Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2017, 80, 3247−3254

Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with Antioxidant and Anti-influenza Activities Bruno M. Bizzarri,† Lorenzo Botta,† Eliana Capecchi,† Ignacio Celestino,‡ Paola Checconi,§ Anna T. Palamara,‡,§ Lucia Nencioni,*,§ and Raffaele Saladino*,† †

Department of Ecology and Biology, University of Tuscia, Via C. De Lellis, Viterbo, 01100, Italy IRCCS, San Raffaele Pisana, Telematic University, Rome, 00163, Italy § Department of Public Health and Infectious Diseases, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia−Fondazione Cenci Bolognetti, Rome, 00161, Italy ‡

S Supporting Information *

ABSTRACT: Different catechol and pyrogallol derivatives have been synthesized by oxidation of coumarins with 2iodoxybenzoic acid (IBX) in DMSO at 25 °C. A high regioselectivity was observed in accordance with the stability order of the incipient carbocation or radical benzylic-like intermediate. The oxidation was also effective in water under heterogeneous conditions by using IBX supported on polystyrene. The new derivatives showed improved antioxidant effects in the DPPH test and inhibitory activity against the influenza A/PR8/H1N1 virus. These data represent a new entry for highly oxidized coumarins showing an antiviral activity possibly based on the control of the intracellular redox value.

C

production of reactive oxygen species (ROS).23 Innovative strategies to design anti-influenza drugs are based on the inhibition of these intracellular pathways.24,25 Antiviral compounds so far developed mainly act by inhibition of the viral coating step involving the glycoprotein neuraminidase (NA).26 However, the efficacy of these antiviral compounds is often limited by toxicity and by the inevitable selection of drug-resistant viral mutants.27 Over the last years, we reported that catechol derivatives are active against DNA and RNA viruses.28 In particular, tocopheryl hydroquinone derivatives were active against influenza A virus by regulation of the internal redox potential of the cell.29,30 This approach could offer important advantages, including broad-spectrum efficacy, antigenic properties, and the reduced probability to select drug-resistant viral strains. In order to evaluate the role of the oxidation state of coumarins in antiviral activity, we report here the use of 2iodoxybenzoic acid (IBX)31 for the regioselective synthesis of new derivatives bearing catechol and pyrogallol moieties. IBX affects the ortho-hydroxylation of phenol to catechol with a reactivity similar to that of polyphenol oxidases.32 The high regioselectivity is a consequence of the intramolecular oxygen transfer from iodine(V) in a λ5-iodanyl intermediate to the ortho-position of the phenolic group.33 The antioxidant activity

oumarins are a large class of secondary metabolites found in plants, bacteria, and fungi1 and are characterized by a 2H-1-benzopyran-2-one core structure.2 They show several pharmacological properties such as anticoagulant,3 antineurodegenerative,4 anticancer,5 and antimicrobial activities.6 Coumarins are also effective against virus replication, including the inhibition of the human immunodeficiency virus type 1 (HIV-1),7 hepatitis C virus (HCV),8 bovine viral diarrhea virus (BVDV),9 herpes simplex virus,10 and influenza A virus.11a−c In association with these biological effects, coumarins bearing the catechol pharmacophore, such as esculetin and 4-methylesculetin, are potent antioxidants12 in lipid peroxidation,13 H2O2induced oxidative cell damage,14,15 and glutathione (GSH) depletion.16 This antioxidant activity is associated with singleelectron (SET) or hydrogen-atom transfer (HAT) to reactive radicals,17 as well as to metal pro-oxidant species.18,19 Influenza virus is a respiratory pathogen belonging to the Orthomyxoviridae family that causes large recurrent epidemics with high mortality and periodic, unpredictable pandemics. After uncoating, the ribonucleoprotein capsids (vRNPS) are released in the cytosol and transported to the host-cell nucleus, where they undergo transcription and replication. In the late phase, vRNPS are exported into the cytosol to be assembled with the other structural proteins and packaged into progeny virions.20 Influenza A virus modulates the intracellular redox of the host cell by different mechanisms,21 including an increase in oxidative stress, depletion of intracellular GSH,22 and © 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 1, 2017 Published: December 13, 2017 3247

