A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity

Article abstract of DOI:10.21577/0103-5053.20180153

Novel isatinspirooxazine derivatives were designed and synthesized as potential anti-proliferative agents. The new compounds were obtained from aldol condensation reactions between isatin and 3-(hydroxyimino)butan-2-one in the presence of an organic base in order to generate an aldol adduct, followed by cyclization in trifluoroacetic acid, providing the desired isatinspirooxazines in 30 to 80percent yield. All the synthesized compounds, including the starting material and the synthetic intermediates, were tested for in vitro anti-proliferative activity against cell lines MCF-7 and MDA-MB231 (breast cancer) and A549 (lung cancer), highlighting the compound 4-methyl,5'-methyl-spiro[(5-aza-4-eno-3-one-cyclohexane)-1,3'-(1H-indol-one)] with an IC₅₀ (half maximal inhibitory concentration) = 0.34 μM, more potent than the reference drug, doxorubicin (IC₅₀ = 1.88 μM), in breast cancer line MDA-MB231.

Full text of DOI:10.21577/0103-5053.20180153

http://dx.doi.org/10.21577/0103-5053.20180153
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-12, 2018
Short Report
Printed in Brazil - ©2018 Sociedade Brasileira de Química
A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro
Anti-Proliferative Activity
Iara S. Santos,a,b Fabiana S. Guerra,c Lucas F. Bernardino,a Patrícia D. Fernandes,c
Lidilhone Hamerskib and Bárbara V. Silva*,a
aInstituto de Química (IQ), Universidade Federal do Rio de Janeiro (UFRJ),
Av. Athos da Silveira Ramos, 149, Cidade Universitária, Ilha do Fundão,
21949-900 Rio de Janeiro-RJ, Brazil
bInstituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro,
Avenida Carlos Chagas Filho, 373, Edifício do CCS, Bloco H, Cidade Universitária,
21941-902 Rio de Janeiro-RJ, Brazil
cLaboratório de Farmacologia da Dor e da Inflamação, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Edifício do CCS,
Bloco J, Cidade Universitária, 21941-902 Rio de Janeiro-RJ, Brazil
Novel isatinspirooxazine derivatives were designed and synthesized as potential
anti-proliferative agents. The new compounds were obtained from aldol condensation reactions
between isatin and 3-(hydroxyimino)butan-2-one in the presence of an organic base in order to
generate an aldol adduct, followed by cyclization in trifluoroacetic acid, providing the desired
isatinspirooxazines in 30 to 80% yield. All the synthesized compounds, including the starting
material and the synthetic intermediates, were tested for in vitro anti-proliferative activity against
cell lines MCF-7 and MDA-MB231 (breast cancer) and A549 (lung cancer), highlighting the
compound 4-methyl,5’-methyl-spiro[(5-aza-4-eno-3-one-cyclohexane)-1,3’-(1H-indol-one)] with
an IC50 (half maximal inhibitory concentration) = 0.34 μM, more potent than the reference drug,
doxorubicin (IC50 = 1.88 μM), in breast cancer line MDA-MB231.
Keywords: isatinspirooxazine, anti-proliferative activity, aldol adduct
Introduction
Isatin (1H-indole-2,3-dione) is a small, versatile and
widely applicable pharmacological molecule. These
characteristics make isatin and its derivatives attractive
to many research groups as resources for chemical and
pharmacological studies. This molecule and its analogues
display diverse types of biological activity, such as
trypanocidal, anticonvulsant, antimicrobial, antiprotozoal
and anti-inflammatory activities.2-8
Furthermore, isatin and its derivatives are effective
against several cancer cell lines, capable of reducing the
proliferation of cancer cells through the inhibition of several
proteins, particularly protein kinases.9-12
The oxazines are heterocyclic derivatives of 2H- and
4H-pyran. They are formed by a six-membered ring that
contains an oxygen and nitrogen heteroatom.13 Oxazines
and their derivatives have broad pharmacological
activity, including antimalarial, antimicrobial, anti-HIV,
antidepressant and anticancer activities.14-21
Cancer is the second largest cause of death globally,
and despite technological and social development, it
is estimated that in 2018, in the United States alone,
1,735,350 new cases of cancer and 609,640 cases of cancer
deaths will be identified.Additionally, there is an alarming
rise in the incidence of new types of cancer, highlighting
the issue as a public health problem for health systems
worldwide. Although the number of new anticancer
therapies has increased in the past decade, the mechanisms
for decreasing the incidence of cancer remains unclear.
