Regioselective IBX-Mediated Synthesis of Coumarin Derivatives with Antioxidant and Anti-influenza Activities

Article abstract of DOI:10.1021/acs.jnatprod.7b00665

Different catechol and pyrogallol derivatives have been synthesized by oxidation of coumarins with 2-iodoxybenzoic 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.

Full text of DOI:10.1021/acs.jnatprod.7b00665

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
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 2-
iodoxybenzoic 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.
2
3
oumarins are a large class of secondary metabolites found
production of reactive oxygen species (ROS). Innovative
1
C
in plants, bacteria, and fungi and are characterized by a
strategies to design anti-influenza drugs are based on the
2
24,25
2
H-1-benzopyran-2-one core structure. They show several
inhibition of these intracellular pathways.
3
pharmacological properties such as anticoagulant, antineur-
Antiviral compounds so far developed mainly act by
inhibition of the viral coating step involving the glycoprotein
neuraminidase (NA). However, the efficacy of these antiviral
compounds is often limited by toxicity and by the inevitable
selection of drug-resistant viral mutants.
Over the last years, we reported that catechol derivatives are
active against DNA and RNA viruses. In particular,
tocopheryl hydroquinone derivatives were active against
4
5
6
odegenerative, anticancer, and antimicrobial activities.
26
Coumarins are also effective against virus replication, including
the inhibition of the human immunodeficiency virus type 1
7
8
27
(
(
HIV-1), hepatitis C virus (HCV), bovine viral diarrhea virus
9 10
11a−c
BVDV), herpes simplex virus, and influenza A virus.
In
2
8
association with these biological effects, coumarins bearing the
catechol pharmacophore, such as esculetin and 4-methylescu-
1
2
13
letin, are potent antioxidants in lipid peroxidation, H O -
2
2
influenza A virus by regulation of the internal redox potential
14,15
induced oxidative cell damage,
depletion. This antioxidant activity is associated with single-
electron (SET) or hydrogen-atom transfer (HAT) to reactive
and glutathione (GSH)
29,30
of the cell.
This approach could offer important advantages,
1
6
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 2-
iodoxybenzoic acid (IBX) for the regioselective synthesis of
new derivatives bearing catechol and pyrogallol moieties. IBX
1
7
18,19
radicals, as well as to metal pro-oxidant species.
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
31
affects the ortho-hydroxylation of phenol to catechol with a
3
2
reactivity similar to that of polyphenol oxidases. The high
regioselectivity is a consequence of the intramolecular oxygen
transfer from iodine(V) in a λ5-iodanyl intermediate to the
33
2
0
ortho-position of the phenolic group. The antioxidant activity
virions.
Influenza A virus modulates the intracellular redox of the
21
host cell by different mechanisms, including an increase in
Received: August 1, 2017
2
2
oxidative stress, depletion of intracellular GSH, and
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00665
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
of the new derivatives was evaluated by the 2,2′-diphenylpi-
crylhydrazyl (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
a
IBX
entry substrate
product
solvent
time
2 h
T (°C) yield (%)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
6
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
rt
66
48
1
6
2 h
24 h
7 days
2 h
2 h
24 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
2 h
60 °C
rt
1
6
65
RESULTS AND DISCUSSION
■
1
6
rt
43
IBX-Mediated Oxidation of Coumarins. IBX is usually
used in different organic solvents depending on the nature and
b
b
1
6
rt
61
2
7
rt
59
34
reactivity of the system. 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
2
7
rt
44
2
7
rt
50
3
8
rt
58
3
5
substrate. Moreover, DMSO shows a dielectric constant high
0
1
2
3
4
5
6
7
8
9
0
4
9
rt
67
36
enough to ensure the solubility of coumarins. Initially, 6-
c
5
10 (11)
rt
54(21)
75
hydroxycoumarin (1) and 7-hydroxycoumarin (umbelliferone)
12
10
11
1
13
14
15
6
rt
(
2) were used as substrates. The appropriate coumarin (1.0
rt
78
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
rt
68
d
d
d
d
d
d
60 °C
60 °C
60 °C
60 °C
60 °C
60 °C
20
1
6
H O
2
56
(
6) and 7,8-dihydroxycoumarin (daphnetin) (7), respectively,
1
6
H O
2
53
in variable yields, as well as unreacted substrate (Scheme 1).
1
6
H O
2
54
1
6
H O
2
51
Scheme 1. Oxidation of Coumarins to Catechol-Type
Derivatives
1
6
H O
2
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
(
b
room temperature. Reaction performed with an excess of IBX (2.0
equiv). Yield of the 6,7-dihydroxycoumarin (11). Oxidation
c
d
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
6
.69 ppm (H-8, J8,7 = 9.0 Hz, 1H), instead of the two singlets of
,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
37
with reported data. 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
38
incipient carbocation or radical intermediate. 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
39a−d
flavanones with IBX.
