Synthesis, biological evaluation, and NMR studies of 3-fluorinated derivatives of 3′,4′,5′-trihydroxyflavone and 3′,4′,5′-trimethoxyflavone

Article abstract of DOI:10.1016/j.bmcl.2020.127720

Flavones are valuable scaffolds in medicinal chemistry, especially as they display activity as antioxidants and neuroprotective agents. The need to incorporate a fluorine atom on flavones has driven much of the recent synthetic work in this area. We now r

Full text of DOI:10.1016/j.bmcl.2020.127720

Journal Pre-proofs
Synthesis, biological evaluation, and NMR studies of 3-fluorinated derivatives
of 3',4',5'-trihydroxyflavone and 3',4',5'-trimethoxyflavone
Maali D. Alshammari, Pavel V. Kucheryavy, Nicole M. Ashpole, David A.
Colby
PII:
S0960-894X(20)30831-3
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127720
BMCL 127720
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
15 September 2020
18 November 2020
23 November 2020
Please cite this article as: Alshammari, M.D., Kucheryavy, P.V., Ashpole, N.M., Colby, D.A., Synthesis,
biological evaluation, and NMR studies of 3-fluorinated derivatives of 3',4',5'-trihydroxyflavone and 3',4',5'-
trimethoxyflavone, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127720
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Graphical Abstract.
Synthesis, biological evaluation, and NMR
studies of 3-fluorinated derivatives of 3',4',5'-
trihydroxyflavone and 3',4',5'-
Leave this area blank for abstract info.
trimethoxyflavone
Maali D. Alshammari, Pavel V. Kucheryavy, Nicole M. Ashpole, and David A. Colby*
O
O
1
O
O
8
F
OCH3
OCH3
OH
OH
OCH3
OH
EC50 = 71 g/mL (DPPH)
(–) neuroprotective effects
EC50 = 0.24 g/mL (DPPH)
(+) neuroprotective effects

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis, biological evaluation, and NMR studies of 3-fluorinated derivatives of
3',4',5'-trihydroxyflavone and 3',4',5'-trimethoxyflavone
Maali D. Alshammari, Pavel V. Kucheryavy, Nicole M. Ashpole, and David A. Colby*
Department of BioMolecular Sciences, University of Mississippi, University, Mississippi 38677, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Flavones are valuable scaffolds in medicinal chemistry, especially as they display activity as
antioxidants and neuroprotective agents. The need to incorporate a fluorine atom on flavones has
driven much of the recent synthetic work in this area. We now report a route for the production of
3-fluorinated derivatives of 3',4,'5-trihydroxyflavone and 3',4',5'-trimethoxyflavone. Biological
evaluation of these agents, along with their non-fluorinated counterparts, demonstrate that
antioxidant activity may be enhanced whereas neuroprotective activity is conserved. Also, the 3-
fluoro-3',4,'5-trihydroxyflavone can act as an NMR probe to detect structural changes during its
action as a radical scavenger.
Keywords:
flavone
fluorine
neuroprotection
antioxidant
NMR
2009 Elsevier Ltd. All rights reserved.
Introducing a fluorine atom into a biologically active molecule
is a valuable approach in the process of drug discovery. This atom
has a high electronegativity, a small size, and forms a strong bond
with carbon, and these effects can improve the pharmacological
and pharmacokinetic qualities of a lead structure.1,2 Flavones are
secondary metabolites found in many fruits and vegetables, and
these molecules display many biological activities, including
action as antioxidants, which makes them a valuable scaffold in
medicinal chemistry.3 Synthetic methods for the construction of
fluorinated flavones have appeared from Fuchigami,4 Dolbier,5
Rozen,6 and Zhang,7 and all of these reports have focused on the
incorporation of a fluorine atom at the 3-position of flavone.8 This
position is the most difficult to access on the flavone skeleton,
because all of the other sites can be modified by standard
halogenation methods for substituted benzenes.
with a 4'-fluorine displays a reduced rate of scavenging of reactive
oxygen species of 55% compared to the natural product of 99%.11
These data provided the basis for this study to investigate the
effects of fluorination on the anti-oxidant and neuroprotective
activities of flavones.
