Three Component One-Pot Synthesis and Antiproliferative Activity of New [1,2,4]Triazolo[4,3-a]pyrimidines

Abstract

A series of new [1,2,4]triazolo[4,3-a]pyrimidine derivatives was prepared using a one-pot three-component synthesis from 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate. The compound structures were confirmed by IR, ¹H-NMR, ¹³C-NMR, HRMS and X-ray analyses. The biological activity of these compounds as antitumor agents was evaluated. Their antitumor activities against cancer cell lines (MDA-MB-231 and MCF-7) were tested by the MTT in vitro method. Among them, compounds 4c and 4j displayed the best antitumor activity with IC₅₀ values of 17.83 μM and 19.73 μM against MDA-MB-231 and MCF-7 cell lines, respectively, compared to the Cisplatin reference.

1. Introduction

Heterocyclic compounds have received special attention in organic chemistry due to their presence in many natural products and their diverse biological properties [1]. Among them, pyrimidines and their derivatives occupy an important place due to their biological importance for medicinal chemistry [2,3,4].

A review of the literature also revealed that substituted 1,2,4-triazoles and their bridged heterocyclic derivatives have attracted particular attention over the last two decades due to their wide range of therapeutic properties [5]. Fusion of the 1,2,4-triazole ring with the pyrimidine ring gives rise to the formation of bicyclic heterocycles called 1,2,4-triazolopyrimidines (TPs), where the common nitrogen of the triazole and pyrimidine occupy the ring junction. These triazole-fused pyrimidines have been the subject of increasing interest for their various important pharmaceutical properties [6,7,8]. They appear in many synthetic pharmacophores that possess antiparasitic, antimicrobial, anticancer and antibiotic activities [9,10,11]. In particular, many triazolo[4,3-a]pyrimidines exhibited antiproliferative effects against HePG-2 and MCF-72a cell lines [12] and cytotoxic activity on a human hepatic carcinoma cell line (HEPG2) [13]. For example, 1,2,4-triazolo[4,3-a]pyrimidines A and B were reported to show antibacterial activity [14,15], and compound B showed higher DNA photocleavage activity [16] and has been described as a potent apoptotic inducer [17] (Figure 1).

Note also that the use of triazolopyrimidines in drug design has recently been reported [18]. In addition, this class of compounds has also been of great importance in the field of agriculture due to their remarkable activities as herbicides [19]. Consequently, TPs have inspired excellent reviews in the literature over the years on their chemistry and synthetic methods [20,21,22,23,24].

Despite the many syntheses described in the literature of 1,2,4-triazolo[1,5-a]pyrimidines, only a limited number of syntheses of [1,2,4]triazolo[4,3-a]pyrimidines have been reported. Generally, the most commonly used syntheses are cyclization of 3-ethoxycarbonyl-2-hydrazinylpyrimidines [25]; or by reactions of 2-hydrazinylpyrimidines with different reagents such as benzoyl chloride, CS2, formic acid [12], acetic anhydride, ethyl chloroformate and triethyl orthoformate [13]; or by oxidation of 2-(2-benzylidenehydrazinyl)-pyrimidines with Br₂ in AcOH [26]; or by the reaction of 3,4-dihydropyrimidine-2(1H)-thiones with ethyl 2-(arylhydrazinylidene)-2-haloacetate [27]. However, the reported methods generally suffer from some drawbacks, such as harsh reaction conditions and little flexibility offered. Therefore, the development of an efficient and economical method for the synthesis of [1,2,4]triazolo[4,3-a]pyrimidines involving available starting materials is highly desired.

In the continuity of the syntheses developed within our group using condensed pyrazoles, which have proven to be of biological interest [28,29,30,31], we report herein the three-component synthesis of [1,2,4]triazolo[4,3-a]pyrimidines in a one-pot manner and preliminary results for in vitro antiproliferative evaluation. Due to their flexibility to assemble several reagents in a short time and transform them into compounds of interest in a one-pot procedure [32,33], multi-component reactions (MCRs) have become very popular in the development of new biologically active compounds. Additionally, MCRs are favored for their experimental simplicity, their atom-/step economy, their reduced reaction time and the high yields produced compared to classical chemistry [34]. Note that syntheses of some condensed triazolopyrimidine derivatives using multicomponent one-pot methods have also been reported [35,36].

2. Results and Discussion

2.1. Chemistry

In our initial investigation, we sought to optimize the conditions for this multicomponent reaction using aminotriazole 1a, benzaldehyde 2a, and ethyl acetoacetate as starting reagents for the reaction model, by varying the catalyst and the solvent (Table 1).

In our first attempts, we performed the reaction using 10 mol% of paratoluene sulphonic acid (APTS) as a catalyst and H₂O as a solvent under reflux for 24 h, but only traces of the desired product 4a were observed (Table 1, entry 1). Interestingly, when water was replaced by ethanol at reflux, the desired product 4a was isolated in 75% yield (Table 1, entry 2). Under the same conditions but replacing APTS with HCl, the expected product 4a was formed in a low yield (45%) (Table 1, entry 3). This decrease in yield was even more pronounced when acetic acid was used as a catalyst (Table 1, entry 4). The use of a basic catalyst such as piperidine proved unsuccessful (Table 1, entry 5). In the absence of any solvent and catalyst, and by heating until the reaction mixture had completely melted, the desired product 4a was isolated with a yield not exceeding 10% (Table 1, entry 7). A reduction in the reaction time to 18 h at the reflux of ethanol in the presence of 10 mol% of APTS resulted in an incomplete conversion rate. The use of a polar aprotic solvent such as acetonitrile did not give complete satisfaction since the yield of the isolated product 4a was only 4 50% (Table 1, entry 6). Consequently, following the results recorded in Table 1, the best conditions for obtaining compound 4a with the best yield were the use of APTS as a catalyst (10 mol%) at the reflux of ethanol for 24 h (Table 1, entry 2).

Based on the optimized reaction conditions, a series of new ethyl multisubstituted-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylates 4a–n were therefore synthesized using 5-amino-1-phenyl-1H-1,2,4-triazoles 1a–b, aromatic aldehydes 2a–g and ethyl acetoacetate 3 (Scheme 1).