In the latter case the oxidation
takes place invariably on the benzylic-type position next to the
4
0
fused ring which is more stabilized as an incipient carbocation
41
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
or radical 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 P-
42
450 enzymes. Compound 7 has been previously isolated from
43
1
, entries 2−5, 7, and 8). Moreover, the oxidation of
the stem and root bark of Daphne giraldii Nitsche.
compounds 1 and 2 with IBX in solvents such as CH Cl /
The oxidation was then applied to a large panel of
compounds, namely, 4,7-dihydroxycoumarin (4-hydroxyumbel-
liferone) (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
2
2
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
B
DOI: 10.1021/acs.jnatprod.7b00665
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Scheme 2. Regioselectivity Observed in the Oxidation of Compounds 1 and 2
multistep procedure requiring time-consuming protection/
Scheme 3. Synthesis of Pyrogallol-Type Derivatives 14 and
15
4
4a,b
deprotection steps.
In addition, compound 9 was recently
45
synthesized using Cr(NO ) by microwave irradiation.
3
3
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
46
due to the de-O-methylation activity of IBX. In this latter
case, the delivery of the oxygen atom from the λ5-iodanyl cyclic
intermediate occurred on the carbon atom bearing the methoxy
4
7
substituent, followed by elimination of methanol. The
oxidation was effective also in the case of the aminocoumarin
1
2 to afford the corresponding 7-amino-8-hydroxy-4-trifluor-
omethylcoumarin (13) (m/z 246.03; elemental analysis C,
8.97; H, 2.47; F, 23.23; N, 5.75; O, 19.57) in good yield
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 polymer-
supported IBX-amide, a readily recoverable and reusable
oxidant. The oxidation of 1 with IBX supported on
polystyrene (PSS-IBX) was analyzed as a representative
example. 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. Water shows limited swelling
properties toward polystyrene, being the most recognized green
solvent for environmentally friendly processes. IBX-mediated
oxidations in H O have been reported as an alternative to
organic solvents. In accordance with these data, the oxidation
of 1 in H O with PSS-IBX afforded 6 in 56% yield (Table 1,
entry 16). Under these experimental conditions, PSS-IBX
4
(75%) (Table 1, entry 12).
Attention was next focused on the synthesis of pyrogallol-
48
type coumarins. A review 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
5
2
49
53
(14) in good yield (78%), 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
54
Next, we focused on the oxidation of esculetin (11). The
55
5,6,7-trihydroxycoumarin (15), previously isolated from an
51
extract of the roots of Pelargonium sidoides, 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-
2
56
2
C
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Journal of Natural Products
Article
Scheme 4. Mechanism Proposed for the Synthesis of Coumarin 14
6
0
retained the reactivity for at least a further four runs (Table 1,
entries 17−19).
and the cell proliferation assay (MTT) 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.
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
57
scavenging properties. 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
−5
prepared DPPH solution (6 × 10 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
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% (IC ) is shown in Table 3.
Table 2. 2,2-Diphenylpicrylhydrazyl (DPPH) Radical
Scavenging Properties of Coumarin Derivatives 1, 2, 6−11,
and 13−15
50
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.
a
entry
compound
IC50
1
2
3
4
5
6
7
8
9
6-hydroxycoumarin 1
>200
>200
19.6
27.2
24.1
22.0
39.2
37.7
9.3
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
1
0
1
8.7
In the latter case better results were obtained in H O warming
2
1
2.5
from room temperature (rt) to 60 °C. Catechol and pyrogallol
derivatives showed increased antioxidant activity and an anti-
influenza A/PR8/H1N1 inhibition effect compared to mono-
hydroxycoumarins. 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.
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
58
(
Table 2, entries 3−8 versus entries 1 and 2). The antioxidant
activity in catechol derivatives was 6 > 9 ≥ 8 > 7 > 11 > 10, in
59
accordance with data previously reported. 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
The range of concentrations of coumarin derivatives that
were effective against influenza in our model is in line with that
11b
evaluated. Briefly, cells were plated at a concentration of 2 ×
reported by Wang et al. 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
5
1
(
0 /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
61
references. Microscopic examination, Trypan blue exclusion,
drug. Owing to their antioxidant properties, the coumarin
D
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Article
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).