New anticancer therapies include new chemotherapeutic
agents, but severe side effects persist. Thus, there is a
constant need to develop alternative anticancer drugs with
minimal side effects.1
*e-mail: barbara@iq.ufrj.br

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A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity J. Braz. Chem. Soc.
In continuation of our interest in the synthesis and
bromo-, iodo-, fluoro-, nitro-, and methyl-groups at the
5 and/or 7 positions of the aromatic ring (1a-i). These
compounds were synthesized according to previous
reports.26-29
The continuous SAR studies on isatin have shown that
C-3-substituted isatins have several biological activities.
Examples include the hydrazone, imine and hydrazide
moieties, which exhibited specific and potent receptor
tyrosine kinases (RTKs) and cyclin-dependent kinases
(CDKs) inhibition.30,31
biological activities of isatin derivatives, we describe
herein the synthesis of a novel series of isatin derivatives
containing the 4H-1,2-oxazin-4-one nucleus, and the
in vitro evaluation of cytotoxic activity of all products,
including the key intermediates, against three human
cancer cell lines.
Results and Discussion
Chemistry
Therefore, isatins (1a-i) were condensed with
3-(hydroxyimino)butan-2-one (4) in tetrahydrofuran, and
in the presence of a catalytic amount of diethylamine or
triethylamine, to directly yield the aldol adduct derivatives
(Table 1).
In the reactions with diethylamine,32,33 there was total
consumption of the starting material over periods that varied
from 7 to 21 days at room temperature. The different yields
can be justified by the different solubility of the products
in the liquid-liquid extraction solvent (ethyl acetate).
Compounds 5a, 5b and 5i were obtained in the lowest
yields, while 5d showed excellent yields.
Reactions with substrates (1e-g), containing two
chlorine or bromine atoms at positions 5 and 7, respectively,
and a nitro group at position 5 of the aromatic ring,
failed when we used the diethylamine base. In this
cases, 2-(2-aminophenyl)-N,N-diethyl-2-oxoacetamides
were obtained in 50, 65 and 70% yield by employing
The anticancer role of isatin derivatives is
well-established in the literature. The most significant
is sunitinib malate (Sutent®), used in the treatment of
advanced renal carcinoma, gastrointestinal stromal tumors,
and pancreatic neuroendocrine tumors. This compound is
a multikinase inhibitor targeting VEGFR-1, VEGFR-2,
PDGFRb, and c-Kit.22,23 Furthermore, the pharmaceutical
company Boehringer reported a new isatin-based triple
angiokinase inhibitor, BIBF1120 II, in phase III clinical
trials for non-small cell lung cancer.24,25 The fluorine atom at
C-5 of sunitinib malate is essential to its activity, and several
structure-activity relationship (SAR) studies revealed that
substitution at position 5 was favored over positions 4, 6 or
7, providing greater anti-oncogenic activity.10,11
For these reasons, we decided to use as our starting
material isatin and its derivatives containing chloro-,
Table 1. Reaction conditions and yields for the synthesis of aldol adduct derivatives
1
5
R’
H
R’’
H
Base
Et2NH
Et2NH
Et2NH
Et2NH
Et3N
Temperature
time
7 d
Yield / %
1a
5a
5b
5c
5d
5e
5f
r.t.
r.t.
47
44
57
91
45
65
70
61
36
1b
Br
Cl
I
H
9 d
1c
H
r.t.
7 d
1d
H
r.t.
8 d
1e
Cl
Br
NO2
F
Cl
Br
H
reflux
reflux
reflux
r.t.
13 h
13 h
12 h
7 d
1f
Et3N
1g
5g
5h
5i
Et3N
1h
H
Et2NH
Et2NH
1i
Me
H
r.t.
21 d
Rr.t..t:.:rorooommtetemmppeeraratuturere..

Vol. 00, No. 00, 2018
Santos et al.
3
substrates 1e, 1f and 1g, respectively. The formation of
the oxoacetamides involves the opening of the heterocycle
ring, i.e., nucleophilic attack of the pair of electrons
from diethylamine to the amide carbonyl (C-2). At first
this was not expected, since the amide carbonyl (C-2)
is less electrophilic than the ketone (C-3). However, the
highly electron withdrawing effect of the two chlorine
or bromine atoms and the nitro group probably increased
the electrodeficiency of both carbonyls. The nucleophilic
attack on C-3 is reversible, while attack on C-2 leads to a
nucleophilic acylic substitution.
The exchange of this for the trimethylamine, a
non-nucleophilic base, and heating under reflux conditions
afforded the desired products in moderate yields.
