One-pot three-component synthesis of new triazolopyrimidine derivatives bearing indole moiety as antiproliferative agents

Mohamed A. A. Radwan1,2*, Fahad M. Alminderej1, Hala E. M. Tolan3, Hanem M. Awad4 1Department of Chemistry, College of Science, Qassim University, Buraydah, Kingdom of Saudi Arabia. 2Applied Organic Chemistry Department, National Research Center, Dokki, 12622, Egypt. 3Department of Photochemistry, National Research Centre, Dokki, 12622, Cairo, Egypt. 4Tanning Materials and Leather Technology Department, National Research Centre, Dokki, Cairo, 12311, Egypt.


MATERIALS AND METHODS
Chemicals were obtained from Merck and Sigma-Aldrich. Melting point (°C) was measured on the XT-5 microscopic apparatus. C, H, and N analyses and infrared (IR) spectra, which were measured using an iS10 spectrometer (ν in cm −1 ) using KBr disk, were carried out at Cairo University. MS (EI) m/z analysis was carried out via a Thermo Scientific DCQII. 1 H and 13 C nuclear magnetic resonance (NMR) were carried out in dimethyl sulfoxide (DMSO-d 6 ) on a Bruker (400 MHz) Ascend -Magnets | Bruker spectrometer.

In vitro cell culture
The human colorectal carcinoma (HCT-116) and human breast adenocarcinoma (MCF-7), (MDA-MB-231), and (A549) cell lines were acquired from the American Type Culture Collection (ATCC, Rockville, MD) and preserved in the DMEM-F12 medium which was complemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The cells were grown at 37°C in a moistened atmosphere of 5% CO 2 .

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) antiproliferative assay
The antiproliferative activity on HCT-116, MCF-7, MDA-MB-231, and A549 human cancer cells and retinal pigment ephitilial-1 (RPE-1) human normal cells was assessed by the MTT assay, which depends on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in feasible cells Emam et al., 2017;Eman et al., 2017). Cells were first distributed in a 96-well sterile microplate (2 × 10 4 cells/well) and incubated at 37°C in DMSO with diverse concentrations of each established compound or doxorubicin for 48 hours in a serum-free medium. After incubation, the media were carefully discarded, and 40 µl of MTT (2.5 mg/ml) was added to individual wells and then incubated for an extra 4 hours. The purple formazan dye crystals were solubilized by adding 200 µl of DMSO. The absorbance was measured at 570 nm via a SpectraMax Paradigm multimode microplate reader. The comparative cell viability was stated as the mean ratio of viable cells related to the unprocessed control cells. All trials were conducted in triplicate and repeated on three diverse days. The values were signified as mean ± SD. The efficacious doses for 50% of the cancerous cells population (ED 50 ) and their toxic doses in 50% of the noncancerous cells population were evaluated by Probit analysis by the Statistical Package for the Social Sciences (IBM Corp., Armonk, NY).
The MCR was carried out in the presence of different solvents and bases to check the effect on the rate of reaction and to find out the standard optimized reaction conditions When the MCR was completed in ethanol without a catalyst, despite heating for 12 hours (Table 1, entry 1), there was no product formation.
Subsequently, we catalyzed a reaction by triethylamine in different solvents (Table 1, entries 1-6). Then, we studied the reactions in various basic conditions (Table 1, entries 7-12). The reaction results showed that organic bases gave good results, while the yield of the reaction in the presence of inorganic strong bases, like KOH and NaOH, is not satisfactory. Surprisingly, K 2 CO 3 did not furnish the product.In conclusion, the best condition for this reaction is to soak in DMF in 0.25 mol triethylamine for 10 hours at 120°C (Table 1, entry 6).
As a result of the overhead reaction, a possible mechanism was proposed for the construction of triazolopyrimidine. First, triethylamine activated the nucleophilic reaction of 3-indolyl-3-oxopropanenitrile 3 via hydrogen bonding to excite carboxaldehyde 1. The initiation of carboxaldehyde by H-bond enhances the electrophilicity of the carboxaldehyde and improves the building of a transitional A (Reddi et al., 2015). Intermediate A and 3-amino-1,2,4-triazole 2 then undergo Michael's reaction to form intermediate B via intramolecular cyclization reaction to form a C-N bond. Subsequently, compound 4 was obtained by autoxidation (Scheme 3).
The structure of the newly synthesized compounds (4a-i) was identified via the interpretation of spectral studies and mass analysis (experimental units), e.g., the IR of compound 4a Scheme 1. Synthesis of triazolopyrimidine under different conditions of base, solvent, temperature, and time.

