Design, synthesis of novel pyranotriazolopyrimidines and evaluation of their anti-soybean lipoxygenase, anti-xanthine oxidase, and cytotoxic activities

Abstract

The synthesis of 14-(aryl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-yl) acetamidoximes 2a–e has been accomplished by reaction of 2-acetonitrile derivatives 1a–e with hydroxylamine. Cyclocondensation reaction of precursors 2a–e with some elctrophilic species such as ethylorthoformate, acetic anhydride, and methyl-acetoacetate provided the new oxadiazole derivatives 3a–e, 4a–e, and 5a–e, respectively. On the other hand, the reaction of precursors 2a–e with 2-chloropropanoyl chloride afforded the new acetimidamides 6a–e which evolve under reflux of toluene to the new oxadiazoles 7a–e. The synthetic compounds were screened for their anti-xanthine oxidase, anti-soybean lipoxygenase, and cytotoxic activities. Moderate to weak xanthine oxidase and soybean lipoxygenase inhibitions were obtained but significant cytotoxic activities were noted. The most cytotoxic activities were recorded mainly (i) 5a was the most active (IC₅₀=4.0 μM) and selective against MCF-7 and (ii) 2a was cytotoxic against the four cell lines with selectivity for MCF-7 and OVCAR-3 (IC₅₀=17 and 12 μM, respectively) while 2e is highly selective against OVCAR-3 (IC₅₀=10 μM).

• Amidoximes
• cytotoxic activity
• soybean lipoxygenase inhibition
• triazolo[1,5-c]pyrimidine,1,2,4-oxadiazole
• xanthine oxidase inhibition

Introduction

In recent years, pyrimidine derivatives attracted organic chemists due to their widespread potential biological and chemotherapeutic activities1. In this framework, triazolopyrimidines, as well as their condensed derivatives are very attractive targets on account of their diverse pharmacological activities, such as antimicrobial2, antimalaria3, antifungal4, macrophage activation5, xanthine oxidase inhibitors (Figure 1-I)6, anti-inflammatory (Figure 1-II)7, and they have proved to be promising cytotoxic agents (Figure 1-III)8.

Figure 1. Structures of compounds bearing triazolopyrimidine moiety.

On the other hand, oxadiazole derivatives possess various biological activities such as immunosuppressive9, antimicrobial10, and antitumor11. Especially, 1,2,4-oxadiazole fragments are beneficial for interacting with DNA12, antibacterial13, antioxidant (Figure 2-I)14, anti-inflammatory (Figure 2-II)15 and cytotoxic (Figure 2-III)16. This phenomenon gives us an important information that 1,2,4-oxadiazole may be a powerful core to construct functional groups in the drug design.

Figure 2. Structures of compounds bearing oxadiazole moiety.

In addition, association of triazolopyrimidine ring with different heterocyclic fragments gives rise to a new class of hybrid molecules possessing improved activity17–21. In this context and as a continuation of our previous work on the synthesis of new fused pyrimidin scaffolds22,23 and wishing to access to bioactive and why not multi-bioactive molecules, we report here the synthesis of amidoximes 2a–e, which allied to a class of precursors having proven to exhibit numerous biological activities such as antimalarial24, antileishmanial25, antioxidant26, anti-inflammatory27, and cytotoxic activities28, using the naphtopyranyl moiety as a support, and their utility as building blocks in the synthesis of some novel hybrid molecules 3, 4, 5, and 7 (Scheme 1) bearing in their structures fragments described as antioxidant, anti-inflammatory, and cytotoxic agents, as indicated above, such triazolopyrimidine and 1,2,4-oxadiazole systems.

Scheme 1. The design of target compounds. a (R = H), b (R = CH₃), c (R = OCH₃), d (R = Cl), e (R = C₂H₅).

The synthetic compounds were then screened for their in vitro xanthine oxidase, soybean lipoxygenase inhibition, and cytotoxic activity.

Materials and methods

General experimental procedures

All reactions were monitored by thin layer chromatography (TLC) using aluminum sheets of Merck silica gel 60 F₂₅₄, 0.2 mm. Melting temperatures were determined on an electrothermal 9002 apparatus and were reported uncorrected. NMR spectra were recorded on a Bruker AC-300 spectrometer at 300 MHz (¹H) and 75 MHz (¹³C). All chemical shifts were reported as δ values (ppm) relative to residual non deuterated solvent. Mass spectra were acquired with a LCT Premier XE (Waters, ESI technique, positive mode) mass spectrometer. The starting materials 1 were prepared according to the literature and their structures were established on the basis of their spectroscopic data22,23.

Synthesis

Synthesis of amidoximes 2a–e

The cyano compounds 1a–e (10 mmol) were added to a solution of NH₂OH.HCl (12 mmol in 2 ml water) and Na₂CO₃ (12 mmol in 2 ml water) in dioxane (15 ml). The resulting mixture was heated under reflux for 20 h. After cooling, the mixture was diluted with water and left to stand at room temperature for 1 h. The precipitated solid was collected by filtration, washed with water, then dried and crystallized from ethanol to afford the amidoximes 2a–e.

2–(14-phenyl-14H-naphto[2,1-b]pyrano[3,2,e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetamidoxime 2a

Gray solid, Yield: 65%; m.p.: >300 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ 9.59 (s, 1H, -OH), 9.10 (s, 1H, H₅), 7.06–8.11 (m, 11 H, H_arom), 6.30 (s, 1H, H₁₄), 5.52 (s, 2H, -NH₂), 3.63 (s, 2H, -CH₂); ¹³C NMR (DMSO-d₆, 75 MHz): δ 165.7, 153.1, 152.3, 148.9, 147.9, 143.1, 139.8, 131.1, 130.2, 129.8, 128.5, 128.1, 127.3, 126.9, 125.1, 123.5, 117.5, 115.0, 102.1, 36.8, 31.0. ESI-HRMS [M + H]⁺calcd. for (C₂₄H₁₉N₆O₂)⁺: 423.1569, found: 423.1579.

