Synthesis of purine homo-N-nucleosides modified with coumarins as free radicals scavengers^*

Abstract

Cross metathesis (CM) of 9-butenylpurines with 4-butenyloxycoumarin in the presence of Grubbs 2nd generation catalyst under MW irradiation resulted to conjugated compounds containing homo-N-nucleosides and coumarins. Analogous derivatives received by the CM reaction of 9-butenyl-6-piperidinylpurine with 6- or 7-butenyloxycoumarins, allyloxycoumarins or coumarinyl acrylate. These compounds were tested in vitro for their antioxidant activity and they present significant scavenging activity. The presence of a pentenyloxy moiety, the attachment position on coumarin ring as well as a purine homo-N-nucleoside group are considered as important structural features.

Keywords::

• Modified homo-N-nucleosides
• purines
• coumarins
• cross metathesis
• microwaves
• antioxidant activity

Introduction

Nucleosides and modified nucleosides represent classes of compounds that possess very interesting biological activities^1–9 especially antiviral, anticancer, antimetabolic. Such activities appears also in acyclic nucleosides^4,5 bearing an alkyl chain in the place of sugar moiety. The acyclovir (I) is an antiviral drug; the adefovir (II) is in use for the treatment of the Hepatitis B virus (HBV) infections; the tenofovir (III) is the most important component in the anti-HIV drug cocktails¹. The AP23464 (IV) and (V) are potent inhibitors of Src and Abl tyrosine kinases, which regulate angiogenesis¹⁰. The hydroxamic acids VI have been synthesized as inhibitors of adenylyl cyclase¹¹ whereas fluorinated pyranonucleoside analogues of N⁴-benzoylcytosine and N⁶-benzoyladenine have been tested for antioxidant activity¹². Compounds like VII show cardiostimulant and antiarrhythmic activities¹³.

Coumarin derivatives, naturally occurring or synthetic, exhibit a broad range of biological activities^14–19 including anticoagulant, antibiotic, antifungal, antipsoriasis, cytotoxic, anti-HIV, anti-inflammatory, antioxidant, antithrombin etc. The geiparvarin (VIII) and their derivatives exhibit antitumorial activity^20,21 and they also appeared as MAO-B inhibitors²². The auraptene (IX) is a chemopreventative agent against different kinds of cancer and an antiiflammatory agent²³. In terms of structural characteristics all the above mentioned compounds include a linker–substituent (Figure 1).

Figure 1  Modified nucleosides and coumarin derivatives with important biological activities.

In continuation to our previous studies on coumarin derivatives^16,24–29 and on modified nucleosides^30,31 and taking under consideration the linker’s presence and its contribution to the biological activity of molecules I–IX, we decided to synthesize a new series of conjugated molecules as modified nucleosides, combining the coumarin, the purine and a spacer moieties. The new compounds will be studied as free radical scavengers and lipid peroxidation inhibitors. Free radicals and the consequent lipid peroxidation could trigger coagulation and inflammation. Evidence points toward extensive cross-talk between coagulation and inflammation. It has been proved that inflammation plays an important role in post-thrombolytic complications. In patients with acute myocardial infarction³², inflammation is induced by thrombolytic therapy. Thrombin, a proteolytic enzyme can elicit many inflammatory responses in microvascular endothelium. Its multiple role in thrombosis makes thrombin an important target for the therapeutic agents designed for thrombus prevention³³.

For our syntheses we will apply the powerfull olefin metathesis^34–37, utilizing its application the cross-metathesis (CM^36,38–40). The olefin metathesis reaction has been emerged the last decade in the nucleoside chemistry⁴¹, while with the CM have been synthesized especially a few acyclic nucleosides^42,43 and dinucleosides^44–47. Hetero-dinucleosides have been prepared in one case by CM under MW irradiation in shorter reaction times and better selectivity of heterodimer over the homodimer⁴⁸. Generally dramatic improvement of yields and reaction times have been reported in the MW-assisted CM reactions^49–51. For those reasons we studied the new reactions under MW irradiation. The studied reactions and the obtained products are depicted in Schemes 1, 2. The derived compounds are examined as possible lipoxygenase (LOX) inhibitors, as potential inhibitors of lipid peroxidation and thrombin, as well as hydroxyl radical scavengers. The results will be discussed in terms of the structural characteristics of the compounds and the role of the combined moieties will be delineated.

Materials and methods

Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. IR spectra were obtained with a Perkin-Elmer 1310 spectrophotometer as KBr pellets or Nujol mulls. NMR spectra were recorded on a Bruker AM 300 (300 MHz and 75 MHz for ¹H and ¹³C, respectively) using CDCl₃ as solvent and TMS as an internal standard. J values are reported in Hz. Mass spectra were determined on a LCMS-2010 EV Instrument (Shimadzu) under Electrospray Ionization (ESI) conditions. Microanalyses were performed on a Perkin-Elmer 2400-II Element analyzer. Silica gel No 60, Merck A.G. was used for column chromatography. All the chemicals used were of analytical grade and commercially available. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), nordihydroguairetic acid (NDGA), Trolox, Soybean Lipoxygenase, linoleic acid sodium salt, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), thrombin, tosyl-Gly-Pro-Arg-pNA are purchased from the Aldrich Chemical Co. & Sigma, Milwaukee, WI, USA. UV-Vis spectra were obtained on a Perkin-Elmer Lambda 20 beam spectrophotometer and on a Hitachi U-2001 spectrophotometer. The MW experiments were performed in a Biotage (Initiator 2.0) scientific MW oven.

Synthesis

Compounds 4a⁵², 8⁵³ and 10⁵⁴ were prepared according to the literature.

General procedure for the butenylation of purines. 6-Piperidinylpurine (1a²⁹) (0.53 g, 2.61 mmol), 4-bromo-1-butene (0.705 g, 0.536 mL, 5.22 mmol) and anhydrous K₂CO₃ (0.72 g, 5.22 mmol) were suspended in 10 mL dry DMF and the mixture was refluxed under N₂ for 15 h. The resulting precipitate was filtered while hot and washed with CH₂Cl₂. The filtrate was evaporated and subjected to column chromatography [silica gel, hexane /EtOAc (1:1)] to give, from the faster moving band, compound 1a (0.47 g, 70% yield).

