Antiproliferative activity of novel isatinyl/indanyl nitrones (INs) as potential spin trapping agents of free radical intermediates

Article abstract of DOI:10.1039/c7md00537g

A series of ketonitrones derived from isatin and indanone (INs) were synthesized and evaluated for their antiproliferative activities against several human cancer cell lines. Then, the antioxidant properties of these substrates were measured by the DPPH test to report their biological activity in terms of their spin trapping action. In particular, one substrate has showed very high biological and scavenging activity, probably due to the strong correlation between its spin trapping activity and structure.

Full text of DOI:10.1039/c7md00537g

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DOI: 10.1039/C7MD00537G
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ARTICLE
Antiproliferative Activity of Novel Isatinyl/Indanyl Nitrones (INs)
as Potential Spin Trapping of Free Radical Intermediates
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a,c
a
Loredana Maiuolo,* Giordana Feriotto, Vincenzo Algieri, Monica Nardi, Beatrice Russo, Maria
d
a
a
e
a
Luisa Di Gioia , Emilia, Furia, Matteo Antonio Tallarida, Carlo Mischiati, Antonio De Nino*
DOI: 10.1039/x0xx00000x
Abstract A series of ketonitrones derived from isatin and indanone (INs) were synthesized and evaluated for their
antiproliferative activities against several human cancer cell lines. Then, antioxidant properties of these substrates was
measured by DPPH test to report the biological activity with their spin trapping action. In particular, one substrate has
showed very high both biological and scavenging activity, probably due to strong correlation between spin trapping
activity and structure.
www.rsc.org/
their anti-cancer action is not known. It is probable that the ability
to scavenge radical intermediates that are produced during disease
Introduction
Small organic molecules have proven to be invaluable tools for processes, including cancer, is the basis for their anti-cancer
4
investigating biological systems, but there is still much to learn from activity. Moreover, their action on important membrane enzymes
1-3
their use.
and as anti-inflammatory agents seems to contribute to the
Nitrones are small molecules that have general chemical formula X- magnification of their antioxidant activity.
CH=NO-Y. Their structural nature confers them their “spin-trap” Isatin and oxindole derivatives have found wide application in
10
ability for trapping free radical intermediates (R•), forming stable medicinal chemistry, including as potential anticonvulsant, cyclin-
4
11
12
13
radical adducts (X-CHR=NO•-Y). In fact, studies of spin trapping dependent kinase 2-inhibitors,
poxvirus,
ectomelia,
14
15
methods involved the reaction of nitrones with reactive free rhinovirus, HIV-1 and the corona-virus responsible for severe
1
6
radicals such as hydroxyl (•OH), lipid alkoxy (•OL) or lipid acute respiratory syndrome (SARS) . Moreover, the insertion of an
hydroxyperoxyl (•OOL) radicals, observing the formation of more oxindole nucleus in spirocompounds has recently emerged because
stable radicals that can be classified and quantified by Electron Spin of their prevalence in various natural products and biologically ac-
1
7
Resonance (ESR) or Electron Paramagnetic Resonance (EPR) tive molecules. In fact, the key of their activity seems to be
5-7
spectroscopy.
combination of a spiro carbon and a variously substituted oxindole
Considering a) that the biological systems may actively produce core. Therefore, the oxindole moiety could be considered as a
reactive oxigen species (ROS) and reactive nitric oxide species useful tool in drug discovery.
(
RNS), b) that specific oxidation products are produced from It is generally been recognized that the incorporating of different
reaction between biological molecules and ROS and RNS, c) that bioactive scaffolds into one molecule is the powerful strategy to
ROS and RNS play an important role in many pathologic diseases, construct substrates with structural novelty and biological
18
nitrones and, in particular, PBN-nitrones where X is a phenyl group potential. In an attempt to generate novel molecular entities with
and Y is a tert-butyl group were recently noted as therapeutics for amplified spin trapping and antiproliferative activity on cancer cells,
8-9
their wide spread anti-cancer activity. The mechanistic basis of we synthesized some isatin and indanone ketonitrones, conjugating
the simultaneous presence of an oxindole-like ring and a nitrone
portion.
a.
