One-Pot Multicomponent Synthesis and Cytotoxic Evaluation of Novel 7-Substituted-5-(1H-Indol-3-yl)Tetrazolo[1,5-a] Pyrimidine-6-Carbonitrile

A series of novel 7-substituted-5-(1H-indol-3-yl)tetrazolo[1,5-a]pyrimidine-6-carbonitrile was synthesized via a one-pot, three-multicomponent reaction of appropriate aldehydes, 1H-tetrazole-5-amine and 3-cyanoacetyl indole in catalytic triethylamine. The cytotoxic activity of the new synthesized tetrazolopyrimidine-6-carbonitrile compounds was investigated against HCT-116, MCF-7, MDA-MB-231, A549 human cancer cell lines and one human healthy normal cell line (RPE-1) using the MTT cytotoxicity assay. Compounds 4h, 4b, 4c, 4i and 4a showed potent anticancer activities against human colon cancer. Additionally, all the compounds showed potent anticancer activities on human lung cancer.

To improve the reaction condition, the classical MCRs were carried out in different solvents and alkali at an appropriate temperature. The MCRs were mainly performed in ethanol with no catalyst, which furnishes no products, even though it was refluxed for 48 h (entry 1, Table 1). Next, we have done the MCRs in catalytic Et3N in ethanol, acetonitrile, 1,4-dioxane, toluene and dimethylformamide (DMF) (entries 2-6, Table 1). Concerning the classification of the solvent, the greatest products are attained through DMF. Subsequently, we examined the MCRs in the existence of basic catalysts, for example, piperidine, 4-(dimethylamino)pyridine (DMAP), pyridine, KOH, NaOH and K2CO3 (entries 7-12, Table 1).
To improve the reaction condition, the classical MCRs were carried out in different solvents and alkali at an appropriate temperature. The MCRs were mainly performed in ethanol with no catalyst, which furnishes no products, even though it was refluxed for 48 h (entry 1, Table 1). Next, we have done the MCRs in catalytic Et 3 N in ethanol, acetonitrile, 1,4-dioxane, toluene and dimethylformamide (DMF) (entries 2-6, Table 1). Concerning the classification of the solvent, the greatest products are attained through DMF. Subsequently, we examined the MCRs in the existence of basic catalysts, for example, piperidine, 4-(dimethylamino)pyridine (DMAP), pyridine, KOH, NaOH and K 2 CO 3 (entries 7-12, Table 1).
Products exhibited that organic bases had a respectable catalytic result, but inorganic strong alkali (KOH, NaOH) released the less catalytic outcome. However, the inorganic weak base (K 2 CO 3 ) could not supply target products. Finally, the best situation was found in this technique in DMF as a solvent, and 25 mol % triethylamine as a catalyst at 120 • C, for 10 h (Table 1). The results were recorded in Table 1.   The construction of targets 4a-i were recognized by the study of spectral data and mass analysis (Experimental part) as characterized for structure 4a: the IR of 4a unveiled absorptions at 3397 and 2223 cm −1 representative -NH-and cyano groups, respectively, but the amino (-NH2) and carbonyl (-C=O) signals are lost. Our 1 H NMR chart revealed a broad singlet at δ 12.23 of -NH groups (D 2 O exchangeable) and aromatic signals at δ 7.21-8. 43. The 13 C NMR spectrum and mass spectra also supported the structural assignment (m/z 371.80, M + ).
From the above results, a probable mechanism is suggested for the production of 7-substituted-5-(1H-indol-3-yl)tetrazolopyrimidine-6-carbonitrile in the presence of triethylamine (TEA). Firstly, TEA initiates aldehydes 1 through hydrogen binding to start the nucleophilic addition of 3-cyanoacetyl indole 3. Activation of the starting aldehydes by hydrogen bonding increases the electrophilicity of the aldehyde and supports the production of the corresponding intermediate A (Knoevenagel product) [47] with compound 3. This adduct undergoes a Michael type addition reaction with 1H-tetrazol-5-amine 2 to yield an adduct B intermediate. After that, intermediate B underwent intramolecular cyclization leading to the C-N bond formation followed by the auto-oxidation leading to the formation product 4, Scheme 2. The construction of targets 4a-i were recognized by the study of spectral data and mass analysis (Experimental part) as characterized for structure 4a: the IR of 4a unveiled absorptions at 3397 and 2223 cm −1 representative -NH-and cyano groups, respectively, but the amino (-NH2) and carbonyl (-C=O) signals are lost. Our 1 H NMR chart revealed a broad singlet at δ 12.23 of -NH groups (D2O exchangeable) and aromatic signals at δ 7.21-8. 43. The 13 C NMR spectrum and mass spectra also supported the structural assignment (m/z 371.80, M + ).
From the above results, a probable mechanism is suggested for the production of 7-substituted-5-(1H-indol-3-yl)tetrazolopyrimidine-6-carbonitrile in the presence of triethylamine (TEA). Firstly, TEA initiates aldehydes 1 through hydrogen binding to start the nucleophilic addition of 3cyanoacetyl indole 3. Activation of the starting aldehydes by hydrogen bonding increases the electrophilicity of the aldehyde and supports the production of the corresponding intermediate A (Knoevenagel product) [47] with compound 3. This adduct undergoes a Michael type addition reaction with 1H-tetrazol-5-amine 2 to yield an adduct B intermediate. After that, intermediate B underwent intramolecular cyclization leading to the C-N bond formation followed by the autooxidation leading to the formation product 4, Scheme. 2.

