Synthesis and in vitro antiproliferative activity of flavone and 6-hydroxyflavone oxime ethers derivatives

Article abstract of DOI:10.21577/0103-5053.20170128

Herein we report the synthesis of a series of O-alkyl oximes of flavone and 6-hydroxyflavone using a simple experimental protocol under solvent free conditions with yields up to 87percent. Cytotoxicity of all compounds was evaluated against MDA-MB-231, PC

Full text of DOI:10.21577/0103-5053.20170128

http://dx.doi.org/10.21577/0103-5053.20170128
J. Braz. Chem. Soc., Vol. 29, No. 1, 177-184, 2018.
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis and in vitro Antiproliferative Activity of Flavone and 6‑Hydroxyflavone
Oxime Ethers Derivatives
a
a
a
b
a
John E. Díaz, Diana C. Martinez, Lina V. López, Gina M. Mendez, Ricardo Vera and
,
a
Alix E. Loaiza*
a
Departamento de Química, Facultad de Ciencias, Pontificia Universidad Javeriana,
Cra. 7 No. 40‑62, Edificio Carlos Ortiz, 110231561 Bogotá, Colombia
b
Facultad de Medicina, Universidad de Ciencias Aplicadas y Ambientales,
Calle 222 No. 55‑37, 111166003 Bogotá, Colombia
Herein we report the synthesis of a series of O-alkyl oximes of flavone and 6-hydroxyflavone
using a simple experimental protocol under solvent free conditions with yields up to 87%.
Cytotoxicity of all compounds was evaluated against MDA-MB-231, PC-3, A-549 and MRC-5
cells. IC50 values for two compounds were determined to be in the range 28.7-47.8 μM against
all tested cell lines. Oxime ethers derivatives showed IC50 values between 28.7 and 49.5 μM
against MDA-MB-231, while the best activity was obtained for 6-hydroxyflavone with an IC50
of 3.4 μM against this cell line. Compounds containing the substituent hydroxyl at the position
six of flavone system displayed the best antiproliferative activity over MDA-MB-231 cells, being
necessary this group to improve the sensibility on this type of cells. The antiproliferative activity
of 6-hydroxyflavone is drastically diminished when the carbonyl group of flavone is changed by
an oxime ether.
Keywords: synthesis, oxime ethers, flavone, 6-hydroxyflavone, antiproliferative activity
Introduction
hydroxylamine, and 4-thionflavone or 4-thionflavone
methiodide as precursos. Previous attempts using
6
Oxime ethers are useful and versatile compounds in
organic synthesis, they are commonly easily prepared
and relatively stable to moisture; thus, they can be stored
directly hydroxylamine and a flavone conduced to
3-o-hydroxyphenyl-5-phenylisooxazole instead of the
7
oxime.
1
8
under air during long periods. Oxime is a pharmacophore
Meshcheryakova et al. reported the synthesis of some
group present in a variety of compounds with diverse
flavone oxime ether derivatives using 4,4-dichloroflavene
or 4-thionflavone and O-alkyl hydroxylamines, and the
evaluation of their pharmacological activity revealed
biological activities, for instance, some α,β-unsaturated
2
3
oximes are known as anticancer, immunosuppressive,
and antibacterial agents.
4
9
that they act on the central nervous system. Green et al.
On the other hand, flavone scaffold is an important core
in many compounds displaying a variety of pharmacological
properties that depend on their substitution patterns. The
wide range of biological activities of flavones has attracted
great interest in the synthesis of derivatives with the
achieved the synthesis of 2-arylchromenone oximes using
Ollis or Lawesson methods; and patented their use as
inhibitors of protein kinases.
Schann et al. synthesized 2-heteroarylchromenone
oximes as allosteric modulators of metabotropic glutamate
receptors, via the synthesis of O-t-butyl oxime ethers
employing microwave (MW) irradiation in methanol, and
further deprotection. They also prepared some of these
compounds directly with hydroxylamine under MW, with
poor yields.
1
0
5
objective of developing new therapeutic agents.
Although oxime ether derivatives of flavones could
display relevant biological activity, there are only a few
reports about these kind of compounds, suggesting that
these derivatives are not as promptly prepared. The first
synthesis of a flavone oxime was reported in 1952 using
Therefore, this work was undertaken with the
aim of extending the knowledge about the synthesis,
characterization and applications of flavone oxime ethers.
*e-mail: aloaiza@javeriana.edu.co

1
78
Synthesis and in vitro Antiproliferative Activity of Flavone and 6-Hydroxyflavone Oxime Ethers Derivatives
J. Braz. Chem. Soc.
We prepared and evaluated the antiproliferative activity
of twelve derivatives containing O-alkyl, O-benzyl and
O-allyl moieties.
A fifth experiment (entry 5), employing 6 equivalents
of both O-methylhydroxylamine hydrochloride and dry
pyridine at 140 °C was carried out in order to displace
the equilibrium towards the formation of oxime ether 3a.
After 2 h under these conditions, the starting material was
not completely consumed, but the desired oxime ether was
Results and Discussion
1
Chemistry
observed in 75% by H NMR.
Guided by the previous result, and with the aim of
finding the molar ratio of the reagents that allowed to obtain
the highest yield of 3a, we performed several experiments
varying the equivalents of both 2a and pyridine. Finally
we found that conditions shown in entry 6, afforded the
maximum amount of product (75% calculated by H NMR,
54% isolated), with the lowest equivalents of starting
materials.
In order to obtain the target compounds by a
previously optimized protocol, we carried out experiments
using the model reaction between flavone (1a) and
O-methylhydroxylamine hydrochloride (2a) in pyridine as a
solvent and a base (Table 1, entry 1). Under these conditions,
no evidence of the formation of compound 3a was observed
after 18 h, and the flavone was mostly recovered.