9-(But-3-en-1-yl)-6-(piperidin-1-yl)-9H-purine (2a). Oil, IR (Neat): 3080, 2920, 2840, 1575, 1545, 1470 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.63–1.80 (m, 6H), 2.62 (q, 2H, J = 6.9 Hz), 4.23 (t, 6H, J = 6.9 Hz), 5.05 (d, 1H, J = 17.3 Hz), 5.06 (d, 1H, J = 11.1 Hz), 5.70–5.86 (m, 1H), 7.70 (s, 1H), 8.34 (s, 1H); ¹³C-NMR (CDCl₃) δ 24.9, 26.2, 34.1, 43.1, 46.4, 118.3, 120.0, 133.8, 138.0, 150.9, 152.5, 154.1; MS (ESI): 258 [M+H]^+.; Anal. Calcd for C₁₄H₁₉N₅: C, 65.34; H, 7.44; N, 27.22. Found: C, 65.23; H, 7.54; N, 27.08.

9-(But-3-en-1-yl)-6-(pyrrolidin-1-yl)-9H-purine (2b). [70% Yield, prepared from 6-pyrrolidinylpurine³³], m.p. 56–58°C (EtOH), IR (Nujol): 3060, 1580 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.97–2.12 (m, 4H), 2.63 (q, 2H, J = 6.9 Hz), 3.70–4.20 (m, 4H), 4.24 (t, 2H, J = 6.9 Hz), 5.06 (dd, 1H, J₁ = 1.5 Hz, J₂ = 17.4 Hz), 5.07 (dd, 1H, J₁ = 1.5 Hz, J₂ = 10.8 Hz), 5.72–5.87 (m, 1H), 7.69 (s, 1H), 8.37 (s, 1H); ¹³C-NMR (CDCl₃) δ 25.5, 34.1, 43.1, 48.1, 118.2, 120.3, 133.7, 138.7, 150.2, 152.8, 153.2; MS (ESI): 244 [M+H]^+.; Anal. Calcd for C₁₃H₁₇N₅: C, 64.17; H, 7.04; N, 28.78. Found: C, 64.42; H, 6.79; N, 28.67.

9-(But-3-en-1-yl)-6-chloro-9H-purine (2c). (75% Yield, prepared from 6-chloropurine), oil, IR (Neat): 3058, 2910, 2835, 1580, 1547, 1490 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.71(q, 2H, J = 6.9 Hz), 4.42 (t, 2H, J = 6.9 Hz), 5.04 (dd, 1H, J₁ = 1.2 Hz, J₂ = 17.1 Hz), 5.08 (dd, 1H, J₁ = 1.2 Hz, J₂ = 10.2 Hz), 5.72–5.87 (m, 1H), 8.18 (s, 1H), 8.75 (s, 1H); ¹³C-NMR (CDCl₃) δ 33.4, 43.5, 118.5, 131.2, 132.7, 145.0, 150.4, 151.4, 151.5; MS (ESI): 209/211 [M+H]^+.; Anal. Calcd for C₉H₉N₅Cl: C, 51.81; H, 4.36; N, 26.85. Found: C, 51.76; H, 4.14; N, 26.98.

General procedure for the butenylation of coumarins. 6-Hydroxy-4-methylcoumarin (3b) (0.25 g, 1.42 mmol), 4-bromo-1-butene (0.192 g, 0.146 ml, 1.42 mmol) and anhydrous K₂CO₃ (0.35 g, 2.53 mmol) were added in 10 ml dry acetone and the mixture was refluxed for 20 h. More 4-bromo-1-butene (96 mg, 0.073 ml, 0.71 mmol, totally 2.13 mmol) and anh. K₂CO₃ (0.175g, 1.27mmol, totally 3.8 mmol) were added and the reflux continued for 28 h. The resulting precipitate was filtered while hot and washed with acetone. The filtrate was evaporated and subjected to column chromatography [silica gel, hexane /EtOAc (3:1)] to give, from the faster moving band, compound 4b (0.229 g, 70% yield).

6-(But-3-en-1-yloxy)-4-methyl-2H-chromen-2-one (4b). M.p. 74–76°C (EtOAc/ hexane), IR (Nujol): 3070, 1705, 1575 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.40 (s, 3H), 2.57 (q, 2H, J = 6.7 Hz), 4.06 (t, 2H, J = 6.7 Hz), 5.14 (dd, 1H, J₁ = 1.6 Hz, J₂ = 12.9 Hz), 5.19 (dd, 1H, J₁ = 1.6 Hz, J₂ = 17.2 Hz), 5.84–5.99 (m, 1H), 6.31 (s, 1H), 7.03 (d, 1H, J = 2.9 Hz), 7.10 (dd, 1H, J₁ = 2.9 Hz, J₂ = 8.9 Hz), 7.26 (d, 1H, J = 8.9 Hz); ¹³C-NMR (CDCl₃) δ 18.6, 33.5, 68.1, 108.7, 115.4, 117.2, 117.8, 119.1, 120.4, 134.1, 147.9, 151.8, 155.3, 160.5; MS (ESI): 253 [M+Na]^+., 231 [M+H]^+.; Anal. Calcd for C₁₄H₁₄O₃: C, 73.03; H, 6.13. Found: C, 73.08; H, 5.86.