2–(14-(4-methylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl) acetamidoxime 2b

Gray solid, Yield: 59%; m.p.: >300 °C; ¹H NMR (C₅D₅N-d₅, 300 MHz): δ 10.81 (s, 1H, -OH), 7.96 (s, 1H, H₅), 6.20–7.50 (m, 10H, H_arom), 5.71 (s, 1H, H₁₄), 5.59 (s, 2H, -NH₂), 3.43 (s, 2H, -CH_2-), 1.26 (s, 3H, -CH₃); ¹³C NMR (C₅D₅N-d₅, 75 MHz): δ 168.2, 155.4, 155.0, 150.7, 142.4, 141.0, 138.5, 133.6, 133.1, 131.8, 131.2, 130.7, 130.4, 129.3, 127.1, 125.9, 125.7, 119.6, 117.5, 105.1, 39.2, 33.8, 22.3. ESI-HRMS [M + H]⁺calcd. for (C₂₅H₂₁N₆O₂)⁺: 437.1726, found: 437.1724.

2–(14-(4-methoxylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl) acetamidoxime 2c

Gray solid, Yield: 66%; m.p.: >300 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ 9.57 (s, 1H, -OH), 9.08 (s, 1H, H₅), 6.75–8.10 (m, 10 H, H_arom), 6.25 (s, 1H, H₁₄), 5.51 (s, 2H, -NH₂), 3.63 (s, 2H, -CH_2-), 3.61 (s, 3H, -OCH₃); ¹³C NMR (DMSO-d₆, 75 MHz): δ 165.7, 158.0, 152.9, 152.3, 149.0, 147.8, 139.7, 135.2, 131.1, 130.3, 129.7, 129.1, 128.5, 127.3, 125.0, 123.5, 117.5, 115.2, 113.9, 102.4, 54.9, 36.0, 31.0,. ESI-HRMS [M + H]⁺calcd. for (C₂₅H₂₁N₆O₃)⁺: 453.1675, found: 453.1685.

2–(14-(4-chlorophenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl) acetamidoxime 2d

Gray solid, Yield: 70%; m.p.: >300 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ 9.59 (s, 1H, -OH), 9.07 (s, 1H, H₅), 7.26–8.08 (m, 10 H, H_arom), 6.35 (s, 1H, H₁₄), 5.52 (s, 2H, -NH₂), 3.63 (s, 2H, -CH_2-); ¹³C NMR (DMSO-d₆, 75 MHz): δ 165.7, 153.0, 152.2, 148.9, 147.9, 141.9, 140.0, 131.1, 130.2, 130.1, 130.0, 128.6, 128.5, 127.4, 125.2, 123.4, 117.5, 114.4, 101.6, 36.3, 31.0. ESI-HRMS [M + H]⁺calcd. for (C₂₄H₁₈ClN₆O₂)⁺: 457.1180, found: 457.1169.

2–(14-(4-ethylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl) acetamidoxime 2e

Gray solid, Yield: 62%; m.p.: >300 °C; ¹H NMR (DMSO-d₆, 300 MHz): δ 9.59 (s, 1H, -OH), 9.09 (s, 1H, H₅), 7.00–8.12 (m, 10H, H_arom), 6.28 (s, 1H, H₁₄), 5.52 (s, 2H, -NH₂), 3.61 (s, 2H, -CH_2-), 2.43 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 1.03 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (DMSO-d₆, 75 MHz): δ 165.7, 153.0, 152.3, 148.9, 147.9, 142.3, 140.4, 139.8, 131.1, 130.2, 129.8, 128.5, 127.9, 127.3, 125.1, 123.5, 117.5, 115.2, 102.3, 36.4, 31.0, 27.5, 15.2. ESI-HRMS [M + H]⁺calcd. for (C₂₆H₂₃N₆O₃)⁺: 451.1882, found: 451.1871.

Synthesis of 2-((1,2,4-oxadiazol-3′-yl)methyl)-14-aryl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidines 3a-c and 3e

A dispersion of compounds “2a–c and 2e” (0.5 mmol) and ethyl orthoformate (3 mmol) in toluene (5 ml) was refluxed for 96 h. The solvent was evaporated and the residue was separated by column chromatography (silica gel, CH₂Cl₂/EtOAc 9:1) to afford compounds 3a–c and 3e.

2-((1,2,4-oxadiazol-3′-yl)methyl)-14-phenyl-14H-naphto [2,1-b]pyrano[3,2-e] [1,2,4]triazolo [1,5-c]pyrimidine 3a

White solid, Yield: 40%; m.p.: 252 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 8.75 (s, 1H, H_5′), 7.13–8.00 (m, 11H, H_arom), 6.37 (s, 1H, H₁₄), 4.55 (s, 2H, -CH₂-); ¹³C NMR (CDCl₃, 75 MHz): δ 166.0, 165.1, 164.6, 154.0, 153.5, 148.6, 142.4, 138.1, 131.6, 130.8, 129.9, 128.6, 128.3, 127.3, 127.2, 125.2, 123.5, 117.5, 114.9, 103.6, 37.6, 26.5. ESI-HRMS [M + H]⁺calcd. for (C₂₅H₁₇N₆O₂)⁺: 433.1413, found: 433.1420.

2-((1,2,4-oxadiazol-3′-yl)methyl)-14-(4-methylphenyl)-14H-naphto[2,1-b] pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 3b

White solid, Yield: 45%; m.p.: 254 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 8.76 (s, 1H, H_5′), 7.00–8.00 (m, 10H, H_arom), 6.35 (s, 1H, H₁₄), 4.55 (s, 2H, -CH₂-), 2.21 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 166.0, 165.1, 164.6, 154.0, 153.5, 148.6, 139.5, 138.0, 136.9, 131.6, 130.9, 129.8, 129.3, 128.6, 128.2, 127.3, 125.2, 123.5, 117.5, 115.1, 103.8, 37.2, 26.5, 20.9. ESI-HRMS [M + H]⁺calcd. for (C₂₆H₁₉N₆O₂)⁺: 447.1569, found: 447.1580.

2-((1,2,4-oxadiazol-3′-yl)methyl)-14-(4-methoxyphenyl) -14H-naphto[2,1-b] pyrano[3,2-e] [1,2,4]triazolo[1,5-c] pyrimidine 3c

White solid, Yield: 41%; m.p.: 260 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 8.76 (s, 1H, H_5′), 6.71–8.00 (m, 10H, H_arom), 6.33 (s, 1H, H₁₄), 4.55 (s, 2H, -CH₂-), 3.68 (s, 3H, -OCH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 166.0, 165.1, 164.6, 158.6, 153.9, 153.5, 148.6, 138.0, 134.7, 131.6, 130.8, 129.8, 129.4, 128.6, 127.3, 125.1, 123.5, 117.5, 115.1, 114.0, 103.8, 55.1, 36.7, 26.5. ESI-HRMS [M + H]⁺calcd. for (C₂₆H₁₉N₆O₃)⁺: 463.1519, found: 463.1515.