3-F = similar inhibition of
telomerase activity
O
C
5
5-F, 6-F, or 7-F =
enhances anti-
proliferative activity
3
6
7
A
3’
4’
O
1
B
4’-F = reduces anti-
oxidant activity
5’
Figure 1. Structure–activity relationships of the fluorination of flavones.
Our
initial
non-fluorinated
targets
were
3',4',5'-
trimethoxyflavone (1) and 3',4',5'-trihydroxyflavone (2), because
each acts as an antioxidant and serves as free radical scavenger in
a DPPH assay.12,13 Flavone 1 was synthesized according to the
route in the literature as shown in Figure 2.12 Briefly, 2-
hydroxyacetophenone 3 and trimethoxybenzoyl chloride 4 were
coupled in pyridine to give the ester 5 in 73% yield. Treatment of
5 with KOH in pyridine at 65 °C produced the 1,3-diketone 6
through a Baker-Venkataraman rearrangement. Cyclization of 6
by refluxing acetic acid and hydrochloric acid gave the 3',4',5'-
trimethoxyflavone 1 in 71% yield.12 In order to improve this
transformation, we found that iron(III) chloride in CH2Cl2 at rt
promotes the cyclization of 6 to 1 in a higher yield of 89%.14
Finally, complete demethylation of 1 was achieved using
Flavones display a three-ring structure of 2-phenyl-4H-
chromen-2-one and are labelled with A-, C-, and B-rings. Limited
structure–activity data for fluorination of flavones have appeared
in the literature and are summarized in Figure 1.9–11 The placement
of fluorine at the 3-position of 3',4',7,8-tetrahydroxyflavone
provides similar inhibition of telomerase activity compared to the
non-fluorinated counterpart.9 Sale and coworkers created
fluorinated derivatives at the 5-, 6-, and 7-positions of the
flavonols, robinetin, myricetin, quercetin, kaempferol, and fisetin,
and these modifications typically enhanced cytotoxicity against
human prostate carcinoma (22Rv1) cells compared to the parent
natural products.10 Flavonols are subclass of flavones that have a
3-hydroxy group. A synthetic derivative of the flavone, chrysin,

hydrobromic acid at 100 °C and gave the 3',4',5'-trihydroxyflavone
Figure 3. Synthesis of 3-fluoro-3',4',5'-trimethoxyflavone (7) and 3-fluoro-
3',4',5'-trihydroxyflavone (8).
2 in quantitative conversion.15
O
O
O
O
The DPPH assay is routinely used to characterize potential
antioxidant activity, and the flavones 1, 2, 7, and 8 were tested in
this assay to determine effects of radical scavenging (Table 1).18
Vitamin C (L-ascorbic acid) was used as the positive control, and
a 1:1 solution of ethanol-acetone provided the optimal solubility
for the flavones in assay. Serial dilutions of each of the test
substrate were treated with DPPH (a stable free radical) across 20
minutes and then absorbance was measured. Six replicates were
performed at each concentration, and EC50 values were calculated
with the standard error. Flavones 1 and 2 displayed EC50 values
(g/mL) of 71 and 0.33, respectively, which are similar to reported
values.12,13 The fluoroflavones 7 and 8 displayed improved activity
as radical scavengers with EC50 values of 37 and 0.24,
respectively. The results support that fluorination at the 3-position
of the flavone scaffold may enhance antioxidant activity. These
structure–activity data at the 3-position correlate well with the
similar increases in potency in the telomerase assay observed by
Menichincheri and coworkers.9 The trihydroxyflavone 2 and the
3-fluorinated trihydroxyflavone 8 display the most promising
activity as antioxidants.
OCH3
pyridine
rt, 73%
Cl
+
OH
OCH3
OCH3
OCH3
OCH3
3
4
5
O
O
OH
OCH3
AcOH, HCl,
100 °C, 71%
KOH,
pyridine
OCH3
OCH3
65 °C, 81%
(or FeCl3, 89%)
O
OH
OCH3
6
O
HBr,
100 °C
OCH3
OCH3
OH
O
O
quant.