7-(But-3-en-1-yloxy)-4-methyl-2H-chromen-2-one (4c). (74% Yield, prepared from 7-hydroxy-4-methylcoumarin), m.p. 68–69°C (EtOAc/hexane), IR (Nujol): 3070, 1710, 1595 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.36 (s, 3H), 2.56 (q, 2H, J = 6.6 Hz), 4.04 (t, 2H, J = 6.6 Hz), 5.13 (dd, 1H, J₁ = 1.5 Hz, J₂ = 10.2 Hz), 5.18 (dd, 1H, J₁ = 1.5 Hz, J₂ = 17.4 Hz), 5.83–5.97 (m, 1H), 6.06 (s, 1H), 6.71 (d, 1H, J = 2.4 Hz), 6.82 (dd, 1H, J₁ = 2.4 Hz, J₂ = 8.7 Hz), 7.44 (d, 1H, J = 8.7 Hz); ¹³C-NMR (CDCl₃) δ 18.5, 33.2, 67.7, 101.4, 111.8, 112.5, 113.5, 117.3, 125.4, 133.8, 152.4, 155.2, 161.0, 161.9; MS (ESI): 253 [M+Na]^+., 231 [M+H]^+.; Anal. Calcd for C₁₄H₁₄O₃: C, 73.03; H, 6.13. Found: C, 73.27; H, 6.19.

Synthesis of 4-methyl-2-oxo-2H-chromen-6-yl acrylate (12). 6-Hydroxy-4-methyl-2H-chromen-2-one (3b) (0.5 g, 2.84 mmol) was dissolved in 20 ml dry acetone. Acryloyl chloride (0.257 g, 0.15 ml, 2.84 mmol) and anhydrous K₂CO₃ (0.392 g, 2.84 mmol) were added and the mixture was refluxed for 15 h. The resulting precipitate was filtered while hot and washed with acetone. The filtrate was evaporated and subjected to column chromatography [silica gel, hexane /EtOAc (3:1)] to give, from the faster moving band, compound 12 (0.57 g, 87% yield), m.p. 137–139°C (hexane/EtOAc); IR (Nujol): 3060, 1720, 1700, 1580 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.04 (s, 3H), 6.08 (dd, 1H, J₁ = 1.2 Hz, J₂ = 10.5 Hz), 6.34 (s, 1H), 6.35 (dd, 1H, J₁ = 10.5 Hz, J₂ = 17.4 Hz), 6.65 (dd, 1H, J₁ = 1.2 Hz, J₂ = 17.4 Hz), 7.30 (dd, 1H, J₁ = 2.4 Hz, J₂ = 9.0 Hz), 7.37 (d, 1H, J = 9.0 Hz), 7.39 (d, 1H, J = 2.4 Hz); ¹³C-NMR (CDCl₃) δ 18.6, 115.8, 117.2, 118.1, 120.6, 125.2, 127.5, 133.2, 146.6, 151.1, 151.6, 160.3, 164.4; MS (ESI): 253 [M+Na]^+., 231 [M+H]^+.; Anal. Calcd for C₁₃H₁₀O₄: C, 67.82; H, 4.38. Found: C, 67.77; H, 4.24.

General procedure for the CM reactions. Dry CH₂Cl₂ (2 ml) was placed in a tube (10 ml) for the Biotage Initiator 2.0, MW oven, equipped with a Teflon-coated stirring bar; the compounds 4a⁵² (22 mg, 0.1 mmol) and 2a (77 mg, 0.3 mmol) were added followed by the catalyst 5 (8.5 mg, 10 mol%), argon was flushed in the tube and the mixture was irradiated at 100°C for 2 h (checked by TLC every 30 min). Another portion of 5 (3.5 mg, 4 mol%, totally 12 mg, 14 mol%) was added and the solution was irradiated for further 1 h. After cooling and evaporation of the solvent the residue was separated by column chromatography [silica gel No 60, hexane/EtOAc (3:1)] to give after the elution of unreacted starting materials the compound 6a (38 mg, 86% yield).

4-{[(3E)-6-(6-piperidin-1-yl-9H-purin-9-yl)-hex-3-en-1-yl]oxy}-2H-chromen-2-one (6a). M.p. 91–92°C (EtOAc/hexane), IR (Nujol): 3060, 1700, 1610, 1570, 980 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.64–1.80 (m, 6H), 2.53 (q, 2H, J = 6.3 Hz), 2.63 (q, 2H, J = 6.9 Hz), 4.03 (t, 2H, J = 6.3 Hz), 4.22 (t, 6H, J = 6.9 Hz), 5.46–5.67 (m, 2H), 5.62 (s, 1H), 7.23–7.34 (m, 2H), 7.54 (t, 1H, J = 8.7 Hz), 7.66 (s, 1H), 7.75 (d, 1H, J = 8.1 Hz), 8.32 (s, 1H); ¹³C-NMR (CDCl₃) δ 24.8, 26.1, 31.7, 33.0, 43.2, 46.3, 68.4, 90.5, 115.7, 116.7, 119.9, 122.9, 123.8, 128.6, 128.9, 132.3, 137.8, 150.8, 152.4, 153.4, 153.9, 162.8, 165.4; MS (ESI): 468 [M+Na]^+., 446 [M+H]^+.; Anal. Calcd for C₂₅H₂₇N₅O₃: C, 67.40; H, 6.11; N, 15.72. Found: C, 67.59; H, 6.24; N, 15.83.

4-{[(3E)-6-(6-pyrrolidin-1-yl-9H-purin-9-yl)-hex-3-en-1-yl]oxy}-2H-chromen-2-one (6b). (89% Yield), m.p. 137–139°C (EtOAc/hexane), IR (Nujol): 3050, 1705, 1620, 1580, 965 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.92–2.10 (m, 4H), 2.54 (q, 2H, J = 6.4 Hz), 2.64 (q, 2H, J = 6.9 Hz), 3.67–4.18 (m, 4H), 4.01 (t, 2H, J = 6.4 Hz), 4.23 (t, 2H, J = 6.9 Hz), 5.46–5.68 (m, 2H), 5.60 (s, 1H), 7.22–7.34 (m, 2H), 7.53 (t, 1H, J = 7.3 Hz), 7.66 (s, 1H), 7.72 (d, 1H, J = 8.0 Hz), 8.34 (s, 1H); ¹³C-NMR (CDCl₃) δ 27.0, 31.7, 33.1, 43.2, 48.3, 68.5, 90.5, 115.8, 116.8, 120.4, 122.9, 123.8, 128.7, 128.9, 132.3, 138.7, 150.2, 151.7, 152.7, 153.6, 162.8, 165.4; MS (ESI): 454 [M+Na]^+., 432 [M+H]^+.; Anal. Calcd for C₂₄H₂₅N₅O₃: C, 66.81; H, 5.84; N, 16.23. Found: C, 66.72; H, 5.92; N, 16.18.