2-((1,2,4-oxadiazol-3′-yl)methyl)-14-(4-ethylphenyl) -14H-naphto[2,1-b]pyrano[3,2-e] [1,2,4]triazolo[1,5-c] pyrimidine 3e

White solid, Yield: 39%; m.p.: 222 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 8.75 (s, 1H, H_5′), 7.00–8.00 (m, 10H, H_arom), 6.37 (s, 1H, H₁₄), 4.55 (s, 2H, -CH₂-),2.43 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 1.03 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 166.0, 165.0, 164.5, 154.1, 153.4, 148.6, 143.1, 139.7, 138.0, 131.6, 130.9, 129.8, 129.1, 128.6, 128.2, 128.1, 127.3, 125.2, 123.5, 117.5, 115.2, 103.8, 37.2, 28.3, 26.4, 15.1. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₁N₆O₂)⁺: 461.1726, found: 461.1715.

Synthesis of 2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14-aryl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo [1,5-c] pyrimidines 4a-e

A mixture of 2a–e (0.5 mmol) and excess of acetic anhydride (2 ml) in toluene (10 ml) was refluxed for 4 h. The solvent was evaporated and the residue was separated by column chromatography (silica gel, CH₂Cl₂/EtOAc 9.5:0.5) to give compounds 4a–e.

2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14-phenyl-14H-naphto[2,1-b] pyrano[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidine 4a

Yellow solid, Yield: 51%; m.p.: 196 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.03 (s, 1H, H₅), 7.06–7.95 (m, 11H, H_arom), 6.37 (s, 1H, H₁₄), 4.44 (s, 2H, -CH₂-), 2.59 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 176.5, 166.1, 164.2, 153.5, 152.8, 148.1, 141.9, 137.6, 131.1, 130.3, 129.4, 128.1, 127.8, 126.9, 126.7, 124.7, 123.0, 117.0, 114.4, 103.1, 37.1, 26.1, 11.8. ESI-HRMS [M + H]⁺calcd. for (C₂₆H₁₉N₆O₂)⁺: 447.1569, found: 447.1580.

2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-methylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine 4b

Yellow solid, Yield: 56%; m.p.: 194 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 7.02–7.99 (m, 10H, H_arom), 6.37 (s, 1H, H₁₄), 4.46 (s, 2H, -CH₂-),2.62 (s, 3H, -CH₃), 2.21 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 176.6, 166.1, 164.1, 153.6, 152.8, 148.0, 139.0, 137.6, 136.4, 131.1, 130.3, 129.3, 128.8, 128.1, 127.7, 126.9, 124.7, 123.0, 117.0, 114.6, 103.2, 36.6, 26.0, 20.5, 11.9,. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₁N₆O₂)⁺: 461.1726, found: 461.1733.

14-(4-methoxyphenyl)-2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14H-naphto[2,1-b] pyrano[3,2-e][1,2,4]triazolo [1,5-c]pyrimidine 4c

Yellow solid, Yield: 59%; m.p.: 172 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.04 (s, 1H, H₅), 6.71–7.96 (m, 10H, H_arom), 6.33 (s, 1H, H₁₄), 4.45 (s, 2H, -CH₂-), 3.68 (s, 3H, -OCH₃), 2.61 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 176.5, 166.2, 164.4, 158.0, 153.3, 153.0, 148.0, 137.5, 134.3, 131.1, 130.4, 129.2, 128.9, 128.1, 126.8, 124.6, 123.0, 117.0, 114.6, 103.3, 54.6, 36.2, 26.1, 11.8. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₁N₆O₃)⁺: 477.1675, found: 477.1661.

14-(4-chlorophenyl)-2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14H-naphto[2,1-b] pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine 4d

Yellow solid, Yield: 49%; m.p.: 160 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.07 (s, 1H, H₅), 7.14–7.93 (m, 10H, H_arom), 6.37 (s, 1H, H₁₄), 4.45 (s, 2H, -CH₂-), 2.62 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 177.1, 166.6, 164.9, 153.9, 153.3, 148.6, 140.8, 138.3, 133.1, 131.6, 130.7, 130.1, 129.7, 128.7, 128.1, 127.5, 125.3, 123.3, 117.5, 114.3, 103.0, 37.0, 26.6, 12.3. ESI-HRMS [M + H]⁺calcd. for (C₂₆H₁₈ClN₆O₂)⁺: 481.1180, found: 481.1165.

2-((5′-methyl-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-ethylphenyl)-14H-naphto[2,1-b] pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine 4e

Yellow solid, Yield: 56%; m.p.: 178 °C; ¹H NMR (CDCl₃, 300 MHz): δ 8.97 (s, 1H, H₅), 6.93–7.92 (m, 10H, H_arom), 6.29 (s, 1H, H₁₄), 4.38 (s, 2H, -CH₂-), 2.63 (s, 3H, -CH₃), 2.45 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 1.04 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 176.6, 166.1, 164.1, 148.1, 142.6, 139.2, 137.5, 131.1, 130.4, 129.2, 128.1, 127.7, 127.6, 126.8, 124.7, 123.0, 117.0, 114.7, 103.3, 36.6, 27.8, 26.0, 14.6, 11.9. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₃N₆O₂)⁺: 475.1882, found: 475.1893.

Synthesis of 1–(3-((14-aryl-14H-naphto[2,1-b]pyrano [3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl)methyl)-1,2,4-oxadiazol-5′-yl) propan-2-ones 5a-e

A dispersion of compounds 2a–e (0.5 mmol) and methyl acetoacetate (1 mmol) in toluene (7 ml) was refluxed for 2 h. The solvent was evaporated and the residue was chromatographed (column, silica gel, CH₂Cl₂/EtOAc, 9:1) to give compounds 5a–e.

1–(3-((14-phenyl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl) methyl)-1,2,4-oxadiazol-5′-yl) propan-2-one 5a

Brown solid, Yield: 49%; m.p.: 210 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.02 (s, 1H, H₅), 7.06–7.94 (m, 11H, H_arom), 6.35 (s, 1H, H₁₄), 4.47 (s, 2H, -CH₂-),4,05 (s, 2H, -CH₂-CO), 2.29 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 198.0, 173.0, 166.4, 164.1, 153.5, 152.9, 148.1, 141.9, 137.6, 131.1, 130.3, 129.3, 128.1, 127.8, 126.8, 126.7, 124.7, 123.0, 117.0, 114.4, 103.1, 41.0, 37.1, 29.4, 26.1. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₁N₆O₃)⁺: 489.1675, found: 489.1683.