Cytotoxicity Screening
Nine compounds 4a-i were screened in vitro on HCT-116, MCF-7, MDA-MB-231 and A549 human cancer cells, as well as RPE-1 human normal cells via the MTT assay. The measurements of viable cells were considered and compared to the control. Activities of these compounds on the five human cell lines were compared to that of doxorubicin or 5-fluorouracil as reference drugs. Compounds 4a-i suppressed the five human cell types in a dose-dependent manner (Figures 3-7). Regarding HCT-116, both Figure 3 and Table 2 display that three compounds (4b, 4c and 4h, respectively) had more potent cytotoxic activities compared to that of doxorubicin.
In addition, two compounds (4i and 4a, respectively) had comparable cytotoxic activities and the rest of the compounds had less cytotoxic activities compared to doxorubicin. In the case of MCF-7 cells, two tested compounds (4e and 4c, respectively) had slightly less anticancer activities compared to that of doxorubicin ( Figure 4 and Table 2); the rest of the compounds had significantly less cytotoxic effect on MCF-7 cancer cells compared to that of the reference drug. In case of MDA-MB-231, all investigated compounds had significantly less anticancer effects compared to that of the reference drug 5-fluorouracil ( Figure 5 and Table 2). In the case of A549 cancer cells, six compounds (4a, 4b, 4d, 4c, 4e and 4f, respectively) had more potent cytotoxic activities; three compounds (4g, 4i and 4h, respectively) had comparable cytotoxic effect compared to that of doxorubicin ( Figure 6 and Table 2). In the case of RPE-1 human normal cells, four compounds (4d, 4c, 4e and 4b, respectively) showed more cytotoxic effect; four compounds (4g, 4a, 4f and 4h, respectively) had comparable cytotoxic effect, and one compound (4i) had significantly less cytotoxic activities compared to doxorubicin. The IC 50 s indicated in Table 2 reveal that all the nine compounds have strong cytotoxic effect on cancer cells rather than on normal cells.
Furthermore, From the above-mentioned results, one can conclude that, compounds 4b, 4c, 4h, 4i and 4a are potent anticancer candidate drugs on the human colon cancer, respectively; all the nine compounds are potent anticancer candidate drugs on the human lung cancer; compounds 4c, 4e and 4h are specifically good anticancer candidate drugs on hormone-dependent human breast cancer rather than on the hormone-independent human breast cancer.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 13 In addition, two compounds (4i and 4a, respectively) had comparable cytotoxic activities and the rest of the compounds had less cytotoxic activities compared to doxorubicin. In the case of MCF-7 cells, two tested compounds (4e and 4c, respectively) had slightly less anticancer activities compared to that of doxorubicin ( Figure 4 and Table 2); the rest of the compounds had significantly less cytotoxic effect on MCF-7 cancer cells compared to that of the reference drug. In case of MDA-MB-231, all investigated compounds had significantly less anticancer effects compared to that of the reference drug 5-fluorouracil ( Figure 5 and Table 2). In the case of A549 cancer cells, six compounds (4a, 4b, 4d, 4c, 4e and 4f, respectively) had more potent cytotoxic activities; three compounds (4g, 4i and 4h, respectively) had comparable cytotoxic effect compared to that of doxorubicin ( Figure 6 and Table 2). In the case of RPE-1 human normal cells, four compounds (4d, 4c, 4e and 4b, respectively) showed more cytotoxic effect; four compounds (4g, 4a, 4f and 4h, respectively) had comparable cytotoxic effect, and one compound (4i) had significantly less cytotoxic activities compared to doxorubicin. The IC50s indicated in Table 2 reveal that all the nine compounds have strong cytotoxic effect on cancer cells rather than on normal cells.