OH
OH
1
2
OCH3
Figure 2. Synthesis of 3',4',5'-trimethoxyflavone (1) and 3',4',5'-
trihydroxyflavone (2).
The 3-fluoro-3',4',5'-trimethoxyflavone (7) is the 3-fluorinated
counterpart of 1, and 7 has been synthesized10 but has not been
tested for antioxidant activity. The 3-fluoro-3',4',5'-
trihydroxyflavone (8) is not known in the literature, to our
knowledge, and is the 3-fluorinated analogue of 2. Initially,
fluoroflavone 7 was synthesized according to the route in the
literature as shown in Figure 3.10 The fluorination of the 1,3-
diketone 6 with NFSI in a solvent mixture of CH2Cl2 and CH3CN
is known to produce the intermediate 9 in a low yield (i.e., 37%)
after seven days. After additional investigations, we discovered
that using pyridine as solvent and heating the mixture to 50 °C not
only increased the yield to 72% but also decreased the reaction
time from seven days to 12 h. The compound 9 is then cyclized in
refluxing acetic acid with sulfuric acid or iron(III) chloride at rt to
give the 3-fluoro-3',4',5'-trimethoxyflavone 7.16 During the course
of our synthetic studies, we discovered that treating 6 with two
equivalents of NFSI at rt for 12 h followed by heating the mixture
to 80 °C for 12 h resulted in formation of 7 in 61% isolated yield.
This one-pot transformation is more efficient than the two-step
conversion of 6 to 9 and then 9 to 7. The fluoroflavone 7 is
demethylated using hydrobromic acid at 100 °C to produce in
quantitative yield the 3-fluoro-3',4',5'-trihydroxyflavone 8.17 The
synthetic routes allowed the production of sufficient amounts of
compounds for biological evaluation as antioxidants and
neuroprotective agents.
Table 1. DPPH assay data for flavones 1, 2, 7 and 8.18
compd
DPPH EC50 (g/mL)a
vitamin C
0.16 ± 0.03
71.66 ± 0.04
0.33 ± 0.01
37.14 ± 0.02
0.24 ± 0.04
1
2
7
8
a Values are given with the standard error.
Flavones are currently investigated as source of potential
neuroprotective agents, because new lead structures are needed for
many neurodegenerative disorders.19 The flavones 1, 2, 7, and 8
were tested in oxidative glutamate toxicity assays with rat cortical
neurons (Figure 4).20,21 The neurons were treated with glutamate
in the presence of a test substrate and compared to the controls,
DMSO and MK801 (GluN receptor antagonist). Glutamate
provides an excitotoxic insult to the neurons, and the cells are
incubated for 24 h before the addition of the MTS reagent used to
evaluate cell viability. The production of formazan dye by viable
neurons is measured, and the data were compared the controls to
determine statistical significance using a one-way ANOVA with
post hoc Dunnett's test (Figure 4A). The trihydroxyflavone 2 and
the 3-fluorinated trihydroxyflavone 8 display neuroprotective
effects similar to MK801, and these data also correspond to the
antioxidant activity in the DPPH assay. As glutamate excitation
induces calcium imbalances and reactive oxygen stress that leads
to neuronal death, the ability of these compounds to reduce
reactive-oxygen species (ROS) generation in the presence of
glutamate stimulation was assessed (Figure 4B). Test substrate
(i.e., 1, 2, 7, or 8) was compared to the vehicle-control treated
(DMSO) and glutamate-stimulated neurons. Only the 3-
fluorinated trihydroxyflavone 8 reduces levels of ROS below
baseline in the rat cortical neurons. These data suggest a potential
beneficial effect for 3-fluorination when compared to the non-
fluorinated counterpart 1 which only displays activity in the MTS
assay.
NFSI (1 equiv.),
CH2Cl2/CH3CN
OH
O
OH
rt, 37% (ref 10)
OCH3
OCH3
6
F
NFSI (1 equiv.), pyridine
50 °C, 72%
OCH3
9
NFSI (2 equiv.), pyridine
rt then 80 °C, 61%
(one pot, two steps)
O
F
FeCl3
HBr, 100 °C
OCH3
OCH3
quant.