As illustrated in Scheme 1, the three-component reaction is compatible with a wide range of aromatic aldehydes containing electron donating or electron withdrawing substituents. Indeed, aldehydes carrying electron donor groups such as CH₃, C₂H₅ or electron withdrawing groups such as Cl and F were easily condensed on 5-aminotriazoles 1a–b in the presence of ethyl acetoacetate, providing access directly to the desired compounds in good to excellent yields. In addition, this reaction was also compatible with aldehydes substituted by a strongly electron-donating group such as OCH₃ or a strongly electron-withdrawing group such as NO₂. Likewise, the use of an aldehyde substituted by two chlorine atoms on the aromatic ring successfully led to the desired products 4g and 4n with respective yields of 85 and 80%.

As also observed, the efficiency of this multicomponent reaction was not affected by the nature of these substituents. Similarly, there was no significant difference in the yields of the reactions depending on the nature of the substituents, either on the aminotriazole or on the aromatic aldehyde.

Based on the above results and drawing inspiration from the results reported in the literature [37,38], we hypothesized that the mechanism of the three-component one-pot reaction could be as follows (Scheme 2).

The reaction begins with an attack of the pi doublet of the enol form of ethyl acetoacetate 3 on the carbonyl of the aromatic aldehyde leading subsequently, after the elimination of a molecule of water, to the corresponding arylidene according to a known Knoevenagel-type reaction. After that, the reaction can proceed along two different pathways:

In pathway a, there is a 1,4-Michael-type addition of a doublet of the amino group of aminotriazole 1 on the most electrophilic carbon of the arylidene, leading to a reaction intermediate, which undergoes intramolecular lactamization followed by dehydration to lead to compound 4′.

In pathway b, it is the doublet of nitrogen 4 of 5-amino-1-phenyl-1H-1,2,4-triazole 1 that attacks the electrophilic carbon of the arylidene, which, after intramolecular cyclization followed by dehydration, leads to isomer 4. This regioselectivity of the reaction appears logical insofar as the Michael addition involves the most nucleophilic nitrogen atom of the aminotriazole 1.

To elucidate the real structures of the synthesized compounds 4a–n, we carried out a 2D NMR HMBC sequence on compound 4f, since one-dimensional NMR does not make it possible to decide between these two isomers of position 4′ and 4. We observed on the one hand a correlation between the C3 carbon and the H5 proton, and on the other hand a correlation between C5 and the methyl group protons attached to the C7 carbon (see Figure S27 in the Supplementary Materials), thus proving the unique formation of isomer 4.

In addition to evidence from 2D NMR spectroscopic analysis, X-ray crystallographic analyses of single crystals of compounds 4a and 4f strongly confirmed the structures of the cyclo-condensation compounds 4a–n (see Figure S2 and the Supplementary Materials for X-ray data of 4a and 4f). The ORTEP diagrams shown in Figure 2 clearly show the formation of type 4 compounds, thus unambiguously confirming the high regioselectivity of this reaction [39].

Next, the biological potential of the synthesized compounds 4a–n as antitumor agents was subsequently evaluated.

2.2. Biological Activity: Anticancer Activity In Vitro

All target compounds 4a–n were evaluated for their antitumor activity in vitro by the MTT method. The IC₅₀ values against MDA-MB-231 and MCF-7 (human breast cancer cell lines) are summarized in Table 2.

Table 2 shows that the synthesized triazolopyrimidines 4a–n exhibited inhibition of the proliferative activity of MDA-MB-231 and MCF-7 cancer cells. These compounds make it possible to improve the efficacy of the antitumor response compared with Cisplatin, particularly as the most potent inhibitory molecules are those that have a more significant decrease in IC₅₀ values. These values are 17.83 μM and 20.33 μM for compound (4c) bearing R = CH₃, R₁ = H, R₂ = CH₃O, and 20.97 μM and 19.73 μM for compound (4j) bearing R = CH₂-CH₃, R₁ = H, R₂ = CH₃O, respectively, _P2 times and 4 times more potent than Cisplatin (one of the most effective FDA-approved treatments for breast cancer [40]). It appears, in this case, that the presence of the CH₃O moiety in the aryl group increases antitumoral activity.

The most potent compounds (4c and 4j) will be the subject of more biological experiments leading to protein target identification and guided by docking studies in order to determine the mechanisms that induced tumor cell death. An entire investigation of cell death and the cell cycle of treated cells is in progress.

Figure 3 and Figure 4 present histograms showing the effect of the synthesized triazolopyrimidine compounds 4a–n on the cell proliferation of the MDA-MB-231 and MCF-7 lines for 48 h.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

All thin layer chromatography (TLC) analyses were performed on type 60 F 254 silica gel n silica-gel-precoated aluminum sheets (Type60 F254, 0.25-mm thickness; from Merck, Darmstadt, Germany) with detection using a UV lamp. Melting points were determined on the Kofler and Buchner bench and were not corrected. Infrared (IR) spectra were recorded using a Perkin Elmer device whose range of precision is from 4000 to 400 cm⁻¹ in powders (dispersed in a KBr pellet). All NMR spectra were recorded using a Brucker AV400 Avance spectrometer (at 400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts are expressed in parts per million (ppm) using TMS as an internal standard in CDCl₃.

5-Amino-1-phenyl-1H-1,2,4-triazole 1a–b was prepared according to the literature [41] by gently refluxing in methanol.

All other reagents and solvents used were analytical grade.

3.1.2. General Procedure for the Synthesis of [1,2,4]triazolo[4,3-a]pyrimidines 4a–n

In a 50 mL flask, (3 mmol) of 5-amino-1-phenyl-1H-1,2,4-triazole 1a–b, (3 × 10⁻³ mol) of aldehyde aromatic 2a–g and (3 × 10⁻³ mol) of ethyl acetoacetate 3 were combined while adding (3 × 10⁻⁴ mol) of APTS as a catalyst in 10 mL of ethanol. The reaction was monitored by TLC with mobile phase ether/hexane (2/1), and after 24 h of heating under reflux, the mixture was left at room temperature. The solid that forms was filtered, washed and then recrystallized with an ethanol/ether mixture.