1
Then, we adapted procedures reported in the literature
for analog structures using microwave irradiation. The
reaction with O-methylhydroxylamine hydrochloride in
Based on the results shown above, we synthesized a
series of oxime ethers of flavone and 6-hydroxyflavone
(3b-l) using the experimental conditions of entry 6. In
order to synthesize oxime ethers 3c-f,i-l, it was necessary
to obtain the O-allyl and O-benzylhydroxylamines 5c-f no
commercially available, via Gabriel synthesis, and their
hydrochlorides 2c-f (Scheme 1).
The E/Z geometries of oxime ethers 3a-l, were
determined by 2D NOESY (nuclear Overhauser effect
spectroscopy) experiments, which allowed to evidence
10
methanol under microwave for 30 minutes (entry 2) did
not give rise to compound 3a; thus, we decided to perform
a new attempt using the former conditions, adding pyridine
as the base (entry 3). This procedure allowed us obtaining
1
the desired oxime ether 3a in a 19% according to H nuclear
magnetic resonance (NMR) spectra of the crude mixture,
as a single isomer.
Due to the low reactivity displayed by
flavone, we employed the conditions reported by
Meshcheryakova et al., that includes the transformation
correlations between hydrogens of O-CH group and the
heterocycle ring, showing that the E isomer was the most
favored product.
2
8
of flavone in 4,4-dichloroflavene (entry 4). The reaction
was monitored by gas chromatography-mass spectrometry
Antiproliferative activity
(
GC-MS) and the dichloride intermediate was detected
after the first step (a), but no evidence of the formation of
a was observed after the second one (b).
Cytotoxicity of all compounds was evaluated against
MDA-MB-231, PC-3,A-549 and MRC-5 cells. Flavone (1a)
3
Table 1. Evaluation of reaction conditions to obtain compound 3a
a
b
entry
Conditions
3a / % (3a / %)
1
2
3
4
5
2a (2.0 equiv.), Py, reflux, 18 h
0
0
2a (2.0 equiv.), MeOH, MW 150 W, 30 min
c
2a (2.0 equiv.), Py (2.0 equiv.), MeOH, MW 150 W, 30 min
(a) SOCl , Et N(dry), CHCl3(dry), reflux, 5 h; (b) 2a (1.2 equiv.), 1.5 h
5 (19)
0
2
3
2a (6.0 equiv.), Py(dry) (6.0 equiv.), 140 °C (oil bath), 2.5 h
2a (2.5 equiv.), Py(dry) (3.5 equiv.), 140 °C (oil bath), 2 h
(75)
54 (75)
d
6
a
b
1
cc
dd
Isolated yield; calculated yield by 1 HH NN MM RR ;; mm ee t thh oo dd AA ;; mm ee t thh oo dd BB ..

Vol. 29, No. 1, 2018
Díaz et al.
179
Scheme 1. (a) K CO , DMSO, room temperature, 2-3 h (87-97%); (b) NH NH .H SO , K CO , Et O, H O, reflux, 5 h (76-90%); (c) HCl(g), Et O;
2
3
2
2
2
4
2
3
2
2
2
(d) Py/Δ (54-87%).
and its oxime derivatives (3a-f) showed no relevant activity
medicine, which contains hydroxyl groups at positions 5,
6 and 7, has motivated to develop studies aimed to verify
the inhibitory effects and the mechanism involved in its
antimetastatic effect against MDA-MB-231 cells. The
at the evaluated concentrations. IC values for compounds
50
3
h,i presenting a hydroxyl and a benzyl group, were
determined to be in the range of 28.7-47.8 μM for all tested
cell lines. Compounds 3g-l containing a hydroxyl group at
sixth position, showed IC between 28.7-49.5 μM against
12
IC value of baicalein (59.5 μM), higher than the IC
50
50
determined for 6-hydroxyflavone in this work over the same
cell line, motivates our interest in continuing the study of
this compound.
50
MDA-MB-231; however, the best activity was obtained
for 6-hydroxyflavone (1b) with an IC of 3.4 μM against
50
the same cell line (Table 2). Previous studies have shown
that 6-hydroxyflavone also exhibits activity over the
leukemia cell lines HL-60 (IC = 2.8 μM) and MOLT-4
It is important to notice that despite the IC50 values
found in this work are not comparable with the reference
drug vincristine (IC = 0.008 μM against MDA-MB-231),
50
50
1
1
(
IC50 = 6.3 μM). It is important to emphasize that no
the search for novel cytotoxic agents with selectivity for a
particular cell line, and the comprenhension of mechanisms
of action, are necessary tasks towards the development of
new drugs that allow to improve the existing anticancer
therapies.
antiproliferative activity was displayed by compound 1b
against healthy fibroblasts from lung (MRC-5).
On the other hand, the antitumor activity of baicalein,
a naturally ocurring flavonoid used in Chinese herbal
Table 2. In vitro IC50 of compounds 1 and 3 against MDA-MB-231, PC-3, A-549 and MRC-5 cells
-1
IC50 / (μmol L )
Compound
R
R1
MDA-MB-231
> 50
PC-3
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
A-549
> 50
MRC-5
> 50
1
1
3
3
3
3
3
3
3
3
3
3
3
3
a
b
a
b
c
d
e
f
H
OH
H
−
−
3.4 ± 0.1
> 50
> 50
> 50
CH3
> 50
> 50
H
CH C H
5
> 50
> 50
> 50
2
6
H
CH -o-BrC H
4
> 50
> 50
> 50
2
6
H
CH CBr=CH
> 50
> 50
> 50
2
2
H
CH −CH=C(CH )
> 50
> 50
> 50
2
3 2
H
CH −CH=CHC H
> 50
> 50
> 50
2
6
5
g
h
i
OH
OH
OH
OH
OH
OH
CH3
49.5 ± 0.8
28.7 ± 2.7
40.4 ± 1.7
34.8 ± 2.2
45.4 ± 0.3
38.7 ± 3.4
> 50
> 50
CH C H
5
47.8 ± 0.6
43.6 ± 2.6
> 50
40.2 ± 0.5
47.4 ± 0.9
> 50
44.5 ± 0.7
39.8 ± 2.7
39.7 ± 2.5
> 50
2
6
CH -o-BrC H
4
2
6
j
CH CBr=CH
2 2
k
l
CH −CH=C(CH )
> 50
> 50
2
3 2
CH −CH=CHC H
> 50
> 50
> 50
2
6
5
The IC50 value was defined as the concentration of the compound which caused a 50% decrease of the cell viability.