DOI: 10.1021/acs.jnatprod.7b00665 J. Nat. Prod. 2017, 80, 3247−3254

Journal of Natural Products

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of the new derivatives was evaluated by the 2,2′-diphenylpicrylhydrazyl (DPPH) radical scavenging test. The antiviral activity against influenza A/PR8/H1N1 virus was measured by the hemagglutination assay. Notably, highly oxidized coumarin derivatives bearing the pyrogallol moiety were the most active antioxidant and antiviral compounds.

Table 1. Synthesis of Catechol- and Pyrogallol-Type Derivatives by Oxidation of Coumarins 1−5, 12, and 14 with IBXa entry

substrate

product

solvent

time

1 2 3 4 5b 6 7 8b 9 10 11 12 13 14 15d 16d 17d 18d 19d 20d

1 1 1 1 1 2 2 2 3 4 5 12 10 11 1 1 1 1 1 1

6 6 6 6 6 7 7 7 8 9 10 (11)c 13 14 15 6 6 6 6 6 6

DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO H2O H2O H2O H2O H2O

2h 2h 24 h 7 days 2h 2h 24 h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h

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RESULTS AND DISCUSSION IBX-Mediated Oxidation of Coumarins. IBX is usually used in different organic solvents depending on the nature and reactivity of the system.34 DMSO was selected as solvent due to its capability to activate IBX by formation of a λ5-iodanyl adduct, in which it acts as a leaving group for the incoming substrate.35 Moreover, DMSO shows a dielectric constant high enough to ensure the solubility of coumarins.36 Initially, 6hydroxycoumarin (1) and 7-hydroxycoumarin (umbelliferone) (2) were used as substrates. The appropriate coumarin (1.0 mmol) was treated with IBX (1.0 equiv) in DMSO (1.0 mL) at different temperatures and reaction times to afford (after the final reduction step) the corresponding 5,6-dihydroxycoumarin (6) and 7,8-dihydroxycoumarin (daphnetin) (7), respectively, in variable yields, as well as unreacted substrate (Scheme 1). Scheme 1. Oxidation of Coumarins to Catechol-Type Derivatives

T (°C) rt 60 rt rt rt rt rt rt rt rt rt rt rt rt 60 60 60 60 60 60

°C

°C °C °C °C °C °C

yield (%) 66 48 65 43 61 59 44 50 58 67 54(21) 75 78 68 20 56 53 54 51 50

a

The oxidations were performed by treating the appropriate coumarin (1.0 mmol) with IBX (1.0 mmol) in DMSO (1.0 mL) for 2.0 h at room temperature. bReaction performed with an excess of IBX (2.0 equiv). cYield of the 6,7-dihydroxycoumarin (11). dOxidation performed with IBX supported on polystyrene (PSS-IBX).

the expected AB doublet at 7.04 ppm (H-7, J7,8 = 9.0 Hz) and 6.69 ppm (H-8, J8,7 = 9.0 Hz, 1H), instead of the two singlets of 6,7-dihydroxycoumarin (esculetin) (11). A similar pattern was observed for coumarin 7, in which case the AB doublet resonated at 7.47 ppm (H-6, J6,5 = 9.0 Hz) and 6.80 ppm (H-5, J5,6 = 9.0 Hz). The spectroscopic data of 7 were in accordance with reported data.37 The IBX-mediated oxidation requires the formation of the λ5-iodanyl cyclic intermediate involving the iodine atom and the phenolic moiety. The regioselectivity in the oxidation of compounds 1 and 2 can be explained by the intramolecular delivery of the λ5-iodanyl oxygen (partially negative) to the ortho-carbon that can form the more stable incipient carbocation or radical intermediate.38 Cations or radicals centered on carbons C-5 and C-8 in compounds 6 and 7, respectively, are of the benzylic type. This order of reactivity is in accordance with the regioselectivity observed in the reaction of naphthalen-2-ol, quinolin-6-ol, flavones, and flavanones with IBX.39a−d In the latter case the oxidation takes place invariably on the benzylic-type position next to the fused ring which is more stabilized as an incipient carbocation40 or radical41 intermediate. Note that the IBX regioselectivity was opposite of that previously reported for the putative cellular formation of 11 from 2, involving the activity of cytochrome P450 enzymes.42 Compound 7 has been previously isolated from the stem and root bark of Daphne giraldii Nitsche.43 The oxidation was then applied to a large panel of compounds, namely, 4,7-dihydroxycoumarin (4-hydroxyumbelliferone) (3), 4-methylumbelliferone (hymecromon) (4), and 7-hydroxy-6-methoxycoumarin (scopoletin) (5), to afford the corresponding catechol-type coumarins 8−10 from acceptable to high yield (Scheme 1; Table 1, entries 9−11). Compound 8 has been previously synthesized using the Hoesch reaction or

