Microwave-assisted synthesis, anti-inflammatory and anti-proliferative activities of new maslinic acid derivatives bearing 1,5- and 1,4-disubstituted triazoles

Abstract

In this work, 40 analogs with a natural maslinic acid core (from Olea europaea L.) and various aromatic azides were synthesized. A regiospecific, facile and practical synthesis of 1,5-triazolyl derivatives by Ru(II)-catalyzed azide-alkyne cycloaddition (RuAAC), and mono-, bis- and tri-1,4-triazolyl derivatives by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) was described. All the reactions were assisted by microwave irradiation avoiding toxic reagents and solvents. The new products were obtained from the reaction mixture by simple purification in almost quantitative yields and the reaction times were in general shorter than those reported in the literature. Their chemical structures were elucidated on the basis of extensive spectroscopic methods including ESI-HRMS, 1D and 2D-NMR. Most of the compounds were evaluated for their anti-inflammatory activity using LPS-stimulated human peripheral blood mononuclear cells (PBMCs) and antiproliferative effects towards cultured murine EMT-6 (Breast) and human SW480 (colon) cancer cell lines.

Keywords:

• Anti-inflammatory
• anti-proliferative
• maslinic acid
• Olea europaea L.
• triazoles

Introduction

Recently, 1,2,3-triazoles have gained special attention in the field of drug discovery due to the growing use of “click” reaction such as ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC)1–3 and copper-catalyzed azide-alkyne cycloaddition (CuAAC)4–6. Moreover, compounds containing a 1,2,3-triazole display a wide range of biological capacities such as anti-HIV7, cytotoxic8 and anti-inflammatory9 activities. On the other hand, natural products and their synthetic derivatives are very important in drug design10,11. Among these, natural pentacyclic triterpenoids and their derivatives are present in several classes of natural and synthetic biologically active compounds12,13. In particular, maslinic acid (1, Figure 1) and its derivatives are of interest because of their wide spectrum of biological activities. Thus, for natural compound 1 an inhibitory activity for glycogen phosphorylases14, protein tyrosine phosphatase 1B15, anti-inflammatory16, anti-tumor17 as well as an anti-HIV-1 activities18 was reported. However, the natural pentacyclic triterpenoid 1 may have utility in other pathological processes for its anti-inflammatory and anti-cancer properties. It has been found to attenuate intracellular oxidative stress via inhibition of NO and H₂O₂ production and reduction of pro-inflammatory cytokine generation in murine19,20. Furthermore, interesting reports from Reyes-Zurita et al.21,22 suggest that compound 1 is able to induce cell death by apoptosis in the colon cancer cell lines HT29 and in Caco-2. Among others, Allouche et al.23 demonstrate that maslinic acid 1 was reported to be cytotoxic for the breast cancer cell line MCF7. Thus, maslinic acid 1 exhibited some selectivity between malignant and nonmalignant cell lines24 and selective parasitostatic activities in mice25. Recent research results correlate the low rate of cancer mortality in Mediterranean countries to a diet rich in olives26,27.

Figure 1. Chemical structure of Maslinic acid 1.

In this context, maslinic acid 1 (Figure 1) which isolated from pomace olive (Olea europaea L.) cultivar: Chemlali, under ultra-sonication conditions with high quantities (17 g (8.5 mg/g DW)) was used herein as a starting material to synthesize new 1,5-disubstituted-1,2,3-triazolo-maslinic acid derivatives through well-established Ru(II)-catalyzed azide-alkyne cycloaddition (RuAAC) and to access to mono, bis- and tri-1,4-triazolyl derivatives via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conducted under microwave conditions. Most obtained compounds were evaluated for their anti-inflammatory and anti-proliferative activities in vitro.

Materials and methods

General experimental procedures

Solvents were distilled and dried using standard methods. Melting points were determined on a Büchi 510 apparatus (Mount Holly, NJ) using capillary tubes. Commercial TLC plates (Silica gel 60, F254, sds) were used to monitor the progress of the reaction. Column chromatography was performed with silica gel 60 (particle size 40–63 μm, sds). HRMS were acquired with a LCT Premier XE (Waters, ESI technique, positive mode) mass spectrometer. For ESI experiments, leucine-enkephaline peptide was employed as the LockSpray lockmass. ¹H (300 MHz, 16–32 scans) and BB-decoupled ¹³C (75 MHz, 256–2048 scans) NMR spectra were recorded at room temperature (rt) on a Bruker AM-300 Fourier Transform spectrometer (Fitchburg, MA) equipped with a 10 mm probe in deuterated chloroform, acetone and pyridine with all chemical shifts (δ), reported in ppm, refereed to residual non-deuterated solvent. Coupling constants were measured in Hz. and signals are using the following abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet, etc.

Chemistry

Synthesis of dipolarophiles 2–6

Method I: To a solution of compound 1 (3 g, 6.3  mmol) in dry DMF (5 mL), sodium hydride (12.6  mmol) and propargyl bromide (18.9  mmol) were added and the reaction mixture was stirred at room temperature for 2 h. Reaction was monitored by TLC. After the completion of the reaction, the residue was diluted with water (300 mL). The mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was chromatographed over silica gel and eluted with ethyl acetate:petroleum ether (2:8 then 3:7) to obtain the alkyl derivatives 2–6 in 44, 5, 15, 11 and 7% yield, respectively. Method II: With the aim to fully propargylate the maslinic acid 1 in C-2, C-3 and C-28 positions, we increased the number of equivalents of NaH (which goes from 2 to 4 equiv.) and propargyl bromide (which goes from 3 to 4 equiv.) and we extended the reaction time from 2 h to 8 h. The same protocol adopted in method I was applied and only the alkyl derivatives 2 and 4–6 was obtained in 25, 23, 20 and 31% yield, respectively.

Propargyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (2). White solid; m.p.: 297–299 °C; ¹H NMR (300 MHz, CDCl₃): δ 5.23 (1H, t, J = 3.6 Hz), 4.61 (1H, dd, J = 15.6; 2.4 Hz), 4.50 (1H, dd, J = 15.6; 2.7 Hz), 3.60 (1H, td, J = 9.3; 2.7 Hz), 2.93 (1H, d, J = 9.6 Hz), 2.80 (1H, dd, J = 13.8; 3.9 Hz), 2.34 (1H, t, J = 2.7 Hz), 1.99 (4H, m), 1.93–1.83 (4H, m), 1.62–1.46 (6H, m), 1.45–1.21 (6H, m), 1.06 (3H, s), 0.96 (3H, s), 0.91 (3H, s), 0.86 (3H, s), 0.83 (3H, s), 0.75 (3H, s), 0.67 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.3, 142.9, 121.9, 83.4, 77.6, 73.8, 68.4, 54.8, 51.1, 47.1, 46.2, 45.9, 45.3, 41.2, 40.7, 38.9, 38.6, 37.8, 36.5, 33.3, 32.5, 32.1, 31.7, 30.1, 28.1, 27.1, 26.7, 25.3, 23.0, 22.9, 22.5, 17.8, 16.6; HRMS (ESI⁺): calculated for (C₃₃H₅₁O₄)⁺[M + H]⁺511.3787, found 511.3778.

(2α)-2-(Propargyloxy)-(3β)-3-hydroxy-olean-12-en-28-oic acid (3). White solid; m.p.: 293–294 °C; ¹H NMR (300 MHz, CDCl₃): δ 5.29 (1H, s), 4.30 (1H, dd, J = 15.9; 2.4 Hz), 4.18 (1H, dd, J = 15.9; 2.4 Hz), 3.58 (1H, td, J = 9.3; 2.7 Hz), 2.93 (1H, d, J = 9.6 Hz), 2.84 (1H, dd, J = 13.8; 3.9 Hz), 2.47 (1H, t, J = 2.4 Hz), 2.05 (2H, m), 1.77–1.47 (8H, m), 1.37–1.23 (10H, m), 1.14 (3H, s), 1.06 (3H, s), 0.99 (3H, s), 0.94 (3H, s), 0.92 (3H, s), 0.85 (3H, s), 0.76 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 183.3, 143.2, 121.7, 80.4, 79.7, 76.5, 73.9, 55.8, 54.5, 47.1, 46.0, 45.3, 42.0, 41.1, 40.4, 38.8, 38.6, 37.7, 33.2, 32.5, 32.1, 31.9, 30.1, 29.2, 28.1, 27.1, 25.4, 23.0, 22.3, 17.6, 16.6, 16.4, 16.0; HRMS (ESI⁺): calculated for (C₃₃H₅₁O₄)⁺[M + H]⁺511.3787, found 511.3794.

Propargyl-(2αro2-(propargyloxy)-(3βro2-(propargolean-12-en-28-oate (4). White solid; m.p.: 296–298 °C; ¹H NMR (300 MHz, CDCl₃): δ 5.22 (1H, s), 4.67 (1H, dd, J = 15.6; 2.4 Hz), 4.56 (1H, dd, J = 15.3; 2.1 Hz), 4.26 (1H, dd, J = 15.9; 2.4 Hz), 4.15 (1H, dd, J = 15.9; 2.1 Hz), 3.54 (1H, td, J = 11.1; 4.2 Hz), 3.06 (1H, d, J = 9.6 Hz), 2.86 (1H, dd, J = 13.8; 3.9 Hz), 2.44 (3H, m), 2.04–1.94 (2H, m), 1.92–1.89 (2H, m), 1.70–1.53 (7H, m), 1.45–1.29 (4H, m), 1.24–1.16 (2H, m), 1.12 (3H, s), 1.04 (3H, s), 0.96 (3H, s), 0.92 (3H, s), 0.89 (3H, s), 0.83 (3H, s), 0.73 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.2, 143.0, 121.8, 80.8, 79.8, 77.6, 76.6, 73.9, 73.9, 55.8, 54.5, 51.1, 47.0, 46.2, 45.3, 42.1, 41.2, 40.7, 38.9, 38.6, 37.7, 33.3, 32.5, 32.1, 31.6, 30.1, 28.2, 27.1, 25.3, 23.1, 23.0, 22.4, 17.6, 16.6, 16.4, 16.0; HRMS (ESI⁺): calculated for (C₃₆H₅₃O₄)⁺[M + H]⁺549.3944, found 549.3938.

Propargyl-(3αropargyl-(3e8-oate-(3den-28-oate aolean-12-en-28-oate (5). White solid; m.p.: 296–298 °C; ¹H NMR (300 MHz, CDCl₃): δ 5.22 (1H, t, J = 3.3 Hz), 4.60 (1H, dd, J = 15.6; 2.4 Hz), 4.49 (1H, dd, J = 15.6; 2.4 Hz), 4.41 (1H, dd, J = 15.9; 2.4 Hz), 4.25 (1H, dd, J = 15.9; 2.4 Hz), 3.72 (1H, td, J = 11.1; 4.2 Hz), 2.79 (1H, dd, J = 13.8; 3.9 Hz), 2.74 (1H, d, J = 9.6 Hz), 2.42 (1H, t, J = 2.4 Hz), 2.33 (1H, t, J = 2.4 Hz), 2.07 (2H, m), 1.91–1.83 (4H, m), 1.59–1.47 (8H, m), 1.34 (2H, m), 1.30–1.22 (4H, m), 1.10 (3H, s), 0.94 (3H, s), 0.88 (3H, s), 0.85 (3H, s), 0.82 (3H, s), 0.73 (3H, s), 0.66 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.2, 142.8, 121.9, 93.0, 80.3, 77.6, 74.1, 73.9, 68.0, 61.0, 54.8, 51.1, 47.1, 46.2, 45.3, 41.2, 40.7, 39.7, 38.9, 37.5, 33.3, 32.5, 32.1, 31.7, 30.1, 28.4, 27.1, 26.4, 25.3, 23.1, 22.9, 22.4, 17.6, 16.6, 16.4, 16.0; HRMS (ESI⁺): calculated for (C₃₆H₅₃O₄)⁺[M + H]⁺549.3944, found 549.3940.