4-{[(3E)-6-(6-chloro-9H-purin-9-yl)-hex-3-en-1-yl]oxy}-2H-chromen-2-one (6c). (76% Yield), m.p. 105–106°C (EtOAc/hexane), IR (Nujol): 3080, 1714, 1645, 1582, 960 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.56 (q, 2H, J = 6.0 Hz), 2.70 (q, 2H, J = 6.6 Hz), 4.07 (t, 2H, J = 6.0 Hz), 4.37 (t, 2H, J = 6.6 Hz), 5.48-5.72 (m, 2H), 5.64 (s, 1H), 7.28 (t, 1H, J = 7.8 Hz), 7.31 (d, 1H, J = 7.8 Hz), 7.55 (t, 1H, J = 7.8 Hz), 7.73 (d, 1H, J = 7.8 Hz), 8.12 (s, 1H), 8.74 (s, 1H); ¹³C-NMR (CDCl₃) δ 31.6, 32.9, 44.0, 68.2, 90.6, 115.6, 116.8, 122.8, 123.8, 128.1, 129.5, 131.6, 132.4, 145.1, 151.1, 151.9, 152.0, 153.3, 162.7, 165.3; MS (ESI): 419/421 [M+Na]^+.; 397/399 [M+H]^+.; Anal. Calcd for C₂₀H₁₇N₄O₃Cl: C, 60.53; H, 4.52; N, 14.12. Found: C, 60.65; H, 4.45; N, 13.98.

9,9′-(3E)-Hex-3-ene-1,6-diylbis(6-chloro-9H-purine (7)). (51% Yield), m.p. 140–141°C (DCM), IR (Nujol): 3080, 1580, 1540, 983 cm⁻¹; ¹H-NMR (CDCl₃) δ 2.58 (q, 2H, J = 6.6 Hz), 4.25 (t, 2H, J = 6.6 Hz), 5.38–5.43 (m, 1H), 8.03 (s, 1H), 8.74 (s, 1H); ¹³C-NMR (CDCl₃) δ 32.8, 43.9, 129.2, 131.7, 144.9, 151.3, 151.8, 152.0; MS (ESI): 411/413/415 [M+Na]^+.; 389/391/393 [M+H]^+.; Anal. Calcd for C₁₆H₁₄Cl₂N₈: C, 49.37; H, 3.63; N, 28.79. Found: C, 49.21; H, 3.69; N, 28.52.

4-Methyl-6-{[(3E)-6-(6-piperidin-1-yl-9H-purin-9-yl)-hex-3-en-1-yl]oxy}-2H-chromen-2-one (6d). (63% Yield), m.p. 109–111°C (EtOAc/hexane), IR (Nujol): 3030, 1710, 1583, 990 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.62–1.78 (m, 6H), 2.41 (s, 3H), 2.46 (q, 2H, J = 6.6 Hz), 2.61 (q, 2H, J = 6.6 Hz), 3.92 (t, 2H, J = 6.6 Hz), 4.22 (t, 6H, J = 6.6 Hz), 5.48–5.65 (m, 2H), 6.29 (s, 1H), 7.00 (d, 1H, J = 2.7 Hz), 7.06 (dd, 1H, J₁ = 2.7 Hz, J₂ = 9.0 Hz), 7.25 (d, 1H, J = 9.0 Hz), 7.69 (s, 1H), 8.33 (s, 1H); ¹³C-NMR (CDCl₃) δ 18.7, 24.5, 25.8, 32.0, 32.7, 42.9, 45.9, 67.7, 108.4, 115.1, 117.4, 118.8, 119.5, 120.1, 128.0, 129.2, 137.9, 147.7, 150.6, 151.5, 151.9, 153.6, 155.0, 160.2; MS (ESI): 482 [M+Na]^+.; Anal. Calcd for C₂₆H₂₉N₅O₃: C, 67.95; H, 6.36; N, 15.24. Found: C, 68.17; H, 6.50; N, 15.12.

4-Methyl-7-{[(3E)-6-(6-piperidin-1-yl-9H-purin-9-yl)-hex-3-en-1-yl]oxy}-2H-chromen-2-one (6e). (74% Yield), m.p. 96–97°C (EtOAc/hexane), IR (Nujol): 3020, 1705, 1605, 1570, 980 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.64–1.78 (m, 6H), 2.39 (s, 3H), 2.47 (q, 2H, J = 6.6 Hz), 2.61 (q, 2H, J = 6.6 Hz), 3.94 (t, 2H, J = 6.6 Hz), 4.22 (t, 6H, J = 6.6 Hz), 5.45–5.63 (m, 2H), 6.13 (s, 1H), 6.78 (d, 1H, J = 2.4 Hz), 6.82 (dd, 1H, J₁ = 2.4 Hz, J₂ = 9.0 Hz), 7.48 (d, 1H, J = 9.0 Hz), 7.68 (s, 1H), 8.33 (s, 1H); ¹³C-NMR (CDCl₃) δ 18.6, 24.8, 26.1, 32.1, 33.1, 43.4, 46.4, 67.7, 101.5, 112.0, 112.6, 113.6, 119.8, 125.5, 127.4, 128.3, 129.3, 138.0, 150.7, 152.4, 153.7, 155.3, 161.2, 161.9; MS (ESI): 482 [M+Na]^+., 460 [M+H]^+.; Anal. Calcd for C₂₆H₂₉N₅O₃: C, 67.95; H, 6.36; N, 15.24. Found: C, 68.06; H, 6.48; N, 15.41.