On the other hand, heterocycles such as the oxazines
and their derivatives have been shown to have a wide
range of pharmacological activity including anticancer.20,21
Thus, in the following step, the new isatin derivatives
were utilized for oxazine derivative addition on C-3. This
strategy could potentially provide compounds that exhibit
anticancer activity.
MDA-MB231 (human mammary epithelial adenocarcinoma
triple negative, ATCC No. HTB-26) by employing the
3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay. The results are summarized in
Table 2. Half maximal inhibitory concentration (IC50)
values are expressed in μM and compared against
doxorubicin as the positive control.
The results obtained for the halogenated, nitro and
methyl isatin derivatives at C-5 and C-7 (Table 2) did
not corroborate those reported in the literature, except
for the mono-halogen compounds (Br, Cl and I) at C-5,
which were cytotoxic to the three cancer cell lines tested.
5-Methyl-isatin (1i) demonstrated a very intriguing result
as, besides cytotoxicity, selectivity of the compound for
the MDA-MB231 cell line (IC50 = 8.84 μM) was observed.
It was inactive against the MCF-7 cell line. This was of
particular interest as both lineages are breast tumor-derived,
but the MDA-MB231 tumor is more aggressive than the
MCF-7 tumor. This compound was also cytotoxic for the
A549 cell line (IC50 = 6.24 μM).
The addition of 3-(hydroxyimino)butan-2-one (4) at C-3
to yield intermediate aldol adducts (5a-i) also generated
compounds that had no significant effect on the cytotoxicity
of this compound on MCF-7, MDA-MB231, and A549
cells. However, the derivative obtained from 5-methyl-
isatin (5i) showed a slight improvement in cytotoxic
activity, and this compound displayed MCF-7 cytotoxicity
(IC50 = 7.14 μM).
The reaction of aldol adducts in trifluoroacetic
acid (TFA) under reflux for 40 min afforded an array of
isatinspirooxazines in moderate to good yields (Scheme 1).
Additionally, aldol adducts (5d) (IC50 = 2.95, 2.79 and
3.66 μM in the MCF-7, MDA-MB231 and A549 lines,
respectively) and (5i) (IC50 = 7.14, 6.31 and 5.43 μM in
the MCF-7, MDA-MB231 and A549 lines, respectively)
exhibited an increase in cytotoxic activity when compared
to their respective starting materials, isatins (1d) and (1i).
The substance (5a) also presented activity superior to (1a).
The last series of compounds tested were the oxazine
derivatives. Again, the mono-halogenated compounds
displayed similar cytotoxicity to their precursors.
However, again the 5-methyl-isatin derivative (6i) showed
an intriguing result: the spirooxazine group at C-3
increased cytotoxic activity specific to the MDA-MB231
cell line (IC50 = 0.34 μM) that was also more potent than
doxorubicin (IC50 = 1.88 μM). This can also be observed
for substance (6b). Though it did not exceed the activity
of the reference standard, it was more active than (1b) and
(5d), indicating the importance of the oxazine nucleus for
these derivatives.
Scheme 1. Synthesis of isatinspirooxazines derivatives.
Biological studies-cytotoxic activity
All compounds, including starting materials, were
evaluated for their cytotoxic activity against MCF-7 (human
mammary epithelial adenocarcinoma,ATCC No. HTB-22),
A-549 (human lung adenocarcinoma,ATCC No. CCL-185),
It is worth mentioning that tumors formed by the
MDA-MB231 lineage are very aggressive, with rapid
proliferation and formation of metastases.Additionally, the
cells are triple negative, i.e., not responsive to hormonal

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A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity J. Braz. Chem. Soc.
