Efficient Green Chemistry Approach for the Synthesis of 1,2,3-Triazoles Using Click Chemistry through Cycloaddition Reaction: Synthesis and Cytotoxic Study

Abstract Herein, we reported the synthesis of diverse N-(substituted phenyl)-2-(4-((dibenzylamino)methyl)-1H-1,2,3-triazol-1-yl)acetamide (6a–6j) through a green chemistry approach using water and eco-friendly catalysts using copper catalyzed click chemistry approach. The optimization of the reaction toward the greener way was discussed through applying the choline azide and ecological suitable solvent/reagent systems. The biological potency of the adducts was described against NCI-60 cancer cell lines in nine cancer panels and four scaffolds with halogens as phenyl ring substituents were found to be most active with negative GI50 values. Supplemental data for this article is available online at


Introduction
Green chemistry shares with click chemistry some of the aforementioned stringent criteria, employing which more efficient and environmentally benign processes can be delineated and implemented. 1 Click chemistry is a conception of organic synthesis that is of paramount importance in modern chemistry. 2,3 The first decade of click chemistry has recently been commemorated; 4 throughout this time, an increasing number of disciplines 5 have taken advantage of the unique benefits offered by CuAAC, the click reaction. The combined use of water as a solvent with metal nanoparticles is a fast-growing area in response to the general upsurge of interest in minimizing the environmental impact of chemistry. 6,7 Preformed organic azides and terminal alkynes are used as starting materials in the first case; in the second case, a multicomponent approach is tackled from different azido precursors, sodium azide, and terminal alkynes. It must be pointed out that although this account is focused on heterogeneous catalysis, the fundamental contribution of homogeneous catalysis to the advance of click chemistry must not be disregarded; speeding up the CuAAC reaction at room temperature and decreasing the amounts of copper to levels of a few parts per million are some praiseworthy achievements of homogeneous CuAAC. 8,9 In fact, a recent highlight identified specific criteria for the categorization of reactions in polymer chemistry as click reactions that can serve as a guidepost in this area. 10 Although Cu(I) may be obtained directly from the utilization of Cu(I) salts and coordination complexes. 11 The Cu(I) species necessary for the CuAAC reaction is often generated in situ by employing Cu(II) in conjunction with a reducing agent such as ascorbate. 12 Additionally, 1,2,3-triazoles and other compounds have been employed as stabilizing ligands for Cu(I), inhibiting the oxidation and disproportionation of the ion while retaining its catalytic activity. 13 Owing to the cytotoxicity associated with Cu(I), several groups have endeavored to minimize the copper concentration while maintaining high reaction rates. 14,15 Dibenzyl amine (DBA) motifs are important compounds with extensive application in rubber compounds, fine chemicals, corrosion inhibitors, and drug formulation. 16,17 Now significant research efforts made in this direction, synthetic routes, such as direct base-promoted mono-Nalkylation or alkylation amination have been developed for this synthesis of DBA motifs. 18 Dibenzyl amine derivative is clinical important from its use as a pharmacologic vehicle for anticonvulsant activity and many more in recent research. 19,20 Heterocyclic compounds containing more numbers of nitrogen plays an important role in agrochemical and pharmaceuticals. 21 It should be noted in recent research published by Aisa et al., the advances in click chemistry, various synthetic methodologies for the 1,2,3-triazole scaffolds (e.g., derivatives, hybrids, and conjugates) have been used in medicinal chemistry. 22 Various biological screenings were conducted 23,24 which led to the identification of anticancer, 25-27 antimicrobial, 28 and many properties of the studied 1,2,3-triazole-bearing hybrids. Moreover, triazole-linked derivatives were used widely in peptides to mimic a trans-amide bond, despite their hazardous effects on native peptide activity. 29 Recently, the 'linker' property of 1,2,3-triazoles was demonstrated, and a novel class of 1,2,3triazole-containing hybrids and conjugates was synthesized and evaluated as lead compounds for diverse biological targets. 22,30 Thus, because of the above factors and the recent focus of researchers on 1,2,3-triazole compounds, we have discussed the greener route for the synthesis and highlighted the cytotoxic activity of diverse 1,2,3-triazole hybrids having four methylene groups as "linkers" in a single core.