O
rt, 84%
7
O
OCH3
F
OH
O
OH
8
OH

converts to an ortho-quinone during the process.22 Sawai and
coworkers demonstrated that 13C NMR can observe the formation
of the two carbonyl groups of this ortho-quinone intermediate
upon the addition of DPPH to individual polyphenols present in
tea leaves (Camellia sinesis).23, 24 Another report more recently
recorded the formation of the ortho-quinone from phenolic acids
treated with DPPH using 1H NMR.25 We investigated if an ortho-
quinone intermediate could be observed from the fluoroflavone 8
following its quenching of free radicals. Accordingly, two
equivalents of DPPH were added to a solution of fluoroflavone 8
in DMSO–d6 and 13C NMR data were acquired after 20 min
(Figure 5). The appearance of three new carbonyl signals at 197,
194, and 193 ppm suggest that ring B of the flavone has changed
in the process. The data from a DEPTQ135 analysis confirmed the
quaternary nature of these carbon atoms.26 The carbonyl signal
assigned to the 4-position of the flavone 8 (i.e., 169 ppm) also
decreased in the 13C NMR, and it is important to note that none of
the other peaks assigned to the parent flavone 8 disappeared
following the addition of two equivalents of DPPH. In order to
assign the new carbonyl peaks to the product, data was acquired
by 1D selective HSQMBC NMR and verified that the carbonyl
signals at 194 and 193 were on the same structure, which we
assigned to the ortho-quinone.26 Additionaly, the experiment was
analyzed by mass spectrometry (i.e., ESI–TOF), and three peaks
were observed in the negative ion mode at 285.020, 287.028, and
287.036.26 The major peak at 287.036 is assigned to the starting
material. The minor peak at 285.020 corresponds to the ortho-
quinone, whereas the minor peak at 287.028 likely arises from a
partially oxidized by-product. These data suggest that the ortho-
quinone can form from 8, but there is not a complete conversion
of the catechol into the quinone.
A)
1.0
0.8
0.6
0.4
0.2
0.0
*
*
*
1
d
1
2
7
8
O
M
0
e
t
S
8
a
l
K
M
u
D
m
i
t
s
n
u
+ glutamate stimulation
B)
4000
3000
2000
1000
*
d
1
2
7
8
O
e
t
S
a
l
M
u
D
m
i
t
s
n
u
+ glutamate stimulation
Figure 4. Results from neuroprotective assays for flavones 1, 2, 7, and 8 with
rat cortical neurons followed by stimulation with the excitotoxin, glutamate.
A) Viability was determined after 24 h with the reagent, MTS. MK801 and
DMSO are controls. B) A reactive-oxygen species (ROS) dye was added
immediately after glutamate stimulation. DMSO is the control.
The scavenging of radical species by flavones is commonly
attributed to the presence of hydroxyl groups on the ring B, which
0.015 mmol compd 8
0.030 mmol DPPH
DMSO–d6
O
F
OH
DPPH
O
8
(
)
O
O
C=O C=O
3’
0.015 mmol compd 8
DMSO–d6
4
O
5
10
3
F
7
8
6
7
2’
2
2’
3’
9
OH
6
O
9
1’
8
4’
4’
5
OH
3
1’
10
OH
4
2
8
210
200
190
180
170
160
150
140
130
120
110
f1 (ppm)
Figure 5. 13C NMR spectra of 3-fluoro-3’,3’,4’-trihydroxyflavone 8 in DMSO–d6 after 20 minutes in the absence and presence of the DPPH. Data was collected
on a Bruker Avance III HD spectrometer at 125 MHz at rt. Peaks corresponding to compd 8 are numbered whereas peaks for DPPH are labelled with a triangle.