Table 2. Cytotoxic effect of compounds against MCF-7, MDA-MB, and A549 tumor human cell lines
IC50a / μM
MCF-7
> 30.0
7.29
MDA-MB231
> 30.0
7.67
A549
6.01
5.23
3.62
5.20
NT
Human blood leukocytes
> 100.0
> 100.0
> 100.0
> 100.0
NT
Erythrocytesb
> 100.0
> 100.0
> 100.0
> 100.0
NT
1a
1b
1c
3.87
4.47
1d
5.97
20.44
> 30.0
> 30.0
> 30.0
> 30.0
8.84
1e
> 30.0
> 30.0
> 30.0
> 30.0
> 30.0
6.69
1f
NT
NT
NT
1g
NT
NT
NT
1h
NT
NT
NT
1i
6.24
5.31
5.14
5.64
3.66
NT
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
NT
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
> 100.0
NT
5a
> 30.0
> 30.0
6.6
5b
10.97
6.63
5c
5d
2.95
2.79
5e
> 30.0
> 30.0
> 30.0
> 30.0
7.14
> 30.0
> 30.0
> 30.0
> 30.0
6.31
5f
NT
5g
NT
5h
NT
5i
5.43
8.53
5.91
5.50
6.86
NT
6a
> 30.0
5.58
14.62
2.66
6b
6c
19.3
6.09
6d
> 30.0
> 30.0
> 30.0
> 30.0
> 30.0
> 30.0
4.49
25.81
> 30.0
> 30.0
> 30.0
> 30.0
0.34
6e
6f
NT
6g
NT
6h
NT
6i
13.2
Doxorubicinc
1.83
0.55
aData are presented as half maximal inhibitory concentration (IC50) values (μM) obtained by nonlinear regression for all cell lines from three independent
experiments; bconcentration of compound that induced erythrocyte lysis; cdoxorubicin was used as positive control. Only compounds with an IC50 value
lower than 10 μM for at least one cell line were considered active; NT: not tested.
treatments, implying limitations in available therapeutic
approach for patients with this type of tumor. This has
a greater degree of clinical implication than for tumors
formed by MCF-7 cells, making 6i a promising prototype
molecule for the treatment of this type of cancer.
All substances tested showed a lytic effect at > 100 μM,
indicating that a concentration greater than 100 μM would
be necessary for cell death and that these derivatives may
be selective agents against tumor cells.
In general, our biological results suggest that the
insertion of hydrophobic substituents and electron-
withdrawing groups such as chlorine, bromine, and
iodine at the 5-position of the aromatic ring make isatins
and derivatives more cytotoxic; this is in agreement
with the literature.10,11 However, the most active 6i has
a methyl group, an electron donor by inductive effect
or via hyperconjugation. In a review, Barreiro et al.34
described that the methyl group is able to block metabolic
active sites, thereby increasing the biological stability of
many compounds and generally contributing to increased
lipophilicity, making the molecule less water soluble.
Conclusions
In this work, isatinspirooxazines derivatives were
designed, synthesized and evaluated against the breast
cancer lineages MCF-7 and MDA-MB231, and some
were evaluated using the lung cancer line A549. These
types of cancer are responsible for high mortality in the
modern world.
The anti-proliferative screening results, with the
exception of the substances containing fluorine atoms,
suggested that the attachment of halogens to the aromatic
ring of the isatin nucleus results in more active derivatives

Vol. 00, No. 00, 2018
Santos et al.
5
against the investigated cell lines. However, oxazine (6i),
which has a methyl group attached to the aromatic ring,
was the most active against the MDA-MB231 cell line
compared to all other synthesized substances.
It should be noted that, as the new compounds
investigated are structurally simple and easily accessible,
they represent a very promising starting point for the design
and synthesis of more potent agents for the treatment of
cancer. Mechanism of action studies of these compounds
are underway.
(6.4 g). The reaction mixture was kept at room temperature
under magnetic stirring. The progress of the reaction was
monitored by thin layer chromatography. After the total
consumption of the starting material, the reaction mixture
was poured into ice and the precipitate formed was vacuum-
1
filtered.35 Orange solid; yield 70%; H NMR (400 MHz,
DMSO-d6) d 6.88 (d, J 8.0 Hz, 1H, H-7), 7.65 (s, 1H, H-4),
7.73 (d, J 8.0 Hz, 1H, H-6); 13C NMR (100 MHz, DMSO-d6)
d 114.76 (CH), 120.03 (C), 127.36 (CH), 140.53 (CH),
150.08 (C), 159.45 (C), 183.68 (C); 13C NMR DEPT-135
(100 MHz, DMSO-d6) d 114.76 (CH), 127.36 (CH),
140.52 (CH); IR (KBr) νmax / cm-1 3488, 3209, 3075, 1751,
1708, 1614, 1469, 1448, 1292, 1272, 844.
Experimental
General procedures
5-Chloro-1H-indoline-2,3-dione (1c)
All the compounds obtained in this work were
characterized by spectroscopic and spectrometric methods.
