram negative bacteria E. coli according to the agar gel diffusion method. 52 0.1 mL of the bacterial suspension was added and spread to the Muller Hinton agar plates by cotton swab and leave the plates for drying at 37 ºC for 30 minutes followed by punching wells of 6 mm with the help of a sterile crock borer in appropriate diameter(6) mm under aseptic condition . DMSO and control DMSO 60 μL of each concentration were placed in the labeled wells respectively then incubated in the incubator at 37 ° C for 24 hours and the inhibition zone of bacteria (mm) was measured. The antibiogram sensitivity of these bacteria were tested using Levofloxacin, Amoxicillin clavulanic acid, Amikacin and Ciprofloxacin.

Result and Discussion: Synthesis and Characterization:
The general route of the synthesis is shown in Scheme (1). In the first step, D-mannose (5) was treated with dry acetone in the presence of concentrated sulfuric acid at room temperature for 4 hours to yield a mixture of α and β-isomers of 2,3:5,6-di-O-isopropylidene-D-mannofuranose (6) in a good yield 75%.70 .The significant stretching bands of FT-IR spectrum showed at ν 3437 cm −1 (O−H), 2985 cm −1 and 2906 cm −1 for aliphatic (C−H). 1 H NMR spectrum verified signals of α and β anomer and the ration of α anomer is the predominant. Four singlets appeared at 1.46 ppm, 1.45 ppm, 1.37 ppm and 1.32 ppm is a good proof of two isopropylidene group formation. 13 C NMR also afforded another evidence of the formation of compound 2 by the appearance of six signals at 112.8 ppm, 109.2 ppm, 27.0 ppm, 26.0 ppm, 25.3 ppm and 24.6 ppm corresponding to two isopropylidene protecting groups. Also, a base peak at m/z 283.1152 assigned to the suggested formula. Secondly, 2C-(hydroxyl methyl)-2,3:5,6-di-Oisopropylidene-D-manofuranose (7) was synthesized via aldol condensation of compound 6 with aq. formaldehyde in the presence of K2CO3 and excess of KOH to gain in approximately high yield 80 %. This protocol was developed by Tan et al., 2016 71 who reported a new extension of work to gain the brunched sugar with quaternary center generating transformation and solved the mysterious retardation attributed to previous work introduced by Ho 1979 89 , the formation of formic acid by a Cannizzaro reaction led to lower the basicity of this reaction. The excess amount of KOH was successfully accelerated the condensation and shorted the reaction time from 48 hours to 100 min in addition to consume the starting materials completely as well as the isolation / purification became easier. FT-IR spectrum of compound 7 afforded good evidence as two hydroxyl groups appeared at ν 3450 cm −1 and 3311 cm −1 . Two epimers A and B were detected in the 1H NMR spectrum in 2:1 ratio. The most important evidence of the formation of compound 6 is the appearance of four at δ 3.98 ppm, 3.85 ppm, 3.79 ppm and 3.76 ppm corresponding to (CH2OH) of two epimers A and B in addition to the disappearance of H-2 signal at 4.61 ppm. The formation of diol 7 is also approved by 13 C NMR when a signal appeared at 63.8 ppm and 62.9 ppm attributed to the extra branched carbon of two epimers. Subsequently, Williamson etherification of the sugar diols were carried to gain dipropargyl ether 8 by using propargyl bromide and NaOH as heterogeneous catalysts in DMF under SN2 conditions to give compound 4 in a moderate yield 60%. 43 The formation of compound 4 was investigated by FT-IR as hydroxyl bands disappeared and both acetylenic proton and triple bond bands were assigned at 3286 cm −1 and 2117 cm −1 respectively. The attachment of the propargyl moieties to the sugar derivative was assigned by the appearance of two new doublet of doublet at 4.28 ppm and 4.21 ppm attributed to two protons of the methylene of the anomeric propargyl. However, the methylene group of the propargyl attached the position 2′ appeared as a doublet with J = 2.4 Hz. Furthermore, a multiplet centered at 2.41 ppm corresponding to two terminal alkyne protons was also observed in 1 H NMR spectrum (Fig. 1). The two propargyls were also assigned by the appearance of six new signals at 79.7 ppm, 79.1 ppm, 74.7 ppm, 74.6 ppm, 59.1 ppm, 54.8 ppm in 13 C NMR spectrum (Fig. 2). It is important to mention that only one anomer formed after the propargylation of the diol 7 as viewed by the NMR spectra of compound 8 (Figs.  1−4). The formation of compound 8 is confirmed by the appearance of a base peak at m/z 389.1570 corresponding to the formula [M + Na] + in HRMS.
In a parallel step, alkyl halides a and b were converted to the corresponding alkyl azides 2a and 2b under SN2 nucleophilic substitution reaction while the aryl azides 4a and 4b were prepared through direct conversion of the corresponding aryl amines c and d to diazonium salt via one-put diazotization with NaNO2 and p-TsOH in aqueous solution that followed with sodium azide.
The high to excellent yields of the azides 2a and 2b, 4a and 4b about 90% was gained. The significant and strong azide bands of FT-IR spectrum showed around ν 2100 cm −1 (N≡N) for alkyl and aryl azides, and the disappeared of both NH2 bands in aromatic amines. Proton NMR spectra of compounds 2a and 2b showed the multiplet signals at 1.37-1.27 ppm attributed to H10′ and H12′-H3′, in addition to signals at 3.3 ppm for H1′ and 1.6 ppm for H2′. 13 Carbon NMR spectra showed 12 and 10 signals for the corresponding compounds 2b and 2a. The compounds 4a and 4b showed a clear signal between 7 to 8 ppm which attributed to the H-aromatic of aryl azides. In addition, the six signals in 13 C NMR spectra for six C-aromatic provided excellent evidence for azides formation. The following scheme describes the overall synthetic route of the targeted compounds:

Scheme 1. Synthesis of Mannose-based bis-1,2,3-triazoles
Finally, regioselective ligation between the sugar alkyne 8 as precursor of and the collection of azides 6a-d have been carried out via Cu-catalyzed Azide Alkyne-1,3-dipolarcycloaddition Click reaction to afford the targeted bis-1,2,3-triazoles 9a-d using Cu(I) as catalysts that was produced in situ by treating the CuSO4.5H2O with the reducing agent Na ascorbate, Compounds 9a-d were isolated in very good yields 80-87%. The constructions of these compounds were also confirmed by NMR spectroscopy furthermore to other techniques. Owing to facilitate the detection of NMR spectra, numbering of compounds 9b and c is shown below: Beside the sugar and aromatic azide signals, there was an important singlet at 7.63, 7.57ppm and 8.38, 8.30, 8.05 7.99 ppm which clearly referred to the 2H of triazoles for each compounds 9a,b, and 9c,d consequently. In the same way, 13 C NMR spectra supported the structures of bistriazoles. The signals at 144.3 and 122.7 ppm, for example, were attributed to the 2C of (CH=CN-triazole) of triazole heterocycle 9a, respectively. All assignments of proton and carbon NMR were based on COSY and HSQC. The formation of compounds 9a-d is confirmed by the appearance of a base peak at m/z 755.5041, 811.5660, 717.2230, 765.0956 corresponding to the formula [M + Na] + in HRMS. Biological Activities: Anticancer Activity The cytotoxic effect of compounds 9a and 9b against AMJ13 cells was studied. The antiproliferative activity of compounds 9a and 9b was tested by studying their ability to inhibit the proliferation of AMJ13 cell line. The results of this study showed there is cytotoxic activity of 9a and 9b compounds against the AMJ13 cell line and the results is concentration dependent manner as shown in Fig. 2 (a and b) as well as in Table 1.       Antibacterial Activity: The antibacterial activity of the prepared bistriazoles 9a-d was verified against pathogenic Gram-positive bacteria S. aureus and Gramnegative bacteria E. coli via the agar well diffusion method. DMSO was used as control. As shown in Table 2, all compounds 9a-d did not give any activity at against Gram positive bacteria S. aureus. In contrast, all the mentioned compounds exhibited moderate to good activity at the concentrations 50-400 µg /mL respectively. On the other hand, compound 9a with C10 in the aliphatic chain and 9c with substituted aryl exhibited maximum antibacterial activity compared to the other measured compounds. The antibacterial activity of bistriazoles 9a-d can be recognized to their performance as glycoconjugate mimics 54 and biosurfactants analogs the asymmetrical structure of the whole molecule or the protein-binding properties. 55 Table 2

Conclusion:
Four bis-1,2,3-triazole derivatives 9a−9d have been synthesized starting from the readily available monosaccharide (D-Mannose) using convenient reaction conditions particularly the azide-alkyne cycloaddition click reaction. The synthesized compounds were fully identified via modern spectroscopic techniques showing their high purity. These compounds were tested against pathogenic G+ bacteria S. aureus and Gbacteria E. coli. All compounds demonstrated no activity against the mentioned types of bacteria. However, compound 9a and 9c demonstrated excellent inhibition zones ~16 mm and 13 mm respectively at concentrations of 50−400 µg/mL. On the other hand, triazole derivatives 9a and 9b were screened against breast cancer AMJ13 cells line and they exhibited remarkable activity IC50 of 167.64 µg/mL 171.61 µg/mL respectively.