Material and method
All the chemicals and solvents for the synthesis were purchased from Merck Chemical Company; dibenzyl amine was purchased from Sigma-Aldrich Chemical Co. particularly. The reaction was carried out by standard techniques exclusive of moisture. Thin-layer chromatography (TLC) was carried out on plates percolated with silica gel 60F254 (E. Merck) and spots were detected with iodine vapor or visualized in UV light (254 and 365 nm). The open-end capillary method was used to determine the melting points of the synthesized compound and results were reported and were uncorrected. IR spectra of all compounds were recorded on the IR Affinity 1S spectrometer (Shimadzu). The 1 H NMR and 13 C NMR spectra were recorded on Bruker Spectrophotometer-400 MH Z using DMSO-d 6 as the solvent and trimethyl silane (TMS) as the internal reference. Mass spectra were recorded on an Agilent GCMS-QP 2010 spectrometer. Elemental analysis was carried out on Euro EA 3000 elemental analyzer. The solvent was evaporated using a rotary evaporator.
General procedure for the synthesis of N,N-dibenzylprop-2-yn-1-amine (3a) A solution of dibenzyl amine (200 mg, 0.001 mol, 1 eq.) was added in 10 mL of the dry round bottom flask containing water (2 mL) as a solvent, and to this anhydrous K 2 CO 3 powder (480 mg, 0.0035 mol, 3.5 eq.) was charged. It was stirred at 65 C for 30 minutes. It was charged with propargyl bromide (120 mg, 0.001 mol, 1 eq.) under heating conditions. The reaction mixture was further heated at 40 C for 2 hours. The completion of the reaction was monitored by thin-layer chromatography using n-hexane: ethyl acetate (4:6) as a mobile phase. After completion of the reaction, it was cooled to room temperature (RT) and further stirred for 30 minutes. The reaction mixture was poured onto crushed ice and continues string for 30 minutes. The light red colored oily product was isolated through extraction in ethyl acetate solvent (twice). The crude oily mass was collected after evaporation of solvent using a rotary evaporator.
General procedure for the synthesis of 2-(4-((dibenzylamino)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-nitrophenyl) acetamide (6a-6j) For the synthesis of the final product (6a-6j), the previously prepared intermediate compound 3a (1 eq.) was charged in the DMF: H 2 O (1:1 ratio) media. It was stirred well for 10 minutes to get a clear solution. The acyl azide (1 eq.), sodium ascorbate (1.5 eq.), and CuSO 4 . 5H 2 O (1.5eq.) were added and the mixture was stirred at RT for 12 hours. TLC was used to monitor the progress of the reaction using two various mobile phase systems. After completion of the reaction, it was poured into ice crushed water and was extracted to ethyl acetate to isolate the oily crude mass after solvent evaporation. The entire series of compounds were purifying by column chromatography on Silica gel (60-120 Mesh) using hexane: ethyl acetate mobile phase system.
The entire compounds of the series were isolated under the above-stated procedure and confirmed by various spectroscopic techniques.  13