Propargyl-(2α,3β) − 2,3 − βισ(propargyloxy)-olean-12-en-28-oate (6). White solid; m.p.: 296–298 °C; ¹H NMR (300 MHz, CDCl₃): δ 5.23 (1H, s), 4.60 (1H, dd, J = 15.6; 2.4 Hz), 4.50 (1H, dd, J = 15.6; 2.4 Hz), 4.41 (1H, dd, J = 15.9; 2.4 Hz), 4.34 (1H, dd, J = 15.9; 2.4 Hz), 4.17 (2H, d, J = 2.1 Hz), 3.59 (1H, td, J = 11.1; 4.2 Hz), 2.82 (1H, d, J = 9.6 Hz), 2.79 (1H, dd, J = 13.8; 3.9 Hz), 2.33 (2H, m), 2.25 (1H, m), 1.96–1.83 (4H, m), 1.60–1.46 (8H, m), 1.34–1.20 (8H, m), 1.10 (3H, s), 0.94 (3H, s), 0.88 (3H, s), 0.85 (3H, s), 0.81 (3H, s), 0.73 (3H, s), 0.66 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.3, 142.9, 121.9, 89.2, 80.6, 77.7, 77.6, 73.8, 73.8, 73.2, 73.1, 60.2, 56.7, 54.8, 51.1, 47.1, 46.2, 45.3, 44.2, 41.2, 40.7, 39.5, 38.9, 37.4, 33.5, 33.3, 32.1, 31.7, 30.1, 29.1, 27.1, 26.4, 25.3, 23.1, 22.5, 17.8, 17.0, 16.5, 15.9; HRMS (ESI⁺): calculated for (C₃₉H₅₅O₄)⁺[M + H]⁺587.4100, found 587.4092.

General procedure for the RuAAC reaction

To a solution of alkyne 2 (0.1 g, 0.2  mmol) in DMF (1 mL), Cp*RuCl(PPh₃)₂ (5 mol %) was added at room temperature. To this mixture, aryl azide (0.4  mmol) was added and the reaction mixture was subjected to microwave irradiations at 250 W completed within 4–8 minutes. The crude mixture was diluted with water (75 mL) then extracted with EtOAc (3 × 25 mL) and the combined organic layer was dried over sodium sulfate, concentrated in vacuo and purified through a column chromatography to give pure the 1,5-triazolyl derivatives 8a–g in 83–98% yields.

(1-Phenyl-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8a). Yellowish gum; m.p.: 218–219 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.77 (1H, s), 7.53–7.42 (5H, m), 5.19 (1H, t, J = 3.3 Hz), 5.09 (1H, d, J = 13.5 Hz), 5.01 (1H, d, J = 13.5 Hz), 3.63 (1H, td, J = 10.8; 4.2 Hz), 2.91 (1H, d, J = 9.6 Hz), 2.76 (1H, dd, J = 11.8; 3.8 Hz), 2.24 (2H, m), 1.93–1.75 (6H, m), 1.61–1.24 (12H, m), 1.23 (3H, s), 1.03 (3H, s), 0.89 (3H, s), 0.86 (3H, s), 0.81 (3H, s), 0.74 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.8, 143.4 136.7, 136.2, 131.4, 129.8, 129.6, 129.6, 124.7, 124.7, 122.6, 83.9, 68.9, 55.2, 53.7, 47.4, 46.9, 46.3, 45.7, 41.7, 41.2, 39.3, 39.1, 38.2, 33.7, 32.6, 31.9, 30.6, 29.3, 28.6, 27.5, 25.8, 23.5, 23.4, 18.2, 16.8, 16.7, 16.5, 14.0; HRMS (ESI⁺): calculated for (C₃₉H₅₆N₃O₄)⁺[M + H]⁺630.4271, found 630.4274.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8b). Dark red solid; m.p.: 222–224 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.83 (1H, s), 7.47 (2H, dd, J = 6.9; 2.1 Hz), 7.06 (2H, dd, J = 6.9; 2.1 Hz), 5.27 (1H, t, J = 3.3 Hz), 5.12 (1H, d, J = 13.5 Hz), 5.06 (1H, d, J = 13.8 Hz), 3.90 (3H, s), 3.71 (1H, td, J = 10.8; 3.9 Hz), 3.01 (1H, d, J = 9.6 Hz), 2.86 (1H, dd, J = 8.8; 3.4 Hz), 2.00–1.86 (4H, m), 1.69–1.56 (6H, m), 1.52–1.44 (6H, m), 1.29–1.22 (4H, m), 1.27 (3H, s), 1.13 (3H, s), 1.04 (3H, s), 1.01 (3H, s), 0.96 (6H, s), 0.83 (3H, s), 0.55 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.3, 160.3, 142.9, 134.7, 132.0, 128.6, 125.7, 125.7, 122.0, 114.2, 114.2, 83.4, 68.4, 55.1, 54.7, 53.3, 47.0, 46.3, 45.8, 45.2, 41.2, 40.7, 38.8, 38.6, 37.7, 33.8, 33.2, 32.4, 32.0, 31.8, 30.1, 29.1, 28.0, 27.0, 25.3, 24.4, 22.6, 17.7, 16.3, 15.2; HRMS (ESI⁺): calculated for (C₄₀H₅₈N₃O₅)⁺[M + H]⁺660.4376, found 660.4381.

(1-(4-Chlorophenyl)-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8c). Dark orange solid; m.p.: 235–237 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.84 (1H, s), 7.51–7.44 (4H, m), 5.25 (1H, t, J = 3.6 Hz), 5.14 (1H, d, J = 13.2 Hz), 5.10 (1H, d, J = 13.2 Hz), 3.67 (1H, td, J = 10.8; 4.2 Hz), 2.98 (1H, d, J = 9.6 Hz), 2.81 (1H, dd, J = 11.2; 4.1 Hz), 2.08–1.82 (6H, m), 1.68–1.54 (8H, m), 1.51–1.37 (6H, m), 1.25 (3H, s), 1.01 (3H, s), 0.98 (3H, s), 0.93 (3H, s), 0.90 (3H, s), 0.79 (3H, s), 0.50 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.3, 142.8, 135.5, 135.2, 133.9, 131.9, 129.4, 129.4, 125.4, 125.4, 122.1, 83.4, 68.3, 54.7, 53.1, 46.9, 46.4, 45.4, 45.2, 41.2, 40.7, 38.8, 38.6, 37.7, 33.2, 32.4, 32.0, 31.8, 30.1, 29.1, 28.1, 27.0, 27.1, 25.3, 22.9, 23.3, 18.1, 17.0, 16.7; HRMS (ESI⁺): calculated for (C₃₉H₅₅ClN₃O₄)⁺[M + H]⁺664.3881, found 664.3885.

(1-(4-Bromophenyl)-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8d). White solid; m.p.: 276–277 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.86 (1H, s), 7.53–7.46 (4H, m), 5.23 (1H, t, J = 3.3 Hz), 5.16 (1H, d, J = 13.5 Hz), 5.08 (1H, d, J = 13.5 Hz), 3.69 (1H, td, J = 11.1; 4.2 Hz), 3.02 (1H, d, J = 9.6 Hz), 2.86 (1H, dd, J = 11.2; 4.1 Hz), 2.05–1.83 (6H, m), 1.68–1.64 (6H, m), 1.58–1.50 (4H, m), 1.46–1.30 (4H, m), 1.25 (3H, s), 1.01 (3H, s), 0.98 (3H, s), 0.93 (3H, s), 0.90 (3H, s), 0.79 (3H, s), 0.51 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.5, 142.9, 135.5, 135.3, 134.0, 132.0, 129.4, 129.4, 125.4, 125.4, 122.1, 83.3, 68.4, 54.8, 53.7, 47.0, 46.4, 45.8, 45.2, 41.2, 40.8, 38.8, 38.6, 37.8, 33.2, 32.4, 32.0, 31.8, 30.1, 29.1, 28.1, 27.0, 27.1, 25.3, 22.8, 23.5, 18.3, 17.2, 16.5; HRMS (ESI⁺): calculated for (C₃₉H₅₅BrN₃O₄)⁺[M + H]⁺708.3376, found 708.3379.

(1-(4-Nitrophenyl)-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8e). Yellow solid; m.p.: 308–310 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.81 (1H, s), 7.51–7.40 (4H, m), 5.22 (1H, t, J = 3.0 Hz), 5.14 (1H, d, J = 13.5 Hz), 5.06 (1H, d, J = 13.5 Hz), 3.66 (1H, td, J = 10.5; 3.9 Hz), 3.00 (1H, d, J = 9.6 Hz), 2.87 (1H, dd, J = 11.2; 4.4 Hz), 2.09–1.87 (4H, m), 1.69–1.66 (4H, m), 1.63–1.58 (2H, m), 1.56–1.41 (6H, m), 1.39–1.28 (4H, m), 1.25 (3H, s), 1.01 (3H, s), 0.98 (3H, s), 0.93 (3H, s), 0.90 (3H, s), 0.79 (3H, s), 0.53 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.5, 144.5, 143.6, 142.9, 142.0, 131.8, 128.6, 128.6, 124.0, 124.0, 122.9, 83.6, 68.7, 54.8, 53.7, 47.0, 46.4, 45.8, 45.2, 41.2, 40.8, 38.8, 38.6, 37.8, 33.2, 32.4, 32.0, 31.8, 30.1, 29.1, 28.1, 27.0, 27.1, 25.3, 22.9, 23.6, 18.0, 17.5, 16.0; HRMS (ESI⁺): calculated for (C₃₉H₅₅N₄O₆)⁺[M + H]⁺675.4122, found 675.4125.

(1-(3-Methylphenyl)-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8f). White solid; m.p.: 326–327 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.84 (1H, s), 7.47–7.34 (4H, m), 5.28 (1H, t, J = 3.3 Hz), 5.16 (1H, d, J = 13.5 Hz), 5.10 (1H, d, J = 13.5 Hz), 3.66 (1H, td, J = 10.5; 3.9 Hz), 3.01 (1H, d, J = 9.6 Hz), 2.87 (1H, dd, J = 9.2; 3.8 Hz), 2.47 (3H, s), 2.04–7.86 (6H, m), 1.69–1.52 (8H, m), 1.50–1.33 (6H, m), 1.13 (3H, s), 1.04 (3H, s), 0.95 (3H, s), 0.90 (6H, s), 0.83 (3H, m), 0.55 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 176.8, 143.4, 139.9, 135.8, 135.4, 132.3, 130.5, 129.3, 125.3, 122.5, 121.7, 83.9, 68.9, 55.2, 53.7, 47.5, 46.8, 46.3, 45.7, 41.7, 41.2, 39.3, 39.1, 38.2, 33.7, 32.9, 32.5, 32.3, 31.9, 30.6, 29.6, 28.5, 27.5, 25.8, 23.5, 23.4, 23.0, 22.6, 21.3, 18.2; HRMS (ESI⁺): calculated for (C₄₀H₅₈N₃O₄)⁺[M + H]⁺644.4427, found 644.4431.

(1-Naphthalen-1-yl-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (8 g). Eggplant solid; m.p.: 335–337 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.09 (1H, d, J = 7.6 Hz), 7.98 (1H, d, J = 7.8 Hz), 7.94 (1H, s), 7.61–7.51 (4H, m), 7.24 (1H, d, J = 8.4 Hz), 5.18 (1H, t, J = 2.7 Hz), 4.96 (1H, d, J = 13.4 Hz), 4.91 (1H, d, J = 13.8 Hz), 3.69 (1H, td, J = 10.5; 3.9 Hz), 2.99 (1H, d, J = 9.6 Hz), 2.68 (1H, dd, J = 13.5; 4.2 Hz), 2.04 (1H, m), 1.93 (3H, m), 1.77–1.60 (4H, m), 1.43–1.18 (12H, m), 1.12 (3H, s), 1.05 (3H, s), 0.93 (3H, s), 0. 86 (3H, s), 0.84 (3H, s), 0.83 (3H, s), 0.49 (3H, s); 13C NMR (75 MHz, CDCl₃): δ 176.1, 143.2, 142.7, 133.6, 133.4, 131.1, 128.2, 128.1, 128.1, 127.2, 126.2, 124.4, 124.4, 121.9, 121.6, 83.4, 68.5, 55.0, 53.4, 47.0, 46.3, 45.3, 41.2, 40.8, 38.3, 38.2, 37.9, 36.4, 33.3, 32.5, 31.9, 30.1, 27.6, 27.1, 26.6, 25.2, 23.1, 22.8, 22.5, 17.7, 16.5, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₄₃H₅₈N₃O₄)⁺[M + H]⁺680.4427, found 680.4429.