These synthetic and mechanistic studies demonstrate the utility
of fluorination of the 3-position on the flavone backbone. Anti-
oxidant activity may be enhanced from this structural change
whereas as neuroprotective activity is conserved. An improved
synthetic route for the installation of the 3-fluorine was developed
to ensure production of sufficient quantities for biological

resultant mixture was extracted with EtOAc (6 mL × 3). The organics
were dried over Na2SO4 and concentrated under reduced pressure. Silica
and neutral alumina (1:1 SiO2 /Al2O3) flash chromatography (7:3
hexanes/EtOAc) afforded the title compound 7 as a yellow solid (108 mg)
in 61% yield: mp 175–176 °C; 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J
= 8.1 Hz, 1H), 7.71 (t, J = 7.0 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.42 (t,
J = 7.5 Hz, 1H), 7.24 (s, 2H), 3.94 (s, 9H); 13C NMR (100 MHz, CDCl3)
δ 170.7 (d, JCF = 16.9 Hz, 1C), 154.9, 153.3 (2C), 150.6 (d, JCF = 23.1 Hz,
1C), 145.9 (d, J = 248.0 Hz, 1C), 141.0 (d, JCF = 1.8 Hz, 1C), 133.9,
125.8, 125.1, 124.1 (d, JCF = 7.0 Hz, 1C), 123.7 (d, JCF = 5.1 Hz, 1C),
118.1, 105.6 (d, JCF = 8.4 Hz, 2C), 61.0, 56.3 (2C); 19F NMR (470 MHz,
CDCl3) –161.31; IR (film) νmax 1725, 1685, 1036 cm–1; HRMS (ESI–
TOF) m/z calcd for C18H15FO5Cs (M+Cs)+ 462.9958, found 462.9949. All
characterization data were identical with the reported data in Ref 10.
17. 3-Fluoro-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one 8: A mixture of
7 (10 mg, 0.03 mmol) in hydrobromic acid (1 mL) was heated in a sealed
tube at 100 °C for 24 h. The reaction mixture was cooled to rt and
concentrated under reduced pressure to afford the title compound as a
evaluation. NMR analysis of the 3-fluorinated flavone in the
presence of a free radical source suggests that quenching occurs
by conversion to the ortho-quinone. Future work is focused on
enhancing the sensitivity of these probes for conducting
mechanistic investigations on cultured neurons following the
application of excitotoxins and will be reported in due course.
Acknowledgments
These studies were conducted with funding from the National
Institute of Health (NIH) and the authors acknowledge National
Institute of General Medical Sciences (P20GM104932 and
P20GM130460). Fellowship support for M.D.A. was provided by
the University of Hail, Saudi Arabia. The Bioanalytical Core of
the GlyCORE at the University of Mississippi is also
acknowledged for assistance with the acquisition of mass
spectrometry data.
1
yellow solid (8.6 mg) in quantitative yield: mp 280–282 °C; H NMR
(500 MHz, DMSO-d6) δ 8.11 (dd, J = 8.0, 1.7 Hz, 1H), 7.86 (t, J = 7.5
Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.07 (s, 2H),
4.12 (br s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 169.4 (d, JCF = 16.1
References and notes
Hz, 1C), 154.5, 151.2 (d, JCF = 23.1 Hz, 1C), 146.2 (2C), 145.1 (d, JCF
=
242.0 Hz, 1C), 137.5, 134.4, 125.3, 124.9 (d, JCF = 3.3 Hz, 1C), 123.6 (d,
JCF = 7.0 Hz, 1C), 118.4, 117.8 (d, JCF = 5.1 Hz, 1C), 107.4 (d, JCF = 8.1
Hz, 2C); 19F NMR (376 MHz, DMSO-d6) δ –165.2; IR (film) νmax 3655,
1685, 1646, 1037 cm–1; HRMS (ESI–TOF) m/z calcd for C15H9FO5 (M)–
287.0356, found 287.0360.
1.
2.
Meanwell, N. A. J. Med. Chem. 2018, 61, 5822–5880.
Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N.
A. J. Med. Chem. 2015, 58, 8315–8359.
Singh, M.; Kaur, M.; Silakari, O. Eur. J. Med. Chem 2014, 84, 206–239.