Ethyl 3,7-dimethyl-1,5-diphenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4a), Yellow powder, yield: 841 mg (75%), Mp 143–145 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1691, ν_C=N: 1640. ¹H NMR (400 MHz, CDCl₃): δ = 1.25 (t, J = 7.1 Hz, 3H, CH₃), 2.19 (s, 3H, CH₃-C3), 2.54 (s, 3H, H10), 4.06–4.16 (m, 2H, O-CH₂), 6.18 (s, 1H, H5), 7.30–7.34 (m, 4H), 7.38–7.40 (m, 2H), 7.48 (t, J = 7.9 Hz, 2H), 8.20 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.6 (C=O), 159.7 (C7), 148.5 (C3), 144.8 (C8a), 142.9, 137.6, 129.0, 128.7, 128.4, 127.2, 126.1, 120.0, 97.9 (C6), 59.5 (O-CH₂), 57.6 (C5), 25.2 (C10), 14.4 (CH₃), 11.1 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₂H₂₃N₄O₂: 375.1821; Found: 375.1812.

Ethyl5-(4-chlorophenyl)-3,7-dimethyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4b), Yellow powder, yield: 796 mg (65%), Mp 141–143 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1685, ν_C=N: 1645. ¹H NMR (400 MHz, CDCl₃): δ = 1.26 (t, J = 6.4 Hz, 3H, CH₃), 2.19 (s, 3H, CH₃-C3), 2.53 (s, 3H, H10), 4.10–4,12 (m, 2H, O-CH₂), 6.16 (s, 1H, H5), 7.29–7.32 (m, 5H), 7.48–7.52 (m, 2H), 8.19 (d, J = 7.4 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.5 (C=O), 159.9 (C7), 148.4 (C3), 144.6 (C8a), 141.4, 137.5, 134.2, 129.0, 128.9, 128.5, 126.2, 120.0, 97.6 (C6), 59.6 (O-CH₂), 57.0 (C5), 25.3 (C10), 14.4 (CH₃), 11.0 (CH_3-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₂H₂₂ClN₄O₂: 409.1431; Found: 409.1424.

Ethyl 5-(4-methoxyphenyl)-3,7-dimethyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4c), Yellow powder, yield: 727 mg (60%), Mp 165–167 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1692, ν_C=N: 1642. ¹H NMR (400 MHz, CDCl₃): δ = 1.25 (t, J = 6.5 Hz, 3H, CH₃ ), 2.20 (s, 3H, CH₃-C3), 2.53 (s, 3H, H10), 3.79 (s, 3H, OCH₃), 4.09–4.13 (m, 2H, O-CH₂), 6.13 (s, 1H, H5), 6.83 (d, J = 7.6 Hz, 2H), 7.31 (d, J = 8.2 Hz, 3H), 7.46–7.49 (m, 2H), 8.19 (d, J = 7.3 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.7 (C=O), 159.5 (C17), 159.3 (C7), 148.4 (C3), 144.8 (C8a), 137.6, 135.5, 129.0, 128.4, 126.0, 120.0, 113.9, 98.2 (C6), 59.5 (O-CH₂), 57.0 (C5), 55.3 (OCH₃), 25.2 (C10), 14.4 (CH₃), 11.1 (CH-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₅N₄O₃: 405.1927; Found: 405.1920.

Ethyl 3,7-dimethyl-1-phenyl-5-(p-tolyl)-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4d), Yellow powder, yield: 780 mg (67%), Mp 143–145 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1688, ν_C=N: 1643.5. ¹H NMR (400 MHz, CDCl₃): δ = 1.25 (t, J = 6.9 Hz, 3H, CH₃), 2.20 (s, 3H, CH₃-C3), 2.33 (s, 3H, CH₃), 2.54 (s, 3H, H10), 4.109–4.13 (m, 2H, O-CH₂), 6.14 (s, 1H, H_-5), 7.12 (d, J = 7.4 Hz, 2H), 7.27 (d, J = 7.4 Hz, 3H), 7.47 (t, J = 7.8 Hz, 2H), 8.19 (d, J = 7.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.6 (C=O), 159.5 (C7), 148.5 (C3), 144.8 (C8a), 140.1, 138.2, 137.7, 129.3, 129.0, 127.0, 126.0, 120.0, 98.1 (C6), 59.4 (O-CH₂), 57.3 (C5), 25.2 (C10), 21.1 (CH₃), 14.4 (CH₃), 11.0 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₅N₄O₂: 389.1978; Found: 389.1970.

Ethyl 3,7-dimethyl-5-(4-nitrophenyl)-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4e), Yellow powder, yield: 1005 mg (80%), Mp 164–166 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1684, ν_C=N: 1644. ¹H NMR (400 MHz, CDCl₃): δ = 1.27 (t, J = 7.1 Hz, 3H, CH₃), 2.19 (s, 3H, CH₃-C3), 2.53 (s, 3H, H10), 4.11–4.17 (m, 2H, O-CH₂), 6.31 (s, 1H, H5), 7.32 (t, J = 7.4 Hz, 1H), 7.49 (t, J = 7.4 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 8.18–8.21 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.3 (C=O), 160.6 (C7), 149.2 (C3), 148.3, 147.8, 144.3 (C8a), 137.4, 129.1, 128.0, 126.4, 124.1, 120.1, 96.9 (C6), 59.8 (O-CH₂), 56.9 (C5), 25.4 (C10), 14.4 (CH₃), 11.0 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₂H₂₂N₅O₄: 420.1672; Found: 420.1664.

Ethyl 5-(4-fluorophenyl)-3,7-dimethyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4f), Yellow powder, yield: 882 mg (75%), Mp 181–183 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1691, ν_C=N: 1647. ¹H NMR (400 MHz, CDCl₃): δ = 1.25 (t, J = 7.0 Hz, 3H, CH₃), 2.20 (s, 3H, CH₃-C3), 2.53 (s, 3H, H10), 4.07–4.17 (m, 2H, O-CH₂ ), 6.17 (s, 1H, H5 ), 7.01 (t, J = 8.4 Hz, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.37 (dd, J = 7.9, 5.5 Hz, 2H), 7.48 (t, J = 7.8 Hz, 2H), 8.19 (d, J = 7.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.5 (C=O), 162.5 (C17), (d, J = 247.5 Hz), 159.7 (C7), 148.4 (C3), 144.6 (C8a), 139.0 (d, J = 3.2 Hz), 137.6, 129.0, 128.9 (d, J = 8.3 Hz), 126.2, 120.0, 115.6 (d, J = 21.7 Hz), 97.9 (C6), 59.5 (O-CH₂), 56.9 (C5), 25.2 (C10), 14.4 (CH₃), 11.0 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₂H₂₂FN₄O₂: 393.1727; Found: 393.1719.