1
80
Synthesis and in vitro Antiproliferative Activity of Flavone and 6-Hydroxyflavone Oxime Ethers Derivatives
J. Braz. Chem. Soc.
Conclusions
Method B (entry 6)
A mixture of flavone or 6-hydroxyflavone 1a-b
(1.0 mmol), an appropriate O-alkyl hydroxilamine
hydrochloride (2a-e) (2.5 mmol), and dry pyridine
(3.5 mmol), was stirred at 140 °C for 2-4 h. The reaction
mixture was treated with distilled water and extracted
with dichloromethane. The organic phase was dried
with Na SO , filtered and concentrated under reduced
The synthesis of twelve flavone and 6-hydroxyflavone
oxime ether derivatives was achieved with reasonably good
yields as a single isomer (E), using a simple experimental
protocol. Changing the carbonyl group of flavone for an
oxime ether did not increase the cytotoxic activity of the
derivatives against the tested line cells. Hydroxyl group at
the position six of flavone system is necessary to display
activity against MDA-MB-231 since all compounds
containing this substituent showed antiproliferative
activity over this cell line. The antiproliferative activity
of 6-hydroxyflavone is drastically diminished when the
carbonyl group of flavone is changed by an oxime ether.
2
4
pressure. The organic combined were purified by column
chromatography (silica gel, hexane:CH Cl 4:1 or CH Cl )
to provide products 3a-l.
2
2
2
2
2‑Phenyl‑4H‑chromen‑4‑one O‑methyloxime (3a)
1
Yield 54%; mp 57-58 °C; H NMR (300 MHz, CDCl )
3
d 4.05 (s, 3H, OCH ), 7.08 (s, 1H, C=CH), 7.30-7.32 (m,
3
Experimental
2H, H-6, 7 Ar), 7.42-7.49 (m, 4H, H-8, H-3’, 4’, 5’ Ar),
7
.89-7.92 (m, 2H, H-2’, 6’Ar), 8.04 (dd, 1H, J 8.1, 1.5 Hz,
13
Chemistry
H-5Ar); C NMR (75 MHz, CDCl ) d 62.06 (OCH ), 93.81
3 3
(
C=CH), 117.53 (C-6 Ar), 118.37 (C-4a Ar), 123.01 (C-5
1
13
H and C NMR spectra were acquired on a Bruker
Ar), 124.65 (C-7 Ar), 125.71 (C-2’, 6’ Ar), 128.65 (C-8
Ar), 130.25 (C-4’ Ar), 130.43 (C-3’, 5’ Ar), 132.89 (C-1’
Ar), 143.84 (C-8a Ar), 151.91 (C=N), 155.23 (C=CH).
Mass spectrum, m/z (relative intensity (I ), %): 251 [M]
(72), 236 (15), 206 (100). Found, m/z: 252.1025 [M + H] ,
Avance spectrometer (300 and 75 MHz, respectively) in
CDCl or CD OD. Chemical shifts and configuration were
3
3
+
assigned with the help of HSQC-edit (heteronuclear single
quantum correlation), HMBC (heteronuclear multiple bond
correlation), COSY (correlation spectroscopy) and NOESY
experiments. High-resolution mass spectra (HRMS)
were recorded on an Agilent 6520 q-TOF-MS instrument
with orthogonal ESI (electrospray ionization). GC-MS
analyses were performed on an Agilent 6850 series II gas
chromatograph coupled to an Agilent 5975B VL mass
spectrometer (electron ionization, 70 eV) equipped with
split/splitess inlet (split relation 15:1, 260 °C),Agilent 6850
series automatic injector, and Agilent HP-5MS column
rel
+
C H NO . Calculated, m/z: 252.1025.
16
14
2
2‑Phenyl‑4H‑chromen‑4‑one O‑benzyloxime (3b)
1
Yield 70%; mp 69-70 °C; H NMR (300 MHz, CDCl )
3
d 5.30 (s, 2H, OCH ), 7.14 (s, 1H, C=CH), 7.21-7.37 (m,
8H, H-6, 7, 8, 2’’, 3’’, 4’’, 5’’, 6’’Ar), 7.39-7.52 (m, 3H,
H-3’, 4’, 5’Ar), 7.87-7.91 (m, 2H, H-2’, 6’Ar), 8.05 (dd,
1H, J 8.1, 1.5 Hz, H-5 Ar); C NMR (75 MHz, CDCl )
d 76.22 (OCH ), 94.07 (C=CH), 117.52 (C-6 Ar), 118.42
2
1
3
3
2
(
8
3
30 m × 0.25 mm × 0.25 μm); initial oven temperature
0 °C for 1 min, then a temperature ramp of 10 °C min to
20 °C (hold 3 min); total run time 28 min. Melting points
(C-4a Ar), 123.14 (C-7 Ar), 124.64 (C-5 Ar), 125.77
(C-2’, 6’ Ar), 127.75 (C-2’’, 6’’ Ar), 128.30 (C-3’’, 5’’
Ar), 128.38 (C-4’’ Ar), 128.66 (C-8 Ar), 130.27 (C-4’,
Ar), 130.46 (C-3’, 5’Ar), 132.88 (C-1’Ar), 138.30 (C-1’’
-1
were determined on a Thermo Fisher Scientific Rochford
100 apparatus.
9
Ar), 144.14 (C-8a Ar), 151.92 (C=N), 155.14 (C=CH).
+
Mass spectrum, m/z (I , %): 327 [M] (36), 236 (46),
rel
+
Synthesis of oxime ether derivates 3a‑l
206 (100). Found, m/z: 328.1337 [M + H] , C H NO .