1–(3-((14-(4-methylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl)methyl)-1,2,4-oxadiazol-5′-yl) propan-2-one 5b

Brown solid, Yield: 40%; m.p.: 210 °C; ¹H NMR (CDCl₃, 300 MHz): δ 8.94 (s, 1H, H₅), 6.89–7.87 (m, 10H, H_arom), 6.23 (s, 1H, H₁₄), 4.47 (s, 2H, -CH₂-), 3,98 (s, 2H, -CH₂-CO), 2.22 (s, 3H, -CO-CH₃), 2.10 (s, 3H, -CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 198.5, 173.5, 167.0, 164.7, 153.9, 153.5, 148.6, 139.6, 138.0, 136.9, 131.6, 130.9, 129.8, 129.3, 128.6, 128.2, 127.3, 125.1, 123.5, 117.5, 115.1, 103.8, 41.5, 37.1, 29.9, 26.7, 21.0. ESI-HRMS [M + H]⁺calcd. for (C₂₉H₂₃N₆O₃)⁺: 503.1832, found: 503.1819.

1–(3-((14-(4-methoxyphenyl)-14H-naphto[2,1-b]pyrano [3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl)methyl)-1,2,4-oxadiazol-5′-yl) propan-2-one 5c

Brown solid, Yield: 44%; m.p.: 202 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.03 (s, 1H, H₅), 6.70–7.88 (m, 10H, H_arom), 6.31 (s, 1H, H₁₄), 4.48 (s, 2H, -CH₂-), 4,07 (s, 2H, -CH₂-CO), 3.67 (s, 3H, -OCH₃), 2.31 (s, 3H, -CO-CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 198.5, 173.5, 166.9, 164.6, 153.8, 153.5, 148.5, 138.0, 134.7, 131.6, 130.8, 129.7, 129.4, 128.6, 128.5, 127.3, 125.1, 123.5, 117.5, 115.1, 113.9, 103.8, 55.0, 41.5, 36.7, 29.8, 26.6. ESI-HRMS [M + H]⁺calcd. for (C₂₉H₂₃N₆O₄)⁺: 519.1781, found: 519.1793.

1–(3-((14-(4-chlorophenyl)-14H-naphto[2,1-b]pyrano [3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl)methyl)-1,2,4-oxadiazol-5′-yl) propan-2-one 5d

Brown solid, Yield: 44%; m.p.: 224 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.07 (s, 1H, H₅), 7.14–7.92 (m, 10H, H_arom), 6.37 (s, 1H, H₁₄), 4.49 (s, 2H, -CH₂-),4,09 (s, 2H, -CH₂-CO), 2.33 (s, 3H, -CO-CH₃); ¹³C NMR (CDCl₃, 75 MHz): δ 198.0, 173.0, 166.4, 164.1, 153.5, 148.1, 140.3, 137.8, 132.6, 131.1, 130.2, 129.6, 129.2, 128.3, 128.2, 127.0, 124.8, 122.8, 117.0, 113.8, 102.5, 41.0, 36.5, 29.4, 26.1. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₀ClN₆O₃)⁺: 523.1285, found: 523.1292.

1–(3-((14-(4-ethylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c] pyrimidin-2-yl)methyl)-1,2,4-oxadiazol-5′-yl) propan-2-one 5e

Brown solid, Yield: 53%; m.p.: 198 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.02 (s, 1H, H₅), 7.00–7.98 (m, 10H, H_arom), 6.35 (s, 1H, H₁₄), 4.48 (s, 2H, -CH₂-),4,09 (s, 2H, -CH₂-CO), 2.48 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 2.30 (s, 3H, -CO-CH₃), 1.11 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 197.9, 173.0, 166.4, 163.9, 153.6, 152.8, 148.1, 142.6, 139.2, 137.5, 131.1, 130.4, 129.2, 128.0, 127.7, 127.6, 126.8, 124.7, 123.0, 117.0, 114.7, 103.3, 41.0, 36.6, 29.4, 27.7, 26.1, 14.6. ESI-HRMS [M + H]⁺calcd. for (C₃₀H₂₅N₆O₃)⁺: 517.1988, found: 517.1979.

Synthesis of N′-((2-chloropropanoyl)oxy)-2–(14-aryl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetimidamides 6a–e

2-chloropropanoyl chloride (1 mmol) was added dropwise during 1 h to a stirred dispersion of compounds 2a–e (1 mmol) and Et₃N (1 mmol) in CH₂Cl₂ (10 ml) and the mixture was stirred vigorously at room temperature for 15 min. the filtrate after removing of the Et₃N.HCl was concentrated under reduced pressure then crystallized from petroleum ether/CH₂Cl₂ (1v:1v) to give compounds 6a–e.

N′-((2-chloropropanoyl)oxy)-2–(14-phenyl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4] triazolo[1,5-c]pyrimidin-2-yl) acetimidamide 6a

Creamy white solid, Yield: 71%; m.p.: 135 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 7.10–7.97 (m, 11H, H_arom), 6.31 (s, 1H, H₁₄), 5.70 (s, 2H, -NH₂), 4.56 (q, 1H, CH, J = 6.9 Hz), 3.97 (s, 2H, -CH₂-), 1.78 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 166.6, 163.6, 155.1, 153.5, 152.7, 147.9, 142.9, 137.7, 131.1, 130.3, 129.5, 128.1, 127.9, 126.9, 124.8, 122.9, 117.0, 114.1, 102.8, 51.1, 37.4, 29.7, 21.1. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₂ClN₆O₃)⁺: 513.1442, found: 513.1449.

N′-((2-chloropropanoyl)oxy)-2–(14-(4-methylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetimidamide 6b

Creamy white solid, Yield: 72%; m.p.: 188 °C; ¹H NMR (CDCl₃, 300 MHz): δ 8.98 (s, 1H, H₅), 6.93–7.91 (m, 10H, H_arom), 6.22 (s, 1H, H₁₄), 5.67 (s, 2H, -NH₂), 4.48 (q, 1H, CH, J = 6.9 Hz), 3.89 (s, 2H, -CH₂-), 2.13 (s, 3H, -CH₃), 1.70 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 167.0, 163.6, 155.5, 154.3, 148.3, 139.5, 138.1, 137.2, 131.7, 130.8, 130.0, 129.4, 128.7, 128.2, 127.5, 125.3, 123.4, 117.5, 114.8, 103.5, 51.6, 37.5, 30.0, 21.6, 20.9. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₄ClN₆O₃)⁺: 527.1598, found: 527.1585.