Furthermore, From the above-mentioned results, one can conclude that, compounds 4b, 4c, 4h, 4i and 4a are potent anticancer candidate drugs on the human colon cancer, respectively; all the nine compounds are potent anticancer candidate drugs on the human lung cancer; compounds 4c, 4e and 4h are specifically good anticancer candidate drugs on hormone-dependent human breast cancer rather than on the hormone-independent human breast cancer.     In addition, two compounds (4i and 4a, respectively) had comparable cytotoxic activities and the rest of the compounds had less cytotoxic activities compared to doxorubicin. In the case of MCF-7 cells, two tested compounds (4e and 4c, respectively) had slightly less anticancer activities compared to that of doxorubicin ( Figure 4 and Table 2); the rest of the compounds had significantly less cytotoxic effect on MCF-7 cancer cells compared to that of the reference drug. In case of MDA-MB-231, all investigated compounds had significantly less anticancer effects compared to that of the reference drug 5-fluorouracil ( Figure 5 and Table 2). In the case of A549 cancer cells, six compounds (4a, 4b, 4d, 4c, 4e and 4f, respectively) had more potent cytotoxic activities; three compounds (4g, 4i and 4h, respectively) had comparable cytotoxic effect compared to that of doxorubicin ( Figure 6 and Table 2). In the case of RPE-1 human normal cells, four compounds (4d, 4c, 4e and 4b, respectively) showed more cytotoxic effect; four compounds (4g, 4a, 4f and 4h, respectively) had comparable cytotoxic effect, and one compound (4i) had significantly less cytotoxic activities compared to doxorubicin. The IC50s indicated in Table 2 reveal that all the nine compounds have strong cytotoxic effect on cancer cells rather than on normal cells.
Furthermore, From the above-mentioned results, one can conclude that, compounds 4b, 4c, 4h, 4i and 4a are potent anticancer candidate drugs on the human colon cancer, respectively; all the nine compounds are potent anticancer candidate drugs on the human lung cancer; compounds 4c, 4e and 4h are specifically good anticancer candidate drugs on hormone-dependent human breast cancer rather than on the hormone-independent human breast cancer.      In addition, two compounds (4i and 4a, respectively) had comparable cytotoxic activities and the rest of the compounds had less cytotoxic activities compared to doxorubicin. In the case of MCF-7 cells, two tested compounds (4e and 4c, respectively) had slightly less anticancer activities compared to that of doxorubicin ( Figure 4 and Table 2); the rest of the compounds had significantly less cytotoxic effect on MCF-7 cancer cells compared to that of the reference drug. In case of MDA-MB-231, all investigated compounds had significantly less anticancer effects compared to that of the reference drug 5-fluorouracil ( Figure 5 and Table 2). In the case of A549 cancer cells, six compounds (4a, 4b, 4d, 4c, 4e and 4f, respectively) had more potent cytotoxic activities; three compounds (4g, 4i and 4h, respectively) had comparable cytotoxic effect compared to that of doxorubicin ( Figure 6 and Table 2). In the case of RPE-1 human normal cells, four compounds (4d, 4c, 4e and 4b, respectively) showed more cytotoxic effect; four compounds (4g, 4a, 4f and 4h, respectively) had comparable cytotoxic effect, and one compound (4i) had significantly less cytotoxic activities compared to doxorubicin. The IC50s indicated in Table 2 reveal that all the nine compounds have strong cytotoxic effect on cancer cells rather than on normal cells.
Furthermore, From the above-mentioned results, one can conclude that, compounds 4b, 4c, 4h, 4i and 4a are potent anticancer candidate drugs on the human colon cancer, respectively; all the nine compounds are potent anticancer candidate drugs on the human lung cancer; compounds 4c, 4e and 4h are specifically good anticancer candidate drugs on hormone-dependent human breast cancer rather than on the hormone-independent human breast cancer.