The nuclear magnetic resonance spectra of hydrogen
(1H NMR) and carbon (13C NMR) were collected on a
Bruker apparatus, XRD-400 model, operating at 200, 300 or
500 MHz for 1H nuclei and 50, 75 or 125 MHz for 13C. The
solvent used was deuterated dimethyl sulfoxide (DMSO-d6,
d 2.50, q, J 1.9 Hz and 1H chemical shift of HOD (chemical
shift of H2O) d 3.3, s) or deuterated chloroform (CDCl3, d
7.26, s, and 1H chemical shift of HOD d 1.5, s). Chemical
shifts (d) were expressed in parts per million (ppm) values
and coupling constants (J) in hertz (Hz). The relative
areas of the hydrogen signals were obtained by electronic
integration, and the multiplicity of absorption bands was
indicated according to the convention: s (singlet), brs (broad
singlet), d (doublet), doublet of doublets (dd), t (triplet), q
(quartet), quint (quintet), sext (sextet) and m (multiplet).
The quaternary carbons of compounds, described by the
letter q, were differentiated from CH, CH2 and CH3 with the
aid of distortionless enhancement by polarization transfer
(DEPT)-135.
A solution of 9 mmol (2.09 g) of trichloroisocyanuric acid
(TCCA) in 12 mL of concentrated sulfuric acid (225.3 mmol)
was prepared in an Erlenmeyer flask. Subsequently, 20 mmol
(2.94 g) of isatin (1a) was added to the solution at 0 °C.
The mixture was kept under magnetic stirring for 15 min.
The mixture was poured onto crushed ice. The obtained
precipitate was filtered under vacuum into a Büchner
funnel and washed with ice water.26 Orange solid; yield
70%; 1H NMR (500 MHz, CDCl3 + DMSO-d6) d 6.92 (d,
J 10.0 Hz, 1H, H-7), 7.54 (d, J 10.0 Hz, 1H, H-4), 7.61 (dd,
J 10.0 and 5.0 Hz, 1H, H-6), 11.15 (s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6) d 114.33 (CH), 119.59 (C), 124.60
(CH), 127.29 (C), 137.74 (CH), 149.69 (C), 159.62 (C),
183.82 (C); 13C NMR DEPT-135 (125 MHz, DMSO-d6) d
114.32 (CH), 124.59 (CH), 137.74 (CH); IR (KBr) νmax / cm-1
3479, 3093, 3066, 1760, 1702, 1616, 1469, 1452, 1284, 846.
5-Iodo-1H-indoline-2,3-dione (1d)
In a 500 mL flask containing 80 mL of methanol and
an equal amount of KICl2 solution (freshly prepared),
27 mmol (4 g) of isatin (1a) was added. This reaction was
stirred under magnetic stirring for five days, after which
time another 20 mL of methanol and 20 mL of KICl2
solution was added, and the suspension remained under
stirring for another three days. The reaction medium
was filtered and washed with water to exhaustion. The
product was recrystallized from ethanol and the final yield
The exact masses of compounds were obtained using
a quadrupole-time-of-flight mass spectrometer (MS,
Micromass, UK) in positive electrospray ionization (ESI)
mode, using formic acid for the ionization of the substances
and concentrated phosphoric acid for equipment calibration.
Melting points of the products were determined on a
Mel-Temp apparatus using glass capillaries. Values were
not corrected.
1
was 86%.29 Red solid; yield 86%; H NMR (400 MHz,
CDCl3 + DMSO-d6) d 6.73 (d, J 12.0 Hz, 1H, H-7), 7.70
(s, 1H, H-4), 7.80 (d, J 12.0 Hz, 1H, H-6), 11.09 (s, 1H,
NH); 13C NMR (100 MHz, CDCl3 + DMSO-d6) d 85.40 (C),
115.06 (CH), 119.96 (C), 133.00 (CH), 146.25 (CH),
150.52 (C), 158.86 (C), 183.47 (C); 13C NMR DEPT-135
(100 MHz, CDCl3 + DMSO-d6) d 115.06 (CH), 133.0 (CH),
146.25 (CH); IR (KBr) νmax / cm-1 3467, 3239, 3091, 1747,
1731, 1604, 1459, 1438, 885.
Synthesis of isatins
5-Bromo-1H-indoline-2,3-dione (1b)
In an Erlenmeyer flask, 40 mmol isatin (5.9 g) was
solubilized in a mixture of ethanol (120 mL) and distilled
water (40 mL) with subsequent addition of 40 mmol bromine

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A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity J. Braz. Chem. Soc.