2
2
18
2
Calculated, m/z: 328.1338.
Method A (entry 3)
A solution of flavone 1a (1.0 mmol), O-methyl
hydroxylamine hydrochloride (2.0 mmol), and pyridine
2‑Phenyl‑4H‑chromen‑4‑one O‑(2‑bromobenzyl)oxime (3c)
1
Yield 74%; mp 82-83 °C; H NMR (300 MHz, CDCl )
3
(
1
2.0 mmol) in methanol (6.6 mL), was irradiated at
50 W for 30 minutes. The reaction mixture was treated
d 5.37 (s, 2H, OCH ), 7.16-7.36 (m, 6H, C=CH, H-6, 7
Ar, H-4’’, 5’’, 6’’ Ar), 7.45-7.54 (m, 4H, H-8, 3’, 4’, 5’
2
with distilled water and extracted with dichloromethane.
The organic phase was dried with Na SO , filtered and
Ar), 7.60 (d, 1H, J 8.7 Hz, H-3’’ Ar), 7.91-7.92 (m, 2H,
13
H-2’, 6’Ar), 8.03 (dd, 1H, J 7.5, 0.3 Hz, H-5Ar); C NMR
(75 MHz, CDCl ) d 75.39 (OCH ), 94.00 (C=CH), 117.50
2
4
concentrated under reduced pressure. The crude mixture
was purified by column chromatography using silica gel
and hexane:CH Cl (4:1), yielding 5% of 3a.
3
2
(C-6 Ar), 118.30 (C-4a Ar), 122.84 (C-Br), 123.24 (C-5
Ar), 124.66 (C-7 Ar), 125.79 (C-2’, 6’ Ar), 127.30 (C-4’’
2
2

Vol. 29, No. 1, 2018
Díaz et al.
181
Ar), 128.68 (C-5’’Ar), 128.94 (C-3’, 5’Ar), 129.65 (C-6’’
Ar), 130.32 (C-4’Ar), 130.53 (C-8 Ar), 132.55 (C-3’’Ar),
Ar), 130.30 (C-4’Ar), 130.48 (C-7 Ar), 132.86 (C-1’Ar),
133.03 (HC=CHPh), 136.83(C-1’’Ar), 144.04 (C-8a Ar),
151.92 (C=N), 155.14 (C=CH). Found, m/z: 354.1496
132.89 (C-1’), 137.94 (C-1’’Ar), 144.57 (C-8aAr), 151.91
+
(
[
[
C=N), 155.38 (C=CH). Mass spectrum, m/z (I , %): 405
[M + H] , C H NO . Calculated, m/z: 354.1494.
rel
20 20
2
79
+
M( Br)] (12), 236 (52), 206 (100). Found, m/z: 406.0446
+
M + H] , C H BrNO . Calculated, m/z: 406.0443.
6‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑methyloxime
3g)
2
2
17
2
(
1
2
‑Phenyl‑4H‑chromen‑4‑one O‑(2‑bromoallyl)oxime (3d)
Yield 54%; mp 149-150 °C; H NMR (300 MHz,
CDCl ) d 4.30 (s, 3H, OCH ), 6.93 (dd, 1H, J 9.0, 2.9 Hz,
1
Yield 72%; mp 79-80 °C; H NMR (300 MHz, CDCl )
3
3
3
d 4.83 (s, 2H, OCH ), 5.67 (s, 1H, BrC=CH), 5.97 (s, 1H,
BrC=CH), 7.16 (s, 1H, C=CH), 7.22-7.32 (m, 2H, H-6,
H-7 Ar), 6.96 (s, 1H, C=CH), 7.19 (d, 1H, J 9.0 Hz, H-8
Ar), 7.31 (d, 1H, J 2.9 Hz, H-5 Ar), 7.40-7.50 (m, 3H,
H-3’, 4’, 5’Ar), 7.70-7.80 (m, 2H, H-2’, 6’Ar); C NMR
2
1
3
7
Ar), 7.45-7.49 (m, 4H, H-8, 3’, 4’, 5’ Ar), 7.91-7.92
(
m, 2H, H-2’, 6’ Ar), 8.04 (dd, 1H, J 7.8, 1.2 Hz, H-5
(75 MHz, CDCl ) d 60.66 (OCH ), 92.00 (C=CH), 106.50
3
3
1
3
Ar); C NMR (75 MHz, CDCl ) d 77.45 (OCH ), 93.79
(C-5 Ar), 118.30 (C-7 Ar), 118.40 (C-4a Ar), 118.69 (C-8
Ar), 125.18 (C-2’, 6’ Ar), 128.39 (C-3’, 5’ Ar), 129.92
(C-4’Ar), 132.78 (C-1’Ar), 143.92 (C-8aAr), 145.54 (C-6
Ar), 154.23 (C=N), 155.17 (C=CH). Found, m/z: 268.0975
3
2
(
(
C=CH), 117.49 (BrC=CH ), 117.55 (C-6 Ar), 118.05
2
C-4aAr), 123.24 (C-5Ar), 124.72 (C-7Ar), 125.81 (C-2’,
6
’ Ar), 128.71 (C-3’, 5’ Ar), 129.66 (BrC=CH ), 130.42
2
+
(C-4’Ar), 130.68 (C-8Ar), 132.79 (C-1’Ar), 144.92 (C-8a
[M + H] , C H NO . Calculated, m/z: 268.0974.
16
14
3
Ar), 151.96 (C=N), 155.31 (C=CH). Mass spectrum, m/z
7
9
+
(
I , %): 355 [M( Br)] (13), 236 (36), 206 (100). Found,
6‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑benzyloxime
(3h)
rel
+
m/z: 356.0287 [M + H] , C H BrNO . Calculated, m/z:
1
8
15
2
1
3
56.0286.