4-{[(2E)-5-(6-piperidin-1-yl-9H-purin-9-yl)-pent-2-en-1-yl]oxy}-2H-chromen-2-one (9). (63 % Yield), m.p. 83–85°C (EtOAc/hexane), IR (Nujol): 3020, 1700, 1615, 1575, 975 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.67–1.80 (m, 6H), 2.74 (q, 2H, J = 7.2 Hz), 4.20–4.37 (m, 6H), 4.58 (d, 2H, J = 5.1 Hz), 5.62 (s, 1H), 5.78 (dt, 1H, J₁ = 5.1 Hz, J₂ = 15.4 Hz), 5.92 (dt, 1H, J₁ = 7.2 Hz, J₂ = 15.4 Hz), 7.21–7.37 (m, 2H), 7.55 (t, 1H, J = 8.4 Hz), 7.71 (s, 1H), 7.80 (d, 1H, J = 8.4 Hz), 8.35 (s, 1H); ¹³C-NMR (CDCl₃) δ 22.7, 26.2, 31.9, 42.9, 46.6, 69.1, 90.9, 115.7, 116.8, 119.8, 123.1, 123.9, 126.4, 131.9, 132.4, 138.1, 144.2, 153.5, 155.4, 160.4, 162.8, 165.1; MS (ESI): 454 [M+Na]^+., 432 [M+H]^+.; Anal. Calcd for C₂₄H₂₅N₅O₃: C, 66.81; H, 5.84; N, 16.23. Found: C, 66.72; H, 5.73; N, 16.45.

4-Methyl-6-{[(2E)-6-(6-piperidin-1-yl-9H-purin-9-yl)-pent-2-en-1-yl]oxy}-2H-chromen-2-one (11). (58% Yield), m.p. 81–82°C (EtOAc/hexane), IR (Nujol): 3010, 1703, 1620, 1565, 970 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.65–1.83 (m, 6H), 2.39 (s, 3H), 2.70 (q, 2H, J = 6.6 Hz), 4.26 (t, 6H, J = 6.6 Hz), 4.47 (d, 2H, J = 5.1 Hz), 5.73 (dt, 1H, J₁ = 5.1 Hz, J₂ = 15.6 Hz), 5.84 (dt, 1H, J₁ = 6.6 Hz, J₂ = 15.6 Hz), 6.29 (s, 1H), 6.99 (d, 1H, J = 2.7 Hz), 7.05 (dd, 1H, J₁ = 2.7 Hz, J₂ = 9.0 Hz), 7.24 (d, 1H, J = 9.0 Hz), 7.67 (s, 1H), 8.33 (s, 1H); ¹³C-NMR (CDCl₃) δ 18.6, 24.9, 26.2, 31.9, 43.2, 46.1, 68.8, 109.2, 115.6, 117.1, 118.0, 119.2, 120.0, 128.7, 137.9, 139.9, 147.9, 148.1, 150.2, 151.3, 152.0, 155.6, 160.1; MS (ESI): 468 [M+Na]^+.,460 [M+H]^+.; Anal. Calcd for C₂₅H₂₇N₅O₃: C, 67.40; H, 6.11; N, 15.72. Found: C, 67.65; H, 6.26; N, 15.67.

4-Methyl-2-oxo-2H-chromen-6-yl(2E)-5-(6-piperidin-1-yl-9H-purin-9-yl)pent-2-enoate (13). (34% Yield), m.p. 89–91°C (EtOAc/hexane), IR (Nujol): 3070, 1702, 1630, 1560, 975 cm⁻¹; ¹H-NMR (CDCl₃) δ 1.63–1.85 (m, 6H), 2.41 (s, 3H), 2.92 (q, 2H, J = 6.9 Hz), 4.18–4.32 (m, 4H), 4.38 (t, 2H, J = 6.9 Hz), 6.05 (d, 1H, J = 15.6 Hz), 6.32 (s, 1H), 7.15 (dt, 1H, J₁ = 6.9 Hz, J₂ = 15.6 Hz), 7.23–7.37 (m, 3H), 7.71 (s, 1H), 8.35 (s, 1H); ¹³C-NMR (CDCl₃) δ 18.6, 22.7, 26.1, 31.9, 42.2, 46.4, 115.8, 117.1, 118.1, 119.9, 120.5, 122.8, 123.2, 125.2, 137.6, 146.7, 147.9, 151.0, 151.7, 157.9, 160.4, 161.7, 164.0; MS (ESI): 460 [M+H]^+.; Anal. Calcd for C₂₅H₂₅N₅O₄: C, 65.35; H, 5.48; N, 15.24. Found: C, 66.31; H, 5.53; N, 15.09.

Biological assay

In vitro experiments

In the in vitro assays each experiment was performed at least in triplicate and the standard deviation of absorbance was less than 10% of the mean.

Determination of the reducing activity of the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH)³⁰

To a solution of DPPH in absolute ethanol (final concentration 0.05 mM) the tested compounds (stock solutions in DMSO) was added (final concentration 0.05 and 0.1 mM). Ethanol was used as control solution. After 20 and 60 min at room temperature the absorbance was recorded at 517 nm (Table 1) and compared with the reference compound NDGA.

Table 1.  Interaction-reducing activity with DPPH (RA%); competition with DMSO for hydroxyl radical (HO˙ %); inhibition of lipid peroxidation (AAPH%); in vitro inhibition of soybean lipoxygenase (LOX); in vitro inhibition of thrombin (% Thr).

Compounds	clog P	RA(%)0.1mM 20/60 min ± SD	HO˙(%)0.1mM ± SD	AAPH(%)0.1mM ± SD	LOX(%)0.1mM ± SD	Thr(%)0.1mM ± SD
6a	3.98	25/31 ± 0.2	88 ± 3.3	no	2 ± 0.1	Nt
6b	3.42	11/15 ± 0.1	no	26 ± 0.4	35 ± 0.5	nt
6c	3.08	6/14 ± 0.05	35 ± 1.1	31 ± 0.6	38 ± 1.5	nt
6d	4.34	no/no	100 ± 2.5	73 ± 1.8	32 ± 0.8	no
6e	4.34	11/16 ± 0.01	no	34 ± 0.2	35 ± 1.1	nt
9	3.65	14/63 ± 1	72 ± 2.4	48 ± 1.6	45 ± 1.4	11 ± 0.03
11	4.01	28/33 ± 1.2	no	77 ± 2.3	47 ± 2	no
13	3.19	49/61 ± 0.8	79 ± 1.6	no	10 ± 0.2	nt
NDGA	81/83 ± 1.7	84 ± 2.1
Trolox	73 ± 1.2	64 ± 3
NAPAP	100 ± 4

no, no activity under the experimental conditions; nt, not tested; ±SD ≤ 10%.