Hou, Y.; Higashiya, S.; Fuchigami, T. J. Org. Chem. 1999, 64, 3346–
3349.
Médebielle, M.; Keirouz, R.; Okada, E.; Shibata, D.; Dolbier, W. R.
Tetrahedron Lett. 2008, 49, 589–593.
Vints, I.; Rozen, S. J. Org. Chem. 2014, 79, 7261–7265.
Wang, R.; Han, J.; Li, C.; Zhang, J.; Liang, Y.; Wang, T.; Zhang, Z. Org.
Biomol. Chem. 2018, 16, 2479–2488.
For a review see O’Leary, E. M.; Jones, D. J.; O’Donovan, F. P.;
O'Sullivan, T. P. J. Fluorine Chem. 2015, 176, 93–120.
Menichincheri, M.; Ballinari, D.; Bargiotti, A.; Bonomini, L.; Ceccarelli,
W.; D'Alessio, R.; Fretta, A.; Moll, J.; Polucci, P.; Soncini, C.; Tibolla,
M.; Trosset, J.-Y.; Vanotti, E. J. Med. Chem. 2004, 47, 6466–6475.
3.
4.
18. DPPH assay. The assay measures the presence of the DPPH (2,2-
diphenyl-1-picrylhydrazyl) radical, which is a stable radical with an
absorbance between 515–528 nm. Stock solutions of test compounds
were prepared at 1 mg/mL in 1:1 ethanol-acetone and a 0.2 M solution of
DPPH in the same solvent system was also made. All solutions were
stored at 3 °C in the dark. Serial dilutions of 100 L of the test compound
were added to a 96-well plate, and six replicates were prepared for each
dilution. Next, the dilutions were treated with 100 L of the DPPH stock
solution, and the plate was incubated at rt in the dark for 20 min.
Absorbance was measured at 517 nm. Vitamin C (ascorbic acid) was the
positive control. The antioxidant activity of the flavones is expressed as
an EC50 value (with the standard error) defined by the concentration in
g/mL that inhibits the formation of DPPH radicals by 50%.
5.
6.
7.
8.
9.
10. Britton, R. G.; Horner-Glister, E.; Pomenya, O. A.; Smith, E. E.; Denton,
R.; Jenkins, P. R.; Steward, W. P.; Brown, K.; Gescher, A.; Sale, S. Eur.
J. Med. Chem 2012, 54, 952–958.
19. Ayaz, M.; Sadiq, A.; Junaid, M.; Ullah, F.; Ovais, M.; Ullah, I.; Ahmed,
J.; Shahid, M. Front. Aging Neurosci. 2019, 11, 155.
11. Chen, Y.-H.; Yang, Z.-S.; Wen, C.-C.; Chang, Y.-S.; Wang, B.-C.; Hsiao,
C.-A.; Shih, T.-L. Food Chem. 2012, 134, 717–724.
12. For the antioxidant activity and synthesis of 3',4',5'-trimethoxyflavone (1)
see: Singh, M.; Kaur, M.; Vyas, B.; Silakari, O. Med. Chem. Res. 2018,
27, 520–530.
13. For the antioxidant activity of 3',4',5'-trihydroxyflavone (2) see:
Mahfoudi, R.; Djeridane, A.; Benarous, K.; Gaydou, E. M.; Yousfi, M.
Bioorg. Chem. 2017, 74, 201–211.
14. Zubaidha, P. K.; Hashmi, A. M.; Bhosale, R. S. Het. Commun. 2005, 11,
97–100.
20. Ashpole, N. M.; Hudmon, A. Mol. Cell. Neurosci. 2011, 46, 720–730.
21. Neuroprotective assay. The 96-well plates were coated with 60 L of
0.01% poly-L-lysine, incubated for 24 h, and then washed with distilled
water. Sprague-Dawley rat cortical neurons were harvested and cultured
for 24 h in a 96-well plate with 100 L growth media that includes
neurobasal media, L-glutamine, fetal bovine serum (FBS), and pen strep.