Yield 58%; mp 150-151 °C; H NMR (300 MHz,
CDCl ) d 5.20 (s, 2H, OCH ), 6.95 (dd, 1H, J 9.0, 3.0 Hz,
3
2
2
‑Phenyl‑4H‑chromen‑4‑one O‑(3‑methylbut‑2‑en‑1‑yl)
H-7 Ar), 7.05 (s, 1H, C=CH), 7.18 (d, 1H, J 9.0 Hz,
H-8 Ar), 7.28-7.35 (m, 6H, H-5, 2’’, 3’’, 4’’, 5’’, 6’’ Ar),
7.37-7.45 (m, 3H, H-3’, 4’, 5’Ar), 7.81-7.84 (m, 2H, H-2’,
oxime (3e)
1
Yield 64%; H NMR (300 MHz, CDCl ) d 1.82 (s, 3H,
3
13
CH ), 1.84 (s, 3H, CH ), 4.75 (d, 2H, J 7.0 Hz, OCH ),
6’Ar); C NMR (75 MHz, CDCl ) d 75.84 (OCH ), 92.70
3 2
3
3
2
5
7
1
4
.51 (t, 1H, J 7.0 Hz, HC=C(CH ) ), 7.10 (s, 1H, C=CH),
(C=CH), 106.96 (C-5 Ar), 118.37 (C-4a Ar), 118.62 (C-7
Ar), 119.08 (C-8 Ar), 125.67 (C-2’, 6’Ar), 127.66 (C-2’’,
6’’ Ar), 128.08 (C-3’’, 5’’ Ar), 128.24 (C-4’’ Ar), 128.53
(C-3’, 5’Ar), 130.16 (C-4’, Ar), 132.85 (C-1’Ar), 138.02
(C-1’’ Ar), 145.12 (C-8a Ar), 145.85 (C-6 Ar), 153.86
3
2
.20-7.26 (td, 1H, J 7.2, 1.3 Hz, H-6 Ar), 7.28-7.32 (dd,
H, J 8.0, 1.3 Hz, H-8 Ar), 7.40-7.54 (m, 4H, H-7, H-3’,
’, 5’ Ar), 7.86-7.96 (m, 2H, H-2’, 6’ Ar), 8.06 (dd, 1H,
13
J 7.95, 1.3 Hz, H-5Ar); C NMR (75 MHz, CDCl ) d 18.28
3
+
(
(
(
(
(
(
CH ), 25.94 (CH ), 70.91 (OCH ), 94.14 (C=CH), 117.48
C-8Ar), 118.64 (C-4aAr), 120.60 (HC=C(CH ) ), 123.06
C-5 Ar), 124.57 (C-6 Ar), 125.72 (C-2’, 6’ Ar), 128.60
C-3’, 5’ Ar), 130.16 (C-4’ Ar), 130.30 (C-7 Ar), 132.97
C-1’Ar), 137.57 (HC=C(CH ) ), 143.63 (C-8aAr), 151.91
(C=N), 156.54 (C=CH). Found, m/z: 344.1287 [M + H] ,
3
3
2
C H NO . Calculated, m/z: 344.1287.
3
2
22 18
3
6‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑(2‑bromobenzyl)
oxime (3i)
3
2
+
1
C=N), 154.92 (C=CH). Found, m/z: 306.1494 [M + H] ,
Yield 76%; mp 123-124 °C; H NMR (300 MHz,
C H NO . Calculated, m/z: 306.1494.
CDCl ) d 5.28 (s, 2H, OCH ), 6.95 (dd, 1H, J 8.9, 3.0 Hz,
2
0
20
2
3
2
H-7 Ar), 7.11 (s, 1H, C=CH), 7.15-7.26 (m, 2H, H-8, 4’’,
Ar), 7.28-7.39 (m, 2H, H-5, 5’’ Ar), 7.41-7.48 (m, 3H,
H-3’, 4’, 5’ Ar), 7.51 (d, 1H, J 7.6 Hz, H-6’’ Ar), 7.58
(d, 1H, J 7.6 Hz, H-3’’ Ar), 7.81-7.91 (m, 2H, H-2’, 6’
2
‑Phenyl‑4H‑chromen‑4‑one O‑cinnamyloxime (3f)
Yield 60%; mp 68-69 °C; H NMR (300 MHz, CDCl )
1
3
d 4.93 (d, 2H, J 6.2 Hz, OCH ), 6.49-6.62 (dt, 1H, J 16.0,
2
13
6
7
5
8
.2 Hz, HC=CHPh), 6.76 (d, 1H, J 16.0 Hz, HC=CHPh),
.16 (s, 1H, C=CH), 7.22-7.56 (m, 11H, H-6, 7, 8, 3’, 4’,
’, 2’’, 3’’, 4’’, 5’’, 6’’Ar), 7.87-7.97 (m, 2H, H-2’, 6’Ar),
Ar); C NMR (75 MHz, CDCl ) d 74.89 (OCH ), 92.29
3 2
(C=CH), 106.73 (C-5 Ar), 118.32 (C-4a Ar), 118.39 (C-7
Ar), 118.93 (C-8 Ar), 122.52 (C-Br), 125.35 (C-2’, 6’Ar),
127.16 (C-5’’), 128.45 (C-3’, 4’, 5’ Ar), 128.91 (C-4’’
Ar), 129.66 (C-3’’ Ar), 132.28 (C-6’’ Ar), 132.78 (C-1’
Ar), 137.64 (C-1’’ Ar), 145.00 (C-8a Ar), 145.66 (C-6
Ar), 154.54 (C=N), 155.50 (C=CH). Found, m/z: 422.0398
13
.09 (dd, 1H, J 7.9, 1.2 Hz, H-5 Ar); C NMR (75 MHz,
CDCl ) d 74.97 (OCH ), 94.00 (C=CH), 117.55 (C-8 Ar),
3
2
1
(
1
18.44 (C-4aAr), 123.14 (C-5Ar), 124.68 (C-6Ar), 125.76
C-2’, 6’Ar), 125.94 (HC=CHPh), 126.65 (C-2’’, 6’’Ar),
27.76 (C-4’’Ar), 128.59 (C-3’, 5’Ar), 128.68 (C-3’’, 5’’
+
[M + H] , C H BrNO . Calculated, m/z: 422.0392.