General procedure for the synthesis of mono-1,4-triazolyl derivatives 9a–g under CuAAC condition

Under solvent-free conditions, 0.1 g (0.2  mmol) of dipolarophile 2, Copper(I) iodide (0.5 equiv.) and triethylamine (1 equiv.) were added at room temperature. To this mixture, aryl- azide (0.4  mmol) was added and the reaction mixture was subjected to microwave irradiations at 200 W completed within 2–4 minutes. The crude mixture was extracted with EtOAc (3 × 25 mL) and the combined organic layer was dried over sodium sulfate, concentrated under reduced pressure and purified through a column chromatography or by recrystallization, to give pure 9a–g in 90–99% yields.

(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9a). Yellow paste; m.p.: 202–204 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.01 (1H, s), 7.70 (2H, dd, J = 7.5; 1.5 Hz), 7.50 (2H, dd, J = 7.2; 1.5 Hz), 7.42 (1H, m), 5.25 (3H, m), 3.15 (1H, dd, J = 11.2; 4.8 Hz), 2.87 (1H, dd, J = 7.7; 3.8 Hz), 1.94 (2H, m), 1.80 (2H, dd, J = 8.7; 3.5 Hz), 1.63–1.45 (12H, m), 1.34 (2H, m), 1.26 (4H, m), 1.08 (3H, s), 0.94 (3H, s), 0.89 (3H, s), 0.86 (3H, s), 0.74 (3H, s), 0.72 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 143.7, 143.5, 136.5, 129.7, 129.7, 129.7, 128.8, 122.5, 120.5, 120.5, 78.9, 57.3, 55.2, 47.5, 46.7, 45.8, 41.6, 41.4, 39.2, 38.7, 38.4, 36.9, 33.6, 33.0, 32.6, 32.4, 30.6, 29.6, 28.0, 27.6, 27.1, 25.7, 23.9, 23.8, 22.9, 18.2, 16.7, 15.5, 15.1; HRMS (ESI⁺): calculated for (C₃₉H₅₆N₃O₄)⁺[M + H]⁺630.4271, found 630.4274.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9b). Dark purple solid; m.p.: 280–281 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.96 (1H, s), 7.62 (2H, d, J = 7.8 Hz), 7.02 (2H, d, J = 8.1 Hz), 5.25 (3H, m), 3.87 (3H, s), 3.20 (1H, dd, J = 10.2; 4.2 Hz), 2.84 (1H, dd, J = 8.7; 3.8 Hz), 2.00 (2H, m), 1.80 (2H, dd, J = 9.0; 3.0 Hz), 1.63–1.45 (10H, m), 1.33–1.19 (8H, m), 1.10 (3H, s), 0.97 (3H, s), 0.91 (3H, s), 0.89 (3H, s), 0.78 (3H, s), 0.75 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 159.3, 143.1, 143.0, 129.8, 122.2, 122.9, 121.6, 121.6, 114.2, 114.2, 78.4, 56.8, 55.1, 46.9, 46.2, 45.3, 41.1, 40.8, 38.7, 38.2, 37.8, 36.4, 33.2, 32.6, 32.0, 31.8, 30.1, 29.2, 27.6, 27.1, 26.6, 25.2, 23.1, 22.8, 22.4, 17.7, 16.2, 15.0, 14.7; HRMS (ESI⁺): calculated for (C₄₀H₅₈N₃O₅)⁺[M + H]⁺660.4376, found 660.4378.

(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9c). Dark red solid; m.p.: 286–287 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.69 (2H, dd, J = 6.9; 2.7 Hz), 7.51 (2H, dd, J = 6.6; 3.0 Hz), 5.28 (3H, m), 3.21 (1H, dd, J = 10.8; 4.2 Hz), 2.87 (1H, dd, J = 9.2; 3.8 Hz), 2.01 (1H, m), 1.80 (2H, dd, J = 7.8; 2.4 Hz), 1.68–1.45 (12H, m), 1.29 (3H, m), 1.18 (4H, m), 1.11 (3H, s), 0.97 (3H, s), 0.92 (3H, s), 0.90 (3H, s), 0.78 (3H, s), 0.76 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.8, 143.5, 135.5, 134.6, 129.9, 129.9, 129.0, 122.5, 121.6, 121.6, 116.2, 78.9, 57.2, 55.1, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 38.7, 38.4, 36.9, 33.8, 33.0, 32.6, 32.4, 31.8, 30.6, 28.0, 27.6, 27.1, 25.7, 23.6, 23.3, 22.9, 18.2, 16.7, 15.5; HRMS (ESI⁺): calculated for (C₃₉H₅₅ClN₃O₄)⁺[M + H]⁺664.3881, found 664.3884.

(1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9d). White solid; m.p.: 294–296 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.01 (1H, s), 7.65 (2H, dd, J = 8.1; 3.4 Hz), 7.50 (2H, dd, J = 7.8; 3.2 Hz), 5.29 (3H, m), 3.21 (1H, dd, J = 10.8; 4.2 Hz), 2.87 (1H, dd, J = 9.2; 3.8 Hz), 1.99 (2H, m), 1.80 (2H, dd, J = 7.9; 2.8 Hz), 1.71–1.55 (10H, m), 1.33–1.27 (4H, m), 1.18 (4H, m), 1.11 (3H, s), 0.97 (3H, s), 0.92 (3H, s), 0.90 (3H, s), 0.78 (3H, s), 0.76 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 143.7, 136.1, 133.9, 129.2, 129.2, 129.0, 122.5, 120.8, 120.8, 115.9, 78.7, 56.8, 55.5, 47.6, 46.7, 45.8, 41.9, 41.3, 39.4, 38.7, 38.8, 36.7, 33.9, 33.3, 32.9, 32.4, 31.8, 30.6, 28.7, 27.9, 27.4, 25.7, 23.6, 23.3, 22.9, 18.2, 16.8, 15.2; HRMS (ESI⁺): calculated for (C₃₉H₅₅BrN₃O₄)⁺[M + H]⁺708.3376, found 708.3379.

(1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9e). Yellow solid; m.p.: 298–300 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.60 (2H, d, J = 7.9 Hz), 7.00 (2H, d, J = 8.1 Hz), 5.28 (3H, m), 3.23 (1H, dd, J = 10.8; 3.8 Hz), 2.87 (1H, dd, J = 9.6; 4.0 Hz), 2.01 (1H, m), 1.83 (2H, dd, J = 7.8; 2.6 Hz), 1.68–1.44 (12H, m), 1.29 (3H, m), 1.22 (4H, m), 1.10 (3H, s), 0.98 (3H, s), 0.92 (3H, s), 0.90 (3H, s), 0.79 (3H, s), 0.76 (3H, s), 0.49 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.6, 143.1, 135.0, 134.4, 129.6, 129.6, 129.0, 122.4, 121.4, 121.4, 116.8, 78.9, 56.8, 55.0, 47.2, 46.6, 45.5, 41.3, 41.0, 39.2, 38.7, 38.2, 36.9, 33.8, 33.0, 32.7, 32.4, 31.8, 30.9, 28.2, 27.6, 27.0, 25.7, 23.9, 23.3, 22.9, 18.2, 16.8, 15.9; HRMS (ESI⁺): calculated for (C₃₉H₅₅N₄O₆)⁺[M + H]⁺675.4122, found 675.4124.

(1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9f). Dark red solid; m.p.: 288–290 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.55 (1H, s), 7.50 (1H, d, J = 8.1 Hz), 7.39 (1H, t, J = 7.5 Hz), 7.25 (1H, d, J = 7.5 Hz), 5.28 (3H, m), 3.23 (1H, dd, J = 10.8; 4.2 Hz), 2.87 (1H, dd, J = 9.2; 3.8 Hz), 2.44 (3H, s), 1.95 (1H, td, J = 10.2; 3.8 Hz), 1.83 (2H, dd, J = 8.7; 3.3 Hz), 1.67–1.43 (10H, m), 1.38 (8H, m), 1.12 (3H, s), 1.04 (3H, s), 0.97 (3H, s), 0.94 (3H, s), 0.78 (3H, s), 0.75 (3H, s), 0.67 (1H, m), 0.45 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 143.6, 143.5, 139.9, 136.9, 129.5, 129.4, 122.6, 122.4, 121.2, 117.6, 78.9, 57.3, 55.1, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 38.7, 38.4, 36.9, 33.8, 33.0, 32.6, 32.4, 31.8, 30.6, 29.6, 28.0, 27.6, 27.1, 25.7, 23.6, 23.3, 22.9, 18.2, 16.7, 15.5; HRMS (ESI⁺): calculated for (C₄₀H₅₈N₃O₄)⁺[M + H]⁺644.4427, found 644.4432.

(1-Naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate (9 g). Eggplant solid; m.p.: 286–287 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.03–7.94 (3H, m), 7.65–7.50 (5H, m), 5.38 (1H, d, J = 12.9 Hz), 5.30 (1H, d, J = 13.8 Hz), 5.26 (1H, s), 3.17 (1H, dd, J = 10.8; 5.1 Hz), 2.88 (1H, dd, J = 13.5; 4.2 Hz), 1.94 (1H, m), 1.80 (2H, m), 1.71–1.57 (3H, m), 1.60–1.47 (8H, m), 1.42–1.37 (2H, m), 1.28–1.19 (6H, m), 1.12 (3H, s), 0.96 (3H, s), 0.91 (3H, s), 0.88 (3H, s), 0.74 (3H, s), 0.73 (3H, s), 0.54 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 143.1, 142.7, 133.7, 133.0, 129.9, 127.8, 127.8, 127.4, 126.5, 126.2, 124.4, 122.9, 121.9, 121.8, 78.4, 59.8, 57.0, 54.6, 47.0, 46.3, 45.3, 41.2, 40.8, 38.3, 38.2, 37.9, 36.4, 33.3, 32.5, 31.9, 30.1, 27.6, 27.1, 26.6, 25.2, 23.1, 22.8, 22.5, 17.7, 16.5, 15.0, 14.6; HRMS (ESI⁺): calculated for (C₄₃H₅₈N₃O₄)⁺[M + H]⁺680.4427, found 680.4429.

General procedure for the synthesis of bis-1,4-triazolyl derivatives 10a–g and 11a–g under CuAAC condition

Under solvent-free conditions, 0.1 g of bis-alkynes 4 or 5, Copper(I) iodide (0.8 equiv.) and triethylamine (2 equiv.) were added at room temperature. To this mixture, aryl azide (4 equiv.) was added and the reaction mixture was subjected to microwave irradiations at 250 W completed within 4–7 minutes. The crude mixture was extracted with EtOAc (3 × 25 mL) and the combined organic layer was dried over sodium sulfate, concentrated under reduced pressure and purified by recrystallization or silica gel column chromatography and eluted with petroleum ether:ethyl acetate (7:3) to obtain the desired new cycloadducts (10a–g and 11a–g).

(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-3-hydroxy-olean-12-en-28-oate (10a). Yellowish solid; m.p.: 227–228 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.06 (1H, s), 8.00 (1H, s), 7.74 (2H, m), 7.71 (2H, m), 7.56–7.50 (3H, m), 7.48–7.43 (3H, m), 5.32–5.22 (3H, m), 4.88 (1H, d, J = 12.3 Hz), 4.72 (1H, d, J = 12.3 Hz), 3.56 (1H, td, J = 11.2; 4.8 Hz), 3.13 (1H, d, J = 9.3 Hz), 2.87 (3H, m), 2.14–1.85 (4H, m), 1.68–1.48 (8H, m), 1.34 (3H, m), 1.26 (3H, m), 1.09 (3H, s), 1.04 (3H, s), 0.92 (3H, s), 0.89 (3H, s), 0.84 (3H, s), 0.80 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 143.3, 143.2, 143.2, 136.4, 136.3, 129.3, 129.3, 129.2, 129.2, 128.4, 128.4, 122.1, 121.7, 120.1, 120.1, 120.1, 120.0, 120.0, 81.0, 77.9, 61.8, 56.8, 54.5, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.4, 17.6, 16.4, 16.2, 15.9; HRMS (ESI⁺): calculated for (C₄₈H₆₃N₆O₄)⁺[M + H]⁺787.4911, found 787.4908.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-hydroxy-olean-12-en-28-oate (10b). Dark solid; m.p.: 235–237 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.94 (1H, s), 7.87 (1H, s), 7.60 (4H, d, J = 8.7 Hz), 7.03–7.00 (4H, m), 5.30–5.24 (3H, m), 4.86 (1H, d, J = 12.3 Hz), 4.70 (1H, d, J = 12.3 Hz), 3.89 (6H, s), 3.53 (1H, td, J = 11.2; 4.8 Hz), 3.16 (1H, d, J = 9.3 Hz), 2.88 (1H, dd, J = 13.5; 4.2 Hz), 2.84 (2H, m), 2.16–1.88 (4H, m), 1.70–1.49 (6H, m), 1.37 (4H, m), 1.29–1.18(4H, m), 1.10 (3H, s), 1.04 (3H, s), 0.93 (3H, s), 0.89 (3H, s), 0.85 (3H, s), 0.81 (3H, s), 0.47 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.6, 160.3, 158.9, 143.8, 143.8, 143.2, 136.4, 136.3, 134.8, 134.8, 125.8, 125.8, 125.7, 121.7, 121.6, 120.2, 114.3, 114.2, 114.0, 80.9, 77.8, 61.9, 55.1, 55.1, 55.0, 54.0, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 16.4, 16.3, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₅₀H₆₇N₆O₆)⁺[M + H]⁺847.5122, found 847.5120.