Next, the growth media was replaced with media that does not contain
FBS, and the plate was further incubated for one week. The assay is
performed with 8–10 day old rat cortical neurons, and the cultures are
pre-treated with varying concentrations of test compound for 1 h before
the application of glutamate in a concentration-dependent manner as an
excitotoxic insult. After incubation for 24 h, MTS (aqueous one solution)
reagent is added, and the plates are returned to the incubator for 1 h. The
formazan dye is produced by viable neurons and is quantified by
measuring absorbance at 490 nm. Total cell number is also assessed using
microscopy. Then, the data are compared to DMSO and the glutamate
receptor antagonist, MK801, as controls to determine statistical
significance using a one-way ANOVA with post hoc Dunnett's test. A
second set of neurons was treated with test compound and glutamate
stimulation for 1 hr and then loaded and incubated with 5 micromolar H2-
DCFDA (2’,7’-dichlorofluorescin diacetate) in PBS for 60 minutes (per
manufacturer recommendations, ThermoFisher). DCFDA fluorescence
was quantified on the microplate reader with 495/520
excitation/emission.
15. 2-(3,4,5-Trihydroxyphenyl)-4H-chromen-4-one 2: A mixture of 2-(3,4,5-
trimethoxyphenyl)-4H-chromen-4-one
1 (10 mg, 0.03 mmol) in
hydrobromic acid (1 mL) was heated in a sealed tube at 100 °C for 24 h.
The reaction mixture was cooled to rt and concentrated under reduced
pressure to afford the title compound (8.1 mg) in quantitative yield: mp
281–283 °C; 1H NMR (500 MHz, DMF-d6) δ 8.07 (dd, J = 7.9, 1.5 Hz,
1H), 7.83 (td, J = 7.9, 1.6 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.17 (s, 2H),
6.68 (s, 1H), 5.76 (br s, 3H); 13C NMR (100 MHz, DMSO–d6) δ 176.9,
163.7, 155.6, 146.5 (2C), 137.8, 134.2, 125.4, 124.8, 123.4, 120.8, 118.4,
105.8 (2C), 104.9; IR (film) νmax 3448, 1639, 1590, 1037 cm–1; HRMS
(ESI–TOF) m/z calcd for C15H9O5 (M–H)– 269.0450, found 269.0432. All
characterization data were identical with the reported data in: Hiza, A.;
Tsukaguchi, Y.; Ogawa, T.; Inai, M.; Asakawa, T.; Hamashima, Y.; Kan,
T. Heterocycles 2014, 88, 1371–1396.
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26. See Supplementary Material for details.
A
mixture of (E)-2-fluoro-3-hydroxy-1-(2-hydroxyphenyl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one 5 (10 mg, 0.03 mmol), and iron(III)
chloride (0.47 mg , 0.009 mmol) in dichloromethane (1 mL) was stirred
at rt for 24 h. The reaction mixture was extracted with CH2Cl2 (1 mL ×
3). The organics were dried over Na2SO4 and concentrated under reduced
pressure to afford the title compound 7 as a yellow solid (7.9 mg) in 84%
yield. Method 2: A mixture of 6 (176.7 mg, 0.53 mmol), and N-
fluorobenzenesulfonimide (340 mg, 1.06 mmol) in pyridine (4 mL) was
stirred at rt for 12 h, then heated to 80 °C for 12 h. The reaction mixture
was cooled to rt and water (2 mL) was added. Then, the mixture was
cooled to 0 °C and acidified with 2.0 M of aqueous HCl (3 mL). The

Supplementary Material
Supplementary material data associated with this article (1H,
13C, and 19F NMR spectra for 7 and 8, HSQC NMR spectra for 8,
and mass spectrometry data, DEPTQ135 and 1D HSQMBC NMR
spectra for 8 in the presence of DPPH) can be found, in the online
version at http://dx.doi.org/10/1016/j.bmcl.XXXX.XX.XXX.
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Highlights
 3-Fluorinated flavones are created by a one-
pot fluorination/cyclization
 Flavones with 3-fluorination display anti-
oxidant and neuroprotective activity
 Structural changes of 3-fluorinated flavones
from free radicals is observed by NMR