Competition of the tested compounds with DMSO for hydroxyl radicals28

The hydroxyl radicals generated by the Fe³⁺/ascorbic acid system, were detected according to Nash, by the determination of formaldehyde produced from the oxidation of DMSO. The reaction mixture contained EDTA (0.1 mM), Fe³⁺ (167 μM), DMSO (33 mM) in phosphate buffer (50 mM, pH 7.4), the tested compounds at 0.1 mM and ascorbic acid (10 mM). After 30 min of incubation (37°C) the reaction was stopped with CCl₃COOH (17 % w/v) (Table 1). Trolox was used as a standard drug.

Inhibition of linoleic acid lipid peroxidation³⁰

Production of conjugated diene hydroperoxide by oxidation of linoleic acid in an aqueous dispersion is monitored at 234 nm. AAPH is used as a free radical initiator. This assay can be used to follow oxidative changes and to understand the contribution of each tested compound.

Azo compounds generating free radicals through spontaneous thermal decomposition are useful for in vitro studies of free radical production. The water-soluble azo compound AAPH has been extensively used as a clean and controllable source of thermally produced alkylperoxyl free radicals. Ten microlitre of the 16 mM linoleic acid sodium salt solution was added to the UV cuvette containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4 prethermostated at 37°C. The oxidation reaction was initiated at 37°C under air by the addition of 50 μL of 40 mM AAPH solution. Oxidation was carried out in the presence of aliquots (10 μL) in the assay without antioxidant; lipid oxidation was measured in the presence of the same level of DMSO. The rate of oxidation at 37°C was monitored by recording the increase in absorption at 234 nm caused by conjugated diene hydroperoxides. Trolox was used as a reference compound (Table 1).

Soybean lipoxygenase inhibition study in vitro^28–30

In vitro study was evaluated as reported previously. The tested compounds dissolved in ethanol were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 ml of enzyme solution (1/9 × 10⁴ w/v in saline). The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor (Table 1). NDGA was used as a reference.

Inhibition of thrombin³⁰

Tosyl-Gly-Pro-Arg-pNA was used as a substrate for thrombin at 1 mM final concentration. Compounds were dissolved at a final concentration of 0.1 mΜ in a Tris-buffer (0.05 M Tris, 0.154 M NaCl, ethanol 5%, pH 8.0). Three minutes after the addition of bovine thrombin (2.5 unit/mg), the reaction was ended by adding 0.1 ml acetic acid 50%. The absorption of the released p-nitroaniline was measured at 405 nm. NAPAP was used as a standard.

Determination of lipophilicity as clog P

Lipophilicity was theoretically calculated as clog P values in n-octanol-buffer by CLOGP Programme of Biobyte Corp⁶⁷.

Results and discussion

Synthesis

The butenylation of 6-piperidinylpurine (1a)²⁹ with excess of 4-bromo-1-butene in DMF in the presence of K₂CO₃, in analogy to allylation⁵⁵, led after refluxing for 9 h to 9-(but-3-en-1-yl)-6-(piperidin-1-yl)-9H-purine 2a (70%). The CM reaction of 4-(but-3-en-1-yloxy)-2H-chromen-2-one (4a)⁵² with three equivalents of compound 2a seems to increase the ratio of the desired cross metathesis product to the possible homodimer⁴⁶. The reaction is performed in CH₂Cl₂ in the presence of the Grubbs 2nd generation catalyst 5 (14 mol%, added in two portions) under MW irradiation at 100°C over 3 h(Scheme 1) and resulted only to the product 6a in 86% yield, while no homodimerization products detected. From the ¹H-NMR of 6a the ratio E/Z is 3.2/1 with the E-isomer to give in the ¹³C-NMR absorptions for the CH₂–CH=CH–CH₂ at 33.0 and 31.7 ppm, while in the Z-isomer appear, as expected^47,56, at lower values at 29.7 and 27.9 ppm. When we studied the above reaction with 5% of the catalyst 5 under the same conditions the yield for 6a was only 29%.

The CM reaction of 9-but-3-en-1-yl-6-(pyrrolidin-1-yl)-9H-purine (2b) (3 equivalents) with alkene 4a in the presence of the catalyst 5 (14 mol% in two portions) under microwaves for 3 h gave the product 6b (89% yield) (E/Z ratio 3/1 from the ¹H-NMR spectra). The same experiment with 11 mol% of the catalyst 5, loaded at once led to 79% yield of the product 6b. The analogous reaction of alkenes 2c and 4a with the catalyst 5 (14 mol% in two portions) under MW led to heteroalkene 6c (76%) [E/Z ratio 3.1/1, from the ¹H-NMR spectrum] followed in this case by the purine homodimer 7 (51%) [E/Z ratio 3/1, from the ¹H-NMR spectrum]. As we can see from the above reactions the CM of the Type I⁵⁷ alkenes 2a–c (3 equiv.) and 4a proceeds under MW irradiation in high yield by using enough amount of the catalyst 5 (14 mol % loaded in two portions) (Scheme 1).

Scheme 1.  Reagents and conditions: (i) 4-bromo-1-butene, anhydrous K₂CO₃, dry DMF, reflux under Ar₂, 15 h; (ii) 4-bromo-1-butene, anhydrous K₂CO₃, dry acetone, reflux, 20 h; (iii) CH₂Cl₂, 5 (14 mol% in two portions, the second after 2 h), MW, 100°C, 3 h.

The products 6d (63%) and 6e (74%) [E/Z ratio 2.8/1 and ratio 3/1, respectively, from the ¹H-NMR spectra] are isolated from the CM reactions (Scheme 2) of purine derivative 2a with the butenyloxycoumarins 4b,c (prepared in analogy to 4a) in the presence of catalyst 5 (14 mol% in two portions). No CM products are received from the efforts for reaction of coumarin derivative 4c with the 6-chloro-9-vinylpurine in the presence of the catalyst 5 or the Hoveyda-Grubbs 2nd generation catalyst under different ratios of the starting materials and under reflux or MW irradiation (Scheme 2).