(
d , and D O 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
Table 3. Inhibitory Concentration That Reduces Virus Yield
6
2
by 50% (IC ) of Coumarin Derivatives 1, 2, 6, 7, 9, and 12−
50
a
1
5
(doublet of doublets), and m (multiplet). MS data were obtained using
entry
compound
IC50 (μg/mL)
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
1
2
3
4
5
6
7
8
9
6-hydroxycoumarin 1
110
7-hydroxycoumarin 2
110
binary solvent system of 95:5 MeOH/H O. UV detection was
2
5,6-dihydroxycoumarin 6
106.5
145.7
91.5
51.0
47.8
69.9
47.9
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
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
following ion source parameters were used: drying gas (N ) flow, 9
2
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
a
IC50 is the drug concentration (mg/mL) causing 50% inhibition of
the desired activity. Each experiment was conducted in triplicate.
linear gradient elution from 100/0 to 50/50 v/v CH CN (formic acid
3
0
.1% v/v)/H O (formic acid 0.1% v/v) for 23 min. UV detection was
2
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
derivatives could affect intracellular redox-sensitive pathways
useful for viral replication, independently from the variability of
the strains
EXPERIMENTAL SECTION
■
Chemistry. 6-Hydroxycoumarin (1), umbelliferone (2), 4,7-
dihydroxycoumarin (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
was treated with H O (1.0 mL) and an excess of Na S O , and the
2
2
2
4
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 NaHCO (10 mL) and
3
1
13
(
Milan, Italy). H NMR and C NMR spectra were recorded on a
NaCl (10 mL). The combined organic phases were dried over Na SO
2
4
Bruker (400 MHz) spectrometer using CDCl , methanol-d , DMSO-
and concentrated under reduced pressure. The crude was purified by
3
4
E
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flash chromatography (EtOAc/hexanes, 1:1) to afford the target
product.
nm against a solvent blank. The capability to scavenge DPPH radicals
was calculated according to the formula: Scavenging effect (%) = [1
(A A )/A ] × 100, where A is the absorbance of the control solution
5
,6-Dihydroxy-2H-chromen-2-one (6): R = 0.6 (EtOAc/hexanes,
f
1
s
o
o
1
1
8
9
:1); orange oil (yield: 66%); H NMR (400 MHz, methanol-d ) δ
(0.5 mL of EtOH in 3.5 mL of DPPH solution), A is the absorbance
4
1
.23 (d, J = 10 Hz, 1H, CH); 7.04 (d, J = 9 Hz, 1H, Ar); 6.69 (d, J =
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).
13
Hz, 1H, Ar); 6.31 (d, J = 10 Hz, 1H, CH); C NMR (100 MHz,
methanol-d ) δ 162.27, 147.58, 142.05, 140.80, 139.56, 118.63, 112.99,
4
−
1
09.03, 105.67; MS (ESI) m/z [M − H] 177.02; anal for C H O
calcd C, 60.68; H, 3.39; O, 35.93; found C, 60.64; H, 3.39; O, 35.95.
9
6
4
7
,8-Dihydroxy-2H-chromen-2-one (7): R = 0.6 (EtOAc/hexanes,
f
1
1
7
8
:1); orange oil (yield: 59%); H NMR (400 MHz, methanol-d ) δ
4
.89 (d, J = 10 Hz, 1H, CH); 7.47 (d, J = 8 Hz, 1H, Ar), 6.80 (d, J =
13
Hz, 1H, Ar); 6.20 (d, J = 10 Hz, 1H, CH); C NMR (100 MHz,
methanol-d ) δ 162.01, 149.69, 145.26, 132.60, 128.77, 118.75, 112.49,
4
−
1
10.76, 101.10; MS (ESI) m/z [M − H] 177.01; anal for C H O
9
6
4
calcd C, 60.68; H, 3.39; O, 35.93; found C, 60.671; H, 3.39; O, 35.97.
,7,8-Trihydroxy-2H-chromen-2-one (8): Rf = 0.4 (EtOAc/
4
1
hexanes, 1:1); orange oil (yield: 49%); H NMR (400 MHz,
methanol-d ) δ 6.68 (d, J = 8 Hz, 1H, Ar); 6.44 (d, J = 8 Hz, 1H,
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. The cytotoxicity
of each compound was calculated as percentage reduction in viable
cells with respect to the control culture (cells treated with DMSO
alone).
4
13
Ar); 6.36 (s, 1H, CH); C NMR (100 MHz, methanol-d ) δ 166.1,
4
1
62.4, 148.3, 144.8, 135.9, 122.8, 114.0, 111.4, 91.1; MS (ESI) m/z [M
6
0
−
−
H] 193.01; anal for C H O calcd: C, 55.68; H, 3.12; O, 41.21;
9
6
5
found C, 55.63; H, 3.12; O, 41.23.
,8-Dihydroxy-4-methyl-2H-chromen-2-one (9): R = 0.2 (EtOAc/
7
f
1
hexanes, 1:1); brown oil (yield: 51%); H NMR (400 MHz, methanol-
d ) δ 7.17 (d, J = 10 Hz, 1H, Ar); 6.86 (d, J = 10 Hz, 1H, Ar); 6.13 (s,
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.