As reported in Table 1 (entries 1 and 5−8), the highest yields of 6 (66%) and 7 (59%) were obtained at 25 °C after 2 h. A longer reaction time, higher amount of IBX (2.0 equiv), and higher temperature (60 °C) did not increase the yields (Table 1, entries 2−5, 7, and 8). Moreover, the oxidation of compounds 1 and 2 with IBX in solvents such as CH2Cl2/ MeOH (4:1 ratio v/v), EtOAc, or THF did not afford catechol derivatives, confirming the beneficial role of DMSO in the oxidation. The catechol-type coumarins 6 and 7 were obtained with high regioselectivity, since only one of the two possible isomers was isolated from the reaction mixture. Compound 6 showed 3248



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Scheme 2. Regioselectivity Observed in the Oxidation of Compounds 1 and 2

multistep procedure requiring time-consuming protection/ deprotection steps.44a,b In addition, compound 9 was recently synthesized using Cr(NO3)3 by microwave irradiation.45 The regioselectivity observed in the oxidation of compounds 3−5 was similar to that observed in the oxidation of 2, the oxygen atom transfer always occurring at C-8. During the oxidation of 5, esculetin (11) was also isolated as a side-product due to the de-O-methylation activity of IBX.46 In this latter case, the delivery of the oxygen atom from the λ5-iodanyl cyclic intermediate occurred on the carbon atom bearing the methoxy substituent, followed by elimination of methanol.47 The oxidation was effective also in the case of the aminocoumarin 12 to afford the corresponding 7-amino-8-hydroxy-4-trifluoromethylcoumarin (13) (m/z 246.03; elemental analysis C, 48.97; H, 2.47; F, 23.23; N, 5.75; O, 19.57) in good yield (75%) (Table 1, entry 12). Attention was next focused on the synthesis of pyrogalloltype coumarins. A review48 of the antioxidant activity of pyrogallol derivatives showed that pyrogallol methyl ether derivatives possessed promising anticancer activity. First, the IBX-mediated de-O-methylation of fraxetin (10) (Scheme 4) was studied. The reaction performed under reported experimental conditions afforded 6,7,8-trihydroxycoumarin (14) in good yield (78%),49 as well as unreacted substrate (Scheme 4; Table 1, entry 13). A similar de-O-methylation process occurs during the oxidative metabolic degradation of fraxetin in rats by intestinal microflora enzymes.50 Next, we focused on the oxidation of esculetin (11). The 5,6,7-trihydroxycoumarin (15), previously isolated from an extract of the roots of Pelargonium sidoides,51 was obtained as the sole product (68%) (Scheme 3, Table 1, entry 14). The difference in the regioselectivity between compounds 2 and 11 might be explained by HO-6 being more nucleophilic than HO-