Antiproliferative screening
According to the revealed synthetic route, a series of the obtained triazolopyrimidines (4a-4i) was selected for further investigation of their antiproliferative activities in vitro on four human cancer types along with their cytotoxic effects on one nontumorous human healthy type RPE-1 by the MTT test. The proportion of live cells was evaluated and correlated with the control and reference drug doxorubicin. All compounds repressed the five human cell types (Figs. 3-7). In the HCT-116, Table 2 and Figure 3 demonstrate that compounds 4a and 4h had close antiproliferative effects, and the remaining compounds had significantly fewer antiproliferative activities than doxorubicin. While, in the MCF-7, compounds 4i and 4h had a more potent effect, compound 4g had a comparable activity and the rest of the compounds had less antiproliferative properties relative to doxorubicin ( Fig. 4 and Table 2). For MDA-MB-231 human breast cancer, compounds 4c and 4b had a more potent effect; compounds 4h and 4e had equipotent effects; and compounds 4d, 4a, 4g, 4f, and 4i had significantly less antiproliferative effects related to doxorubicin ( Fig. 5 and Table 2). For A549 cancer, compounds 4d, 4i, 4e, 4f, 4g, and 4h had extra antiproliferative actions and compounds 4b, 4c, and 4a had insignificantly lesser antiproliferative effects than doxorubicin ( Fig. 6 and Table 2). In the nontumorous human healthy normal cells (RPE-1), compounds 4f, 4g, and 4e were more toxic; three compounds 4a, 4i, and 4d had insignificant toxic effects; and three compounds 4c, 4b, and, 4h had significant toxic effects related to the reference drug.
These results indicated that all the prepared candidates are effective anticancer compounds in human lung cancer, except for compound 4a, which had significantly less activity; compound 4h had an effective antiproliferative activity on all cancerous types and had the least toxic effect on the noncancerous cells; and compound 4i had the best antiproliferative effect on both MCF-7 and A549 cancerous types, while having a weak antiproliferative effect on both HCT-116 and MDA-MB-231 cancerous types; compounds 4i, 4h, and 4g are efficient antiproliferative medicines on hormone-dependent contrary to the independent human breast cancer.    The therapeutic index (TI) of the compounds, which was calculated by dividing their efficacious dose for 50% of the cancerous cells population (ED 50 ) over their toxic dose in 50% of the noncancerous cells population, is presented in Table 3 (Hatem et al., 2019). From Table 3, it is obtained that the TI ranged from 1.7 to 3.1 compared to 2.8 for the reference drug on human colon cancer. In addition, TI ranged from 1.6 to 6.1 relative to 3.3 for the reference drug on the hormone-dependent human breast adenocarcinoma. While the TI ranged from 1.5 to 3.8 relative to 2.5 for the reference drug on the hormone-independent human breast adenocarcinoma. In addition, TI ranged from 1.8 to 3.1 relative to 2.3 for human lung cancer reference drugs. These results indicate that all synthetic compounds have the same efficacy and safety, or better in some compounds, than the standard drug doxorubicin.

CONCLUSION
The conventional three-component reactions have been used to construct a new series of triazolopyrimidine derivatives with built-in indole moiety. Compared to other traditional methods, this method is advanced in its cost-effectiveness, is easy to set-up, and results in high product quality. Antiproliferative activity of the new compounds has been examined toward four different human cancer cells and one human healthy cell line. Compounds 4a and 4h are active against the human colon cancer; all triazolopyrimidines are active toward MCF-7; 4i, 4h, and 4g are effective anticancer applicants on hormone-dependent instead of hormone-independent MCF-7. Consequently, it was found that the triazolopyrimidine derivatives could be studied for further biological investigation. Moreover, the multicomponent method is promising in the synthesis of many additional heterocyclic compounds.