(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-hydroxy-olean-12-en-28-oate (10c). Dark solid; m.p.: 230–231 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.95 (1H, s), 7.68 (4H, d, J = 8.7 Hz), 7.50 (4H, dd, J = 5.1; 3.3 Hz), 5.30 (1H, m), 5.26 (2H, m), 4.86 (1H, d, J = 12.3 Hz), 4.70 (1H, d, J = 12.3 Hz), 3.55 (1H, td, J = 11.2; 4.8 Hz), 3.12 (1H, d, J = 9.6 Hz), 2.86 (1H, dd, J = 13.5; 4.2 Hz), 2.13–1.85 (6H, m), 1.67–1.44 (8H, m), 1.33 (2H, m), 1.26 (4H, m), 1.10 (3H, s), 1.04 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 144.8, 143.5, 143.2, 134.9, 134.9, 134.1, 134.1, 129.4, 129.4, 129.4, 129.4, 122.4, 122.2, 121.7, 121.7, 121.6, 121.6, 120.3, 81.1, 77.9, 61.9, 55.0, 54.0, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 16.4, 16.3, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₄₈H₆₁Cl₂N₆O₄)⁺[M + H]⁺855.4131, found 855.4127.

(1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxy-olean-12-en-28-oate (10d). White solid; m.p.: 227–229 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.95 (1H, s), 7.70 (4H, d, J = 8.7 Hz), 7.52 (4H, m), 5.31 (1H, t, J = 3.3 Hz), 5.28 (2H, m), 4.87 (1H, d, J = 12.3 Hz), 4.71 (1H, d, J = 12.3 Hz), 3.53 (1H, td, J = 11.2; 4.8 Hz), 3.15 (1H, d, J = 9.6 Hz), 2.83 (1H, dd, J = 13.5; 4.2 Hz), 2.11–1.81 (8H, m), 1.69–1.40 (4H, m), 1.33 (4H, m), 1.23 (4H, m), 1.11 (3H, s), 1.02 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.80 (3H, s), 0.75 (3H, s), 0.48 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 144.8, 143.5, 143.2, 134.9, 134.9, 134.1, 134.1, 129.4, 129.4, 129.4, 129.4, 122.4, 122.2, 121.7, 121.7, 121.6, 121.6, 120.3, 81.1, 77.9, 61.9, 55.0, 54.0, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 16.4, 16.3, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₄₈H₆₁Br₂N₆O₄)⁺[M + H]⁺943.3121, found 943.3118.

(1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxy-olean-12-en-28-oate (10e). Yellowish solid; m.p.: 251–253 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.00 (1H, s), 7.94 (1H, s), 7.77–7.68 (4H, m), 7.55–7.49 (4H, m), 5.31 (1H, t, J = 3.6 Hz), 5.28 (2H, m), 4.85 (1H, d, J = 12.3 Hz), 4.73 (1H, d, J = 12.3 Hz), 3.55 (1H, td, J = 11.2; 4.8 Hz), 3.17 (1H, d, J = 9.6 Hz), 2.81 (1H, dd, J = 13.5; 4.2 Hz), 2.09–1.85 (6H, m), 1.71–1.45 (6H, m), 1.33 (2H, m), 1.29–1.21 (6H, m), 1.11 (3H, s), 1.05 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 144.8, 143.5, 143.2, 134.9, 134.9, 134.1, 134.1, 129.4, 129.4, 129.4, 129.4, 122.4, 122.2, 121.7, 121.7, 121.6, 121.6, 120.3, 81.1, 77.9, 61.9, 55.0, 54.0, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 16.4, 16.3, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₄₈H₆₁N₈O₈)⁺[M + H]⁺877.4612, found 877.4610.

(1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-(3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxy-olean-12-en-28-oate (10f). White solid; m.p.: 211–213 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.96 (1H, s), 7.55 (2H, m), 7.48 (2H, m), 7.41–7.36 (2H, m), 7.24 (2H, m), 5.32–5.21 (3H, m), 4.86 (1H, d, J = 12.3 Hz), 4.71 (1H, d, J = 12.3 Hz), 3.57 (1H, td, J = 11.2; 4.8 Hz), 3.17 (1H, d, J = 9.3 Hz), 2.87 (1H, dd, J = 13.5; 4.2 Hz), 2.45 (3H, s), 2.44 (3H, s), 2.13–1.86 (4H, m), 1.67–1.43 (6H, m), 1.33 (4H, m), 1.29–1.21 (6H, m), 1.11 (3H, s), 1.05 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.45 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.2, 145.2, 143.2, 139.4, 136.5, 136.4, 131.3, 129.0, 129.0, 128.5, 126.5, 122.2, 121.7, 120.7, 120.6, 119.9, 117.0, 113.6, 111.7, 81.0, 77.4, 61.9, 56.8, 54.5, 47.0, 46.2, 45.3, 42.4, 41.2, 40.8, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 20.8, 20.8, 16.4, 16.3, 16.3, 16.1; HRMS (ESI⁺): calculated for (C₅₀H₆₇N₆O₄)⁺[M + H]⁺815.5224, found 815.5220.

(1-Naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-((1-naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methoxy)-3-hydroxy-olean-12-en-28-oate (10 g). dark purple solid; m.p.: 219–220 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.05–7.96 (4H, m), 8.02 (1H, s), 7.91 (1H, s), 7.68–7.53 (10H, m), 5.40 (1H, d, J = 12.6 Hz), 5.34 (1H, d, J = 12.6 Hz), 5.31 (1H, t, J = 3.6 Hz), 4.95 (1H, d, J = 12.3 Hz), 4.81 (1H, d, J = 12.3 Hz), 3.61 (1H, td, J = 11.2; 4.8 Hz), 3.17 (1H, d, J = 9.3 Hz), 2.87 (1H, dd, J = 13.5; 4.2 Hz), 2.15–1.87 (4H, m), 1.75–1.43 (10H, m), 1.39–1.31 (3H, m), 1.25 (2H, m), 1.13 (3H, s), 1.06 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.83 (3H, s), 0.80 (3H, s), 0.57 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 143.9, 142.7, 143.2, 133.7, 133.6, 133.2, 133.0, 133.0, 129.9, 129.9, 128.0, 127.8, 127.7, 127.7, 127.7, 127.7, 127.4, 126.6, 126.2, 124.4, 124.4, 123.0, 122.8, 121.8, 121.7, 81.0, 77.4, 62.0, 57.0, 54.5, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 17.6, 16.6, 16.6, 16.4, 16.0; HRMS (ESI⁺): calculated for (C₅₆H₆₇N₆O₄)⁺[M + H]⁺887.5224, found 887.5221.

(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11a). Yellowish solid; m.p.: 230–232 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.94 (1H, s), 7.73–7.69 (4H, m), 7.54–7.47 (4H, m), 7.45–7.39 (2H, m), 5.28–5.25 (3H, m), 4.98 (1H, d, J = 12.0 Hz), 4.95 (1H, d, J = 12.0 Hz), 3.88 (1H, td, J = 11.2; 4.8 Hz), 2.95 (1H, d, J = 9.3 Hz), 2.87 (1H, dd, J = 13.5; 4.2 Hz), 2.02 (2H, m), 2.00–1.85 (4H, m), 1.70–1.61 (4H, m), 1.64–1.43 (4H, m), 1.34 (4H, m), 1.26 (4H, m), 1.14 (3H, s), 1.10 (3H, s), 0.99 (3H, s), 0.89 (3H, s), 0.83 (3H, s), 0.79 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.2, 143.3, 143.0, 143.0, 136.4, 136.4, 129.2, 129.2, 129.2, 129.2, 129.2, 129.2, 128.3, 128.2, 122.1, 120.1, 120.1, 119.9, 119.9, 94.6, 68.2, 67.2, 56.8, 54.8, 47.0, 46.2, 46.1, 45.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.4, 17.6, 17.0, 16.2, 15.9; HRMS (ESI⁺): calculated for (C₄₈H₆₃N₆O₄)⁺[M + H]⁺787.4911, found 787.4905.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11b). Dark solid; m.p.: 269–270 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.94 (1H, s), 7.86 (1H, s), 7.60 (4H, dd, J = 6.9; 2.4 Hz), 7.00 (4H, dd, J = 6.9; 2.4 Hz), 5.30 (1H, m), 5.27 (1H, d, J = 12.9 Hz), 5.25 (1H, d, J = 12.9 Hz), 4.97 (1H, d, J = 12.3 Hz), 4.94 (1H, d, J = 12.3 Hz), 3.86 (1H, td, J = 11.2; 4.8 Hz), 3.85 (6H, s), 3.16 (1H, d, J = 9.3 Hz), 2.87 (1H, dd, J = 13.5; 4.2 Hz), 2.10–1.86 (4H, m), 1.66–1.48 (8H, m), 1.43 (4H, m), 1.35–1.19 (4H, m), 1.13 (3H, s), 1.03 (3H, s), 0.93 (3H, s), 0.89 (3H, s), 0.85 (3H, s), 0.81 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.2, 159.4, 159.3, 146.0, 143.1, 143.0, 129.9, 129.8, 122.1, 121.9, 121.7, 121.7, 121.5, 121.5, 119.8, 114.2, 114.2, 114.2, 114.2, 94.6, 68.2, 67.3, 56.9, 55.1, 55.1, 54.8, 47.0, 46.2, 46.1, 45.2, 41.1, 40.6, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 22.9, 16.4, 16.3, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₅₀H₆₇N₆O₆)⁺[M + H]⁺847.5122, found 847.5103.

(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11c). Yellowish solid; m.p.: 244–246 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.94 (1H, s), 7.73 (4H, dd, J = 6.9; 2.1 Hz), 7.50 (4H, dd, J = 6.9; 2.4 Hz), 5.29 (1H, m), 5.26 (2H, s), 4.98 (2H, s), 3.86 (1H, td, J = 11.2; 4.8 Hz), 3.12 (1H, d, J = 9.3 Hz), 2.86 (1H, dd, J = 13.5; 4.2 Hz), 2.13–1.85 (4H, m), 1.69–1.41 (10H, m), 1.36 (2H, m), 1.28 (4H, m), 1.10 (3H, s), 1.04 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.46 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 145.1, 144.0, 144.0, 139.9, 139.9, 134.6, 134.5, 129.9, 129.9, 129.9, 129.9, 122.4, 122.4, 121.7, 121.7, 121.6, 121.6, 120.0, 95.0, 68.8, 67.7, 57.2, 55.3, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 18.1, 17.6, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₄₈H₆₁Cl₂N₆O₄)⁺[M + H]⁺855.4131, found 855.4123.

(1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11d). White solid; m.p.: 256–258 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.01 (1H, s), 7.91 (1H, s), 7.76 (4H, dd, J = 6.6; 2.1 Hz), 7.52 (4H, dd, J = 6.6; 2.1 Hz), 5.29 (3H, m), 4.99 (1H, d, J = 12.3 Hz), 4.96 (1H, d, J = 12.03 Hz), 3.89 (1H, td, J = 11.2; 4.8 Hz), 3.14 (1H, d, J = 9.3 Hz), 2.89 (1H, dd, J = 13.5; 4.2 Hz), 2.17–1.89 (4H, m), 1.69–1.46 (8H, m), 1.39 (2H, m), 1.35–1.26 (6H, m), 1.11 (3H, s), 1.05 (3H, s), 0.90 (3H, s), 0.86 (3H, s), 0.82 (3H, s), 0.78 (3H, s), 0.47 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.6, 146.0, 144.2, 144.4, 139.4, 139.4, 134.6, 134.5, 129.9, 129.9, 129.8, 129.8, 122.9, 122.9, 121.5, 121.5, 121.5, 121.5, 119.5, 95.0, 68.8, 67.7, 57.2, 55.3, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 18.1, 17.6, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₄₈H₆₁Br₂N₆O₄)⁺[M + H]⁺943.3121, found 943.3115.