Scheme 3. Synthesis of Pyrogallol-Type Derivatives 14 and 15

7. The 6-hydroxy group is not involved in resonance with the carbonyl group and is more nucleophilic for attack on the hypervalent iodine to form the λ5-iodanyl intermediate. Pyrogallol derivative 15 showed all the expected NMR signals, with a general pattern different from that of 14 (Figure S1-F and G, Supporting Information). Finally, in view of possible large-scale applications, heterogeneous conditions were applied by using polymersupported IBX-amide, a readily recoverable and reusable oxidant.52 The oxidation of 1 with IBX supported on polystyrene (PSS-IBX) was analyzed as a representative example.53 PSS-IBX showed a low reactivity in DMSO, affording 6 in low yield (Table 1, entry 15). The swelling properties of polystyrene in DMSO are probably responsible for the observed low reactivity.54 Water shows limited swelling properties toward polystyrene, being the most recognized green solvent for environmentally friendly processes.55 IBX-mediated oxidations in H2O have been reported as an alternative to organic solvents.56 In accordance with these data, the oxidation of 1 in H2O with PSS-IBX afforded 6 in 56% yield (Table 1, entry 16). Under these experimental conditions, PSS-IBX 3249



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Scheme 4. Mechanism Proposed for the Synthesis of Coumarin 14

and the cell proliferation assay (MTT)60 showed that the compounds did not exert any toxic effect on the cells at any concentrations tested (Figure 1). Next, the cells were infected with influenza A/PR8/H1N1 virus, and, after viral challenge, the cells were treated with different concentrations (ranging from 30 to 120 μg/mL) of each compound. Twenty-four hours postinfection, viral particles released in the supernatant of infected cells were measured by means of hemagglutinating units (HAU). The catechol and pyrogallol derivatives 6, 9, and 13−15 and amino coumarin 12 inhibited viral replication compared to untreated infected cells. The cytotoxicity and HAU activity of the other compounds can be found as SI#2 and SI#3, Supporting Information. The inhibition was dose-dependent, and the effect was more than 50% at higher concentrations compared to untreated infected cells (Figure 2). A slight reduction of the viral replication (about 50%) was also observed by treating infected cells with compounds 1, 2, and 6 at higher concentrations. The required inhibitory concentration of coumarin derivatives able to reduce virus yield by 50% (IC50) is shown in Table 3. It is interesting to note that pyrogallol derivatives 14 and 15 were more active than catechol derivatives 6, 7, and 9. Moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 1 and 2 (Table 3). In conclusion, IBX was an efficient oxidant for the synthesis of catechol and pyrogallol derivatives in DMSO at 25 °C. The oxygen atom transfer from the λ5-iodanyl cyclic adduct occurred selectively on the benzylic-like ortho-position capable of better stabilizing the incipient carbocation or radical intermediate. The oxidation was also performed under heterogeneous conditions using IBX supported on polystyrene. In the latter case better results were obtained in H2O warming from room temperature (rt) to 60 °C. Catechol and pyrogallol derivatives showed increased antioxidant activity and an antiinfluenza A/PR8/H1N1 inhibition effect compared to monohydroxycoumarins. Both the antioxidant activity (DPPH test) and A/PR8/H1N1 inhibition (hemaggluttination assay) increased with an increase in the number of phenolic moieties, pyrogallol-type coumarins being the most active derivatives. These data suggest a plausible relationship between the inhibition of influenza A/PR8/H1N1 virus and the modulation of the internal cell redox state. The range of concentrations of coumarin derivatives that were effective against influenza in our model is in line with that reported by Wang et al.11b Although the available anti-influenza drugs (i.e., adamantanes and neuraminidase inhibitors) are stronger inhibitors, they target specific viral proteins that may undergo mutations, with consequent reduced sensitivity to the drug.61 Owing to their antioxidant properties, the coumarin

retained the reactivity for at least a further four runs (Table 1, entries 17−19). Antioxidant Activity. The in vitro antioxidant activity of catechol-type (6−11) and pyrogallol-type (14 and 15) coumarins was evaluated by analysis of the DPPH radical scavenging properties.57 The DPPH activity of 1 and 2 was evaluated as reference systems. The coumarin derivative was dissolved in EtOH (0.01−100 mg/mL) and added to freshly prepared DPPH solution (6 × 10−5 M in EtOH). The decrease in absorbance (475 nm) was determined at different times until the reaction reached a plateau. The kinetics of the process were analyzed for each concentration tested, and the concentration of DPPH remaining in the steady state was estimated. This value was used to calculate the IC50 (defined as the concentration of substrate (mg/mL) that causes 50% loss of DPPH activity). The results are reported in Table 2. Catechol Table 2. 2,2-Diphenylpicrylhydrazyl (DPPH) Radical Scavenging Properties of Coumarin Derivatives 1, 2, 6−11, and 13−15 entry