Scheme 2.  Reagents and conditions: (i) CH₂Cl₂, 3 (14 mol% in two portions, the second after 2 h), MW, 100°C, 3 h.

The CM reaction of derivative 2a with the 4-allyloxycoumarin (8)⁵³ by treatment with the catalyst 5 (14 mol% in two portions) under MW irradiation resulted to the product 9 (63%). This product seems to be the E-isomer since in the ¹³C-NMR spectrum the shift is only at 69.1 ppm, but nothing at 65.1 ppm as expected for the possible Z-isomer⁴⁵. In the ¹H-NMR spectrum there are also two peaks at 5.78 ppm (dt, 1H, J₁ = 5.1 Hz, J₂ = 15.4 Hz) and 5.92 ppm (dt, 1H, J₁ = 7.2 Hz, J₂ = 15.4 Hz) for the olefinic protons revealing that this is the E-isomer. The reaction of coumarin 8 with the 9-allyl-6-chloropurine in the presence of catalyst 5 under reflux gave no CM products.

The analogous reaction between the purine derivative 2a and the 6-allyloxy-4-methylcoumarin (10)⁵⁴ with 5 (14 mol% in two portions) gave the heteroproduct 11 (58%). This product is the E-isomer since in the ¹H-NMR spectrum there are two peaks at 5.73 ppm (dt, 1H, J₁ = 5.1 Hz, J₂ = 15.6 Hz) and 5.84 ppm (dt, 1H, J₁ = 6.6 Hz, J₂ = 15.6 Hz) for the olefinic protons and in the ¹³C-NMR spectra⁴⁵ there is a shift at 68.8 ppm. In this case no Z-isomer was isolated from the column chromatography. By using a higher loading for the catalyst (16 mol%) but at once, the product 11 has been received in lower yield (52%). The reaction of allyloxycoumarin 10 with the 9-allyl-6-chloropurine (10:1 equivalents), in the presence of the catalyst 5 under reflux resulted to unchanged starting materials.

The CM reaction of derivative 2a and acrylate 12 [prepared from the reaction of 4-methyl-6-hydroxycoumarin (3b) with acryloyl chloride] with the catalyst 5 (14 mol%, in two portions) led to the E-isomer 13 (50%) (J = 15.6 Hz for the coupling of olefinic protons). No Z-isomer was detected in the fractions of column chromatography. When we used 17 mol% of the catalyst 5 (added at once) the isolated yield of the product 13 was only 34%. The reaction of acrylate 12 with the 9-allyl-6-chloropurine in the presence of Hoveyda-Grubbs 2nd generation catalyst or the catalyst 5 under reflux or MW irradiation didn’t resulted to CM products. These reactions gave only the 6-chloro-9-(prop-1-enyl)purine (isomerization product of the purine derivative).

The yields for the above CM reactions of allyloxycoumarins are lower than the analogous reactions with butenyloxycoumarins as expected due to the possible complexation of allylic oxygen to the Ru metal in the intermediate steps. The analogous complexations also of vinylic or allylic nitrogen from purines to the Ru metal, in the intermediate steps, are possibly responsible for no reaction of 9-vinyl- or 9-allylpurine derivatives.

Biological studies

In the present investigation, the compounds 6a–e, 9, 11 and 13 were studied with regard to their antioxidant ability as well as to their ability to inhibit soybean LOX. Compounds with antioxidant properties could be expected to offer protection in thrombosis and inflammation and to lead to potentially effective drugs. Taking into account the multifactorial character of oxidative stress and inflammation, we decided to evaluate the in vitro antioxidant activity of the synthesized molecules using three different antioxidant assays: (a) interaction with the stable free radical DPPH, (b) competition of the tested compounds with dimethyl sulfoxide (DMSO) for hydroxyl radicals and (c) interaction with the water-soluble azo compound AAPH. In view of the differences among the test systems available, the results of a single assay can give only a suggestion on the protective potential of tested compounds.

The DPPH test is very useful in the micromolar range demanding minutes to hours for both lipophilic and hydrophilic samples. In cases where the structure of the electron donor is not known, this method can afford data on the reduction potential of the sample, and hence can be helpful in comparing the reduction potential of unknown materials. The interaction of the examined compounds with the stable free radical DPPH was studied by the use of DPPH⁵⁸ at 0.05 mM and 0.1 mM after 20 and 60 min (Table 1). This interaction indicates their reducing ability in an iron-free system. The compounds did not show any interaction at 0.05 mM. The results showed that this interaction is time dependent but low compared to the reference compound NDGA. This can be attributed to the absence of any easily oxidizable functionalities like the ones (two catechol subunits) present in NDGA which is used as a reference compound. Compounds 9, 13, 11 and 6a present the higher interaction values at 60 min (31–63%). Among the derivatives of subgroup 6a–c, no significant changes are observed. The attachment of hexenyloxy group at position 6-, namely compound 6d, resulted in complete loss of activity. The presence of the ester group in compound 13 improves significantly the reducing ability, whereas in compound 11 the absence of this group leads to complete loss of antioxidant activity. The low interaction may be also due to the fact that they are large molecules and they can’t reach the radical site or due to steric reasons. Lipophilicity is not well correlated with the results. However, comparing the reducing abilities of 6a (clog P = 3.98, 31%) to 9 (clog P = 3.65, 63%) and of 6d (clog P = 4.34, no) to 11 (clog P = 4.01, 33%), it seems that lower lipophilicity (due to smaller aliphatic chain), is correlated with higher antioxidant ability.

It is consistent that rates of reactive oxygen species (ROS) production are increased in most diseases⁵⁹. Cytotoxicity of O₂-. and H₂O₂ in living organisms is mainly due to their transformation into HO., reactive radical metal complexes and ¹O₂. During the inflammatory process, phagocytes generate the superoxide anion radical at the inflamed site and this is connected to other oxidizing species as HO. Hydroxyl radicals are among the most reactive oxygen species and are considered to be responsible for some of the tissue damage occurring in inflammation.