Dipartimento di Chimica e Tecnologie Chimiche, Via P. Bucci, cubo 12C,
Università della Calabria, 87036 Rende (CS), Italy
Dipartimento di Chimica e Chimica Farmaceutica, via L. Borsari 46, Università
degli Studi di Ferrara, 44121 Ferrara, Italy
Dipartimento di Agraria, Università Telematica San Raffaele, Roma, Via di Val
Cannuta, 247, 00166, Italy.
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Edificio
Polifunzionale, Università della Calabria, 87036 Rende (CS), Italy.
Dipartimento di Scienze Biomediche e Chirurgico Specialistiche, Via L. Borsari 46,
Università di Ferrara, 44121 Ferrara, Italy
b.
c.
d.
e.
Results and discussion
In literature, few examples of synthetic procedures of nitrones of
oxindole derivatives have been reported thus far.
19-22
Recently, we
have realized an environmental-friendly approach to synthesize
aldo- and ketonitrones by solvent-free condensation of alkyl- or
arylhydroxylamines hydrochlorides with aromatic aldehydes under
23
†
Footnotes relating to the title and/or authors should appear here.
microwave irradiation.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Herein, we report a practical and stereoselective synthesis of
isatinyl and indanyl nitrones (INs) starting from substituted
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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hydroylamines 1a-c and isatin derivatives 2a-c or indanone 3a-b, as Compounds 4c-e inhibited the proliferation of osteosaVirecwoAmrtiacleMOGnl6in3e
cells compared to untreated cells (Fig. 1A, ICDOIa:b10o.u10t3797/Cµ7MM)D.00537G
showed in table 1. Then, we investigated the antiproliferative
activity of these substrates on MG63 and TE85 human
osteosarcoma cell lines and K562 human erythroleukemic cell line,
and we next sought to correlate it with their antioxidant properties
measured by DPPH assay.
50
In Figure 1B are shown the antiproliferative activities of 4a and 4c-d
nitrones on TE85 cells (IC50 about 32 µM). The active compounds
are less effective in inhibiting the proliferation of K562 cells, with
the exception of 4e nitrone with an IC50 of about 78 µM (Fig. 1C).
These results together have shown that the indanyl derivatives do
not possess antiproliferative activity on the tumour cells analyzed,
as well as the derivative 4b with the phenyl substituent. By
contrast, isatinyl nitrones containing methyl or benzyl groups
The target compounds 4a-e and 5a-c were prepared following the
23
our previously reported methodology that consists of the co-
grinding of the ketone and hydroxylamine in a mortar, followed by
transfer of the mixture without use of solvents in an appropriate possess antiproliferative activity.
vessel and further mixing of the solids in a vortex. Finally, the
mixture is placed in a microwave oven without use of solvent and is
irradiated at 600 W or 400 W for a variable time in the order of
minutes.
Table 1. Synthesis of isatinyl and indanyl nitrones
1
Entry
1
R
R
Y
X
MW
W)
Time
(min)
10
Product
Yield
%)
(
(
Me
Ph
5-NO
2
2
NH
CO
CO
CO
CO
CO
600
600
600
600
600
400
400
400
4a
4b
4c
4d
4e
5a
5b
5c
95
92
92
95
92
82
83
85
a
2
H
NH
10
3
4
5
6
7
Bn
Bn
Bn
Me
Me
Bn
5-NO
H
NH
13
NH
12
H
NMe
12
H
CH
CH
CH
2
2
2
CH
CH
CH
2
30
5-F
H
2
2
28
8
30
a
Ketone/alkylhydroxylamine ratio1:2 for all substrates except for entry 2 with ratio 1:3.
The variously substituted products 4a-e and 5a-c were obtained in
high yields (83-95%). The minor reactivity of indanone cycle induced
us both to reduce the MW power and to increase the reaction
times. In all cases, nitrones were obtained as single isomers, (E) 4a-
e and (Z) 5a-c respectively, as confirmed by NOESY experiments. In
particular, for nitrone 4b the formation of only a single product, E-
isomer, is observed respect to Z/E mixture obtained by other
24
synthetic methodologies.