N′-((2-chloropropanoyl)oxy)-2–(14-(4-methoxyphenyl)-14H-naphto[2,1-b]pyrano[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetimidamide 6c

Creamy white solid, Yield: 80%; m.p.: 140 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.08 (s, 1H, H₅), 6.74–8.00 (m, 10H, H_arom), 6.32 (s, 1H, H₁₄), 5.76 (s, 2H, -NH₂), 4.54 (q, 1H, CH, J = 6.9 Hz), 4.00 (s, 2H, -CH₂-),3.69 (s, 3H, -OCH₃), 1.79 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 167.1, 164.2, 158.0, 155.4, 153.8, 153.5, 148.5, 138.0, 134.7, 131.6, 130.8, 129.7, 129.4, 128.6, 128.5, 127.3, 125.1, 123.5, 117.5, 115.1, 113.9, 103.8, 54.9, 51.6, 37.3, 30.3, 21.6. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₄ClN₆O₄)⁺: 543.1548, found: 543.1537.

N′-((2-chloropropanoyl)oxy)-2–(14-(4-chlorophenyl)-14H-naphto[2,1-b]pyrano[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetimidamide 6d

Creamy white solid, Yield: 79%; m.p.: 170 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.08 (s, 1H, H₅), 7.18–7.93 (m, 10H, H_arom), 6.30 (s, 1H, H₁₄), 5.61 (s, 2H, -NH₂), 4.55 (q, 1H, CH, J = 6.9 Hz), 3.97 (s, 2H, -CH₂-), 1.80 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 167.1, 164.3, 155.5, 154.0, 153.2, 148.4, 140.9, 138.3, 133.2, 131.7, 130.6, 130.3, 129.7, 128.9, 128.8, 127.5, 125.4, 123.2, 117.5, 114.0, 102.9, 51.6, 37.3, 30.3, 21.6. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₁Cl₂N₆O₃)⁺: 547.1052, found: 547.1062.

N′-((2-chloropropanoyl)oxy)-2–(14-(4-ethylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) acetimidamide 6e

Creamy white solid, Yield: 79%; m.p.: 170 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.03 (s, 1H, H₅), 7.01–7.97 (m, 10H, H_arom), 6.26 (s, 1H, H₁₄), 5.67 (s, 2H, -NH₂), 4.53 (q, 1H, CH, J = 6.9 Hz), 3.94 (s, 2H, -CH₂-),2.50 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 1.76 (d, 3H, -CH₃, J = 6.9 Hz), 1.10 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 166.6, 163.6, 155.2, 153.1, 152.8, 147.9, 142.8, 139.3, 137.6, 131.1, 130.3, 129.4, 128.1, 127.8, 127.6, 126.9, 124.7, 123.0, 117.0, 114.3, 103.1, 51.1, 37.0, 29.7, 27.7, 21.1, 14.6. ESI-HRMS [M + H]⁺calcd. for (C₂₉H₂₆ClN₆O₃)⁺: 541.1755, found: 541.1765.

Synthesis of 2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-aryl-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo [1,5-c]pyrimidines 7a–e

A solution of compound 6a–e (0.5 mmol) in toluene (5 ml) was refluxed for 6 h. The solvent was evaporated and the residue was crystallized from Hexane/CH₂Cl₂ (1v:1v) to give compounds 7a–e

2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-phenyl-14H-naphto[2,1-b] pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 7a

Creamy white solid, Yield: 89%; m.p.: 128 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 7.09–7.98 (m, 11H, H_arom), 6.40 (s, 1H, H₁₄), 4.20 (q, 1H, CH, J = 6.9 Hz), 4.50 (s, 2H, -CH₂-), 1.98 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 177.6, 166.4, 163.8, 148.1, 141.8, 137.6, 131.1, 130.3, 129.4, 128.1, 127.8, 126.9, 126.7, 124.7, 122.9, 117.0, 114.4, 103.1, 45.1, 37.0, 26.1, 21.9. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₂₀ClN₆O₂)⁺: 495.1336, found: 495.1324.

2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-methylphenyl)-14H-naphto [2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 7b

Creamy white solid, Yield: 87%; m.p.: 204 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.04 (s, 1H, H₅), 6.99–7.97 (m, 10H, H_arom), 6.33 (s, 1H, H₁₄), 5.20 (q, 1H, CH, J = 6.9 Hz), 4.49 (s, 2H, -CH₂-), 2.16 (s, 3H, -CH₃), 1.98 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 178.0, 166.8, 164.5, 154.3, 153.8, 148.3, 139.5, 138.1, 137.2, 131.7, 130.8, 130.0, 129.4, 128.7, 128.2, 127.5, 125.3, 123.4, 117.5, 114.8, 103.5, 45.1, 37.1, 26.2, 21.8,20.9. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₂ClN₆O₂)⁺: 509.1493, found: 509.1482.

2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-methoxyphenyl)-14H-naphto [2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 7c

Creamy white solid, Yield: 79%; m.p.: 162 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 6.71–7.97 (m, 10H, H_arom), 6.33 (s, 1H, H₁₄), 5.20 (q, 1H, CH, J = 6.9 Hz), 4.50 (s, 2H, -CH₂-), 3.68 (s, 3H, -OCH₃), 1.99 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 178.1, 166.9, 164.3, 158.6, 153.9, 153.4, 148.5, 138.3, 138.0, 134.7, 131.6, 130.8, 129.8, 129.3, 128.8, 128.6, 127.3, 125.2, 123.5, 117.5, 115.1, 114.0, 103.8, 55.1, 45.6, 36.7, 26.6, 22.4. ESI-HRMS [M + H]⁺calcd. for (C₂₈H₂₂ClN₆O₃)⁺: 525.1442, found: 525.1430.