Structure-Activity Relationship
Generally, tetrazolopyrimidine unit have interesting biological activities. All synthesized compounds contain a tetrazolopyrimidine unit based on the indole ring at the 5-position. Compound 4b substituted with 4-nitrophenyl at 7-position has the best anticancer activities against human colon cancer followed by compound 4h which contains the pyrrole ring at the 7-position. In the case of MCF-7 cells, both compound 4e substituted with 2-nitrophenyl at the 7-position and 4c which contains a 4-bromophenyl ring at 7-had slightly less anticancer activities compared to that of doxorubicin. Compound 4a substituted with 4-chlorophenyl at the 7-position has the best anticancer activities against A549 cancer cells. In the case of RPE-1 human normal cells, compound 4d substituted with 2-hydroxyphenyl, followed by compound 4c substituted with 4-bromophenyl, respectively) showed more cytotoxic effect.

Structure-Activity Relationship
Generally, tetrazolopyrimidine unit have interesting biological activities. All synthesized compounds contain a tetrazolopyrimidine unit based on the indole ring at the 5-position. Compound 4b substituted with 4-nitrophenyl at 7-position has the best anticancer activities against human colon cancer followed by compound 4h which contains the pyrrole ring at the 7-position. In the case of MCF-7 cells, both compound 4e substituted with 2-nitrophenyl at the 7-position and 4c which contains a 4-bromophenyl ring at 7-had slightly less anticancer activities compared to that of doxorubicin. Compound 4a substituted with 4-chlorophenyl at the 7-position has the best anticancer activities against A549 cancer cells. In the case of RPE-1 human normal cells, compound 4d substituted with 2-hydroxyphenyl, followed by compound 4c substituted with 4-bromophenyl, respectively) showed more cytotoxic effect.

Structure-Activity Relationship
Generally, tetrazolopyrimidine unit have interesting biological activities. All synthesized compounds contain a tetrazolopyrimidine unit based on the indole ring at the 5-position. Compound 4b substituted with 4-nitrophenyl at 7-position has the best anticancer activities against human colon cancer followed by compound 4h which contains the pyrrole ring at the 7-position. In the case of MCF-7 cells, both compound 4e substituted with 2-nitrophenyl at the 7-position and 4c which contains a 4-bromophenyl ring at 7-had slightly less anticancer activities compared to that of doxorubicin. Compound 4a substituted with 4-chlorophenyl at the 7-position has the best anticancer activities against A549 cancer cells. In the case of RPE-1 human normal cells, compound 4d substituted with 2-hydroxyphenyl, followed by compound 4c substituted with 4-bromophenyl, respectively) showed more cytotoxic effect.

General Methods
All chemicals were purchased from the Merck and Sigma-Aldrich chemical companies (Muskegon, MI, USA) and used without further purification. Melting points were determined on XT-5 microscopic melting-point apparatus (Veb Analytik, Dresden, Germany) and were uncorrected. Infrared (IR) spectra were recorded on the FT Bruker Tensor 27 spectrometer ((Jasco, Easton, PA, USA). Proton nuclear magnetic resonance ( 1 H NMR) and carbon-13 (C13) nuclear magnetic resonance ( 13 C NMR) spectra were obtained from solution in dimethyl sulfoxide (DMSO)-d 6 using a Bruker-400 spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were recorded on AccuTOF LC-Plus from (Jeol, Tokyo, Japan). All melting points were determined using open capillaries on an Electrothermal-9100 (Veb, Analytik, Dresden, Germany) instrument and are uncorrected.

In-Vitro Cytotoxic Activity
Cell cultures of human colorectal carcinoma (HCT)-116, MCF-7 (hormone-dependent human breast adenocarcinoma), MDA-MB-231 (hormone-independent human breast adenocarcinoma), A549 (human lung carcinoma) and human normal Retina pigmented epithelium (RPE)-1 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's modified Eagle medium (DMEM) medium which was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin. The cells were grown at 37 • C in a humidified atmosphere of 5% CO 2 .

MTT Cytotoxicity Assay
The cytotoxicity activity on HCT-116, MCF-7, MDA-MB-231 and A549 human cancer cell lines as well as RPE-1 human normal cells was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in viable cells [48][49][50]. Cells were dispensed in 96-well sterile microplates (5 × 10 4 cells/well), and incubated at 37 • C with a series of different concentrations, in DMSO, of each tested compound or Doxorubicin (positive control) for 48 h in a serum free medium prior to the MTT assay. After incubation, media were carefully removed, 40 µL of MTT (2.5 mg/mL) were added to each well and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 570 nm using a Spectra Max Paradigm Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. The relative cell anti-proliferative was measured according to the following equation: % cytotoxicity = (1 − As/Ab) × 100. Where; As = Absorbance of each sample and Ab = Absorbance of the blank. All experiments were conducted in triplicate and repeated on three different days. All the values were represented as mean ± SD. IC 50 s were determined by probit analysis by SPSS Inc probit analysis (IBM Corp., Armonk, NY, USA).

Conclusions
Briefly, we have a conventional three-constituent reaction to the construction of a completely substituted new series of tetrazolopyrimidine-6-carbonitrile based on indole moiety in the existence of triethylamine as a catalyst and DMF as a solvent.
The significance of this process is important over the additional usual ones, by short times, respectable yields, trivial conditions, cost-effective and relaxed management. Cytotoxic evaluation of novel series of 7-substituted-5-(1H-indol-3-yl)tetrazolopyrimidine-6-carbonitrile was investigated against HCT-116, MCF-7, MDA-MB-231 and A549 human cancer cell lines using the MTT test. From the results indicated in this work, one can conclude that compounds 4h, 4b, 4c, 4i and 4a had potent anticancer activities against human colon cancer, respectively; all the nine compounds had potent anticancer activities on human lung cancer as well.