In an attempt to understand the molecular characteristics and the
effects produced by the modifications introduced in our nitrones on
tumour cell proliferation, nitrones prepared according to this
procedure were evaluated for their antiproliferative activity against
25
human osteosarcoma (MG63 and TE85) and chronic myeloid
26
leukemia (K562) cell lines. Cells were cultured for three days in
the absence or in the presence of increasing concentrations of
nitrones and then their vitality was evaluated by dosing the activity
of the oxidative metabolism by the 3-(4,5- dimethylthiazolyil-2)-2,5-
27
Figure 1. Effect of nitrones 4a-e and 5a-c on cell proliferation.
MG63 (A), TE85 (B) and K562 (C) cells were incubated for three days
in the presence of the indicated compounds. Viable cells were
measured by MTT test and reported as % of untreated control. The
arithmetic mean value ± standard deviation of three experiments
performed in triplicates is shown.
diphenyltetrazolium bromide (MTT) assay.
In Figure 1 the
proliferation of the treated cells was expressed as percentage
compared to un-treated control cells.
The data indicated that not all the nitrones had antiproliferative
activity, since the 5a-c and 4b compounds were completely inactive.
2
| J. Name., 2012, 00, 1-3
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The in vitro antioxidant activity of these ketooxindole nitrones 4a–e As can be seen in the table 2, all theDOcIo: m10p.1o0u39n/dCs7MeDx0h0ib5i3t7eGd
and 5a-c was evaluated by using the stable organic free radical antioxidant activity. The substances can be divided in three main
2
8-31
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity.
groups. The first group is composed by two substances (4c and 4e)
In scheme 1 reaction mechanism for the spin-trapping action of with higher values of antioxidant activity than others. In particular,
nitrones 4a-e and 5a-c with a generic free radical (including DPPH the greatest values were observed for 4e, for which the RSA% is
32
-6
radical) is illustrated, in according to literature. It can be noted equal to 60% at a concentration of 2.45 10 mol/L.
from the scheme below that the reaction produces the highly stable Four substances compose the second group with intermediary
3
3-34
nitroxyl radical 6.
antioxidant activity (4a-b, 5a and 5c). For all of these RSA% was
-6
major than 60% at a concentration equal to 6.25 10 mol/L. In the
third group, only two compounds with lower less antioxidant
activity were included: 4d and 5b. For these last two compounds
O
O
R
R
N
N
R2
-6
RSA% was 80% at a concentration equal to 9.10 10 mol/L. As can
R2
R1
X
+
R1
X
be seen in the table 2 and in figure 2, all nitrones 4a-e and 5a-c
exhibited a radical scavenging activity significantly major than those
of the reference (BHT). We have not investigated concentrations
Y
Y
4
a-e; 5a-c
6
-6
higher than 9.10 10 mol/L as the RSA% trend reaches a plateau.
The RSA% of nitrones 4a-e and 5a-c may be compared with the
value of archetypal (Z) α-phenyl-N-tert-butylnitrone (PBN) reported
X = CO or CH2 ; Y = NH, NMe or CH2
O N
NO2
35
-3
2
2
in literature. Its RSA% is 1.4 ± 0.9 at concentration of 0.5 10
R = Generic free radical or
mol/L calculated through DPPH test, demonstrating an antioxidant
property highly minor respect to our nitrones.
N
N
NO2
DPPH
Scheme 1. Reaction mechanism of nitrones 4a-e and 5a-c with free
radicals
When DPPH reacts with a radical scavenger its maximum
absorbance decreases. A freshly prepared DPPH solution displayed
a deep purple colour with an absorption maximum at 517 nm. The
ethanolic solution of DPPH was added to the solution of the
synthesized compounds in ethanol. After 10-min incubation in dark,
the absorbance was measured by using absolute ethanol as a blank.
BHT (butylated hydroxytoluene) was used as reference. In the
presence of antioxidant the DPPH absorbance decreases. The Figure 2. Graphical presentation of in vitro DPPH radical scavenging
antioxidant activity was calculated as radical scavenging activity activity of compounds relative to the standard antioxidant BHT.