Ethyl 5-(2,4-dichlorophenyl)-3,7-dimethyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4g), Yellow powder, yield: 1129 mg (85%), Mp 179–181 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1687, ν_C=N: 1641. ¹H NMR (400 MHz, CDCl₃): δ = 1.22 (t, J = 7.1 Hz, 3H, CH₃), 2.27 (s, 3H, CH₃-C3), 2.56 (s, 3H, H10), 4.11 (q, J = 7.1 Hz, 2H, O-CH₂), 6.63 (s, 1H, H5 ), 7.23–7.25 (m, 1H), 7.30 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.45–7.50 (m, 3H), 8.16 (d, J = 8.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.2 (C=O), 160.6 (C7), 148.2 (C3), 144.7 (C8a), 139.4, 137.4, 134.8, 131.9, 131.6, 129.2, 129.0, 128.6, 126.3, 120.1, 97.0 (C6), 59.6 (O-CH₂), 53.6 (C5), 25.2 (C10), 14.5 (CH₃), 11.4 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₂H₂₁Cl₂N₄O₂: 443.1042; Found: 443.1034.

Ethyl 3-ethyl-7-methyl-1,5-diphenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4h), Yellow powder, yield: 920 mg (79%), Mp 169–171 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1693, ν_C=N: 1642. ¹H NMR (400 MHz, CDCl₃): δ = 1.21–1.27 (m, 6H, CH₃-C3, CH₃), 2.35–2.42 (m, 1H, CH₂-C3), 2.53 (s, 3H, H10), 2.55–2.63 (m, 1H, CH₂-C3), 4.07–4.16 (m, 2H, O-CH₂), 6.20 (s, 1H, H5 ), 7.26–7.33 (m, 4H), 7.38–7.40 (m, 2H), 7.48 (t, J = 7.9 Hz, 2H), 8.22 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.6 (C=O), 159.7 (C7), 148.9 (C3), 148.6 (C8a), 143.1, 137.7, 129.0, 128.7, 128.3, 127.0, 126.0, 120.1, 97.9 (C6), 59.5 (O-CH₂), 57.4 (C5), 25.2 (C10), 18.7 (CH₂-C3), 14.4 (CH₃), 9.9 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₅N₄O₂: 389.1978; Found: 389.1970.

Ethyl 5-(4-chlorophenyl)-3-ethyl-7-methyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4i), Yellow powder, yield: 861 mg (68%), Mp 148–151 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1691, ν_C=N: 1640. ¹H NMR (400 MHz, CDCl₃): δ = 1.23–1.28 (m, 6H, CH₃-C3, CH₃), 2.34–2.43 (m, 1H, CH₂-C3), 2.52 (s, 3H, H10), 2.54–2.62 (m, 1H, CH₂-C3), 4.08–4.17 (m, 2H, O-CH₂), 6.18 (s, 1H, H5), 7.28–7.34 (m, 5H), 7.48 (t, J = 7.9 Hz, 2H), 8.21 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.5 (C=O), 159.9 (C7), 148.7 (C3), 148.5 (C8a), 141.5, 137.6, 134.2, 129.0, 128.9, 128.4, 126.2, 120.1, 97.6 (C6), 59.6 (O-CH₂), 56.8 (C5), 25.3 (C10), 18.7 (CH₂-C3), 14.4 (CH₃), 9.8 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₄ClN₄O₂: 423.1588; Found: 423.1582.

Ethyl 3-ethyl-5-(4-methoxyphenyl)-7-methyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4j), Yellow powder, yield: 727 mg (58%), Mp 138–140 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1692, ν_C=N: 1648. ¹H NMR (400 MHz, CDCl₃): δ = 1.22–1.27 (m, 6H, CH₃-C3, CH₃), 2.39–2.45 (m, 1H, CH₂-C3), 2.53 (s, 3H, H10), 2.55–2.62 (m, 1H, CH₂-C3), 3.79 (s, 3H, OCH₃), 4.10–4.14 (m, 2H, O-CH₂), 6.15 (s, 1H, H5), 6.83 (d, J = 8.1 Hz, 2H), 7.26–7.31 (m, 3H), 7.48 (t, J = 7.9 Hz, 2H), 8.22 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.7 (C=O), 159.4 (C17), 159.3 (C7), 148.9 (C3), 148.5 (C8a), 137.8, 135.6, 129.0, 128.3, 126.0, 120.0, 113.9, 98.2 (C6), 59.4 (O-CH₂), 56.8 (C3), 55.3 (OCH₃), 25.2 (C10), 18.7 (CH₂-C3), 14.4 (CH₃), 9.9 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₄H₂₇N₄O₃: 419.2083; Found: 419.2075.

Ethyl 3-ethyl-7-methyl-1-phenyl-5-(p-tolyl)-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4k), Yellow powder, yield: 748 mg (62%), Mp 137–139 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1690, ν_C=N: 1645. ¹H NMR (400 MHz, CDCl₃): δ = 1.22–1.28 (m, 6H, CH₃-C3, CH₃), 2.32 (s, 3H, CH₃), 2.40–2.46 (m, 1H, CH₂-C3), 2.53 (s, 3H, H10), 2.55–2.61 (m, 1H, CH₂-C3), 4.05–4.17 (m, 2H, O-CH₂), 6.16 (s, 1H, H5), 7.11 (d, J = 7.4 Hz, 2H), 7.26–7.28 (m, 3H), 7.48 (t, J = 7.9 Hz, 2H), 8.22 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.7 (C=O), 159.5 (C7), 148.9 (C3), 148.6 (C8a), 140.2, 138.1, 137.8, 129.3, 129.0, 126.9, 126.0, 120.1, 98.1 (C6), 59.4 (O-CH₂), 57.1 (C5), 25.2 (C10), 21.1 (CH₃), 18.7 (CH₂-C3), 14.5 (CH₃), 9.9 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₄H₂₇N₄O₂: 403.2134; Found: 403.2127.