2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-chlorophenyl)-14H-naphto [2,1-b]pyrano[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 7d

Creamy white solid, Yield: 88%; m.p.: 230 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.05 (s, 1H, H₅), 7.14–7.91 (m, 10H, H_arom), 6.33 (s, 1H, H₁₄), 5.20 (q, 1H, CH, J = 6.9 Hz), 4.50 (s, 2H, -CH₂-), 2.00 (d, 3H, -CH₃, J = 6.9 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 178.1, 167.0, 164.5, 153.8, 153.4, 148.6, 140.8, 138.3, 133.0, 131.6, 130.7, 130.1, 129.7, 128.8, 128.7, 127.5, 125.3, 123.3, 117.5, 114.2, 103.0, 45.6, 37.0, 26.7, 22.4. ESI-HRMS [M + H]⁺calcd. for (C₂₇H₁₉Cl₂N₆O₂)⁺: 529.0947, found: 529.0948.

2-((5′-(1-chloroethyl)-1,2,4-oxadiazol-3′-yl)methyl)-14-(4-ethylphenyl)-14H-naphto[2,1-b]pyrano[3,2-e][1,2,4]triazolo [1,5-c]pyrimidine 7e

Creamy white solid, Yield: 89%; m.p.: 140 °C; ¹H NMR (CDCl₃, 300 MHz): δ 9.04 (s, 1H, H₅), 7.10–7.99 (m, 10H, H_arom), 6.37 (s, 1H, H₁₄), 5.20 (q, 1H, CH, J = 6.9 Hz), 4.50 (s, 2H, -CH₂-), 2.50 (q, 2H, -CH₂-CH_3, J = 8.7 Hz), 1.99 (d, 3H, -CH₃, J = 6.9 Hz), 1.12 (t, 3H, -CH_3, J = 8.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 178.1, 166.9, 164.3, 154.1, 153.4, 148.6, 143.1, 139.7, 138.0, 131.6, 130.9, 129.7, 128.6, 128.2, 128.1, 127.3, 125.1, 123.5, 117.5, 115.2, 103.8, 45.6, 37.1, 28.2, 26.6, 22.4, 15.0. ESI-HRMS [M + H]⁺calcd. for (C₂₉H₂₄ClN₆O₂)⁺: 523.1649, found: 523.1639.

Biological evaluation

Xanthine oxidase inhibition

A solution of 50 μL of each compound, 60 μL of 0.1 mM phosphate buffer (pH = 7.5) and 30 μL of enzyme (xanthine oxidase from bovine milk) solution (0.1 u/ml) prepared from phosphate buffer (pH = 7.5) were prepared before use29. The mixture was added in a 96-well microplate and incubated at 25 °C for 15 min, then 60 μL of substrate solution (150 mM xanthine in the same buffer). The assay mixture was incubated at 25 °C for 30 min and the absorbance was determined at 290 nm. A blank was prepared in the same manner. Result is expressed as the percentage inhibition of xanthine oxidase in the above system, calculated as (1-B/A) × 100, where A and B are the activities of the enzyme without and with test material. The tested compounds were dissolved initially in DMSO, followed by dilution with the buffer; the final concentration of DMSO was less than 0.5%. Allopurinol was used as positive control.

Soybean lipoxygenase inhibition

The anti-soybean lipoxygenase activity of the compounds was determined as described by Bekir et al.30 with some modifications. Various concentrations of 20 μL of each compound was mixed individually with sodium phosphate buffer (pH 7.4) containing soybean lipoxygenase and 60 μL of linoleic acid (3.5 mM). However, the blank does not contain the substrate but will be added 30 μL of buffer solution. All compounds were re-suspended in the DMSO followed by dilution in the buffer so that the DMSO does not exceed 1%. The mixture was incubated at 25 °C for 10 min, and the absorbance was determined at 234 nm. The absorption change with the conversion of linoleic acid to 13-hydroperoxyoctadeca-9, 11-dienoate (characterized by the appearance of the conjugated diene at 234 nm) was flowed for 10 min at 25 °C. Nordihydroguaiaretic acid (NDGA) was used as positive control. The percentage of enzyme activity was plotted against concentration of each compound.

Cytotoxic activity

The human cancer cell lines (HCT-116, MCF-7, IGROV-1, and OVCAR-3) were used for cytotoxic assay30. The cells were grown in RPMI-1640 medium supplemented with 10% faetal calf serum (Gibco, Grand Island, NY), air and 5% CO₂. The tested compound was added to a medium containing 1 × 10⁶ cells/ml, L-glutamine (2 mM) and gentamycin (50 μg/ml), and kept at 37 °C in a fully humidified atmosphere. After 18 h of incubation at 37 °C in 5% CO₂ incubator, the tubes were centrifuged at 8000 g for 10 min. The supernatant was decanted, and the pellets were taken and washed with 20 mM of phosphate buffered saline solution. Each pellet was dissolved in 100 μL (2 mg/ml) MTT solution in a tube, incubated at 22 °C for 4 h and centrifuged at 8000 g for 10 min. All the pellets were dissolved in 500 μL DMSO and read spectrophotometrically at 500 nm. Doxorubicin (HCT116 and MCF-7) and tamoxifen (IGROV-1 and OVCAR-3) were used as positive control.

Results and discussion

Synthesis

In this work, we prepared naphtopyranotriazolopyrimidine amidoximes 2a–e using cyano compounds 1a–e which was synthesized in three steps from the key intermediates 2-amino-naphto[2,1-b]pyrane-3-carbonitriles according to some previous works22,23. Initially, we carried out the reaction of 2-acetonitrilestriazolopyrimidine derivatives 1a–e with hydroxylamine hydrochloride as a model reaction, in various solvents (ethanol, methanol, and dioxane) and bases (piperidine, triethylamine, and sodium carbonate)31. The progress of the reaction was monitored by TLC and the best yields were obtained when compounds 1a–e, hydroxylamine hydrochloride, and aqueous solution of Na₂CO₃ were heated under reflux of dioxane (Scheme 2).

Scheme 2. Synthesis of compounds 2–7.

The formed amidoximes 2a–e were characterized by their ¹H NMR and ¹³C NMR spectra. In fact, the ¹H NMR spectra of compounds 2a–e showed the presence of new signals at 5.52–5.59 ppm and 9.55–10.81 ppm relative to mobile protons -NH₂ and -OH, respectively. The ¹³C NMR spectra of 2a–e showed the appearance of a new signal at 147.8–147.9 ppm due to amidoxime carbon and the disappearance of the signal relative to the -CN carbon.