22
17
3

1
82
Synthesis and in vitro Antiproliferative Activity of Flavone and 6-Hydroxyflavone Oxime Ethers Derivatives
J. Braz. Chem. Soc.
6
‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑(2‑bromoallyl)
Synthesis of 2‑alkyloxyisoindoline‑1,3‑diones
oxime (3j)
1
Yield 69%; mp 129-130 °C; H NMR (300 MHz,
A solution of N-hydroxyphthalimide (1 mmol), an
CDCl ) d 4.76 (s, 2H, OCH ), 5.61 (s, 1H, BrC=CH), 5.93
alkyl bromide (0.5 mmol), and K CO (2 mmol) in DMSO
3
2
2
3
(
7
(
s, 1H, BrC=CH), 6.96 (dd, 1H, J 9.0, 3.0 Hz, H-7 Ar),
.07 (s, 1H, C=CH), 7.21 (d, 1H, J 9.0 Hz, H-8 Ar), 7.32
d, 1H, J 3.0 Hz, H-5 Ar), 7.40-7.50 (m, 3H, H-3’, 4’, 5’,
(1 mL), was stirred at room temparature for 2-3 h. The
reaction mixture was treated whit distilled water (10 mL),
filtered, washed whith water and dried to provide products
3c-f.
13
Ar), 7.80-7.90 (m, 2H, H-2’, 6’ Ar); C NMR (75 MHz,
CDCl ) d 77.14 (OCH ), 92.53 (C=CH), 106.99 (C-5 Ar),
3
2
1
1
1
1
17.37 (BrC=CH ), 118.14 (C-4a Ar), 118.64 (C-8 Ar),
19.30 (C-7Ar), 125.62 (C-2’, 6’Ar), 128.57 (C-3’, 5’Ar),
2‑((2‑Bromobenzyl)oxy)isoindoline‑1,3‑dione (4c)
2
1
Yield 92%; mp 159-160 °C; H NMR (300 MHz,
29.42 (BrC=CH ), 130.27 (C-4’, Ar), 132.78 (C-1’ Ar),
CDCl ) d 5.37 (s, 2H, OCH ), 7.25 (td, 1H, J 7.5, 1.3 Hz,
2
3
2
45.76 (C-6 Ar), 145.88 (C-8a Ar), 153.93 (C=N), 155.80
H-4’ Ar), 7.37 (t, 1H, J 7.5 Hz, H-5’ Ar), 7.56 (d, 1H,
J 7.5 Hz, H-3’Ar), 7.66 (dd, 1H, J 7.5, 1.3 Hz, H-6’Ar),
7.72-7.79 (m, 2H, H-4, 7 Ar), 7.79-7.86 (m, 2H, H-5, 6
+
(C=CH). Found, m/z: 372.0235 [M + H] , C H BrNO .
18
15
3
Calculated, m/z: 372.0235.
1
3
Ar); C NMR (75 MHz, CDCl ) d 78.67 (OCH ), 123.55
3
2
6
2
‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑(3‑methylbut‑
‑en‑1‑yl)oxime (3k)
(C-5, 6 Ar), 124.55 (C-Br), 127.65 (C-5’ Ar), 128.33
(C-3a, 1a Ar), 130.72 (C-4’Ar), 131.75 (C-6’Ar), 132.89
(C-3’ Ar), 133.66 (C-1’ Ar), 134.47 (C-4, 7 Ar), 163.38
1
Yield 67%; mp 82-84 °C; H NMR (300 MHz, CDCl )
3
7
9
+
d 1.77 (s, 3H, CH ), 1.80 (s, 3H, CH ), 4.74 (d, 2H,
(C=O). Mass spectrum, m/z (I , %): 331 [M( Br)] , 169
3
3
rel
J 7.0 Hz, OCH ), 5.56 (t, 1H, J 7.0 Hz, HC=C(CH ) ), 7.00
(100), 76 (22).
2
3 2
(
(
dd, 1H, J 8.9, 2.8 Hz, H-7 Ar), 7.06 (s, 1H, C=CH), 7.19
d, 1H, J 8.9 Hz, H-8Ar), 7.43-7.51 (m, 4H, H-5, 3’, 4’, 5’
2‑((2‑Bromoallyl)oxy)isoindoline‑1,3‑dione (4d)
1
3
1
Ar), 7.80-7.92 (m, 2H, H-2’, 6’Ar); C NMR (75 MHz,
Yield 97%; mp 110-111 °C; H NMR (300 MHz,
CDCl ) d 18.30 (CH ), 25.98 (CH ), 70.71 (OCH ), 93.24
CDCl ) d 4.85 (s, 2H, OCH ), 5.79 (s, 1H, BrC=CH), 6.16
3
3
3
2
3
2
(
C=CH), 107.54 (C-5 Ar), 118.67 (C-4a Ar), 118.96 (C-7
(s, 1H, BrC=CH), 7.75-7.80 (m, 2H, H-4, 7 Ar), 7.85-7.89
13
Ar), 119.22 (C-8 Ar), 120.26 (HC=C(CH ) ), 125.36
(m, 2H, H-5, 6 Ar); C NMR (75 MHz, CDCl ) d 81.08
3
2
3
(
1
C-2’, 6’ Ar), 128.61 (C-3’, 5’ Ar), 130.22 (C-4’ Ar),
32.89 (C-1’ Ar), 138.08 (HC=C(CH ) ), 144.74 (C-8a
(OCH ), 122.82 (BrC=CH ), 123.71 (C-5, 6 Ar), 124.66
2
2
(BrC=CH), 128.70 (C-3a, 1a Ar), 134.70 (C-4, 7 Ar),
3
2
7
9
+
Ar), 146.30 (C-6 Ar), 152.87 (C=N), 155.35 (C=CH).