Ethyl 3-ethyl-7-methyl-5-(4-nitrophenyl)-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4l), Yellow powder, yield: 1104 mg (85%), Mp 161–163 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1691, ν_C=N: 1643. ¹H NMR (400 MHz, CDCl₃): δ = 1.24–1.29 (m, 6H, CH₃-C3, CH₃), 2.30–2.39 (m, 1H, CH₂-C3), 2.52 (s, 3H, H10), 2.55–2.63 (m, 1H, CH₂-C3), 4.08–4.18 (m, 2H, O-CH₂), 6.32 (s, 1H, H5), 7.31–7.33 (m, 1H), 7.50 (t, J = 7.9 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 8.18–8.22 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.4 (C=O), 160.6 (C7), 149.3 (C3), 148.4 (C8a), 148.4, 147.7, 137.5, 129.1, 127.9, 126.4, 124.1, 120.1, 96.9 (C6), 59.8 (O-CH₂), 56.7 (C5), 25.4 (C10), 18.7 (CH₂-C3), 14.4 (CH₃), 9.8 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₄N₅O₄: 434.1828; Found: 434.1820.

Ethyl 3-ethyl-5-(4-fluorophenyl)-7-methyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4m), (CH₃-C7)¹H NMR (400 MHz, CDCl₃): δ = 1.23–1.27 (m, 6H, CH₃-C3, CH₃), 2.36–2.44 (m, 1H, CH₂-C3), 2.53 (s, 3H, H10), 2.55–2.61 (m, 1H, CH₂-C3), 4.07–4.17 (m, 2H, O-CH₂), 6.19 (s, 1H, H5), 7.00 (t, J = 8.5 Hz, 2H), 7.30–7.33 (m, 1H), 7,36 (dd, J = 8.5, 5.3 Hz, 2H), 7.48 (t, J = 7.9 Hz, 2H), 8.21 (d, J = 7.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.6 (C=O), 162.5 (C17) (d, J = 247.2 Hz), 159.7 (C7), 148.7 (C3), 148.5 (C8a), 139.1 (d, J = 3.2 Hz), 137.7, 129.0, 128.8 (d, J = 8.4 Hz), 126.1, 120.1, 115.6 (d, J = 21.7 Hz), 97.9 (C6), 59.5 (O-CH₂), 56.7 (C5), 25.2 (C10), 18.7 (CH₂-C3), 14.4 (CH₃), 9.9 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₄FN₄O₂: 407.1883; Found: 407.1877.

Ethyl 5-(2,4-dichlorophenyl)-3-ethyl-7-methyl-1-phenyl-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (4n), Yellow powder, yield: 1094 mg (80%), Mp 179–181 °C; IR (KBr): ν_max/cm⁻¹ ν_C=O: 1685, ν_C=N: 1642. ¹H NMR (400 MHz, CDCl₃): δ = 1.21–1.29 (m, 6H, CH₃-C3, CH₃), 2.38–2.48 (m, 1H, CH₂-C3), 2.56 (s, 3H, H10), 2.66–2.75 (m, 1H, CH₂-C3), 4.09–4.14 (m, 2H, O-CH₂), 6.63 (s, 1H, H5), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.45–7.50 (m, 3H), 8.18 (d, J = 7.7 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 166.2 (C=O), 160.6 (C7), 148.9 (C3), 148.3 (C8a), 139.5, 137.5, 134.7, 131.5, 129.1, 129.0, 128.5, 126.2, 120.2, 97.1 (C6), 59.5 (O-CH₂), 53.5 (C5), 25.2 (C10), 18.8 (CH₂-C3), 14.5 (CH₃), 10.2 (CH₃-C3). HRMS (ESI): m/z [M + H]⁺ Calcd for C₂₃H₂₃Cl₂N₄O₂: 457.1198; Found: 457.1193.

3.2. Pharmacology

We adopted the same method as Msalbi et al. [42]. Compounds were solubilized in DMSO and serially diluted with cell culture media just before use.

For MCF-7 and T47D, the cell lines are obtained from the Institut Pasteur in Tunis (Tunisia). For line MDA-MB-231, it is obtained from our colleague Mohamed Abdelkarim, Faculty of Medicine of Tunis. This line was the subject of two publications by our colleague.

Human breast cancer cell lines MDA-MB-231 (ER-, PR-, HER2-) and MCF7 (ER+, PR+, HER2-) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 50 IU/mL penicillin and 50 mg/mL streptomycin and maintained in an incubator at 37 °C in a humidified atmosphere at 5% CO₂. The MTT test was performed to determine the effect of our chemical compounds on breast cancer cell lines MDA-MB-231 and MCF7 compared to Cisplatin. A 96-well microtiter plate containing 0.1 mL DMEM/well was seeded with 8.103 of each cell line. After 24 h of culture, the cells were treated with these compounds and Cisplatin for 48 h with different concentrations ranging from 0 to 100 µM. After exposure, the medium was removed and MTT solution was added (5 mg/mL) to each well containing 100 µL of DMEM. After 4 h, a formazan precipitate was dissolved in 100 µL/well 10% SDS and recorded at 570 nm. The IC₅₀ value was defined as cell viability ratio (%) = (OD treated/OD untreated) × 100. All the experiments were carried out in triplicate with six measurements for each concentration tested.

4. Conclusions

In conclusion, we have developed a simple and efficient strategy for preparation of [1,2,4]triazolo[4,3-a]pyrimidine derivatives through a one-pot three-component reaction from easily available 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate using 10 mol% APTS as a catalyst. All synthesized products are new and have been isolated with good yields. The regioselectivity of this developed condensation was confirmed by 2D NMR analysis and X-ray crystallographic analyses. Fourteen of the newly synthesized compounds were screened for their biological activities and found to be more active against the tumor cell lines MDA-MB-321 and MCF-7 compared to Cisplatin. The results showed that among this series, compounds 4c and 4j exhibited the best antitumor activity against breast cancer with respective IC₅₀ values of 17.83 μM, and 19.73 μM. These will be taken into account in further study.

