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

INTRODUCTION

The construction of both scaffolds triazole and pyrimidine rings with a built-in indole moiety might have promising biological activity. Multicomponent reactions (MCRs) are a wide range of synthetic techniques that can be used to achieve this target candidate (Cioc et al., 2014; Dekamin et al., 2013; Liu et al., 2016; Peng et al., 2017; Ravichandiran et al., 2017; Rotstein et al., 2014). Compared to the direct synthesis method, the MCR method is appreciated for its cost-effectiveness, ease of set-up, and high production. Currently, many nitrogen heterocyclic structures (mainly indole units with multiple biological activities) are prepared by MCRs (Allen et al., 2017; Anand et al., 2017; Carbajales et al., 2017; Gribble, 2000). The unique indole backbone is considered to be the most widespread compound that is common in medical advances. Similarly, an indole is a substructure of many natural medical activities (Gribble, 2010). In addition, several indole compounds have attracted more consideration owing to their biological properties, such as their antimicrobial, anticancer, and antiviral activities (Bao et al., 2005; Endo et al., 2007; Sakemi and Sun, 1991; Yang et al., 2004). In such an indole, a framework, triazole (Duran et al., 2002; Gujjar et al., 2009; Holla et al., 2005; Sztanke et al., 2008) and pyrimidine (Kim et al., 2012; Selvaraj and Rajesh, 2016) ring, is well identified as the key structure of several therapeutic scaffolds as they display diverse bioactivities. Fused heterocycles are widely considered due to their share in the different steps of drug discovery, and they often function as basic central units in many therapeutic chemistry programs (Coxon et al., 2017; Taylor et al., 2014). Among them, the triazolopyrimidinone unit, a purine analog, is well known for displaying some biological activities, such as anti-inflammatory activity, ulcerogenic properties, serotonin antagonist, analgesic, antimicrobial, antifungal, cytotoxic, and antitumor activities (Boechat et al., 2012; Hafez et al., 2008; Huang et al., 2012; Lakamoska et al., 2009; Wang et al., 2017; Zhang et al., 2007; Zhao et al., 2007). Moreover, triazolopyrimidines have the greatest valuable construction base for the synthesis of bioactive drugs (Fig. 1) (Łakomska et al., 2013), which possess many pharmacological measures, such as antitumor (Marwaha et al., 2012), antimalarial (Luo et al., 2013), antimicrobial, (Abdel-Aziem et al., 2012), and anti-inflammatory activities (Khera et al., 2011), inhibition of the kinase insert domain-covering receptor (kill/death ratio kinase) (Ashour et al., 2013), and antifungal properties (Chen et al., 2008). They are known to show anticancer, anti-Alzheimer’s, antihypertensive, leishmanicidal cardiac stimulant, antipyretic, potential herbicidal, and anti-HBV activities (Gujjar et al., 2009). The previous biological history of triazolopyrimidine is evident in Trapidil (Fischer, 2008) and Cevipabulin (El-Gendy et al., 2008). Recently, triazolopyrimidines have been realized to modify AChE inhibition, amyloid βaggregation inhibition, anti-inflammation, and are noted as a probable anti-Alzheimer’s agent (Kumar et al., 2016). Generally, heterocyclic compounds have many applications (Adole et al., 2019, 2020; Bisht et al., 2018; Chobe et al., 2014; Kamble et al., 2016). As an extension of our work regarding the direction on the synthesis of bioactive indolyl molecules (El-Nezhawy et al., 2016, 2009a, 2009b; Fakhr et al., 2004, 2009; Ghorab et al., 2008; Muhammad et al., 2019; Radwan and El-Sherbiny, 2007; Radwan and Eman, 2009; Radwan et al., 2007; Radwan et al., 2009a, 2009b, 2020) (Fig. 2), we describe herein a multicomponent synthesis and antitumor estimation of the novel triazolopyrimidine-6-carbonitrile derivatives via the reaction of 3-aminotriazole, suitable aldehydes, and 3-cyanoacetyl indole.

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. ¹H and ¹³C nuclear magnetic resonance (NMR) were carried out in dimethyl sulfoxide (DMSO-d₆) on a Bruker (400 MHz) Ascend - Magnets | Bruker spectrometer.