4
13
1
1
1
6
H, CH); 2.44 (s, 3H, CH ); C NMR (100 MHz, methanol-d ) δ
3
4
62.17, 155.07, 149.40, 126.07, 115.34, 112.92, 111.10, 109.75, 102.05,
−
7.38; MS (ESI) m/z [M − H] 191.03; anal for C H O calcd C,
10
8
4
2.50; 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):
1
R = 0.6 (EtOAc/hexanes, 1:1); orange oil (yield: 34%); H NMR
f
(
400 MHz, methanol-d ) δ 7.36 (d, J = 8 Hz, 1 H, Ar); 6.66 (d, J = 8
4
13
Hz, 1H, Ar); 6.44 (s, 1H, CH); C NMR (100 MHz, methanol-d4)
δ 159.86, 154.58, 141.0 (q CF , J = 62.9 Hz, J = 31.6 Hz), 139.26,
3
1
2
1
35.88, 123.81, 118.33, 117.03, 112.70, 99.44; MS (ESI) m/z [M +
+
H] 246.03; anal for C H F NO calcd C, 48.99; H, 2.47; F, 23.25; N,
10
6
3
3
5
.71; O, 19.58; found C, 48.97; H, 2.47; F, 23.23; N, 5.75; O, 19.57.
,6,7-Trihydroxy-2H-chromen-2-one (14): Rf = 0.4 (EtOAc/
5
1
hexanes, 1:1); orange oil (yield: 45%); H NMR (400 MHz,
DMSO-d ) δ 7.12 (d, J = 9.6 Hz, 1H, CH); 6.88 (s, 1H, Ar);
6
13
6
1
.09 (d, J = 9.6 Hz, 1H, CH); C NMR (100 MHz, methanol-d ) δ
4
61.18, 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 C H O calcd C, 55.68; H,
.12; O, 41.21; found C, 55.66; H, 3.12; O, 41.23.
,7,8-Trihydroxy-2H-chromen-2-one (15): R = 0.4 (EtOAc/
9
6
5
3
6
f
1
hexanes, 1:1); orange oil (yield: 68%).; H NMR (400 MHz,
ASSOCIATED CONTENT
■
DMSO-d ) δ 7.92 (d, J = 12, 1H, CH); 6.76 (d, J = 12 Hz, 1H,
6
13
*
S
Supporting Information
CH); 6.39 (s, 1H, Ar); C NMR (100 MHz, methanol-d ) δ
4
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00665.
1
61.23, 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 C H O calcd C, 55.68; H,
.12; O, 41.21; found C, 55.69; H, 3.11; O, 41.19.
9
6
5
3
Oxidation of Coumarins with Heterogeneous IBX: General
1
D NMR spectra of compounds 6−10 and 13−15; MTT
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.
cytotoxicity and influenza virus A/Puerto Rico/8/34
H1N1 data of compounds 3−5, 7, 8, 10, and 11 (PDF)
The polymer was recovered by simple filtration, H O (1.0 mL) and an
2
excess of Na S O were added, and the mixture was left stirring for 15
2
2
4
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 Na SO , and
AUTHOR INFORMATION
■
Corresponding Authors
(L. Nencioni) Tel: +39 06 4468622. Fax: +39 06 4468625. E-
mail:lucia.nencioni@uniroma1.it.
(R. Saladino) Tel: +39 0761 357284. Fax: +39 0761 357242.
2
4
*
concentrated under reduced pressure to afford the target product. PSS-
IBX was regenerated by treating the filtered resin with a solution of
tetrabutylammonium oxone and methanesulfonic acid according to the
*
62
E-mail: saladino@unitus.it.
reported procedure.
Biological Section. DPPH Radical Scavenging Capacity Assay.
Samples were serially diluted to various concentrations in EtOH
ORCID
Raffaele Saladino: 0000-0002-4420-9063
Notes
The authors declare no competing financial interest.
(
×
0.01−100 mg/mL) and added to freshly prepared DPPH solution (6
−5
10 M in EtOH). The mixture was shaken vigorously and left
standing for 30 min in the dark. The absorbance was measured at 517
F
DOI: 10.1021/acs.jnatprod.7b00665
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
23) Amatore, D.; Sgarbanti, R.; Aquilano, K.; Baldelli, S.; Limongi,
ACKNOWLEDGMENTS
■
D.; Civitelli, L.; Nencioni, L.; Garaci, E.; Ciriolo, M. R.; Palamara, A. T.
Cell. Microbiol. 2015, 17, 131−145.
(
This work was supported by FILAS project “MIGLIORA” of
Latium Region (Italy) and Ateneo grants.
24) (a) Palamara, A. T.; Nencioni, L.; Aquilano, K.; De Chiara, G.;
Hernandez, L.; Cozzolino, F.; Ciriolo, M. R.; Garaci, E. J. Infect. Dis.
2
005, 191, 1719−1729. (b) Matarrese, P.; Nencioni, L.; Checconi, P.;
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