Author Contributions

Conceptualization, F.C. and S.A.; methodology, M.B.H. and D.M.; validation, B.J., M.A. and F.C.; formal analysis, H.A. and F.E.; investigation, M.A.; writing—original draft preparation, M.A.; writing—review and editing, M.A.; visualization, B.J.; supervision, F.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data set presented in this study is available in this article.

Acknowledgments

We thank the “Departement d’Analyses Chimiques et Medicales” (Tours, France) for the chemical analyses.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of all compounds are available from the authors.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures, Schemes and Tables

Figure 1. Some examples of biologically active [1,2,4]triazolo[4,3-a]pyrimidines.

Scheme 1. Synthesis of multi-substituted [1,2,4]triazolo[4,3-a]pyrimidines 4a–n.

Scheme 2. Plausible mechanism for the formation of [1,2,4]triazolo[4,3-a]pyrimidines 4a–n.

Figure 2. X-ray single-crystal structures of compounds 4a and 4f.

Figure 3. Antiproliferative activity via MDA-MB-231 cell lines.

Figure 4. Antiproliferative activity via MCF-7 cell lines.

Table 1

Optimization of reaction conditions for substituted [1,2,4]triazolo[4,3-a]pyrimidines 4a.

[Image omitted. Please see PDF.]
Entry	Solvent	Catalyst	Yield (%) a
1	H2O	APTS	Trace
2	Ethanol	APTS	75
3	Ethanol	HCl	45
4	Ethanol	Acetic acid	10
5	Ethanol	Piperidine	Trace
6	CH3CN	APTS	50
7	Fusion state	-	10

^a Isolated yield.

Table 2

IC₅₀ of compounds 4a–n and Cisplatin against MDA-MB-231 and MCF7 after 48 h of treatment activities.

Compound	IC50 (μM)		Compound	IC50 (μM)
	MDA-MB-231	MCF-7		MDA-MB-231	MCF-7
4a	41.30 ± 1.8	43.86 ± 3.1	4h	>100	>100
4b	38.31 ± 2	36.28 ± 3.3	4i	43.88 ± 1	>100
4c	17.83 ± 2	20.33 ± 1.7	4j	23.97 ± 2.7	19.73 ± 1.5
4d	44.63 ± 0.3	34.20 ± 1	4k	53.45 ± 4.9	48.45 ± 1.1
4e	62.26 ± 1.7	>100	4l	>100	>100
4f	43.79 ± 1.8	32.09 ± 2.8	4m	84.43 ± 3.8	>100
4g	39.57 ± 0.3	51.26 ± 0.7	4n	58.34 ± 3.4	46.34 ± 2
Cisplatin	38.6 ± 0.8	83.1 ± 1.3	Cisplatin	38.6 ± 0.8	83.1 ± 1.3

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28093917/s1, Figures S1–S26: 1H and 13C NMR spectra of 4a–n; Figure S27: 2D NMR HMBC sequence of compound 4f; Figure S28: FT-IR spectrum of 4n; Copies of HRMS report (in French).

References

1. Jha, M.; Guy, S.; Chou, T.-Y. Microwave assisted synthesis of indole-annulated dihydropyrano [3,4-c]chromene derivatives via hetero-Diels–Alder reaction. Tetrahedron. Lett.; 2011; 52, pp. 4337-4341. [DOI: https://dx.doi.org/10.1016/j.tetlet.2011.06.052]

2. Pinheiro, S.; Pinheiro, E.M.C.; Muri, E.M.F.; Pessôa, J.C.; Cadorini, M.A.; Greco, S.J. Biological activities of [1,2,4]Triazolo[1,5-a]pyrimidines and analogs. Med. Chem. Res.; 2020; 29, pp. 1751-1776. [DOI: https://dx.doi.org/10.1007/s00044-020-02609-1]

3. Wang, S.; Yuan, X.-H.; Wang, S.-Q.; Zhao, W.; Chen, X.-B.; Yu, B. FDA-approved pyrimidine-fused bicyclic heterocycles for cancer therapy: Synthesis and clinical application. Eur. J. Med. Chem.; 2021; 214, 113218. [DOI: https://dx.doi.org/10.1016/j.ejmech.2021.113218] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33540357]

4. Rai, U.S.; Isloor, A.M.; Shetty, P.; Vijesh, A.M.; Prabhu, N.; Isloor, S.; Thiageeswaran, M.; Hoong-Kun, F. Novel chromeno [2,3-b]pyrimidine derivatives as potential anti-microbial agents. Eur. J. Med. Chem.; 2010; 45, pp. 2695-2699.

5. Zafar, W.; Sumrra, S.H.; Chohan, Z.H. A review: Pharmacological aspects of metal based 1,2,4-triazole derived Schiff bases. Eur. J. Med. Chem.; 2021; 222, 113602. [DOI: https://dx.doi.org/10.1016/j.ejmech.2021.113602]

6. Salgado, A.; Varela, C.; Collazo, A.M.G.; Pevarello, P. Differentiation between [1,2,4]Triazolo[1,5-a]pyrimidine and [1,2,4]Triazolo[4,3-a]pyrimidine regioisomers by ¹H-¹⁵N HMBC experiments. Magn. Reson. Chem.; 2010; 48, pp. 614-622. [DOI: https://dx.doi.org/10.1002/mrc.2634]

7. Fischer, G. Recent progress in 1,2,4-Triazolo[1,5-a]pyrimidine chemistry. Adv. Heterocycl. Chem.; 2008; 95, pp. 143-219.