(1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11e). Yellowish solid; m.p.: 218–220 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.03 (1H, s), 7.95 (1H, s), 7.75–1.71 (4H, m), 7.52–7.49 (4H, m), 5.28 (3H, m), 4.98 (2H, s), 3.89 (1H, td, J = 11.2; 4.8 Hz), 3.12 (1H, d, J = 9.3 Hz), 2.91 (1H, dd, J = 13.5; 4.2 Hz), 2.11–1.89 (6H, m), 1.71–1.55 (6H, m), 1.46 (4H, m), 1.31 (4H, m), 1.12 (3H, s), 1.04 (3H, s), 0.92 (3H, s), 0.86 (3H, s), 0.81 (3H, s), 0.75 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 145.1, 144.0, 144.0, 139.9, 139.9, 134.6, 134.5, 129.9, 129.9, 129.9, 129.9, 122.4, 122.4, 121.7, 121.7, 121.6, 121.6, 120.0, 95.0, 68.8, 67.7, 57.2, 55.3, 47.0, 46.2, 45.2, 42.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 23.0, 18.1, 17.6, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₄₈H₆₁N₈O₈)⁺[M + H]⁺877.4612, found 877.4604.

(1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1-(3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11f). White solid; m.p.: 261–263 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s), 7.93 (1H, s), 7.55 (2H, s), 7.50 (2H, d, J = 8.1 Hz), 7.42–7.36 (2H, td, J = 7.8; 1.5 Hz), 7.25 (2H, d, J = 8.1 Hz), 5.31 (1H, m), 5.29 (1H, d, J = 12.6 Hz), 5.25 (1H, d, J = 12.6 Hz), 5.01 (1H, d, J = 12.3 Hz), 4.95 (1H, d, J = 12.3 Hz), 3.87 (1H, td, J = 11.2; 4.8 Hz), 3.17 (1H, d, J = 9.3 Hz), 2.88 (1H, dd, J = 13.5; 4.2 Hz), 2.45 (6H, s), 2.04 (2H, m), 1.89 (2H, m), 1.67–1.43 (10H, m), 1.36 (2H, m), 1.25–1.17 (4H, m), 1.11 (3H, s), 1.05 (3H, s), 0.97 (3H, s), 0.93 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.45 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.2, 143.7, 143.5, 143.5, 139.9, 139.9, 137.0, 136.9, 129.6, 129.6, 129.5, 129.5, 121.6, 122.4, 121.3, 121.2, 120.1, 117.6, 117.5, 95.1, 77.2, 68.7, 67.8, 57.3, 55.3, 47.1, 45.3, 42.4, 41.2, 40.8, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.6, 23.4, 22.9, 21.3, 21.3, 18.1, 17.6, 16.7, 16.5; HRMS (ESI⁺): calculated for (C₅₀H₆₇N₆O₄)⁺[M + H]⁺815.5224, found 815.5218.

(1-Naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2-hydroxy-3-((1 naphthalen-1-yl -1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (11 g). Dark purple solid; m.p.: 244–245 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.05–7.91 (4H, m), 8.01 (1H, s), 7.97 (1H, s), 7.67–7.52 (10H, m), 5.41–531 (3H, m), 5.09 (1H, d, J = 12.6 Hz), 5.03 (1H, d, J = 12.6 Hz), 3.92 (1H, td, J = 11.2; 4.8 Hz), 3.04 (1H, d, J = 9.6 Hz), 2.90 (1H, dd, J = 13.5; 4.2 Hz), 2.15–1.87 (6H, m), 1.75–1.43 (8H, m), 1.35(3H, m), 1.26 (2H, m), 1.13 (3H, s), 1.06 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.83 (3H, s), 0.80 (3H, s), 0.57 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 143.6, 142.7, 143.2, 133.7, 133.6, 133.2, 133.0, 133.0, 129.9, 129.9, 128.0, 127.8, 127.7, 127.7, 127.7, 127.7, 127.4, 126.6, 126.2, 124.4, 124.4, 123.0, 122.4, 122.3, 122.2, 95.2, 77.2, 68.8, 67.8, 57.6, 55.4, 47.6, 46.8, 45.8, 41.7, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 17.6, 16.6, 16.6, 16.4, 16.0; HRMS (ESI⁺): calculated for (C₅₆H₆₇N₆O₄)⁺[M + H]⁺887.5224, found 887.5220.

General procedure for the synthesis of tri-1,4-triazolyl derivatives 12a–g under CuAAC condition

Maslinic acid-tri-alkyne 6 (0.1 g, 0.17  mmol) and the appropriate azide (1  mmol) were dissolved in 3 mL H₂O. While the mixture was being stirred, cuprous iodide (1 equiv) was added, followed by Et₃N (0.5  mmol) at room temperature. The reaction mixture was then subjected to microwave irradiations at 300 W for 5–10 min, after which it was diluted with water and then extracted with ethyl acetate (3 × 50 mL). The organic layer was dried over Na₂SO₄. After removal of solvent in vacuo, the resulting residue was purified by recrystallization or silica gel column chromatography and eluted with petroleum ether:ethyl acetate (8:2) to obtain the desired new products (12a–g).

(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12a). Purple solid; m.p.: 214–216 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.27 (1H, s), 8.07 (1H, s), 8.05 (1H, s), 7.77–7.71 (6H, m), 7.57–7.36 (9H, m), 5.33–5.23 (5H, m), 4.90 (1H, d, J = 12.3 Hz), 4.81 (1H, d, J = 12.3 Hz), 3.73 (1H, td, J = 11.2; 4.8 Hz), 3.03 (1H, d, J = 9.6 Hz), 2.91 (1H, dd, J = 13.8; 4.2 Hz), 2.14–1.85 (6H, m), 1.78–1.48 (11H, m), 1.31 (2H, m), 1.20 (2H, m), 1.12 (3H, s), 1.07 (3H, s), 1.00 (3H, s), 0.89 (3H, s), 0.84 (3H, s), 0.80 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 143.6, 137.1, 137.1, 137.1, 129.7, 129.7, 129.7, 129.6, 129.6, 129.6, 129.6, 129.6, 129.6, 128.8, 128.6, 128.4, 122.5, 122.3, 120.5, 120.4, 120.3, 120.3, 120.3, 120.3, 120.3, 120.3, 90.7, 77.9, 67.5, 63.3, 57.3, 47.5, 46.7, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.4, 17.6, 16.4, 16.2, 15.9; HRMS (ESI⁺): calculated for (C₅₇H₇₀N₉O₄)⁺[M + H]⁺944.5551, found 944.5573.

(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12b). Red solid; m.p.: 255–257 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.16 (1H, s), 7.97 (1H, s), 7.95 (1H, s), 7.65–7.58 (6H, m), 7.05–6.95 (6H, m), 5.32–5.20 (5H, m), 4.90–4.77 (2H, m), 3.87 (6H, s), 3.84 (3H, s), 3.79 (1H, td, J = 11.2; 4.8 Hz), 3.02 (1H, d, J = 9.9 Hz), 2.92 (1H, dd, J = 13.8; 4.2 Hz), 2.14–1.85 (5H, m), 1.78–1.48 (10H, m), 1.31 (4H, m), 1.20 (2H, m), 1.12 (3H, s), 1.07 (3H, s), 1.00 (3H, s), 0.89 (3H, s), 0.84 (3H, s), 0.80 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 159.9; 159.7, 159.6, 146.6, 146.5, 143.6, 143.5, 130.6, 130.4, 122.2, 122.1, 122.1, 121.9, 121.9, 121.9, 121.0, 120.9, 114.8, 114.8, 114.8, 114.7, 114.7, 114.7, 114.7, 114.7, 114.7, 90.7, 77.9, 67.5, 63.3, 57.3, 55.5, 55.6, 55.5, 47.5, 46.7, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.6, 18.2, 17.5, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₆₀H₇₆N₉O₄)⁺[M + H]⁺1034.5868, found 1034.5917.

(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12c). Yellowish solid; m.p.: 234–235 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.20 (1H, s), 7.96 (1H, s), 7.93 (1H, s), 7.65–7.57 (6H, m), 7.44–7.35 (6H, m), 5.23–5.10 (5H, m), 4.90–4.77 (2H, m), 3.67 (1H, td, J = 11.2; 4.8 Hz), 2.95 (1H, d, J = 9.9 Hz), 2.87 (1H, dd, J = 13.8; 4.2 Hz), 2.14–1.85 (5H, m), 1.78–1.48 (10H, m), 1.31 (4H, m), 1.20 (2H, m), 1.12 (3H, s), 1.07 (3H, s), 0.97 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.74 (3H, s), 0.38 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 159.9; 159.7, 159.6, 146.6, 146.5, 143.6, 143.5, 130.6, 130.4, 122.2, 122.1, 122.1, 121.9, 121.9, 121.9, 121.0, 120.9, 114.8, 114.8, 114.8, 114.7, 114.7, 114.7, 114.7, 114.7, 114.7, 90.7, 77.9, 67.5, 63.3, 57.3, 47.5, 46.7, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.6, 18.2, 17.5, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₅₇H₆₇Cl₃N₉O₄)⁺[M + H]⁺1046.4382, found 1046.4411.

(1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12d). White solid; m.p.: 221–223 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.26 (1H, s), 8.01 (1H, s), 7.97 (1H, s), 7.75–7.55 (6H, m), 7.41–7.29 (6H, m), 5.25 (4H, m), 5.21–5.12 (4H, m), 4.93–4.79 (2H, m), 3.71 (1H, td, J = 11.2; 4.8 Hz), 2.99 (1H, d, J = 9.9 Hz), 2.87 (1H, dd, J = 13.8; 4.2 Hz), 2.14–1.85 (5H, m), 1.70–1.55 (6H, m), 1.48–1.38 (4H, m), 1.31 (2H, m), 1.25 (4H, m), 1.12 (3H, s), 1.07 (3H, s), 0.97 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.74 (3H, s), 0.49 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 159.6; 159.3, 159.3, 145.1, 145.1, 151.1, 143.5, 130.6, 130.4, 122.2, 122.1, 122.1, 121.9, 121.9, 121.9, 121.0, 120.9, 115.9, 115.8, 115.8, 1116.0, 116.0, 116.0, 116.2, 116.2, 116.2, 90.7, 77.9, 67.5, 63.3, 57.3, 47.5, 46.7, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.6, 18.2, 17.8, 16.2, 16.0; HRMS (ESI⁺): calculated for (C₅₇H₆₇Br₃N₉O₄)⁺[M + H]⁺1178.2866, found 1178.2897.

(1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12e). Yellowish solid; m.p.: 211–212 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.17 (1H, s), 7.91 (1H, s), 7.90 (1H, s), 7.68–7.55 (6H, m), 7.40–7.31 (6H, m), 5.24–5.16 (5H, m), 4.92–4.78 (2H, m), 3.69 (1H, td, J = 11.2; 4.8 Hz), 2.95 (1H, d, J = 9.9 Hz), 2.87 (1H, dd, J = 13.8; 4.2 Hz), 2.14–1.85 (5H, m), 1.78–1.48 (8H, m), 1.39 (4H, m), 1.22 (4H, m), 1.12 (3H, s), 1.07 (3H, s), 0.97 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.74 (3H, s), 0.44 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 159.9; 159.7, 159.6, 146.6, 146.5, 143.6, 143.5, 130.6, 130.4, 122.2, 122.1, 122.1, 121.6, 121.6, 121.6, 121.0, 120.9, 115.2, 115.2, 115.1, 114.9, 114.8, 114.8, 114.7, 114.7, 114.7, 90.7, 77.9, 67.5, 63.3, 57.3, 47.5, 46.7, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.6, 18.2, 17.5, 16.7, 16.4; HRMS (ESI⁺): calculated for (C₅₇H₆₇N₁₂O₁₀)⁺[M + H]⁺1046.5103, found 1072.5144.