Chemistry
The N,N-dibenzylprop-2-yn-1-amine (3a), which were the precursor necessary for CuAAC click chemistry, were synthesized by reaction of corresponding dibenzyl amine (1) with propargyl bromide in a water media. We used two synthetic ways to accomplish this purpose (Scheme 1). Firstly, anhydrous potassium carbonate was used as a base and water was used as a solvent. The reaction time was 2 h at 40 C, and the yield was 80-90% (Procedure A). In a second way, sodium hydride was used as a base in dried DMF solvent (Procedure B). The procedure B was not much useful here due to dual behaviors of NaH as base as well as a proton donor and due to this, there is the generation of various by-products simultaneously which becomes difficult to work up the process and ultimately loss of the product (Table 1).
For the click chemistry approach, the second important reactant is azide functionality (5a-5j) and instead to use commercially available azide, we had synthesized acyl azide from various arylamine (4a-4j) through two steps in situ procedure, first, react with Bromo acetyl bromide followed by slow addition of freshly prepared choline azide solution in THF (followed the procedure described by Azarnia et al.) 31 as a green substitute of sodium azide to overcome its low solubility in common organic solvent (THF was used for in situ reaction conditions) (Scheme 2).
The final products (6a-6j) were optimized through varying catalyst and temperature and was outlined in Scheme 3 and Table 2. The use of the efficient catalytic system of CuSO 4 .5H 2 O (1.5 mol%) and sodium ascorbate (1.5 mol%) in DMF as a solvent (procedure A, entry 1) made it easy to handle the reaction, even with the solvent-extraction method using the isolated product out of the reaction mixture. On the other hand, reaction time was prolonged until 2 h when the reaction was carried out at room temperature. The yield of (6a) was 97.8 % in this case. The increasing reaction mixture temperature (only to $40 C) led to the formation of oily or tar substances having indefinite structures possibly due to the decomposition of the reactants or products. Ease of workup through preferential crystallization of final product was achieved by using the catalyst of copper(I) iodide (1.5 mol%) in t-BuOH as a solvent with little water as an additive (entry 2, procedure B), However, the reaction time was prolonged until 6 h and a yield of 65.2 % was attained. Copper was supported on microporous metal-organic framework MOF, and in this case, Cu@MOF-5 (developed inhouse by Department of Physics, RK University using stated protocol by Anandhan et al. 32 ) (1.5 mol%) (procedure C, entry 3) gave the best results of catalyst surveyed. The reaction mixture temperature was 78-80 C. On other hand, the use of absolute ethanol brought an outstanding advantage in this entry. After the end time of reaction, the product was isolated simply by adding toluene (by half a volume), heating and filtering out solid catalyst and provided a good method in the click chemistry approach. But due to acute toxicity, it was avoided to follow for the entire library of compounds. The obstruction of the copper salts to isolating products have been avoided. A yield of 97.8% was attained when this procedure was employed. we decided to choose CuSO 4 .5H 2 O (1.5 mol%) to be a catalyst in the synthesis of remained substituted 1,2,3-triazole (6a). Furthermore, Huisgen's 1,3-dipolar cycloaddition between an azide and alkyne proceeds with a mixture of the 1,4-and 1,5-disubstituted triazoles are well studied in literature and our past work confirms that using mentioned mole proportion of cooper catalyst gave only 1,4-isomer. The fundamental steps for the click coupling pathway using cooper catalyst is well described through single crystal study as well 2 D-NMR studies. 27,33  The presence of four methylene bridges was identified using DEPT-135 NMR (Distortionless Enhancement by Polarization Transfer) spectra and it was an advanced technique to confirm the targeted molecular spatial arrangements (Figure 1). The obtained synthetic results with their physical parameters were shown in Table 3.

Spectroscopic analysis
The structure of synthesized compounds 6a-6j was confirmed based on spectral data. The IR spectrum of compound 6a-6j showed a strong absorption band at $3255 cm À1 due to N-H stretching, secondary amine. The absorption band appeared at $3086 cm À1 due to stretching vibrations of aromatic hydrogen. Sharp absorption peak observed at $1666 cm À1 in > C¼O Scheme 2. Green synthetic outline for various acyl azide using choline azide Scheme 3. Click chemistry approach for the synthesis of Triazoles (6a-6j) adducts through 1,3-dipolar cycloaddition reaction. stretching of the carbonyl group. Moreover, the absorption band at $1473, $1087 cm À1 corresponding to C ¼ C, C-N stretching. In 1 H NMR spectra, the appearance of singlet peaks in compounds 6a-6j showed a characteristic value at d ¼ $10.64 ppm due to the presence of an amine group near to aromatic ring. The single proton of -CHdisplayed singlet at d ¼ $8.13 ppm due to the presence of triazole nucleus. The presence of -CH 2 -linkage showed a singlet peak at $5.51 ppm. All aromatic protons appeared multiplet in the region d ¼ $7.65 ppm. The remaining substituents protons were in good agreement with theoretical values. The 13 C NMR added more confirmation to the formation of the targeted motifs. The signal obtained at $48.72 ppm can be assigned to the presence of methylene carbon between -CONH 2 -linkage and triazole ring. The chemical shift details of the carbon in the > C¼O group was seen $166.58 ppm. The mass spectrum revealed a molecular ion peak in compound 6a-6j between at m/z ¼ $429 to $490 in mass spectra, molecular ion peak was in agreement with proposed molecular weight and elemental analysis.