8. Shaaban, M.R.; Saleh, T.S.; Farag, A.M. Synthesis and antimicrobial evaluation of novel pyrazolo[1,5-a]pyrimidine, Triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]benzimidazole derivatives. Heterocycles; 2007; 71, pp. 1765-1777. [DOI: https://dx.doi.org/10.1002/chin.200750171]

9. Boutaleb-Charki, S.; Marín, C.; Maldonado, C.R.; Rosales, M.J.; Urbano, J.; Guitierrez-Sánchez, R.; Quirós, M.; Salas, J.M.; Sánchez-Moreno, M. Copper (II) complexes of [1,2,4]Triazolo[1,5-a]pyrimidine derivatives as potential anti-parasitic agents. Drug. Metab. Lett.; 2009; 3, pp. 35-44. [DOI: https://dx.doi.org/10.2174/187231209787176317]

10. Ruisi, G.; Canfora, L.; Bruno, G.; Rotondo, A.; Mastropietro, T.F.; Debbia, E.A.; Girasolo, M.A.; Megna, B. Triorganotin(IV) derivatives of 7-amino-2-(methylthio)-[1,2,4]Triazolo[1,5-a]pyrimidine-6-carboxylic acid. Synthesis, spectroscopic characterization, in vitro antimicrobial activity and X-ray crystallography. J. Organomet. Chem.; 2010; 695, pp. 546-551. [DOI: https://dx.doi.org/10.1016/j.jorganchem.2009.11.019]

11. El-Gendy, M.M.A.; Shaaban, M.; Shaaban, K.A.; El-Bondkly, A.M.; Laatsch, H. Essramycin: A first triazolopyrimidine antibiotic isolated from nature. J. Antibiot.; 2008; 61, pp. 149-157. [DOI: https://dx.doi.org/10.1038/ja.2008.124]

12. Mohamed, M.M.; Khalil, A.K.; Abbass, E.M.; El-Naggar, A.M. Design, synthesis of new pyrimidine derivatives as anticancer and antimicrobial agents. Synth. Commun.; 2017; 47, pp. 1441-1457. [DOI: https://dx.doi.org/10.1080/00397911.2017.1332223]

13. El-Zahar, M.I.; Abd El-Karim, S.S.; Haiba, M.E.; Khedr, M.A. Synthesis, antitumor activity and molecular docking study of novel benzofuran-2-yl pyrazole pyridine derivatives. Acta Pol. Pharm. Drug Res.; 2011; 68, pp. 357-373.

14. Prakash, O.; Bhardwaj, V.; Kumar, R.; Tyagi, P.; Aneja, K.R. Organoiodine (III) mediated synthesis of 3-aryl/hetryl-5,7-dimethyl-1,2,4-Triazolo[4,3-a]pyrimidines as antibacterial agents. Eur. J. Med. Chem.; 2004; 39, pp. 1073-1077. [DOI: https://dx.doi.org/10.1016/j.ejmech.2004.06.011]

15. Khera, M.K.; Cliffe, I.A.; Mathur, T.; Prakash, O. Synthesis and in vitro activity of novel 1,2,4-Triazolo[4,3-a]pyrimidine oxazolidinone antibacterial agents. Bioorg. Med. Chem. Lett.; 2011; 21, pp. 2887-2889. [DOI: https://dx.doi.org/10.1016/j.bmcl.2011.03.075]

16. Sharma, A.; Kumar, V.; Khare, R.; Gupta, G.K.; Beniwal, V. Synthesis, docking study, and DNA photocleavage activity of some pyrimidinyl hydrazones and 3-(quinolin-3-yl)-5,7-dimethyl-1,2,4- Triazolo[4,3-a]pyrimidine derivatives. Med. Chem. Res.; 2015; 24, pp. 1830-1841. [DOI: https://dx.doi.org/10.1007/s00044-014-1265-9]

17. Kamal, R.; Kumar, V.; Kumar, R.; Bhardwaj, J.K.; Saraf, P.; Kumari, P.; Bhardwaj, V. Design, Synthesis, and Screening of Triazolopyrimidine–Pyrazole Hybrids as Potent Apoptotic Inducers. Arch. Pharm. Chem.; 2017; 350, e1700137. [DOI: https://dx.doi.org/10.1002/ardp.201700137]

18. Oukoloff, K.; Lucero, B.; Francisco, K.R.; Brunden, K.R.; Ballatore, C. 1,2,4-Triazolo[1,5-a]pyrimidines in drug design. Eur. J. Med. Chem.; 2019; 165, pp. 332-346. [DOI: https://dx.doi.org/10.1016/j.ejmech.2019.01.027]

19. Liu, Y.-C.; Qu, R.-Y.; Chen, Q.; Yang, J.-F.; Cong-Wei, N.; Zhen, X.; Yang, G.-F. Triazolopyrimidines as a new herbicidal lead for combating weed resistance associated with acetohydroxyacid synthase mutation. J. Agric. Food Chem.; 2016; 64, pp. 4845-4857. [DOI: https://dx.doi.org/10.1021/acs.jafc.6b00720]

20. Ochoa, C.; Goya, P. Six-membered ring systems: Triazines, tetrazines and fused ring polyaza systems. Prog. Heterocycl. Chem.; 2000; 12, pp. 294-316.

21. Hajos, G.; Riedl, Z. Bicyclic 5–6 systems with one bridgehead (ring junction) nitrogen atom: Three extra heteroatoms 2:1. Compr. Heterocycl. Chem. III; 2008; 11, pp. 671-763.

22. Yet, L. Five-membered ring sysyems: With more than one N atom. Prog. Heterocycl. Chem.; 2009; 21, pp. 224-260.

23. Fizer, M.; Slivka, M. Synthesis of [1,2,4-Triazolo[1,5-a]pyrimidine (microreview). Chem. Heterocycl. Compd.; 2016; 52, pp. 155-157. [DOI: https://dx.doi.org/10.1007/s10593-016-1851-5]

24. Fischer, G. Recent advances in 1,2,4-Triazolo[1,5-a]pyrimidine chemistry. Adv. Heterocycl. Chem.; 2019; 128, pp. 1-101.

