Anticancer Activity of New 1,2,3-Triazole-Amino Acid Conjugates Anticancer Activity of New 1,2,3-Triazole-Amino Acid Conjugates

: A multistep synthesis was developed to prepare new 1,2,3-triazole-amino acid conjugates ( 6 and 7 ). These compounds contain the diaryl ether moiety and were synthesized via S N Ar reaction under mild condition and in good yield. Their structures were conﬁrmed by spectroscopic analyses (HR-MS, NMR, IR). These compounds showed signiﬁcant antiproliferative activity (>30%) toward the breast MCF7 and liver HepG2 cancer cells lines at <10 µ M concentration. Abstract: A multistep synthesis was developed to prepare new 1,2,3-triazole-amino acid conjugates ( 6 and 7 ). These compounds contain the diaryl ether moiety and were synthesized via S N Ar reaction under mild condition and in good yield. Their structures were confirmed by spectroscopic analyses (HR-MS, NMR, IR). These compounds showed significant antiproliferative activity (>30%) toward the breast MCF7 and liver HepG2 cancer cells lines at <10 µM concentration.


Synthesis
The synthesis strategy of the triazole-amino acid conjugates 6 and 7 is depicted in Scheme 1. The synthesis started with an S N Ar reaction between vanillic acid 1 and 1fluoro-4-nitrobenzene in DMSO using KOH as base. The ratio between vanillic acid 1 and KOH is important, and was optimized to improve the yield of compound 2 (Table 1). Two Molbank 2021, 2021, 0 2 of 6 equivalents of KOH was not enough, giving only 59% yield. The best yield (70%) was obtained when 3 to 5 equivalents of KOH were used.

Synthesis
The synthesis strategy of the triazole-amino acid conjugates 6 and 7 is depicted in Scheme 1. The synthesis started with an SNAr reaction between vanillic acid 1 and 1fluoro-4-nitrobenzene in DMSO using KOH as base. The ratio between vanillic acid 1 and KOH is important, and was optimized to improve the yield of compound 2 (Table 1). Two equivalents of KOH was not enough, giving only 59% yield. The best yield (70%) was obtained when 3 to 5 equivalents of KOH were used. KOH Reduction of nitro group of compound 2 using Fe/HCl in EtOH/H2O produced the aniline 3 in 73% yield. This aniline was then converted to azide using a two-step procedure (diazotization and azidation) providing compound 4 in 51% yield. A click reaction was employed for the synthesis of triazole 5 using Sharpless conditions [9]. Compound 5 was obtained in 58% yield. The 1 H-NMR spectrum of compound 5 showed a new singlet at 9.12 ppm (1H) which is the proton of the newly formed triazole ring. In parallel, ethyl ester of natural amino acid L-phenylalanine 8 and glycine 9 were prepared by reacting the corresponding amino acids with SOCl2 in EtOH at 80 °C. Amide coupling between 5 and 8 (or 9) was successful using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagent. The target triazole-amino acid conjugates 6 and Scheme 1. Synthesis of 1,2,3-triazole-amino acid conjugates 6 and 7. Reduction of nitro group of compound 2 using Fe/HCl in EtOH/H 2 O produced the aniline 3 in 73% yield. This aniline was then converted to azide using a two-step procedure (diazotization and azidation) providing compound 4 in 51% yield. A click reaction was employed for the synthesis of triazole 5 using Sharpless conditions [9]. Compound 5 was obtained in 58% yield. The 1 H-NMR spectrum of compound 5 showed a new singlet at 9.12 ppm (1H) which is the proton of the newly formed triazole ring. In parallel, ethyl ester of natural amino acid L-phenylalanine 8 and glycine 9 were prepared by reacting the corresponding amino acids with SOCl 2 in EtOH at 80 • C. Amide coupling between 5 and 8 (or 9) was successful using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagent. The target triazole-amino acid conjugates 6 and 7 were obtained in good yield (57% and 53%, respectively). The structures of compounds 6 and 7 were confirmed by IR, NMR and HR-MS spectroscopies (see Supplementary Materials).

Anticancer Activity of 6 and 7
The two triazole-amino acid conjugates 6 and 7 were tested for their cancer cell antiproliferative activity against the breast and liver cancer cells lines (MCF7 and HepG2) using the natural alkaloid ellipticine, a topoisomerase II inhibitor, as positive control. The results are shown in Table 2. Compound 6 showed similar IC 50 for both cancer cells lines, while compound 7 was more selective toward HepG2 than MCF7. Interestingly, the two compounds significantly inhibited both MCF7 and liver HepG2 cancer cell proliferation by >30% at low concentration (<10 µM). Table 2. Anticancer activity of compounds 6 and 7. MTT cancer cells antiproliferation assay was done using reported protocols from literature [10,11]

Materials
Reagents and solvents were purchased from commercial suppliers and used without further purification. Column chromatography was carried out using Merck Kieselgel 60 silica gel (particle size: 32-63 Å). Analytical TLC was performed using Merck precoated silica gel 60 F-254 sheets.
NMR spectroscopic data were acquired on Bruker Avance III at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR. HR-MS spectra were recorded on a Bruker MICROTOF-Q 10,187 and a LC-MS Thermo, model: UltiMate 3000/ISO EC. Infrared spectra were taken on a SHIMADZU FTIR 8400S (KBr).

MTT Assay for Cell Antiproliferative Activity
The anticancer activity of the synthesized compounds 6 and 7 was evaluated on two human cancer cells lines HepG2 (HB-8065 TM ), and MCF-7 (HTB-22 TM ) which were obtained from the American Type Culture Collection (USA) ATCC using recent reported protocol [12].

Conclusions
An efficient multistep synthetic procedure was reported for preparing new 1,2,3triazole-amino acid conjugated compounds (6 and 7). The structures of these compounds were confirmed by IR, NMR and HR-MS. These conjugates significantly inhibited the breast MCF7 and liver HepG2 cancer cells proliferation of >30% at concentration < 10 µM. Thus, compounds 6 and 7 represent a new class of potential anticancer compounds for further optimization and mechanistic studies. The reported synthetic route can be used to prepare different collections of triazole-amino acid conjugates for screening on diverse pharmacological targets.