compound

IC50a

1 2 3 4 5 6 7 8 9 10 11

6-hydroxycoumarin 1 7-hydroxycoumarin 2 5,6-dihydroxycoumarin 6 7,8-dihydroxycoumarin 7 4,7,8-trihydroxycoumarin 8 7,8-dihydroxy-4-methylcoumarin 9 7,8-dihydroxy-5-methoxycoumarin 10 6,7-dihydroxycoumarin 11 7-amino-8-hydroxy-4-trifluoromethylcoumarin 13 6,7,8-trihydroxycoumarin 14 5,6,7-trihydroxycoumarin 15

>200 >200 19.6 27.2 24.1 22.0 39.2 37.7 9.3 8.7 2.5

a

IC50 is the drug concentration (mg/mL) causing 50% inhibition of the desired activity. Each experiment was conducted in triplicate.

derivatives 6−11 showed a DPPH activity higher than 1 and 2 (Table 2, entries 3−8 versus entries 1 and 2).58 The antioxidant activity in catechol derivatives was 6 > 9 ≥ 8 > 7 > 11 > 10, in accordance with data previously reported.59 The presence of the pyrogallol moiety in derivatives 14 and 15 further increased the DPPH activity (Table 2, entries 10 and 11). Cytotoxicity and Antiviral Activity. The cytotoxicity of coumarin derivatives on A549 cell monolayers was also evaluated. Briefly, cells were plated at a concentration of 2 × 105/mL and after 24 h were treated with various concentrations (range 10−120 μg/mL) of each compound and incubated for the next 24 h. Compounds 1−4 and 10−12 were used as references. Microscopic examination, Trypan blue exclusion, 3250



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Figure 1. Cell proliferation assay data for active compounds 1, 2, 6, 9, and 12−15 evaluated on A549 cell monolayers.

Figure 2. Anti-influenza A/PR8/H1N1 activity of effective coumarin derivatives 1, 2, 6, 9, and 12−15 measured by means of hemagglutinating units (HAU). d6, and D2O as solvents. Chemical shifts (δ) are expressed in parts per million (ppm). Coupling constants J are expressed in hertz (Hz). Spin multiplicities are given as s (singlet), b (broad), d (doublet), dd (doublet of doublets), and m (multiplet). MS data were obtained using an LC/MS Agilent 1100 LC-MSD VL system (G1946C) interfaced with an ESI source (spray voltage of 4.5 kV and nitrogen as sheath gas). Conditions: direct injection with a 0.4 mL/min flow rate using a binary solvent system of 95:5 MeOH/H2O. UV detection was monitored at 254 nm. Mass spectra were acquired in positive mode scanning over the mass range 105−1500 m/z, using a variable fragmentor voltage of 10−70 mV with a 0.4 mL/min flow rate. The following ion source parameters were used: drying gas (N2) flow, 9 mL/min; nebulizer pressure, 40 psig; drying gas temperature, 350 °C. The purity of the final products was 95% or higher, and it was assessed by HPLC-MS, using an Equivalence 3 C18 column (ACEEQV-8977: 150 × 4.6 mm, 5 μm particle size) at a flow rate of 0.6 mL/min with a linear gradient elution from 100/0 to 50/50 v/v CH3CN (formic acid 0.1% v/v)/H2O (formic acid 0.1% v/v) for 23 min. UV detection was monitored at 210 nm. Oxidation of Coumarins with IBX: General Procedures. A solution of the appropriate substrate (1.0 mmol), DMSO (1.0 mL), and IBX (1.0 equiv) was stirred at room temperature for 2 h. During the reaction, different chromatic changes were observed for every substrate (orange, purple, brown). The reaction was monitored by TLC. After the disappearance of the substrate, the reaction mixture was treated with H2O (1.0 mL) and an excess of Na2S2O4, and the solution was left stirring until it became yellow. EtOAc (10 mL) was added, and the two phases were separated. The aqueous phase was further extracted with EtOAc (2 × 10 mL). The combined organic layers were treated with a saturated solution of NaHCO3 (10 mL) and NaCl (10 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude was purified by