25. El Ashry, S.H.; Awad, L.F.; Badawy, M.E.I.; Rabea, E.I.; Ibrahim, N.A.; Abd Al Moaty, M.N. Synthesis, antibacterial, antioxidant, and molecular docking studies of 6-methylpyrimidin-4(3H)-one and oxo-1,2,4-Triazolo[4,3-a]pyrimidine derivative. J. Mol. Struct.; 2022; 1249, 131551. [DOI: https://dx.doi.org/10.1016/j.molstruc.2021.131551]

26. El Ashry, S.H.; Awad, L.F.; Teleb, M.; Ibrahim, N.A.; Abu-Serie, M.M.; Abd Al Moaty, M.N. Structure-based design and optimization of pyrimidine- and 1,2,4-Triazolo[4,3-a]pyrimidine-based matrix metalloproteinase-10/13 inhibitors via Dimroth rearrangement towards targeted polypharmacology. Bioorg. Chem.; 2020; 96, 103616. [DOI: https://dx.doi.org/10.1016/j.bioorg.2020.103616]

27. Bowack, D.A.; Lapworth, A. CLXXIX.-Hydrizino-halides derived from oxalic acid. J. Chem. Soc., Trans.; 1905; 87, pp. 1854-1869. [DOI: https://dx.doi.org/10.1039/CT9058701854]

28. Silpa, L.; Niepceron, N.; Laurent, F.; Brossier, F.; Penichon, M.; Enguehard-Gueiffier, C.; Abarbri, M.; Silvestre, A.; Petrignet, J. Synthesis and evaluation of the anticoccidial activity of trifluoropyrido[1,2-a]pyrimidin-2-one derivatives. Bioorg. Med. Chem. Lett.; 2016; 26, pp. 114-120. [DOI: https://dx.doi.org/10.1016/j.bmcl.2015.11.018]

29. Jismy, B.; Allouchi, H.; Guillaumet, G.; Akssira, M.; Abarbri, M. Concise and efficient access to 5,7- Disubstituted pyrazolo[1,5-a]pyrimidines by Pd-Catalyzed sequential arylation, alkynylation and SNAr reaction. Eur. J. Org. Chem.; 2017; 2017, pp. 6168-6178. [DOI: https://dx.doi.org/10.1002/ejoc.201701024]

30. Jismy, B.; Guillaumet, G.; Akssira, M.; Knez, D.; Abarbri, M. A simple and a practical strategy to synthesize new 5-trifluoromethyl-7-substituted Imidazo[1,2-a]pyrimidines and benzimidazo[1,2-a]pyrimidines, and their biological evaluation test. New. J. Chem.; 2019; 43, pp. 9961-9968. [DOI: https://dx.doi.org/10.1039/C9NJ01982K]

31. Jismy, B.; El Qami, A.; Pislar, A.; Frlan, R.; Kos, J.; Gobec, S.; Knez, D.; Abarbri, M. Pyrimido[1,2-b]indazole derivatives: Selective inhibitors of human monoamine oxidase B with neuroprotective activity. Eur. J. Med. Chem.; 2021; 209, 112911. [DOI: https://dx.doi.org/10.1016/j.ejmech.2020.112911]

32. Deshmukh, M.B.; Salunkhe, S.M.; Patil, D.R.; Anbhule, P.V. A novel and efficient one step synthesis of 2-amino- 5-cyano-6-hydroxy-4-aryl pyrimidines and their anti-bacterial activity. Eur. J. Med. Chem.; 2009; 44, pp. 2651-2654. [DOI: https://dx.doi.org/10.1016/j.ejmech.2008.10.018]

33. John, S.E.; Gulati, S.; Shankaraiah, N. Recent advances in multi-component reactions and their mechanistic insights: A triennium review. Org. Chem. Front.; 2021; 8, pp. 4237-4287. [DOI: https://dx.doi.org/10.1039/D0QO01480J]

34. Weber, L.; Illgen, K.; Almstetter, M. Discovery of new multi component reactions with combinatorial methods. Synlett; 1999; 3, pp. 366-374. [DOI: https://dx.doi.org/10.1055/s-1999-2612]

35. Dandia, A.; Singh, R.; Singh, D.; Arya, K. Facile regioselective green synthesis of Triazolo[4,3-a]pyrimidines in aqueous medium. Lett. Org. Chem.; 2009; 6, pp. 100-105. [DOI: https://dx.doi.org/10.2174/157017809787003124]

36. Ablajan, K.; Kamil, W.; Tuoheti, A.; Sun Wan-Fu, S. An Efficient Three Component One-Pot Synthesis of 5-Amino-7-aryl-7,8-dihydro-[1,2,4]Triazolo[4,3-a]-pyrimidine-6-carbonitriles. Molecules; 2012; 17, pp. 1860-1869. [DOI: https://dx.doi.org/10.3390/molecules17021860]

37. Fedorova, O.V.; Zhidovinova, M.S.; Rusinov, G.L.; Ovchinnikova, I.G. Aminoazoles in the three-component synthesis of 7-substituted 6-ethoxycarbonyl-5-methyl-4,7-dihydroazolo[1,5-a]pyrimidines. Russ. Chem. Bull.; 2003; 52, pp. 1768-1769. [DOI: https://dx.doi.org/10.1023/A:1026052603951]

38. El Rady, E.A. Three-component uncatalyzed eco-friendly reactions for one-pot Synthesis of 4,7-dihydro[1,2,4]Triazolo[1,5-a]pyrimidine derivatives. J. Hetrocycl. Chem.; 2014; 51, pp. 869-875. [DOI: https://dx.doi.org/10.1002/jhet.1771]

39. Deposit Numbers CCDC 2239427(for 4a) and CCDC 2239426 (for 4f) Contain Additional Crystallographic Data for This Article. These Data are Provided Free of Charge by the Joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures. Available online: www.ccdc.cam.ac.uk/structures (accessed on 15 March 2023).

40. Hodeify, R.; Siddiqui, S.S.; Matar, R.; Vazhappilly, C.G.; Merheb, M.; Al Zouabi, H.; Marton, J. Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells. Heliyon; 2021; 7, e06041. [DOI: https://dx.doi.org/10.1016/j.heliyon.2021.e06041]

41. Aouali, M.; Mhalla, D.; Allouche, F.; El Kaim, L.; Tounsi, S.; Trigui, M.; Chabchoub, F. Synthesis, antimicrobial and antioxidant activities of imidazotriazoles and new multicomponent reaction toward 5-amino-1-phenyl[1,2,4]triazole derivatives. Med. Chem. Res.; 2015; 24, pp. 2732-2741. [DOI: https://dx.doi.org/10.1007/s00044-015-1322-z]

42. Msalbi, D.; Elloumi-Mseddi, J.; Hakim, B.; Sahli, E.; Aifa, S. Chemical alternative for cell identification and cross-contamination detection. 3 Biotech.; 2022; 12, 78. [DOI: https://dx.doi.org/10.1007/s13205-022-03144-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35251881]