(
RSA%) as expressed in the equation 1:
For all tested compounds the EC50 values were also calculated by
using GraphPad Prism 5.01 program, as reported in literature (table
RSA% = [(A – A ) / A ] × 100
(1)
0
i
0
36
2). The EC50 is the antioxidant concentration required to obtain a
where A and A are the DPPH absorbance at 517 nm in the absence 50% radical inhibition. As can be seen in table 2 the value of EC50 of
0
i
and presence of the synthesized compounds, respectively. The all eight synthesized nitrones are higher than reference substance
RSA% for ketonitrones 4a–e and 5a-c at five different (BHT).
-6
concentrations (i.e., 1.15, 2.45, 4.00, 6.25 and 9.10, 10 mol/L) of The antioxidant properties of INs seems to converge quite well with
the tested compounds with DPPH at 517 nm was reported in table the biological results, confirming the essentiality of isatinyl ring. In
2.
particular, the presence both of aromatic systems and electron-
donating groups (e.g. methyl group) improve the performance, as
can particularly be seen for 4e substrate.
Table 2. Percentage of in vitro radical scavenging activity of INs
-
6
Concentrations (10 mol/L)
Compounds
1.15
2.45
4.00
1.1±0.5
1.2±0.5
60±1
6.25
72±2
9.10
79±2
EC50
Experimental
4
a
b
-
-
1.2±0.2
1.3±0.2
1.0±0.2
1.8±0.1
0.6±0.3
1.3±0.2
1.8±0.1
1.2±0.2
3.8±0.5
4
1.1±0.5
1.2±0.5
-
69±2
80±2
Commercial starting materials were used without further
purification. Reactions were monitored by TLC using silica plates 60-
F264, commercially available from Merck. H and C NMR spectra
were recorded at 300 MHz and 75 MHz, respectively, in CDCl and
DMSO-d6 using tetramethylsilane (TMS) as internal standard
4c
-
67±2
80±2
4d
4e
5a
5b
1.1±0.5
1.1±0.5
60±1
-
1.2±0.5
68±2
1.2±0.5
70±2
78±2
1
13
1.2±0.5
79±2
3
-
1.1±0.5
1.1±0.5
1.3±0.5
11.7±0.5
60±1
78±2
-
-
1.2±0.5
70±2
79±2
(
Bruker ACP 300 MHz). Chemical shifts are given in parts per million
5c
1.1±0.5
-
1.2±0.5
4.6±0.5
79±2
and coupling constants in Hertz. The stereochemistry were
BHT
23.0±0.5
27.0±0.5
This journal is © The Royal Society of Chemistry 20xx
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established by NOESY experiments. Purity was verified by NMR and ESI (+)-MS: m/z [M+H] calcd for for C H N O 298.0828, found:
15
12
3
4
View Article Online
DOI: 10.1039/C7MD00537G
HPLC spectra.
298.0816.
LC-MS analysis were carried using an Agilent 6540 UHD Accurate -
Mass Q-TOF LC–MS (Agilent, Santa Clara, CA) fitted with a
electrospray ionisation source (Dual AJS ESI) operating in positive
(
E)-N-(2-oxoindolin-3-ylidene)-1-phenylmethanamine oxide 4d
1
ion mode. Chromatographic separation was achieved using a C18 Orange solid, 95% yield (0.80 g). H NMR (300 MHz, DMSO-d ): δ
6
RP analytical column (Poroshell 120, SB-C18, 50 × 2.1 mm, 2.7 μm) 5.89 (s, 2H, CH Bn), 6.91 (d, J = 7.78 Hz, 1H, Ar), 7.10 (dt, J = 0.91,
2
at 30 °C with a elution gradient from 5% to 95% of B over 13 min., A 7.65 Hz, 1H, Ar), 7.25-7.54 (m, 6H, Ar), 8.12 (d, J = 7.65 Hz, 1H, Ar),
13
being H O (0.1% FA) and B CH CN (0.1% FA). Flow rate was 0.4 11.01 (s, 1H, NH), C NMR (75 MHz, DMSO-d ): δ 64.3, 109.8,
2
3
6
ml/min.