Table 3. Inhibitory Concentration That Reduces Virus Yield by 50% (IC50) of Coumarin Derivatives 1, 2, 6, 7, 9, and 12− 15a entry

compound

IC50 (μg/mL)

1 2 3 4 5 6 7 8 9

6-hydroxycoumarin 1 7-hydroxycoumarin 2 5,6-dihydroxycoumarin 6 7,8-dihydroxycoumarin 7 7,8-dihydroxy-4-methylcoumarin 9 7-amino-4-trifluoromethylcoumarin 12 7-amino, 8-hydroxy-4-trifluoromethylcoumarin 13 6,7,8-trihydroxycoumarin 14 5,6,7-trihydroxycoumarin 15

110 110 106.5 145.7 91.5 51.0 47.8 69.9 47.9

a

IC50 is the drug concentration (mg/mL) causing 50% inhibition of the desired activity. Each experiment was conducted in triplicate.

derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains

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EXPERIMENTAL SECTION

Chemistry. 6-Hydroxycoumarin (1), umbelliferone (2), 4,7dihydroxycoumarin (3), 4-methylumbelliferone (4), fraxetin (10), esculetin (11), and 7-amino-4-(trifluoromethyl)coumarin (12) were purchased from Sigma-Aldrich (Milan, Italy). Homogeneous IBX and IBX on polystyrene were commercially available from Sigma-Aldrich (Milan, Italy). 1H NMR and 13C NMR spectra were recorded on a Bruker (400 MHz) spectrometer using CDCl3, methanol-d4, DMSO3251