Figure 1. Biological activity of some of the triazolopyrimidine compounds. [Click here to view]

Figure 2. Some of our previous and present works bearing indole moiety. [Click here to view]

5,7-di(1H-Indol-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carbonitrile (4i)

Yield 61%; mp 296°C–8°C. IR (KBr, cm⁻¹): 3,236–3,265 (2NH), 2,206(CN). ¹H NMR (DMSO-d₆) δ/ppm: 7.31–7.35 (m, 4H, di-indole H5, H6), 7.48 (dd, J = 1.1, 8.1 Hz, 2H, di-indole H7), 8.01–8.02 (dd, J = 1.1, 8.1 Hz, 2H, di-indole H4), 8.18 (s, 1H, triazole), 8.31, 8.32 (ss, 2H, indole-H2), 12.21 (s, NH), 12.23 (s, NH); ¹³C NMR (DMSO-d₆) δ/ppm: 152.06, 151.52 137.81, 137.74, 135.31, 132.33, 131.10, 128.24, 127.03, 124.95, 124.61, 123.47, 122.31, 118.39, 114.47, 112.23, 111.16; MS (EI) m/z (%) 375 [M⁺]. Anal. calcd. for C₂₂H₁₃N₇: C, 70.39; H, 3.49; N, 26.12. Found: C, 70.45; H, 3.42; N, 26.10.

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% CO₂.

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 (Ashraf et al., 2017; Emam et al., 2017; Eman et al., 2017). Cells were first distributed in a 96-well sterile microplate (2 × 10⁴ 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 (ED₅₀) 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).

Scheme 1. Synthesis of triazolopyrimidine under different conditions of base, solvent, temperature, and time. [Click here to view]

RESULTS AND DISCUSSION

Chemistry

The strategy in the presented work is the synthesis of a new series of triazolopyrimidine derivatives via an MCR of proper carboxaldehydes 1, 3-amino-1,2,4-triazole 2, and 3-indolyl-3-oxopropanenitrile 3 (Scheme 1 and Scheme 2).

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, K₂CO₃ 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 exposed bands at 3,312 and 2,212 cm⁻¹ for the NH and cyano functions, respectively, along with the disappearance of NH₂ and C=O groups. Furthermore, ¹H NMR showed a broad peak (exchangeable D₂O) at 12.27 ppm of the −NH group, a single peak at 8.20 ppm of the triazole proton, and aromatic signals at 7.23–8.43 ppm. Also, ¹³C NMR data and mass reinforced a proposed structure for 4a (m/z 370, M+).

Scheme 2. Reaction conditions: aldehyde 1 (1 mmol), 3-amino-1,2,4-triazole 2 (1 mmol), 3-cyanoacetyl indole 3 (1 mmol), and Et3N (0.25 mmol), in DMF at 120°C, 10 hours. [Click here to view]

Table 1. Optimization of the reaction conditions for the synthesis of triazolopyrimidine derivatives 4a. [Click here to view]

Scheme 3. Proposed mechanism of three-component reactions. [Click here to view]

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.

Figure 3. Dose-dependent antiproliferative effects of 4a–4i compounds on HCT-116 cancer cells via the MTT assay. [Click here to view]

Figure 4. Dose-dependent antiproliferative effects of 4a-4i compounds on MCF-7 cancer cells via the MTT assay. [Click here to view]

Figure 5. Dose-dependent antiproliferative effects of 4a–4i compounds on MDA-MB-231 cancer cells via the MTT assay. [Click here to view]

Figure 6. Dose-dependent antiproliferative effects of 4a–4i compounds on A549 cancer cells via the MTT assay. [Click here to view]

Figure 7. Dose-dependent toxic effects of 4a–4i compounds on RPE-1 human normal cells via the MTT assay. [Click here to view]

Table 3. TI of the compounds’ activities on the four cancer types relative to the normal cells according to the results in Table 2. [Click here to view]

Table 2. EC50 of the compounds on the four human cancer types and the TD50 on the noncancerous human normal cells via the MTT assay. [Click here to view]

The therapeutic index (TI) of the compounds, which was calculated by dividing their efficacious dose for 50% of the cancerous cells population (ED₅₀) 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.

ACKNOWLEDGMENTS

We express our appreciation to the National Research Centre and Qassim University for the services provided.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FINANCIAL SUPPORT AND SPONSORSHIP

None.

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