In vitro study for anticancer activity
Single dose-response against NCI-60 cell lines of the ten synthesized compounds was carried out at National Cancer Institute, A division of NIH, USA at a single dose concentration (10 À5 M). The number reported for the One-dose assay is growth relative to the "no-drug control", and   relative to the time zero number of cells and COMPARE analysis was used for screening of the molecules. Experimental criteria: a value of 0 means no net growth, a negative value shows lethality (shows resistivity), and a positive value (Exp. X) shows (100-X) growth inhibition (shows sensitivity). The detailed protocol for the screening was described in our previously published article 34 and the same is also available at https://dtp.cancer.gov/discovery_development/nci-60/ methodology.htm.
The NCI-60 cancer cell lines screen is used for the evaluation of in vitro anticancer activity against nine cancer cell panels. We identified "five" out of nine screening of Leukemia, Non-small cell lung cancer, (NSCLC) colon cancer, CNS, Melanoma, Ovarian, Renal, Prostate and Breast cancer, those five screening are most active for select molecules 6b, 6d, 6h, and 6i because its halogen contains molecules. The report survey gave us the direction that the halogens in triazole scaffolds enhance the anticancer activity with contorted chemistry. More benefit with their GI 50 values is given below in Table 4.
The single dose-response of active molecules among synthesized is summarized in Table 4. We divided the effectiveness of compounds into three categories; the first class was for highly active compounds i.e., 6i and 6b, which were shown the consequential effectiveness in leukemia, nonsmall cell lung cancer, melanoma, and breast cancer in most cell lines. The compound 6i showed major influence in leukemia cancer with the negative value for two cell-lines, K-562 (GI 50 ¼ À55.81) and CCRF-CM (GI 50 ¼ À20.87); one in NSCLC (NCI-H522¼ À72.38); one in colon cancer (SW-620¼ À72.02); two in Melanoma cancer (MALME-3M¼ À72.91, SK-MEL-5¼ À5.51) and one in breast cancer (T-47D¼ À50.87). On the other hand, compound 6b shown efficacy in RPMI-8226 with a À19.60 value. Furthermore, it shown negative growth inhibition in HOP-92: À27.87 (NSCLC), NCI-H23: À2.48 (NSCLC), and MDA-MB-468: À10.40 (Breast cancer) and in the remaining cell-lines found to comparable active with GI 50 values ranging from 0.35 to 18. The second category of the compounds was 6d and 6h due to moderate activity against some of the cancer panels. These two molecules were found to be active in Leukemia, Non-Small Cell Lung Cancer, and Breast Cancer with the GI 50 values good as compared to not active molecules against specific cell-lines (RPMI-8226, HOP-92, HCT-116, MDA-MB-231/ATCC, T-47D, and MDA-MB-468) with average inhibition values) in each panel. The third category of the compounds was 6a, 6c, 6e, 6f, 6g and 6j with very low growth inhibition values.
To explain the differences among the biological activities of these compounds, in this work we pursued to gain insight into the role of the halogen's atom on the chemical reactivity toward cancer cell lines and it was found that the acyl azide ring having fluoro functionalities enhances the activity while those with electron-donating part lower the efficacy in almost all cell-lines. It should be noted here that the chloro at the 4th position also shown potency in three cancer panels while 2-Cl not found to be active. [35][36][37] Based on the above discussion, molecule 6i could be considered as the "Lead molecule" for further screening, and one dose-response graph of described four molecules in nine cancer panels is given in Figure 2.

Conclusions
To sum up, a series of N-(substituted phenyl)-2-(4-((dibenzylamino)methyl)-1H-1,2,3-triazol-1yl)acetamide were synthesized based on selecting decremental fragments and using copper-catalyzed pathways. Ten compounds were synthesized according to the green chemistry approach, which affords high yields and purity. All of the compounds were tested for their activity activities against the NCI-60 cell lines and the results provide are a new idea to synthesize hit molecules similar to "6i" with the slight modification of structure for better efficacy in the cancer field. To summarize, our work shows that simple, nitrogen-rich triazole molecules may be valuable leading structures in the development of anticancer drugs.