5,7-Dichloro-1H-indoline-2,3-dione (1e)
13C NMR (100 MHz, CDCl3 + DMSO-d6) d 113.10 (CH),
117.85 (C), 120.31 (CH), 133.45 (CH), 143.30 (C),
155.71 (C), 159.83 (C), 182.92 (C); 13C NMR DEPT-135
(100MHz, CDCl3 + DMSO-d6) d113.10 (CH), 120.30(CH),
133.45 (CH); IR (KBr) νmax / cm-1 3332, 3093, 1767, 1751,
1620, 1533, 1471, 1335, 748.
A solution of 20 mmol (4.64 g) of trichloroisocyanuric
acid (TCCA), used as the chlorinating agent, in 12 mL
of sulfuric acid was prepared in an Erlenmeyer flask.
Subsequently, 20 mmol (2.94 g) of isatin (1a) was added to
the solution at 0 °C. The mixture was kept under magnetic
stirring for 30 min. The mixture was poured onto crushed
ice. The obtained precipitate was filtered under vacuum
into a Büchner funnel and washed with ice water.26 Orange
solid; yield 80%; 1H NMR (400 MHz, CDCl3 + DMSO-d6)
d 7.39 (s, 1H, H-6), 7.54 (s, 1H, H-4), 11.55 (s, 1H, NH);
13C NMR (100 MHz, CDCl3 + DMSO-d6) d 118.27 (C),
120.07 (C), 123.24 (CH), 128.33 (C), 136.83 (CH),
147.05 (C), 159.32 (C), 182.91 (C); 13C NMR DEPT-135
(100 MHz, CDCl3 + DMSO-d6)d123.24(CH), 136.83(CH);
IR (KBr) νmax / cm-1 3209, 3088, 1757, 1699, 1614, 1454,
1400, 1290, 876.
General procedure for the preparation of fluoro-1H-indoline-
2,3-dione and methyl-1H-indoline-2,3-dione (1h-i)
In an Erlenmeyer flask containing 9 mL of sulfuric acid,
18 mmol of the respective isonitrosoacetanilide formed
from the anilines was slowly added and kept under magnetic
stirring at room temperature. The end of the reaction
was identified by elevated temperature and gradual color
change. The solution was poured onto crushed ice, and the
precipitate formed was vacuum-filtered on Büchner funnel
and washed with ice water.28
5,7-Dibromo-1H-indoline-2,3-dione (1f)
In an Erlenmeyer flask, the isatin (1) 20 mmol (3.0 g)
was solubilized in a mixture of acetic acid (70 mL)
followed by addition of 47 mmol of bromine (7.5 g).
The reaction mixture was heated at 70-80 °C for 1 h.
The progress of the reaction was monitored by thin
layer chromatography. After the total consumption of
the starting material, the reaction medium was poured
onto ice and the precipitate formed was vacuum filtered.
Subsequently the formed product was treated with 50 mL
of 10 M HCl and redissolved.35 Orange solid; yield 70%;
1H NMR (400 MHz, CDCl3 + DMSO-d6) d 7.53 (s, 1H,
H-6), 7.77 (s, 1H, H-4), 11.38 (s, 1H, NH); 13C NMR
(100 MHz, CDCl3 + DMSO-d6) d 106.55 (C), 115.60 (C),
120.51 (C), 126.52 (CH), 142.23 (CH), 149.07 (C),
159.19 (C), 183.02 (C); 13C NMR DEPT-135 (100 MHz,
CDCl3 + DMSO-d6) d 126.52 (CH), 142.23 (CH); IR (KBr)
5-Fluoro-1H-indoline-2,3-dione (1h)
Red solid; yield 85%; 1H NMR (400 MHz,
CDCl3 + DMSO-d6) d 6.89 (dd, J 8.0 and 4.0 Hz, 1H, H-7),
7.27-7.29 (m, 1H, H-6), 7.33-7.38 (m, 1H, H-4), 11.02
(s, 1H, NH); 13C NMR (100 MHz, CDCl3 + DMSO-d6)
d 111.66 (d, J 24.0 Hz, CH), 113.98 (d, J 8.0 Hz, CH),
118.7 (d, J 7.0 Hz, C), 124.99 (d, J 24.0 Hz, CH), 147.45 (C),
158.58 (d, J 236.0 Hz, C), 159.80 (C), 184.36 (C);
13C NMR DEPT-135 (100 MHz, CDCl3 + DMSO-d6)
d 111.66 (d, J 23.0 Hz, CH), 113.88 (d, J 8.0 Hz, CH),
124.4 (d, J 24.0 Hz, CH); IR (KBr) νmax / cm-1 3446, 3228,
3099, 1761, 1738, 1620, 1489, 1288, 1194, 891.