On the other hand, amidoximes 2a–c and 2e were treated with ethylorthoformate to afford the 1,2,4-oxadiazoles 3a–c and 3e32. The reaction was conducted until TLC indicated that the starting materials have been completely converted. The analytical and spectral data are in good agreement with the proposed structures. The ¹H NMR spectra of compounds 3a–c and 3e displayed a singlet in the region 8.75-8.76 ppm corresponding to H_5′ of the oxadiazole ring, also showed the disappearance of signals related to mobile protons -OH and -NH₂ (of compounds 2) (Scheme 2). Treatment of amidoximes 2a–e with acetic anhydride under reflux of toluene gave the 5-methyl-1,2,4-oxadiazoles 4a–e. Analog reaction of compounds 2a–e with an excess of methyl acetoacetate gave the corresponding acetonyl compounds 5a–e. The ¹H NMR spectra of compounds 4a–e and 5a–e showed the disappearance of signals relative to mobile protons -NH₂ and -OH, the presence of new signals at 2.59–2.63 ppm (s, 3H, CH₃) (for compounds 4), 2.22–2.33 ppm (s, 3H, -CO-CH₃) and 3.98–4.09 ppm (s, 2H, -CH₂-CO-) (for compounds 5), while the ¹³C NMR spectra of compounds 4a–e showed new signals at 11.8–12.3 ppm and at 176.5–177.1 ppm relative to carbons -CH₃ et C_5′, respectively. Analysis of ¹³C NMR spectra of compounds 5a–e showed the appearance of signals at 26.1–26.7, 41.0–41.5, 173.0–173.5, and 197.9–198.5 ppm relative to carbons (COCH₃), (-CH₂CO), C_5′, and (C=O), respectively. Reactions of amidoximes 2a–e with 2-chloropropanoyl chloride in the presence of Et₃N resulted to O-acyl derivatives 6a–e. The ¹H NMR spectra of compounds 6a–e showed, in addition to the signals relative to protons introduced by the 2-chloropropanoyl chloride, the presence of new signals at 1.70–1.80 ppm (d, 3H, J = 6.9 Hz) and 4.48-4.56 ppm (q, 1H, J = 6.9 Hz) relative to protons -CH₃ and -CH, respectively and the disappearance of the signal related to the mobile proton -OH. The structures of compounds 6a–e has been also characterized by ¹H and ¹³C NMR data. The exploration of ¹³C NMR spectra allowed to note the appearance of new signals at 21.1–21.6, 51.1–51.6, and 166.6–167.1 ppm attributed to carbons -CH₃, -CH, and C=O, respectively. Heating of the toluene solution of compounds 6a–e resulted to the condensation products 7a–e as indicated from ¹H NMR (no signal of -NH₂ protons) and supported by ESI-HRMS (Scheme 2).

Biological activity

Most of the synthetic compounds were screened for the first time for their xanthine oxidase and soybean lipoxygenase inhibition and cytotoxic activity.

Xanthine oxidase inhibition

According to the results obtained (Table 1), most of the tested compounds have displayed weak anti-xanthine oxidase activity (4.4–25.4% inhibition) compared to allopurinol (81% inhibition at 100 μM) used as a reference compound, while the remaining compounds were inactive. Particularly, compounds 6 are all inactive at the used concentration (100 μM). Despite the presence of the triazolopyrimidine moiety in all tested molecules and the presence of the oxadiazole ring in compounds 3, 4, 5, and 7, no significant activity was obtained. This finding could be explained by the common naphtopyranyl system that may reduce the activity in this case and by the position of the oxadiazole ring in compounds 3, 4, 5, and 7, which probably was not in favor of this activity.

Table 1. Anti-xanthine oxidaseand anti-soybean lipoxygenase activities of compounds 2a–e, 3a–c, 6e, 4a, 4b, 4e, 5a–e, 6a, 6d, 6e, and 7a–e.

Products	% Inhibition of xanthine oxidase (100 μM)	% Inhibition of 5-lipoxygenase (100 μM)
2a	8.3 ± 2.2	10.2 ± 3.0
2b	0.0	21.4 ± 0.6
2c	9.8 ± 2.8	0.0
2d	4.4 ± 0.1	0.0
2e	0.0	19.4 ± 2.8
3a	11.6 ± 3.2	41.4 ± 0.9
3b	8.7 ± 1.9	0.0
3c	13.2 ± 4.8	8.5 ± 2.3
3e	18.8 ± 2.3	44.9 ± 2.7
4a	0.0	44.7 ± 1.8
4b	4.5 ± 1.1	50.2 ± 0.8
4e	24.4 ± 4.5	40.3 ± 2.5
5a	17.2 ± 5.5	30.4 ± 1.2
5b	21.0 ± 5.4	0.0
5c	0.0	0.0
5d	10.0 ± 2.5	30.0 ± 3.1
5e	20.5 ± 2.2	41.4 ± 3.5
6a	0.0	71.1 ± 9.7
6d	0.0	46.6 ± 2.7
6e	0.0	23.4 ± 0.7
7a	7.7 ± 0.3	22.8 ± 2.6
7b	0.0	23.4 ± 2.4
7c	0.0	32.9 ± 4.4
7d	25.5 ± 4.8	38.9 ± 1.6
7e	4.7 ± 0.3	18.8 ± 3.0
NDGA (20 μM)		54.0 ± 4.2
Allopurinol (100 μM)	81.6 ± 1.5

Soybean lipoxygenase inhibition

Soybean lipoxygenase catalyzes deoxygenation of polyunsaturated fatty acids to yield cis, trans-conjugated dienehydroperoxides. The results for soybean lipoxygenase inhibitory (Table 1) revealed that the synthesized compounds showed variable degrees of in vitro percentage inhibition. In fact, the acetimidamide 6a was found to be the most active compound (71% inhibition at 100 μM). The compounds 3a, 3e, 4a, 4b, 4e, 5e, and 6d exhibited a moderate inhibitory activity (40.3–50.2% inhibition at 100 μM). The remaining compounds showed weak inhibitory effects.

This limited activity fails to understand the structure–activity relationship and consider unambiguously that the system attached at C-2 of the triazole ring alone manages the behavior of these compounds against soybean lipoxygenase. The diversification of fragments borne by the triazole ring in a larger series of derivatives could lead to a plausible conclusion.