Found, m/z: 322.1445 [M + H] , C H NO . Calculated,
163.28 (C=O). Mass spectrum, m/z (I , %): 281 [M( Br)] ,
rel
+
+
202 (100), 162 (18). Found, m/z: 281.9761 [M + H] ,
2
0
20
3
m/z: 322.1443.
C H BrNO . Calculated, m/z: 281.9766.
11
9
3
6
‑Hydroxy‑2‑phenyl‑4H‑chromen‑4‑one O‑cinnamyloxime
2‑((3‑Methylbut‑2‑en‑1‑yl)oxy)isoindoline‑1,3‑dione (4e)
1
(3l)
Yield 78%; mp 95-96 °C; H NMR (300 MHz, CDCl ) d
3
1
Yield 87%; mp 83-84 °C; H NMR (300 MHz, CDCl )
1.75 (s, 3H, CH ), 1.79 (s, 3H, CH ), 4.74 (d, 2H, J 7.6 Hz,
3
3
3
d 4.88 (d, 2H, J 6.0 Hz, OCH ), 6.44-6.56 (dt, 1H, J 16.0,
OCH ), 5.55 (t, 1H, J 7.6 Hz, HC=C), 7.71-7.81 (m,
2
2H, H-4, 7 Ar), 7.82-7.89 (m, 2H, H-5, 6 Ar); C NMR
2
13
6
6
.0 Hz, HC=CHPh), 6.71 (d, 1H, J 16.0 Hz, HC=CHPh),
.99 (dd, 1H, J 8.9, 2.9 Hz, C-7), 7.02 (d, 1H, J 8.9 Hz,
(75 MHz, CDCl ) d 18.13 (CH ), 25.97 (CH ), 74.06
3
3
3
C-8), 7.09 (s, 1H, C=CH), 7.24-7.55 (m, 9H, H-5, 3’, 4’,
’, 2’’, 3’’, 4’’, 5’’, 6’’ Ar), 7.84-7.92 (m, 2H, H-2’, 6’
(OCH ), 117.04 (HC=C(CH ) ), 123.45 (C-5, 6Ar), 128.92
2 3 2
5
(C-3a, 1a Ar), 134.41 (C-4, 7 Ar), 143.72 (HC=C(CH ) ),
3 2
1
3
+
Ar); C NMR (75 MHz, CDCl ) d 74.83 (OCH ), 93.12
163.86 (C=O). Found, m/z: 232.0971 [M + H] , C H NO .
13 14 3
3
2
(
C=CH), 107.55 (C-5 Ar), 118.74 (C-4a Ar), 118.92 (C-7
Calculated, m/z: 232.0974.
Ar), 119.11 (C-8 Ar), 125.76 (HC=CHPh, C-2’, 6’ Ar),
1
5
26.62 (C-2’’, 6’’ Ar), 127.74 (C-4’’Ar), 128.56 (C-3’,
’ Ar), 128.64 (C-3’’, 5’’ Ar), 130.24 (C-4’ Ar), 132.91
2‑(Cinnamyloxy)isoindoline‑1,3‑dione (4f)
1
Yield 87%; mp 144-145 °C; H NMR (300 MHz,
(
(
C-1’Ar), 133.10 (HC=CHPh), 136.75 (C-1’’Ar), 144.57
CDCl ) d 4.88 (d, 2H, J 7.0 Hz, OCH ), 6.48 (dt, 1H, J 15.9,
7.0 Hz, HC=CHPh), 6.69 (d, 1H, J 15.9 Hz, HC=CHPh),
3
2
C-8aAr), 146.53 (C-6Ar), 152.94 (C=N), 155.6 (C=CH).
+
Found, m/z: 370.1444 [M + H] , C H NO . Calculated,
m/z: 370.1443.
7.22-7.43 (m, 5H, H-2’, 3’, 4’, 5’, 6’ Ar), 7.71-7.78 (m,
24
20
3
13
2H, H-4, 7 Ar), 7.78-7.86 (m, 2H, H-5, 6 Ar); C NMR
75 MHz, CDCl ) d 78.67 (OCH ), 122.01 (HC=CHPh),
(
3
2

Vol. 29, No. 1, 2018
Díaz et al.
183
1
1
23.56 (C-5, 6 Ar), 126.93 (C-4’Ar), 128.47 (C-2’, 6’Ar),
28.64 (C-3’, 5’Ar), 128.80 (C-3a, 1a Ar), 134.50 (C-4, 7
(dt, 1H, J 15.6, 6.9 Hz, HC=CHPh), 6.83 (d, 1H, J 15.6 Hz,
HC=CHPh), 7.25-7.35 (m, 3H, HAr), 7.40-7.45 (m, 2H, H
13
Ar), 135.77 (C-1’Ar), 137.53 (HC=CHPh), 163.86 (C=O).
Found, m/z: 302.0794 [M + Na] , C H NaNO . Calculated,
m/z: 302.0793.
Ar), C NMR (75 MHz, CDCl -CD OD) d 75.74 (OCH ),
3 3 2
+
119.76 (HC=CHPh), 126.85 (Ar-2,6), 128.61 (Ar-3,5),
128.72 (Ar-5), 135.38(Ar-1), 138.53 (HC=CHPh).