5-Methyl-1H-indoline-2,3-dione (1i)
Red solid; yield 84%; 1H NMR (500 MHz, DMSO-d6)
d 2.21 (s, 3H, CH3), 6.76 (d, J 10.0 Hz, 1H, H-7), 7.27 (s,
1H, H-4), 7.35 (d, J 10.0 Hz, 1H, H-6), 10.90 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) d20.62 (CH3), 112.56 (CH),
118.30 (C), 125.32 (CH), 132.54 (C), 139.32 (CH),
149.08 (C), 159.99 (C), 185.12 (C); 13C NMR DEPT-135
(125 MHz, DMSO-d6) d 20.60 (CH3), 112.52 (CH),
125.29 (CH), 139.28 (CH); IR (KBr) νmax / cm-1 3442, 3284,
3029, 1743, 1716, 1490, 1303, 1128, 829.
ν
max / cm-1 3459, 3180, 3079, 1743, 1608, 1448, 1288, 873.
5-Nitro-1H-indol-2,3-dione (1g)
In an Erlenmeyer flask, a solution containing 20 mmol
(3.00 g) of isatin (1a) was completely solubilized in
40 mL of concentrated sulfuric acid (H2SO4). This solution
was then cooled to 0 °C in an ice bath and a solution of
19 mmol (1.92 g) of potassium nitrate (KNO3) in 4 mL
of concentrated sulfuric acid (H2SO4) was added to the
reaction medium, which in turn was kept under stirring at
0 °C for 30 min. After this time, the reaction was poured
over crushed ice and the yellow precipitate formed was
vacuum-filtered on a Büchner funnel and washed with
ice water.36 Yellow solid; yield 70%; 1H NMR (400 MHz,
CDCl3 + DMSO-d6) d 7.07 (d, J 8.0 Hz, 1H, H-7), 8.23 (s,
1H, H-4), 8.39 (d, J 8.0 Hz, 1H, H-6), 11.66 (s, 1H, NH);
General procedure for the preparation of (2’-oxo-3’-hydroxy-
3’-indolyl)-3-oxo-4-hydroxy-imino-butane (5a-d, 5h-i)
To a round bottom flask 1 mmol of the isatin (1a-i),
2 mmol of 3-(hydroxyimino)butan-2-one (4), 20 mL of
tetrahydrofuran (THF) and 1 mL of diethylamine were
added. The reaction was maintained at room temperature
and was monitored by thin layer chromatography until

Vol. 00, No. 00, 2018
Santos et al.
7
the total consumption of the starting material. Then,
liquid-liquid extraction was performed with ethyl acetate
(3 × 20 mL), the organic phase was dried over anhydrous
sodium sulfate, filtered and concentrated in vacuum to give
a brown solid, which was purified by silica gel column
chromatography.
J 10.0 and 5.0 Hz, 1H, H-6), 7.29 (d, J 5.0 Hz, 1H, H-4),
10.38 (s, 1H, NH), 12.44 (s, 1H, NOH), *signal overlap
occurred of solvent; 13C NMR (125 MHz, DMSO-d6) d 8.19
(CH3), 44.57 (CH2), 73.37 (C), 111.30 (CH), 124.41 (CH),
125.67 (C), 129.15 (CH), 134.24 (C), 142.14 (C), 155.11 (C),
178.39 (C), 195.66 (C); 13C NMR DEPT-135 (125 MHz,
DMSO-d6) d 8.20 (CH3), 44.56 (CH2), 111.30 (CH),
124.41 (CH), 129.16 (CH); IR (KBr) νmax / cm-1 3504, 3207,
1699, 1621, 1483, 1473, 1184, 993; HRMS (ESI) calcd. for
C12H11ClN2NaO4+ [M + Na]+: 305.03050, found: 305.02975,
error (ppm): 2.45; calcd. for C24H22Cl2N4NaO8+ [2M + Na]+:
587.07021, found: 587.07021, error (ppm): 3.40.