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flash chromatography (EtOAc/hexanes, 1:1) to afford the target product. 5,6-Dihydroxy-2H-chromen-2-one (6): Rf = 0.6 (EtOAc/hexanes, 1:1); orange oil (yield: 66%); 1H NMR (400 MHz, methanol-d4) δ 8.23 (d, J = 10 Hz, 1H, CH); 7.04 (d, J = 9 Hz, 1H, Ar); 6.69 (d, J = 9 Hz, 1H, Ar); 6.31 (d, J = 10 Hz, 1H, CH); 13C NMR (100 MHz, methanol-d4) δ 162.27, 147.58, 142.05, 140.80, 139.56, 118.63, 112.99, 109.03, 105.67; MS (ESI) m/z [M − H]− 177.02; anal for C9H6O4 calcd C, 60.68; H, 3.39; O, 35.93; found C, 60.64; H, 3.39; O, 35.95. 7,8-Dihydroxy-2H-chromen-2-one (7): Rf = 0.6 (EtOAc/hexanes, 1:1); orange oil (yield: 59%); 1H NMR (400 MHz, methanol-d4) δ 7.89 (d, J = 10 Hz, 1H, CH); 7.47 (d, J = 8 Hz, 1H, Ar), 6.80 (d, J = 8 Hz, 1H, Ar); 6.20 (d, J = 10 Hz, 1H, CH); 13C NMR (100 MHz, methanol-d4) δ 162.01, 149.69, 145.26, 132.60, 128.77, 118.75, 112.49, 110.76, 101.10; MS (ESI) m/z [M − H]− 177.01; anal for C9H6O4 calcd C, 60.68; H, 3.39; O, 35.93; found C, 60.671; H, 3.39; O, 35.97. 4,7,8-Trihydroxy-2H-chromen-2-one (8): Rf = 0.4 (EtOAc/ hexanes, 1:1); orange oil (yield: 49%); 1H NMR (400 MHz, methanol-d4) δ 6.68 (d, J = 8 Hz, 1H, Ar); 6.44 (d, J = 8 Hz, 1H, Ar); 6.36 (s, 1H, CH); 13C NMR (100 MHz, methanol-d4) δ 166.1, 162.4, 148.3, 144.8, 135.9, 122.8, 114.0, 111.4, 91.1; MS (ESI) m/z [M − H]− 193.01; anal for C9H6O5 calcd: C, 55.68; H, 3.12; O, 41.21; found C, 55.63; H, 3.12; O, 41.23. 7,8-Dihydroxy-4-methyl-2H-chromen-2-one (9): Rf = 0.2 (EtOAc/ hexanes, 1:1); brown oil (yield: 51%); 1H NMR (400 MHz, methanold4) δ 7.17 (d, J = 10 Hz, 1H, Ar); 6.86 (d, J = 10 Hz, 1H, Ar); 6.13 (s, 1H, CH); 2.44 (s, 3H, CH3); 13C NMR (100 MHz, methanol-d4) δ 162.17, 155.07, 149.40, 126.07, 115.34, 112.92, 111.10, 109.75, 102.05, 17.38; MS (ESI) m/z [M − H]− 191.03; anal for C10H8O4 calcd C, 62.50; H, 4.20; O, 33.30; found C, 62.53; H, 4.21; O, 33.28. 7-Amino-8-hydroxy-4-(trifluoromethyl)-2H-chromen-2-one (13): Rf = 0.6 (EtOAc/hexanes, 1:1); orange oil (yield: 34%); 1H NMR (400 MHz, methanol-d4) δ 7.36 (d, J = 8 Hz, 1 H, Ar); 6.66 (d, J = 8 Hz, 1H, Ar); 6.44 (s, 1H, CH); 13C NMR (100 MHz, methanol-d4) δ 159.86, 154.58, 141.0 (q CF3, J1 = 62.9 Hz, J2 = 31.6 Hz), 139.26, 135.88, 123.81, 118.33, 117.03, 112.70, 99.44; MS (ESI) m/z [M + H]+ 246.03; anal for C10H6F3NO3 calcd C, 48.99; H, 2.47; F, 23.25; N, 5.71; O, 19.58; found C, 48.97; H, 2.47; F, 23.23; N, 5.75; O, 19.57. 5,6,7-Trihydroxy-2H-chromen-2-one (14): Rf = 0.4 (EtOAc/ hexanes, 1:1); orange oil (yield: 45%); 1H NMR (400 MHz, DMSO-d6) δ 7.12 (d, J = 9.6 Hz, 1H, CH); 6.88 (s, 1H, Ar); 6.09 (d, J = 9.6 Hz, 1H, CH); 13C NMR (100 MHz, methanol-d4) δ 161.18, 150.63, 149.37, 148.81, 143.53, 112.21, 111.09, 110.91, 103.23; MS (ESI) m/z [M − H]− 193.01; anal for C9H6O5 calcd C, 55.68; H, 3.12; O, 41.21; found C, 55.66; H, 3.12; O, 41.23. 6,7,8-Trihydroxy-2H-chromen-2-one (15): Rf = 0.4 (EtOAc/ hexanes, 1:1); orange oil (yield: 68%).; 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 12, 1H, CH); 6.76 (d, J = 12 Hz, 1H, CH); 6.39 (s, 1H, Ar); 13C NMR (100 MHz, methanol-d4) δ 161.23, 145.81, 145.45, 139.79, 139.67 137.29, 112.32, 110.51, 100.74; MS (ESI) m/z [M − H]− 193.01; anal for C9H6O5 calcd C, 55.68; H, 3.12; O, 41.21; found C, 55.69; H, 3.11; O, 41.19. Oxidation of Coumarins with Heterogeneous IBX: General Procedure. A solution of coumarin 1 (1.0 mmol), the appropriate solvent (1.0 mL), and PSS-IBX (1 equiv) was stirred at 60 °C for 16 h. The polymer was recovered by simple filtration, H2O (1.0 mL) and an excess of Na2S2O4 were added, and the mixture was left stirring for 15 min. EtOAc (10 mL) was added, and the two phases were separated. The aqueous phase was further extracted with EtOAc (2 × 10 mL). The combined organic layers were treated with a saturated solution of NaHCO3 (10 mL) and NaCl (10 mL), dried over Na2SO4, and concentrated under reduced pressure to afford the target product. PSSIBX was regenerated by treating the filtered resin with a solution of tetrabutylammonium oxone and methanesulfonic acid according to the reported procedure.62 Biological Section. DPPH Radical Scavenging Capacity Assay. Samples were serially diluted to various concentrations in EtOH (0.01−100 mg/mL) and added to freshly prepared DPPH solution (6 × 10−5 M in EtOH). The mixture was shaken vigorously and left standing for 30 min in the dark. The absorbance was measured at 517