17
13
3
Synthesis of O‑alkylhydroxylamine hydrochlorides
Antiproliferative activity
To a stirred solution of hydrazine sulfate (1.2 mmol),
K CO (1.2 mmol) and water (2.0 mL) in THF (2.0 mL), was
Cell lines and culture conditions
Cells were obtained from American Type Cell Culture
(ATCC, Bethesda, BD). Tumor derived cells that originated
in multiple tissue sites were maintained as follow: PC-3
(prostate adenocarcinoma), MRC-5 (fibroblasts derived
from lung tissue) and A-549 (lung: NSCLC alveolar basal
epithelial-squamous) in Dulbecco’s modified Eagle’s
medium with high glucose (Lonza), supplemented with
10% (v/v) fetal bovine serum (Biowest), 2 mM L-glutamine,
2
3
added the corresponding O-alkyl-N-hydroxyphthalimide
1.0 mmol), and the mixture was refluxed during 5 h, then
(
distilled water (10 mL) was added. The resulting mixture
was extracted with ethyl ether (3 × 10 mL), the organic
phase was dried with sodium sulfate, filtrated and the
solvent evaporated under reduced pressure. The residue
was chromatographed by CC (column chromatography)
using silica gel, Hex:AcOEt 7:3 to give the corresponding
hydroxylamine. Subsequently the hydroxilamine was
disolved in dried ethyl ether and HCl(g) was bubbled to
obtain the salt (3c-f).
-1
-1
5000 UI mL penicillin and 5 mg mL streptomycin.
MDA-MB-231 (breast adenocarcinoma) was grown
in RPMI 1640 medium (Lonza) supplemented with 10%
-1
serum (Biowest) and 2 mM L-glutamine, 5000 UI mL
-
1
penicillin and 5 mg mL streptomycin. Eight thousand
cells were seeded in 96 well plates and grown in a 5%
O‑(2‑Bromobenzyl)hydroxylamine hydrochloride (2c)
1
Yield 78%; mp 98-99 °C; H NMR (300 MHz, CDCl )
CO atmosphere at 37 °C for 24 h before treatment. Cells
3
2
d 4.76 (s, 2H, OCH ), 7.18 (td, 1H, J 7.5, 1.5 Hz, H-5 Ar),
were screened for mycoplasma contamination before each
experiment, by means of DAPI staining (Invitrogen) and
fluorescence microscopy camera (Motic CamPro 282A).
2
7
.33 (t, 1H, J 7.5 Hz, H-4 Ar), 7.44 (dd, 1H, J 7.5, 1.5 Hz,
13
H-6Ar), 7.57 (d, 1H, J 7.5 Hz, H-3Ar); C NMR (75 MHz,
CDCl ) d 77.14 (OCH ), 123.54 (C-Br), 127.35 (C-5 Ar),
3
2
1
29.31 (C-4Ar), 129.95 (C-6Ar), 132.74 (C-3Ar), 136.90
MTT assay
79
+
(CCH O Ar). Mass spectrum, m/z (I , %): 201 [M( Br)] ,
Cancer cells from breast (MDA-MB-231), prostate
(PC-3) and lung (A-549) and healthy fibroblasts from lung
2
rel
169 (100), 90 (25).
-1
(MRC-5) were seeded in a 96-well plate (5000 cells well )
O‑(2‑Bromoallyl)hydroxylaminehydrochloride (2d)
for 24 h. Cells were then treated with concentrations
in a range of 0 to 50 μM of compounds 1 and 3 (a
1
Yield 87%; mp 153-154 °C; H NMR (300 MHz, CDCl )
3
d 4.30 (s, 2H, OCH ), 5.70 (d, 1H, J 1.2 Hz, BrC=CH), 5.93
compound was considered active if IC ≤ 50 μM). Each
2
50
13
(
d, 1H, J 1.2 Hz, BrC=CH); C NMR (75 MHz, CDCl ) d
compound at each concentration was tested as a triplicate.
After 48 h of incubation, cells were washed and then
3,4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium
bromide (MTT) was added for 4 h. Purple formazan crystals
formed were then dissolved in DMSO and the plates
were read under 570 nm. IC values were determined by
3
7
9.60 (OCH ), 119.05 (BrC=CH), 129.71 (BrC=CH). Mass
2
79
+
spectrum, m/z (I , %): 151 [M( Br)] , 119 (61), 39 (100).
rel
O‑(3‑Methylbut‑2‑en‑1‑yl)hydroxylamine hydrochloride (2e)
1
Yield 76%; mp 160-161 °C; H NMR (300 MHz,
50
acetone-d ) d 1.72 (s, 3H, CH ), 1.74 (s, 3H, CH ), 4.92 (d,
non-linear regression. Vincristine was used as a positive
6
3
3
2
H, J 7.4 Hz, OCH ), 5.37 (t, J 7.4 Hz, 1H, HC=C(CH ) );
control at IC determined by MTT assay under the same
conditions described above.
2
3
2
50
1
3
C NMR (75 MHz, acetone-d ) d 18.20 (CH ), 25.72
6
3
(
(
CH ), 72.12 (OCH ), 116.83 (HC=C(CH ) ), 143.12
3
2
3 2
HC=C(CH ) ).
Supplementary Information
3
2
O‑(3‑Phenyl‑2‑propenyl)‑1‑hydroxylamine hydrochloride
2f)
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
(
1
Yield 90%; mp 176-177 °C; H NMR (300 MHz,
CDCl -CD OD) d 4.72 (d, 2H, J 6.9 Hz, CH ), 6.23-6.34
3
3
2

1
84
Synthesis and in vitro Antiproliferative Activity of Flavone and 6-Hydroxyflavone Oxime Ethers Derivatives
J. Braz. Chem. Soc.
Acknowledgments
6. Baker, W.; Harborne, J. B.; Ollis, W. D.; J. Chem. Soc. 1952,
1
303.
The authors thank Pontificia Universidad Javeriana
PUJ) and COLCIENCIAS for the financial support through
the projects ID 5564 and 120365843351; J. E. D. thanks
PUJ for the teaching assistantship.
7. Gulati, K. C.; Ray, J. N.; Curr. Sci. 1936, 5, 75.
8. Meshcheryakova, L. M.; Tsikalova, T. S.; Orlova, E. K.; Burov,
Yu. V.; Speranskaya, N. P.; Zagorevskii, V.A.; Khim. Farm. Zh.
1976, 10, 37.
(
9
. Green, J.; Aronov, A.; Pierce, A.; US pat. 7547794 B2 2009
(CA2522595A1).
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