Cytotoxic activity

Cytotoxic activity of the most synthesized products tested against the human cell lines HCT-116 (human colon carcinoma), MCF-7 (breast carcinoma), IGROV-1 (human ovarian carcinoma), and OVCAR-3 (human ovarian carcinoma) (Table 2) was assessed using MTT assay, which is reliable to detect proliferation of cells. The cytotoxic effect of the compounds 2c, 2d, 3c, 3e, 4a, 4b, 4e, 6a, 7a, 7b, 7c, 7d, 7e was found limited against the four cell lines (IC50 ≥ 94 μM). The other compounds were active against at least one cell line.

Table 2. Cytotoxic activity of compounds 2a–e, 3a–c, 6e, 4a, 4b,4e, 5a–e, 6a, 6d, 6e, and 7a–e.

	Cytotoxic activity (IC50 μM)
Compounds	OVCAR-3	IGROV-1	HCT-116	MCF-7
2a	12 ± 1	33 ± 2	45 ± 3	17 ± 2
2b	25 ± 2	60 ± 5	32 ± 1	33 ± 1
2c	>100	>100	>100	>100
2d	94 ± 8	>100	>100	>100
2e	10 ± 1	45 ± 3	71 ± 3	>100
3a	56 ± 3	50 ± 4	>100	>100
3b	63 ± 4	>100	>100	>100
3c	>100	>100	>100	>100
3e	97 ± 6	>100	>100	>100
4a	>100	>100	>100	>100
4b	>100	>100	>100	>100
4e	96 ± 11	>100	>100	>100
5a	28 ± 3	63 ± 7	31 ± 3	4 ± 0.2
5b	50 ± 4	>100	34 ± 2	36 ± 2
5c	100 ± 12	>100	38 ± 1	>100
5d	>100	>100	63 ± 4	>100
5e	40 ± 6	>100	25 ± 2	16 ± 1
6a	>100	96 ± 6	>100	>100
6d	13 ± 1	47 ± 3	40 ± 2	>100
6e	16 ± 2	28 ± 2	36 ± 2	51 ± 4
7a	>100	>100	>100	>100
7b	>100	>100	>100	>100
7c	>100	>100	>100	>100
7d	>100	>100	>100	>100
7e	>100	>100	>100	>100
Doxorubicin			1.8 ± 0.2	0.75 ± 0.05
Tamoxifen	1.3 ± 0.1	2.0 ± 0.1

The results obtained showed that the coexistence of the amidoxime function at C-2 of the triazole and the unsubstituted phenyl at C-14 (compound 2a) of para-methylphenyl (compound 2b) and para-ethylphenyl (compound 2e) appears to the origin of the activity of these compounds against most cancer cell lines used. We find that the presence of the methoxy and chlorine both in the para position of the phenyl-C at C-14 are responsible for the loss of activity of the corresponding compounds (2c and 2d, respectively) against all cell lines used.

It appears from our findings that the conversion of the amidoxime function into unsubstituted oxadiazole in compounds 3 theoretically carrying a cytotoxic activity16 and independently of the substituent in the para position on the aromatic ring attached at C-14 resulted in the loss of activity toward all cell lines used.

The presence of the methyl group at C-5′ in oxadiazoles 4a (R = H) and 4b (R = CH₃) attenuated the cytotoxic activity compared to the corresponding derivatives 3a and 3b where the oxadiazole is unsubstituted. The activity of the compound 4e (R = C₂H₅) is held against the cell line OVCAR-3 compared to the third derivative (R = C₂H₅).

Unlike derivatives 3 and 4, the presence of the 2-oxopropyl group at C-5′ of the oxadiazole ring could explain the activity of compound 5a toward OVCAR-3 (IC₅₀=28 ± 3 μM), the compounds 5a, 5b, 5c, and 5e against HCT-116 (IC₅₀=31 ± 3, 34 ± 2, 38 ± 1, and 25 ± 2 μM, respectively) and 5a and 5e toward MCF-7. In the case of HCT-116 cell line, the 2-oxopropyl group in 5c (IC₅₀=38 ± 1 μM) appears impose its effect on that of the methoxy group which appears to be one of responsible of the loss of activity of the analog 3c where the oxadiazole ring is unsubstituted.

In the case of compounds 6, the acylation of the hydroxyl group of the amidoxime function in compounds 2 with the 2-chloropropionic acid chloride did not improve the activity of compounds 2d (R = Cl) and 2e (R = C₂H₅) except against the OVCAR-3 cell line which the IC₅₀ values passes from 10 ± 1 μM (2e) to 16 ± 2 μM (6e). The total inactivity of the derivative 6a compared to the starting amidoxime 2a against all the cell lines used shows the difficulty of explaining the structure–activity relationship, taking into account both the introduced acyl and variable R in para position of the aromatic ring at C-14. In addition, the limited number of derivatives 6 synthesized in this work seems insufficient to advance a plausible explanation.

In the case of compounds 7, the 1-chloroethyl group attached at C-5′ of the oxadiazole and independently of the nature of the R group attached in para position of the aromatic ring at C-14 seems to have no effect against all the cell lines used in comparison to the compounds 3 wherein the oxadizole ring is unsubstituted.

Our findings point out that the chlorine atom in para position of the phenyl at C-14 in compounds 2d, 5d, and 7d also appears responsible for the loss in cytotoxic activity of these compounds.

The compounds 2a, 2e, and 5a that were found to be the most cytotoxics deserve to be tested against other cancer cell lines and to be chemically modified by thinking to replace naphthalene used as a support by other systems that could play a dual role (support and activity carrier).

Conclusion

In this paper, we designed and synthesized 24 novel acetimidamides 6a–e and oxadiazoles 3a–c and 3e, 4a–e, 5a–e, and 7a–e. Their in vitro xanthine oxidase, soybean lipoxygenase inhibition and cytotoxic activities (against four cancer cell lines: HCT-116, MCF-7, IGROV-1, and OVCAR-3) were also evaluated. A moderate xanthine oxidase and soybean lipoxygenase inhibition were obtained but the good cytotoxic activities were recorded. Most compounds bearing a free or esterified amidoxime function exhibited an interesting cytotoxic effect, hence its contribution in this activity. On the other hand, it is well known from the literature that triazolopyrimidines and isoxazoles are among the systems responsible for the cytotoxic activity of several molecules but their coexistence in a single molecule in a determined sequence can eliminate or at least mitigate this activity. The significant cytotoxic activity of compounds 2a, 2e, and 5a may open new ways of research on the amidoxime function, the oxadiazole bearing a 2-oxopropyl fragment without neglecting the triazolopyrimidine moiety and the naphthalene which could be replaced by other systems.

Declaration of interest

The authors report no declarations of interest.