(1-(3-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-(3-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12f). White solid; m.p.: 211–213 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.12 (1H, s), 8.02 (1H, s), 7.97 (1H, s), 7.57 (3H, m), 7.51–7.36 (6H, m), 7.40–7.35 (3H, m), 5.33 (1H, s), 5.30 (2H, m), 5.27 (2H, m), 4.99 (2H, m), 3.89 (1H, td, J = 11.2; 4.8 Hz), 3.17 (1H, d, J = 9.3 Hz), 2.88 (1H, dd, J = 13.5; 4.2 Hz), 2.46 (3H, s), 2.44 (6H, s), 2.06 (4H, m), 1.89 (4H, m), 1.67–1.43 (6H, m), 1.34 (2H, m), 1.25–1.19 (5H, m), 1.11 (3H, s), 1.05 (3H, s), 0.97 (3H, s), 0.93 (3H, s), 0.81 (3H, s), 0.78 (3H, s), 0.45 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.3, 143.7, 143.5, 143.5, 143.4, 139.9, 139.9, 139.8, 137.0, 136.9, 129.6, 129.4, 122.2, 122.1, 122.1, 121.0, 120.9, 115.2, 115.2, 115.1, 114.9, 114.8, 114.8, 114.8, 115.8, 115.7, 115.7, 90.0, 77.8, 67.5, 63.3, 57.3, 47.5, 46.7, 46.5, 46.5, 46.3, 45.8, 44.4, 44.3, 41.2, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 31.9, 30.2, 29.2, 28.2, 27.0, 25.3, 23.1, 23.0, 22.6, 18.2, 17.5, 16.7, 16.0; HRMS (ESI⁺): calculated for (C₆₀H₇₆N₉O₄)⁺[M + H]⁺986.6020, found 986.6053.

(1-Naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methyl-(2α,3β)-2,3-bis((1-naphthalen-1-yl-1H-1,2,3-triazol-4-yl)methoxy)-olean-12-en-28-oate (12 g). Dark purple solid; m.p.: 204–205 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.15–7.99 (12H, m), 7.77–7.64 (6H, m), 7.62–7.55 (6H, m), 5.41–531 (3H, m), 5.09 (2H, d, J = 12.6 Hz), 5.03 (2H, d, J = 12.6 Hz), 3.92 (1H, td, J = 11.2; 4.8 Hz), 3.04 (1H, d, J = 9.6 Hz), 2.90 (1H, dd, J = 13.5; 4.2 Hz), 2.15–1.87 (6H, m), 1.75–1.43 (8H, m), 1.35(3H, m), 1.26 (4H, m), 1.13 (3H, s), 1.06 (3H, s), 0.90 (3H, s), 0.87 (3H, s), 0.83 (3H, s), 0.80 (3H, s), 0.57 (3H, s); ¹³C NMR (75 MHz, CDCl₃): δ 177.5, 143.6, 143.2, 143.2, 143.2, 142.7, 142.7, 133.7, 133.6, 133.2, 133.0, 133.0, 129.9, 129.9, 129.9, 129.9, 129.9, 129.9, 128.0, 127.8, 127.7, 127.7, 127.7, 127.7, 127.4, 126.6, 126.2, 124.4, 124.4, 123.0, 122.4, 122.4, 122.4, 122.4, 122.3, 122.3, 122.3, 122.2, 122.2, 122.2, 93.2, 77.2, 68.8, 67.8, 57.6, 55.4, 47.6, 46.8, 45.8, 41.7, 40.1, 38.8, 38.5, 37.6, 33.2, 32.5, 32.0, 31.6, 29.2, 28.2, 27.0, 25.3, 23.5, 23.2, 17.6, 16.6, 16.6, 16.6, 16.2; HRMS (ESI⁺): calculated for (C₆₉H₇₆N₉O₄)⁺[M + H]⁺1094.6020, found 1094.6061.

Biological assay

Natural pentacyclic triterpenoid (Maslinic acid 1) and most of the synthesized triazoles were dissolved in dimethyl sulfoxide (DMSO) and added into the medium at final concentrations of 10, 30 and 100 μM. The final concentration of DMSO was 0.33% and was determined to have no cytotoxicity.

Determination of pro-inflammatory IL-1β cytokine production in PBMCs

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors by density gradient centrifugation (Pancoll human, d.1.077 g/mL). Recovered PBMCs were washed thrice in Dulbecco’s phosphate buffered saline, seeded in a 96-well microplate at 6.67 × 105 cells/mL in RPMI-1640 containing 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), amphotericin B (250 ng/mL), and incubated at 5% CO₂ and 37 °C. PBMCs were treated with compound 1 and the synthesized triazoles (8b–g; 9a–d,f,g; 10a,c,f,g; 11a–c,f; 12b,c,f,g) (10, 30 and 100 μM) then were stimulated for 24 h with LPS (E. coli, 0128:B12, Sigma) (10 ng/mL)28,29. Three anti-inflammatory standards were tested in the same conditions: ZVAD (5 μM), dexamethasone (DEXA, 1 μM) and prednisolone (PRED, 70 μM). After incubation, the supernatants were harvested and kept at −20 °C until use. Viability of PBMCs was assessed by XTT assay. Secretion IL-1β was measured only in the culture supernatants of PBMCs treated with compounds that showed low toxicity, using a sandwich enzyme-linked immunosorbent assay (ELISA) method (eBioscience, San Diego, CA), according to manufacturer instructions.

Determination of anti-proliferative activity in EMT-6 and SW480 cancer cell lines

Murine mammary carcinoma cell line EMT-6 and human colorectal adenocarcinoma cell line SW480, purchased from American Type Culture Collection (ATCC, Manassas, VA), were cultured in DMEM supplemented with 10% of fetal bovine serum and penicillin (100 U/mL), streptomycin (100 μg/mL), amphotericin B (250 ng/mL) and placed in CO₂ incubator with 5% of CO₂ at 37 °C. Cells were seeded in a 96-well microplate at 3.125 × 10⁴ cells/mL and treated with compounds (10, 30 and 100 μM) for 48 h. Doxorubicin, etoposide, 5-fluorouracil and methotrexate (10 μM) were used as anti-cancer references. The anti-proliferative activity of compounds and drug references were determined using XTT assay30,31. This assay is based on the conversion of the water-soluble XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) inner salt) reagent, in the presence of the electron-coupling reagent PMS (phenazine methylsulfate) to an orange formazan product by metabolically active cells. The amount of formazan produced was detected by measurement of the absorbance at 499 nm and a reference wavelength was used at 660 nm on a microplate reader Infinite M200 Pro TECAN (Männedorf, Switzerland). Cell viability is expressed as a percentage of control, which was taken as 100%.

Results and discussion

Chemistry

Synthesis of maslinic acid-alkyne derivatives 2–6 as dipolarophiles

Taking cue from the literature, we have undertaken a research program directed toward the structural modifications of maslinic acid 1 to fine tune its biological potential as anti-inflammatory and anti-proliferative agent. The synthesis of maslinic acid-alkyne derivatives 2–6 was done on maslinic acid (1) which isolated under green chemistry condition, from pomace olive (Olea europaea L.) cultivar: Chemlali with a large amount (17 g (8.5 mg/g DW) using an ultrasonic bath. The compound 1 was subjected to a propargylation of the hydroxyl groups at C-2, C-3 and carboxylic group at C-28. This reaction was tried under different conditions in dry N,N-dimethylformamide (DMF). Method I: 2 equiv. of NaH was used in the presence of propargyl bromide (3 equiv.) in dry DMF for 2 h at room temperature yielded a mixture of compounds in 82% yield (Table 1). Chromatography on a silica-gel column of this mixture resulted compounds 2 (44%), 3 (5%), 4 (15%), 5 (11%) and 6 (7%). Method II: When the amounts of NaH and propargyl bromide were increased to 4 equiv. of each in dry DMF at room temperature for 8 h, the yield of mixture has been improved (99%). Chromatography on a silica-gel column of this mixture resulted only the alkyl derivatives 2 and 4–6 in 25, 23, 20 and 21% yield, respectively (Table 1).

Table 1. Method I and II for the synthesis of dipolarophiles 2–6^a.

Compounds	Method I (82%)^b	(%)^c	Method II (99%)^b	(%)^c
2		44		25
3	NaH (2 equiv)^d	5	NaH (4 equiv)^d	0
4	propargyl bromide	15	propargyl bromide	23
5	(3 equiv)^d	11	(4 equiv)^d	20
6	2 h	7	8 h	31

^aMaslinic acid (1) in dry DMF as solvent at room temperature.

^b yield of mixture, based on the starting material (compound 1).

^c Isolated yield of compounds (2–6) after column chromatography (EP-EtOAc), based on the maslinicacid (1).

^d Referred to the starting material (compound 1).

The structures of the propargylated compounds 2–6 were unambiguously confirmed by ESI-HRMS and NMR. Thus, the molecular formula of compounds 2 and 3 (C₃₃H₅₀O₄) as determined by ESI-HRMS (m/z 511.3778 and 511.3794, respectively [M + H]⁺, calculated for 511.3787), was in agreement with a monopropargylated structure of maslinc acid 1. While, the molecular formula of compounds 4 and 5 (C₃₆H₅₂O₄) as established by ESI-HRMS (m/z 549.3938 and 549.3940, respectively [M + H]⁺, calculated for 549.3944), indicated that were they derived from the dipropargylation of maslinic acid 1. Moreover, the molecular formula of compound 6 (C₃₉H₅₄O₄) as determined by ESI-HRMS (m/z 587.4092 [M + H]⁺, calculated for 587.4100), was found to be in concordance with the tripropargylation of maslinic acid 1. The position of the alkylation in the obtained derivatives was deduced by simple comparison of their NMR spectral data (¹H and ¹³C) with those of the starting substrate 1.

RuAAC and CuAAC for the synthesis of 1,5- and 1,4-disubstituted triazoles

Aromatic azides were synthesized from the appropriate anilines using a two-step procedure by diazotization with sodium nitrite in acidic conditions followed by displacement with sodium azide. The desired aromatic azides 7a–g were obtained in yields ranging from 85 to 98%.

The thermal [3 + 2] cycloaddition method can suffer from low yields, side-reactions and poor regioselectivity32,33. While “click” reaction mediated preparations of 1,4- and 1,5-disubstituted triazoles have been reported to provide satisfactory yields34,35. The ruthenium-catalyzed version of the reaction (RuAAC), led mainly to 1,5-regioisomers36. Although the Ru(II)-catalyzed Hüisgen 1,3-dipolar cyclization usually proceeds in high yields, this type of reaction has been reported to be highly dependent on the substrate and reaction conditions. At the outset of our studies, we investigated the cyclo-addition reaction between phenylazide 7a and maslinic acid-alkyne 2 with different concentrations of the catalyst Cp*RuCl(PPh₃)₂. The most relevant results of this study are shown in Table 2.

Table 2. Optimization of the 1,3-dipolar cyclization for the ruthenium-catalyzed click reaction^a.

Entry	Mol% of Ru(II)^b	Method	Time (h)	Yield (%)
1	2	110 °C (Toluene)	8	55
2	5	110 °C (Toluene)	8	82
3	10	110 °C (Toluene)	8	78
4	5	rt (Toluene)	48	30
5	5	MW^c (DMF)	0.1	98
6	5	MW^c (H2O)	0.4	Traces
7	5	MW^c (DMF/H2O (2:1))	0.1	86
8	5	MW^c (Solvent free)	0.4	No reaction

Bold in entry highlight the optimal reaction condition.

^a Alkyne 2 (2  mmol) and two equivalents of phenylazide (7a).

^b Referred to the starting alkyne 2.

^c The reaction was performed at 250 W.