118.1, 122.04, 123.9, 128.5, 129.6, 129.1, 131.8, 133.4, 134.2,
MW-assisted reactions were performed in Synthos 3000 instrument 139.6, 161.2. ESI (+)-MS: m/z [M+H] calcd for C H N O 253.0977,
15 13 2 2
from Anton Paar, equipped with a 4x24MG5 rotor and an IR probe found: 253.0977.
as external control of the temperature. 0.3–3 mL. Glass vials sealed
with a dedicated PEEK screw-cup together with a reliable PTFE seal
(
E)-N-(1-methyl-2-oxoindolin-3-ylidene)-1-phenylmethanamine
were used for all reactions. In synthesis of all derivatives the setting
of temperature was always maintained to 180 °C in each
oxide 4e
1
experiment, except for 5a-c where temperature was always Yellow solid, 92% yield (0.76 g). H NMR (300 MHz, DMSO-d ): 3.25
6
maintained to 125 °C.
(s, 3H, CH ), 5.92 (s, 2H, CH ), 7.08 (t, J = 8.26 Hz, 2H, Ar), 7.37-7.50
3 2
13
(
m, 6H, Ar), 8.15 (d, J = 7.34 Hz, 1H, Ar), C NMR (75 MHz DMSO-
d6): δ 26.6, 65.2, 109.2, 117.8, 123.1, 124.0, 129.0, 129.0, 129.59,
32.2, 134.6, 141.5, 160.4. ESI (+)-MS: m/z [M+H] calcd for
C H N O 267.1134, found: 267.1126.
General procedure for synthesis of nitrones 4a-e and 5a-c
1
16 15 2 2
The selected ketone (0.50 g) and appropriate hydroxylamine
derivative (2 eq or 3 eq for N-phenylhydroxylamine) were grinded in
a mortar, placed in apposite vessel and mixed by vortex. The
mixture was transferred to a microwave oven and was irradiated
with the opportune power. After the appropriate time the crude oil
was recrystallized with ethyl acetate (4a-e) or cyclohexane (5a-c).
(
Z)-N-(2,3-dihydro-1H-inden-1-ylidene)methanamine oxide 5a
1
White solid, 82% yield (0.50 g). H NMR (300 MHz, CDCl ): δ 2.90-
3
7
3
.05 (m, 2H, CH ), 3.11-3.22 (m, 2H, CH ), 3.81 (s, 3H, CH ), 7.29-
2 2 3
13
.44 (m, 3H, Ar), 8.84 (d, J = 7.81 Hz, 1H, Ar), C NMR (75 MHz,
CDCl ): δ 28.7, 29.4, 49.6, 124.5, 127.0, 131.0, 134.4, 147.8, 149.7.
3
ESI (+)-MS: m/z [M+H] calcd for C H NO 162.0919, found:
10 12
(
E)-N-(5-nitro-2-oxoindolin-3-ylidene)methanamine oxide 4a
162.0912.
1
Yellow solid, 95% yield, (0.52 g). H NMR (300 MHz, DMSO-d ): 4.28
6
(
8
5
s, 3H, CH ), 7.02 (d, J = 8.73 Hz, 1H, Ar), 8.23 (d, J = 8.73 Hz, 1H, Ar),
3
(
5
Z)-N-(5-fluoro-2,3-dihydro-1H-inden-1-ylidene)methanamine oxide
b
13
.75 (s, 1H, Ar), 11.53 (s, 1H, NH), C NMR (75 MHz, DMSO-d ): δ
6
1.8, 110.2, 118.7, 118.7, 128.0, 133.3, 142.4, 145.2, 161.9. ESI (+)-
1
White solid, 83% yield (0.48 g). H NMR (300 MHz, CDCl ): δ 3.01
(
3
MS: m/z [M+H] calcd for C H N O 222.0515, found: 222.0503.