The competition of compounds with DMSO for ^·OH radicals⁵⁸, generated by the Fe³⁺/ascorbic acid system, expressed as the inhibition of formaldehyde production, was used for the evaluation of their hydroxyl radical scavenging activity. Derivatives 6a, 6d, 9 and 13 highly compete with DMSO (33 mM) at 0.1 mM in comparison to Trolox (Table 1). Perusal of the percentage results shows that the 4-substituted hybrids 6a and 9 as well as the 6-substituted 6d and 13 are more potent. The magnitude of the ring (6- or 5-membered) 6a and 6b seems to influence the competition as well as the attachment position of the hexenyloxy group on the aromatic ring of the coumarin. Thus, the 6-substituted derivative compound 6d is a potent scavenger (100%, Table 1), whereas in the corresponding 7-substituted the competition is dramatically vanished. Small differences are observed between the hexenyloxy- and pentylenyloxy analogues (6a and 9). The presence of the ester group in compound 13 improves significantly the competition, whereas in compound 11 the absence of this group leads to complete loss of hydroxyl radical scavenging activity. Lipophilicity is not correlated with the results. Antioxidants of hydrophilic or lipophilic character are both needed to act as radical scavengers in the aqueous phase or as chain-breaking antioxidants in biological membranes.

In our studies, AAPH was used as a free radical initiator to follow oxidative changes of linoleic acid to conjugated diene hydroperoxide. Azo compounds generating free radicals through spontaneous thermal decomposition are useful for free radical production studies in vitro⁶⁰. The water-soluble azo compound AAPH has been extensively used as a clean and controllable source of thermally produced C-centered radicals. The activity of the radicals produced by the action of AAPH shows a similarity to cellular activities such as lipid peroxidation⁶¹. Effective hydrogen atom transfer (HAT) agents are compounds with high hydrogen atom donating ability⁶². These include: (i) compounds with low heteroatom-H bond dissociation energies and/or (ii) compounds from which hydrogen abstraction leads to sterically hindered radicals and (iii) compounds from which abstraction of hydrogen leads to C-centered radicals stabilized by resonance.

All compounds caused inhibition of lipid peroxidation (LPO), with the exception of compounds 6a and 13. The two most powerful and almost equipotent inhibitors of LPO (compounds 6d and 11) include the same structural characteristics. The only difference is the pentenyl/hexenyloxy chain in 11/6d compounds. Within the 6 subgroup, complete loss is observed when A is piperidinyl residue. No significant differences are observed between compounds 6b and 6c. The 6-substituted derivative, namely compound 6d is more potent (73%) than the corresponding 7-substituted, 6e (34%).

Our compounds were further evaluated for inhibition of soybean lipoxygenase LOX by the UV absorbance based enzyme assay⁶³. While one may not extrapolate the quantitative results of this assay to the inhibition of mammalian 5-LOX, it has been shown that inhibition of plant LOX activity by NSAIDs is qualitatively similar to their inhibition of the rat mast cell LOX and may be used as a simple qualitative screen for such activity. Many flavonoids and other phenolics as well as coumarin derivatives inhibit soybean lipoxygenase. This inhibition is related to their ability to reduce from the iron species in the active site to the catalytically inactive ferrous form⁶⁴. Lipoxygenases oxidize certain fatty acids at specific positions to hydroperoxides that are the precursors of leukotrienes, which contain a conjugated triene structure. Most of the LOX inhibitors are antioxidants or excellent ligands for Fe³⁺ or free radical scavengers, since lipoxygenation occurs via a carbon-centered radical. It is known that soybean lipoxygenase, is inhibited by NSAIDs in a qualitatively similar way to that of the rat mast cell lipoxygenase and may be used in a reliable screen for such activity. NDGA a known inhibitor of soybean LOX has been used as a reference compound. Perusal of percentage inhibition values (Table 1) shows that the percentage inhibition values of all compounds range from 32 to 47, with the exception of 6a and 13 which are very poor inhibitors. There is no evidence for any special structural characteristic that influences the anti-LOX activity within this dataset. For all the compounds the LOX% inhibition values are in agreement with these on lipid peroxidation. The inhibitory activity of the compounds which do not contain an easily oxidizable moiety might be attributed mainly to their ability to attach to the active site of the enzyme and to a lesser degree to their electron donating ability. Although lipophilicity is referred^65,66 as an important physicochemical property for LOX inhibitors, all the above tested derivatives do not follow this concept.

We evaluated the ability of some potent representative structures (6d, 9 and 11) to inhibit thrombin. Compound 9 shows 11% inhibition, while 6d and 11 do not exhibit any activity under the reported experimental conditions.

The above research will be used for the design and the synthesis in the near future of more potent compounds.

Conclusion

In conclusion, we have synthesized acyclic purine N-nucleosides modified with coumarin skeleton by the CM reaction under MW irradiation in moderate to high yields without receiving the possible purine homodimers, by the use of three equivalents excess of purine alkene derivative and the catalyst (14 mol%) added in two portions. Preliminary biological evaluation of compounds containing a linker in a coumarin modified homo-N-nucleoside resulted to significant antioxidant activity mostly for 6d, 9 and 13. Compounds 9 and 13 interact to the free stable radical DPPH and scavenge potently HO˙ radicals. Our studies showed that the presence of a pentenyloxy moiety, the attachment position on coumarin ring as well as a purine homo-N-nucleoside group are important structural features for the antioxidant and anti-LPO activity. This provides an impetus for designing new interesting molecules for the development of a new pharmacophore for disease-modifying agents useful in the treatment of a variety of inflammatory and coronary artery diseases.

Acknowledgements

We would like to thank Dr. A. Leo and C. Hansch^†, as well as to Biobyte Corp. 201 West 4th Str., Suite 204, Claremont CA California, 91711, USA for free access to the C-QSAR program.

Declaration of Interest

The authors report no conflicts of interest.