At the beginning, the reaction conditions were optimized in heating at 110 °C (reflux temperature of toluene) with different catalyst loading (Table 2, entries 1–3), and it was found that 5 mol % Ru(II) was the optimum amount of catalyst for these conditions (entry 2). When the temperature was decreased to room temperature, the yield of reaction, decreased significantly (30%) after even 48 h of reaction (entry 4). It should be noted that some catalyst deactivation has been encountered during long-heating times. Thus, to circumvent this limitation, we decided to work under microwave activation. Interestingly, from the different solvents, solvent/co-solvent mixtures and solvent-free tested under microwave conditions, DMF was found to be the most effective for this reaction (Table 2, compare entries 5–8). Thus, using 5 mol% Cp*RuCl(PPh₃)₂ under microwave heating in DMF, the reaction of dipolarophile 2 with phenylazide 7a, gave (1-phenyl-1H-1,2,3-triazol-5-yl)methyl-(2α,3β)-2,3-dihydroxyolean-12-en-28-oate 8a almost quantitatively in only 6 min of reaction time (Table 2, entry 5). It is interesting to note that anhydrous conditions are not necessary. Indeed, undistilled DMF, or DMF-containing 0.5 equiv. of water can be used to run this RuAAC (entries 5 and 7). While the use of water as solvent results in a significant reduction in yield (Traces) even after 15 min reaction (entry 6). It may be due to the poor solubility of the starting dipolarophile 2.

Under the optimized conditions, a region-specific approach using Huisgen 1,3-dipolar cyclo-addition reaction (RuAAC) of terminal alkyne 2 with various aromatic azides 7a–g in presence of Cp*RuCl(PPh₃)₂ as catalyst resulting into the formation of regiospecific 1,5-substituted-triazolyl derivatives 8a–g in excellent yields (Scheme 1). All the reactions were carried under microwave irradiation (250 W) completed within 4–8 minutes (Table 3). A similar reactivity was observed for most azides (Table 3, entries 1–4, 6 and 7). In contrast, 4-nitrophenylazide showed to be relatively less reactive under the same reaction conditions (Table 3, entry 5), probably due to the presence of a group with a negative mesomeric effect at the 4-position of the phenyl group, which could promote the dimerization of azides to give the corresponding secondary amines37,38.

Scheme 1. Synthesis of the 1,5- and 1,4-triazolyl (8a–g, 9a–g) derivatives. Reagents and conditions: (a) [Cp*RuCl(PPh₃)₂], DMF, Microwave (250 W, 4–8 min), 83–98%. (b) Cuprous iodide (CuI), Et₃N, solvent free, Microwave (200 W, 2–4 min), 90–99%.

Table 3. Maslinic acid-1,5- and 1,4-disubstituted triazolyl derivatives varying at aromatic ring.

		1,5-Regioisomers (RuAAC)		1,4-Regioisomers (CuAAC)
Entry	R	Time (min)	Yield% (Compounds)	Time (min)	Yield% (Compounds)
1		6	98 (8a)	2	99 (9a)
2		4	98 (8b)	2	98 (9b)
3		6	95 (8c)	4	94 (9c)
4		4	96 (8d)	4	94 (9d)
5		8	83 (8e)	4	90 (9e)
6		6	95 (8f)	2	96 (9f)
7		6	96 (8g)	2	95 (9g)

We have then investigated the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) under microwave conditions for the synthesis of 1,4-regiomers 9a–g. The copper (I)-catalyzed of the reaction (CuAAC) leading to the sole 1,4-regioisomers. Working under microwave conditions at 200 W, we first investigated the optimal conditions for the “click” reaction of dipolarophile 2 and phenylazide 7a in presence of triethylamine with different solvent and copper(I) iodide loading (Table 4). A 92% conversion was obtained only after 2 min of reaction with 0.5 equiv. catalyst. The total conversion of starting maslinic acid-alkyne 2 was obtained only after 2 min under solvent-free conditions and in 5 min in DMF for 0.5 equiv. catalyst loading. For lower CuI loading, conversion was not complete, even after 15 min reaction. Moreover, when we worked under H₂O as solvent, the yield decreased (70%) even after 15 min reaction (entry 4). It may be due to the poor solubility of the maslinic acid-alkyne 2 or the possible complexation of the copper species in water.

Table 4. Optimization of CuAAC under microwave conditions for 9a^a.

			Irradiation time^c
Entry	Catalyst loading CuI (equiv)^b	Solvent	2 min	5 min	15 min
1	0.1	DMF	–	–	45%
2	0.3	DMF	–	78%	80%
3	0.5	DMF	92%	100%	–
4	0.5	H2O	–	–	70%
5	0.5	Solvent free	100%	–	–

^a Maslinic acid-alkyne 2 (2  mmol), Et₃N (2  mmol) and two equivalents of phenylazide (7a).

^b Referred to the starting alkyne 2.

^c The reaction was performed at 200 W.

Starting with the maslinic acid-alkyne 2 the synthesis of various 1,4-disubstituted-1,2,3-triazolyl-maslinic acid was performed region-specifically using CuI as catalyst under microwave and solvent-free conditions (Scheme 1). The desired new 1,2,3-triazoles 9a–g were obtained in yields ranging from 90 to 99% (Table 3). This method has the advantage of being simple, low-cost and the products can be obtained from the reaction mixture by simple purification. The new products can be obtained from the reaction mixture in almost quantitative yields and the reaction times were in general shorter than those reported in the literature6.

The synthesized 1,5- and 1,4-disubstituted-1,2,3-triazolyl-maslinic acid (8a–g, 9a–g) were characterized by ¹H/¹³C NMR, DEPT 135, NOESY and ESI-HRMS (Experimental section). The absence of ¹H NMR signals of terminal alkyne 2 at δ_H 2.34 (1H, t, J = 2.7 Hz), and emerging of a singlet at δ_H 7.77–8.09 (H-4 of the triazole ring, for 1,5-isomers, 8a–g) and at δ_H 7.96–8.03 (H-5 of the triazole ring, for 1,4-isomers, 9a–g) provides a good support for the cycloaddition to form 1,5- and 1,4-disubstituted-triazoles. The same features are reflected in ¹³C NMR spectra, where the signal belongs to the terminal carbon of alkyne was disappeared and a new signal belongs to C–H ring carbon, was appeared at δ_C ∼114.2–136.7 after cyclo-addition.

The 1,5-regiochemistry thus adopted in compounds 8a–g was confirmed by NOESY spectra showing NOEs between the H-4_triazole/H_1′ and H_1′/H_arom, and the absence of any NOE between H-4_triazole/H_arom, under RuAAC conditions. While The NOESY spectra of regioisomers 9a–g showed NOEs between H-5_triazole/H_1′ and H-5_triazole/H_arom, and the absence of any NOE between H_1′/H_arom, under CuAAC conditions. Such observations are in perfect agreement with the proposed structures and with that cited in the literature.

CuAAC for the synthesis of bis-1,4-disubstituted triazolyl derivatives

Working under microwave and solvent-free conditions, we first investigated the optimal conditions for the cyclo-addition of bis-alkyne 4 and phenylazide 7a with different concentrations of catalyst (Table 5). A 85% conversion into the desired cyclo-adduct 10a was obtained only after 4 min of reaction with 0.7 equiv. catalyst. The total conversion of starting bis-alkyne 4 was obtained after 6 min with 0.7 equiv. catalyst loading and in 4 min for 0.8 equiv. catalyst loading. For lower catalyst loading, conversion was not complete, even after 12 min reaction (Scheme 2).

Scheme 2. Bis-1,4-disubstituted triazoles 10a–g and 11a–g prepared by the Cu(I)-catalyzed synthesis.

Table 5. Optimization of CuAAC under microwave and solvent free conditions for 10a^a.

	Irradiation timec (min)
Catalyst loading CuI (equiv)^b	4	6	12
0.5	–	–	55%
0.6	–	–	80%
0.7	85%	100%	–
0.8	100%	–	–

^aMaslinic acid-bis-alkyne 4 (2  mmol), Et₃N (4  mmol) and 4 equivalents of phenylazide (7a).

^b Referred to the starting bis-alkyne 4.

^c The reaction was performed at 250 W.

Thus, 0.8 equiv. catalyst, 2 equiv. Et₃N and 4 equiv. of phenylazide 7a under microwave and solvent-free conditions for 4 min allowed the formation of the desired cyclo-adduct 10a. With this optimized condition, we probed the scope of the condensation of bis-alkyne 4 with various aromatic azides. The desired new bis-1,4-disubstituted-1,2,3-triazolyl-maslinic acid 10a–g were obtained in good yields (92–98%). The same methodology was successfully applied to bis-alkyne 5. The latter compound gave the desired new bis-1,4-disubstituted-1,2,3-triazolyl-maslinic acid 11a–g with various aromatic azides. All the reactions were carried under microwave irradiation (250 W) completed within 4–7 minutes (Scheme 2). A similar reactivity was observed for most azides. In contrast, with the electron-deficient azides, reactivity was decreased slightly under the same reaction conditions. Similarly the synthesized new bis-1,4-disubstituted triazoles 10a–g and 11 a–g were characterized by ¹H/¹³C NMR, DEPT 135, NOESY and ESI-HRMS (Experimental section).

CuAAC for the synthesis of tri-1,4-disubstituted triazolyl derivatives

On the contrary of dipolarophiles 2–5, tri-alkyne 6 is fully soluble in water. Interestingly the use of water as solvent, we investigated the reactivity for the cyclo-addition of latter dipolarophile (6) and phenylazide 7a in the presence of triethylamine (3 equiv. referred to the starting tri-alkyne 6) with different catalyst (CuI) loading under microwave heating in water. The addition of CuI as catalyst (1 equiv. referred to the starting maslinic acid-tri-alkyne 6), was essential to obtain the maximum yield of the desired tri-1,4-disubstituted triazolyl 12a in minimum reaction time. For lower catalyst loading (0.8 and 0.9 equiv.), conversion was not complete. With the optimized condition in hand, the dipolarophile 6 was reacted with various aromatic azides, 3 equiv. of triethylamine and 1 equiv. of CuI catalyst. All the reactions were performed at 300 W under microwave conditions in water completed within 5–10 minutes. A similar reactivity was observed for all azides used. The desired new tri-triazoles 12a–g were obtained in yields ranging from 92 to 96% (Scheme 3).

Scheme 3. Tri-1,4-disubstituted triazoles 12a–g prepared by the Cu(I)-catalyzed synthesis.

All the synthesized new tri-1,4-disubstituted triazoles were characterized by ¹H/¹³C NMR, DEPT 135, NOESY and ESI-HRMS (Experimental section).

Pharmacological screening

Anti-inflammatory activity

The anti-inflammatory activity of the tested compounds was studied using LPS-stimulated human peripheral blood mononuclear cells (PBMCs). Three anti-inflammatory references were tested in the same conditions: ZVAD (5 μM), dexamethasone (DEXA, 1 μM) and prednisolone (PRED, 70 μM). Secretion IL-1β was measured only in the culture supernatants of PBMCs treated with compounds that showed no or low toxicity, using a sandwich enzyme-linked immunosorbent assay (ELISA) method (eBioscience, San Diego, CA), according to manufacturer instructions.

As shown in Table 6, the % IL-1β production indicated that most compounds exhibited a significant anti-inflammatory activity. It has been found that all the synthesized triazoles exhibited more potent inhibitory activities than the starting natural compound 1. This finding showed that the introduction of the triazoles moieties in compound 1 considerably improve its anti-inflammatory effect. In the series of 1,5-regioisomers, compounds 8e (p-NO₂) and 8f (m-C₂H₅) were found to be the most actives (% IL-1β production = 42 ± 1 and 46 ± 3 respectively; 100 μM). On the other hand, our findings showed that most of the tested 1,4-regioisomers 9a, 9b, 9d, 9f and 9 g displayed a significant anti-inflammatory activity, the derivative 9d with (p-Br) was found to be the most active (% IL-1β production = 21 ± 1; 100 μM). The comparison of % IL-1β production values of the tested compounds type 8 with those of the derivatives 9 substituted identically at the triazole ring revealed that these latter are more active (% IL-1β production = 40–91 and 21–82, respectively; 100 μM). However, we found that the regiochemistry in two analogs from the series 8 and 9 bearing the same substituent intervenes to reverse the activity, we cite the example of compounds 8d (p-Br) (% IL-1β production = 61 ± 5; 100 μM) and 9d (p-Br) (% IL-1β production = 21 ± 1; 100 μM). This finding showed the importance of both the regiochemistry of the triazole and the nature of the aryl attached to it.

Table 6. Effect of natural pentacyclic triterpenoid (Maslinic acid 1) and the synthesized triazoles on viability and inflammatory cytokine (IL-1β) production in LPS-stimulated PBMCs.