9
8
3
4
dd, J = 5.43 Hz, 11.73 Hz, 2H, CH ), 3.14 (dd, J = 5.55 Hz, 11.97 Hz,
2
2
H, CH ), 6.80-7.12 (m, 2H, Ar), 8.86 (dd, J = 5.79 Hz, 8.64 Hz, 1H,
2
13
(
E)-N-(2-oxoindolin-3-ylidene)aniline oxide 4b
Ar), C NMR (75 MHz, CDCl ): δ 28.8 (d, JCF = 2.28), 29.9, 49.6,
3
1
111.9 (d, J2CF = 23.02), 114.3 (d, J2CF = 22.86), 128.8 (d, J3CF =
Orange red solid, 92% yield, (0.74 g). H NMR (300 MHz, DMSO-d ):
6
9
.03), 130.9 (d, J4CF = 2.02), 148.1, 150.5 (d, J3CF = 8.95), 164.43 (d,
6
7
1
1
.85 (d, J = 7.80 Hz, 1H, Ar), 7.15 (t, J = 7.65 Hz, 1H, Ar), 7.40 (t, J =
.80 Hz, 1H, Ar), 7.45-7.65 (m, 5H, Ar), 8.27 (d, J = 7.65 Hz, 1H, Ar),
0.78 (s, 1H, NH), C NMR (75 MHz, DMSO-d ): δ 109.7, 118.4,
J1CF = 250.96). ESI (+)-MS: m/z [M+H] calcd for C H FNO
10 11
13
180.0825, found: 180.0818.
6
21.7, 123.9, 124.1, 128.7, 130.1, 132.2, 134.4, 140.9, 146.3, 159.9.
ESI (+)-MS: m/z [M+H] calcd for C H N O 239.0821, found:
14 11 2 2
(
5
Z)-N-(2,3-dihydro-1H-inden-1-ylidene)-1-phenylmethanamine oxide
c
2
39.0817.
1
White solid, 85% yield (0.76 g). H NMR (300 MHz, CDCl ): δ 2.91-
3
(
E)-N-(5-nitro-2-oxoindolin-3-ylidene)-1-phenylmethanamine oxide 3.09 (m, 2H, CH ), 3.10-3.23 (m, 2H, CH ), 5.11 (s, 2H, CH Bn), 7.21-
2 2 2
4
c
7.45 (m, 7H, Ar), 7.51 (d, J = 7.16 Hz, 1H, Ar), 8.92 (d, J = 7.63 Hz, 1H,
Ar), C NMR (75 MHz, CDCl ): δ 29.0, 29.2, 66.6, 124.5, 127.1,
3
13
1
Yellow solid, 92% yield (0.64 g). H NMR (300 MHz, DMSO-d ): 5.91
6
1
27.2, 128.2, 128.3, 128.9, 131.1, 133.4, 134.8, 147.7, 149.1. ESI (+)-
(
(
(
s, 2H, CH ), 7.07 (d, J = 8.73 Hz, 1H, Ar), 7.31-7.58 (m, 5H, Ar), 8.28
dd, J = 2.43 Hz, 8.73 Hz, 1H, Ar), 8.84 (d, J = 2.43 Hz, 1H, Ar), 11.70
s, 1H, NH) C NMR (75 MHz, DMSO-d ): δ 65.6, 110.4, 118.8,
2
MS: m/z [M+H] calcd for C H NO 238.1232, found: 238.1230.
16 16
13
6
1
19.0, 128.3, 129.1, 129.1, 129.6, 133.2, 134.1, 142.6, 145.4, 161.9.
4
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Antiproliferative activity evaluation
Acknowledgements
View Article Online
DOI: 10.1039/C7MD00537G
We thank the Italian Ministry of University and Scientific
MG63 and TE85 human osteosarcoma cell lines were maintained in Research (MIUR) for a doctoral grant and the University of
DMEM, while K562 human chronic myeloid leukemia cell line in Calabria for financial support.
RPMI 1640. Culture medium was supplemented with 10% fetal
bovine serum, penicillin (100 U/mL), streptomycin (100 U/mL) and
Notes and references
glutamine (2 mM); incubation was at 37 °C in a 5% CO atmosphere.
2
Compounds were solubilized in DMSO at 40 mM, kept at -80°C in
the dark and diluted in complete medium immediately before use.
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