nm against a solvent blank. The capability to scavenge DPPH radicals was calculated according to the formula: Scavenging effect (%) = [1 (A1As)/Ao] × 100, where Ao is the absorbance of the control solution (0.5 mL of EtOH in 3.5 mL of DPPH solution), A1 is the absorbance in the presence of the appropriate compound in DPPH solution, and As (which is used for error correction arising from unequal color of the sample solutions) is the absorbance of the tested sample solution without DPPH. The scavenging capacity of coumarins against DPPH radicals was determined by IC50 value. IC50 value is the effective concentration at which DPPH radicals are scavenged by 50% and was obtained through interpolation from the nonlinear regression curve. The lower IC50 value indicates higher radical scavenging capacity and vice versa. Cell Cultures. A549 human carcinoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.3 mg/mL glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. All reagents were purchased from Invitrogen (Milan, Italy). Cytotoxicity Assays. The cytotoxicity of 6−11 and 13−15 was estimated on A549 cells by Trypan blue (0.02%) exclusion and by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction (MTT, Sigma-Aldrich, Milan, Italy) assay.60 The cytotoxicity of each compound was calculated as percentage reduction in viable cells with respect to the control culture (cells treated with DMSO alone). Virus Production, Infection, and Titration. Influenza virus A/ Puerto Rico/8/34 H1N1 (PR8 virus) was grown in the allantoic cavities of 10-day-old embryonated chicken eggs. After 48 h at 37 °C, the allantoic fluid was harvested and centrifuged at 5000 rpm for 30 min to remove cellular debris. Confluent monolayers of alveolar basal epithelial A549 cells were challenged for 1 h at 37 °C with PR8 at a multiplicity of infection of 0.0001, incubated for 1 h at 37 °C, washed with phosphate buffered saline (PBS), and incubated with medium supplemented with 2% FBS. Mock infection was performed with the same dilution of allantoic fluid from uninfected eggs. Virus production was determined in the supernatants of infected cells 24 and 48 h postinfection, by measuring the HAU, using human type 0 Rh+ erythrocytes. For the evaluation of the antiviral activity, stock solutions of compounds dissolved in DMSO were diluted in DMEM to final concentrations of 30, 40, 60, 80, and 120 μg/mL. Compounds were added after the adsorption period and maintained in the culture media until the end of the experiments. The highest DMSO concentration present in the culture medium was 0.2%. Control cells were treated with DMSO alone at the same concentration present in the test substance being evaluated.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00665. 1D NMR spectra of compounds 6−10 and 13−15; MTT cytotoxicity and influenza virus A/Puerto Rico/8/34 H1N1 data of compounds 3−5, 7, 8, 10, and 11 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(L. Nencioni) Tel: +39 06 4468622. Fax: +39 06 4468625. Email:[email protected]. *(R. Saladino) Tel: +39 0761 357284. Fax: +39 0761 357242. E-mail: [email protected]. ORCID

Raffaele Saladino: 0000-0002-4420-9063 Notes

The authors declare no competing financial interest. 3252



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ACKNOWLEDGMENTS This work was supported by FILAS project “MIGLIORA” of Latium Region (Italy) and Ateneo grants.

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Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with

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