		Conc. (μM)	% PBMCs viability (/Control)	% IL-1β production (/Control)
DMSO 0.33% + LPS (Control)			100 ± 4	100 ± 2
ZVAD + LPS		5	110 ± 4	42 ± 2
DEXA + LPS		1	81 ± 7	32 ± 2
PRED + LPS		70	67 ± 8	43 ± 4
Entry	Code
1	1	100	14 ± 7	Nd
		30	53 ± 5	109 ± 3
Mono-1,5-disubstituted triazoles
2	8b	100	87 ± 4	91 ± 3
		30	93 ± 5	Nd
3	8c	100	76 ± 3	60 ± 2
		30	74 ± 5	Nd
4	8d	100	94 ± 3	61 ± 5
		30	87 ± 4	Nd
5	8e	100	75 ± 4	42 ± 1
		30	93 ± 3	Nd
6	8f	100	51 ± 7	46 ± 3
		30	99 ± 9	Nd
7	8g	100	93 ± 2	76 ± 1
		30	95 ± 7	Nd
Mono-1,4-disubstituted triazoles
8	9a	100	92 ± 6	62 ± 1
		30	91 ± 6	Nd
9	9b	100	71 ± 5	65 ± 2
		30	75 ± 9	Nd
10	9c	100	83 ± 4	82 ± 4
		30	55 ± 1	Nd
11	9d	100	92 ± 8	21 ± 1
		30	74 ± 2	Nd
12	9f	100	88 ± 10	59 ± 3
		30	97 ± 3	Nd
13	9g	100	97 ± 2	53 ± 3
		30	88 ± 7	Nd
Bis-1,4-disubstituted triazoles
14	10a	100	92 ± 5	71 ± 2
		30	92 ± 3	Nd
15	10c	100	98 ± 4	56 ± 2
		30	94 ± 2	Nd
16	10f	100	66 ± 2	100 ± 4
		30	93 ± 5	Nd
17	10g	100	97 ± 3	95 ± 2
		30	80 ± 5	Nd
18	11a	100	88 ± 5	82 ± 1
		30	82 ± 4	Nd
19	11b	100	74 ± 5	34 ± 2
		30	88 ± 2	Nd
20	11c	100	71 ± 2	74 ± 2
		30	91 ± 5	Nd
21	11f	100	81 ± 5	52 ± 1
		30	83 ± 5	Nd
22	11g	100	95 ± 5	91 ± 3
		30	93 ± 5	Nd
Tri-1,4-disubstituted triazoles
23	12b	100	73 ± 1	43 ± 3
		30	96 ± 2	Nd
24	12c	100	83 ± 5	47 ± 2
		30	85 ± 3	Nd
25	12f	100	40 ± 5	Nd
		30	63 ± 4	23 ± 3
26	12g	100	31 ± 1	Nd
		30	72 ± 5	34 ± 3

Regarding the bis-1,4-disubstituted triazoles type 10, only compounds 10a (H), 10c (p-Cl), showed a relatively-important activity (% IL-1β production = 71 ± 2 and 56 ± 2, respectively; 100 μM) compared to the remaining analogs and maslinic acid 1 (% IL-1β production = 109 ± 3; 30 μM). However, the % IL-1β production exhibited by the bis-1,4-disubstituted triazoles type 11 revealed that compounds with electron donating groups at an aromatic ring are generally more active than compounds without substitutions or substitutions with an electron withdrawing group. In fact, compounds 11b and 11f, with p-OCH₃ and m-Me substitutions at the aromatic ring were the most promising (% IL-1β production = 34 ± 2 and 52 ± 1 respectively; 100 μM). The position of the triazole at C-2 (compounds 10) or C-3 (compounds 11) in maslinic acid 1 is sometimes decisive in this activity, we cite the case of 10f (% IL-1β production = 100 ± 4; 100 μM) and 11f (% IL-1β production = 52 ± 1; 100 μM) where the triazole bears the same aromatic system (m-MePh).

On the other hand, the tested compounds from the series of tri-1,4-disubstituted triazoles 12 were found to be the most potent among the whole synthesized compounds (% IL-1β production = 23–47; 30–100 μM). This finding showed the importance of the number and may be also the position of the triazole moieties to improve the anti-inflammatory activity of maslinic acid 1. Compounds 12f (m-Me) and 12 g with a naphthyl group on the triazole ring showed relatively-important activities (% IL-1β production = 23 ± 3 and 34 ± 3 respectively; 30 μM) compared to the remaining analogs and maslinic acid 1 (% IL-1β production = 109 ± 3; 30 μM).

In vitro anti-proliferative activity

The anti-proliferative activity of maslinic acid 1 and twenty-four of its triazole derivatives (8b–g; 9a–d,f,g; 10a,c,f,g; 11a–c,f; 12b,c,f,g) was studied using cultured murine EMT-6 (Breast) and human SW480 (colon) cancer cell lines. Doxorubicin, etoposide, 5-fluorouracil and methotrexate (10 μM) were used as anti-cancer references in this study. The obtained data revealed that maslinic acid 1 was found to be the most anti-proliferative against EMT-6 (Breast) and SW480 (colon) cancer cell lines (Viability (%/Control) = 5 and 9%, respectively; 100 μM) and most of its triazole derivatives showed moderate to good activities (Table 7). The percentage viability data indicated that most 1,5-regioisomers type 8 exhibited potent anti-proliferative activity. Compound 8d (p-Br) was displayed the most activity in this series against EMT-6 (Breast) (Viability (%/Control) = 13%) and SW480 (colon) (Viability (%/Control) = 34%; 100 μM) cancer cell lines. For the 1,4-regioisomers 9, compounds 9a, 9c and 9d exhibited higher anti-proliferative activity against the two used cancer cell lines at the concentration of 100 μM compared to the other analogs 9b, 9f and 9 g. Indeed, compound 9c (p-Cl) was found to be the most active against both EMT-6 (Breast) (Viability (%/Control) = 6%; 30 μM) and SW480 (colon) (Viability (%/Control) = 10%; 30 μM). In most cases, 1,4-regioisomers type 9 possess better anti-proliferative activity compared to that of 1,5-regioisomers type 8. This finding showed the signification of the region-chemistry of the triazole formed (1,4- or 1,5-) to this activity which may be explained by the approach in space of the aryl group attached to the triazole in relation to the triterpene moiety (Table 7).

Table 7. Effect of natural pentacyclic triterpenoid 1 and the synthesized triazoles on viability of EMT-6 and SW480 cell lines.

			Viability (%/Control)
Tissue:			Breast	Colon
Cell line:		Conc. (μM)	EMT-6	SW480
DMSO 0.33% (Control)			14 ± 2	100 ± 2
Doxorubicin		10	6 ± 0	25 ± 1
Etoposide		10	41 ± 2	70 ± 5
5-fluorouracil		10	26 ± 1	92 ± 2
Methotrexate		10	14 ± 2	68 ± 0
Entry	Code
1	1	100	5 ± 0	9 ± 0
		30	5 ± 0	11 ± 1
		10	100 ± 2	123 ± 2
1,5-disubstituted triazoles
2	8b	100	103 ± 1	122 ± 1
		30	97 ± 0	108 ± 1
		10	96 ± 2	106 ± 6
3	8c	100	24 ± 0	51 ± 1
		30	54 ± 0	61 ± 3
		10	96 ± 1	75 ± 0
4	8d	100	13 ± 1	34 ± 11
		30	108 ± 2	128 ± 3
		10	105 ± 2	118 ± 4
5	8e	100	40 ± 0	99 ± 2
		30	110 ± 3	117 ± 2
		10	108 ± 1	110 ± 0
6	8f	100	43 ± 1	56 ± 3
		30	62 ± 2	73 ± 3
		10	104 ± 3	108 ± 0
7	8g	100	47 ± 4	59 ± 1
		30	102 ± 0	99 ± 0
		10	110 ± 0	120 ± 0
Mono-1,4-disubstituted triazoles
9	9a	100	15 ± 1	45 ± 1
		30	33 ± 1	74 ± 0
		10	92 ± 3	102 ± 1
10	9b	100	40 ± 0	59 ± 3
		30	66 ± 1	86 ± 9
		10	109 ± 0	96 ± 5
11	9c	100	9 ± 0	13 ± 0
		30	6 ± 0	10 ± 0
		10	87 ± 1	19 ± 2
12	9d	100	7 ± 0	13 ± 0
		30	92 ± 0	113 ± 9
		10	108 ± 0	112 ± 11
13	9f	100	39 ± 3	64 ± 2
		30	70 ± 0	77 ± 1
		10	98 ± 0	94 ± 4
14	9g	100	76 ± 4	73 ± 0
		30	87 ± 0	87 ± 0
		10	102 ± 3	93 ± 3
Bis-1,4-disubstituted triazoles
15	10a	100	116 ± 0	103 ± 1
		30	114 ± 0	114 ± 1
		10	113 ± 1	119 ± 0
16	10c	100	118 ± 0	117 ± 0
		30	115 ± 0	115 ± 1
		10	111 ± 4	117 ± 1
17	10f	100	123 ± 0	112 ± 1
		30	117 ± 1	112 ± 2
		10	113 ± 1	111 ± 1
18	10g	100	117 ± 1	116 ± 4
		30	111 ± 2	111 ± 3
		10	112 ± 2	115 ± 1
19	11a	100	115 ± 1	103 ± 1
		30	110 ± 0	100 ± 1
		10	109 ± 1	102 ± 0
20	11b	100	108 ± 0	108 ± 0
		30	108 ± 1	107 ± 1
		10	109 ± 0	105 ± 3
21	11c	100	105 ± 0	116 ± 1
		30	108 ± 2	122 ± 3
		10	110 ± 1	115 ± 8
22	11f	100	104 ± 1	115 ± 1
		30	105 ± 1	109 ± 1
		10	106 ± 0	106 ± 4
Tri-1,4-disubstituted triazoles
24	12b	100	100 ± 4	119 ± 3
		30	100 ± 2	113 ± 4
		10	107 ± 2	107 ± 6
25	12c	100	46 ± 0	123 ± 6
		30	63 ± 6	123 ± 8
		10	100 ± 3	114 ± 4
26	12f	100	8 ± 0	13 ± 0
		30	40 ± 1	70 ± 3
		10	109 ± 1	116 ± 2
27	12g	100	8 ± 0	13 ± 0
		30	90 ± 1	98 ± 4
		10	110 ± 1	111 ± 0

The anti-proliferative activity of the synthesized bis-triazoles 10 and 11 were investigated and they did not exhibit any marked activity. In the series of tri-1,4-disubstituted triazoles 12, only compounds 12f (m-Me) and 12 g (naphthyl) showed potent activity against EMT-6 (Breast) (Viability (%/Control) = 8%; 100 μM) and SW480 (colon) (Viability (%/Control) = 13%; 100 μM) cancer cell lines. The above data show the contribution of the free hydroxyl groups at C-2 and C-3 in maslinic acid 1 to this activity and also show the importance of nature and the number of triazoles fixed in these positions.

Conclusion

In conclusion, we have achieved the low-cost ultrasound irradiation-assisted isolation of maslinic acid 1 (17 g (8.5 mg/g DW)), under green chemistry condition, from pomace olive (Olea europaea L.) cultivar: Chemlali. On the other hand, we used it as starting material to develop an effective, facile and practical procedure for the regio-specific synthesis of 1,5-triazolyl derivatives by Ru(II)-catalyzed azide-alkyne cycloaddition (RuAAC), and mono, bis and tri-1,4-triazolyl derivatives by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), using click-chemistry and microwave irradiation conditions, avoiding toxic reagents and solvents. This method afforded twenty-four maslinic acid triazole derivatives in quantitative yields. Most of the compounds were evaluated for their anti-inflammatory and anti-proliferative activities. Our findings showed the contribution of the nature of the introduced triazole ring and its regio-chemistry to improve in some cases the anti-inflammatory activity of maslinic acid 1 towards LPS-stimulated human peripheral blood mononuclear cells (PBMCs) and the importance of the two free hydroxyl groups in C-2 and C-3 positions to get anti-proliferative activity against EMT-6 (Breast) and human SW480 (colon) cancer cell lines.

Declaration of interest

The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article. This study was financially supported (LR11ES39) by the Ministry of Higher Education and Scientific Research of Tunisia.

Acknowledgements

The authors are grateful to Mrs Amna Benzarti and Miss Nadia Msaddek, NMR service at the Faculty of Monastir, University of Monastir for the 1D and 2D NMR analysis.