(2’-Oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-imino-
butane (5a)
Light brown solid; yield 47%; mp 160-164 ºC; 1H NMR
(400MHz,DMSO-d6)d1.62(s,3H,H-11),3.26(d, J24.0Hz,
1H, H-8a), 3.82 (d, J 24.0 Hz, 1H, H-8b), 5.99 (s, 1H, OH),
6.78 (d, J 8.0 Hz, 1H, H-7), 6.87 (t, J 8.0 Hz, 1H, H-6), 7.13-
7.18 (m, 2H, H-4 and H-5), 10.21 (s, 1H, NH), 12.39 (s,
1H, NOH); 13C NMR (100 MHz, DMSO-d6) d 7.72 (CH3),
44.08 (CH2), 72.86 (C), 109.41 (CH), 121.17 (CH),
123.60(CH),128.96(CH),131.50(C),142.73(C),154.75(C),
178.22 (C), 195.13 (C); 13C NMR DEPT-135 (100 MHz,
DMSO-d6) d 8.16 (CH3), 44.51 (CH2), 109.85 (CH),
121.62 (CH), 124.04 (CH), 129.42 (CH); IR (KBr)
νmax / cm-1 3376, 3245, 3062, 2913, 1704, 1679, 1618,
1471, 1193, 933; high-resolution (HR)MS (ESI) calcd.
for C12H13N2O4+ [M + H]+: 249.08754, found: 249.08685,
error (ppm): 2.73; calcd. for C24H26N4NaO8+ [2M + Na]+:
519.14919, found: 519.14833, error (ppm): 1.65.
5’-Iodo-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5d)
1
Brown solid; yield 91%; mp 162-164 ºC; H NMR
(400 MHz, DMSO-d6) d 1.66 (s, 3H, H-11), 3.33 (d,
J 20.0 Hz, 1H, H-8a), 3.80 (d, J 20.0 Hz, 2H, H-8b)*,
6.12 (s, 1H, OH), 6.66 (d, J 8.0 Hz, 1H, H-7), 7.51 (dd,
J 8.0 and 4.0 Hz, 1H, H-6), 7.54 (d, J 4.0 Hz, 1H, H-4),
10.36 (s, 1H, NH), *signal overlap occurred of solvent;
13C NMR (100 MHz, DMSO-d6) d 8.21 (CH3), 44.58 (CH2),
73.19 (C), 84.37 (C), 112.43 (CH), 132.57 (CH),
134.93 (C), 137.85 (CH), 143.05 (C), 155.11 (C),
178.03 (C), 195.67 (C); 13C NMR DEPT-135 (100 MHz,
DMSO-d6) d 8.21 (CH3), 44.58 (CH2), 112.43 (CH),
132.56 (CH), 137.85 (CH); IR (KBr) νmax / cm-1 3250, 2887,
1714, 1697, 1616, 1443, 1180, 827; HRMS (ESI) calcd. for
C12H11IN2NaO4+ [M + Na]+: 396.96630, found: 396.96525,
error (ppm): 2.64; calcd. for C24H22I2N4NaO8+ [2M + Na]+:
770.94284, found: 770.94151, error (ppm): 1.72.
5’-Bromo-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5b)
1
Brown solid; yield 44%; mp 184-186 ºC; H NMR
(400 MHz, DMSO-d6) d 1.67 (s, 3H, H-11), 3.36 (d,
J 16.0 Hz, 4H, H-8a), 3.82 (d, J 16.0 Hz, 1H, H-8b),
6.16 (s, 1H, OH), 6.77 (d, J 8.0 Hz, 1H, H-7), 7.35 (d,
J 8.0 Hz, 1H, H-6), 7.41 (s, 1H, H-4), 10.39 (s, 1H, NH),
12.46 (s, 1H, NOH); 13C NMR (100 MHz, DMSO-d6)
d 8.20 (CH3), 44.58 (CH2), 73.35 (CH), 111.87 (CH),
113.35 (C), 127.11 (CH), 132.01 (CH), 134.65 (C),
142.57 (C), 155.10 (C), 178.25 (C), 195.67 (C);
13C NMR DEPT-135 (100 MHz, DMSO-d6) d 8.20 (CH3),
44.58 (CH2), 111.86 (CH), 127.11 (CH), 132.00 (CH);
IR (KBr) νmax / cm-1 3485, 3217, 1713, 1697, 1618, 1182,
991; HRMS (ESI) calcd. for C12H11BrN2NaO4+ [M + Na]+:
350.97794, found: 350.97717, error (ppm): 2.19; calcd.
5’-Fluoro-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5h)
1
Brown solid; yield 61%; mp 180-182 ºC; H NMR
(500 MHz, DMSO-d6) d 1.62 (s, 3H, H-11), 3.25 (d,
J 15.0 Hz, 1H, H-8a), 3.76 (d, J 15.0 Hz, 1H, H-8b), 6.12
(s, 1H, OH), 6.72-6.75 (m, 1H, H-7), 6.94-6.98 (m, 1H,
H-6), 7.08 (d, J 10.0 Hz, 1H, H-4), 10.24 (s, 1H, OH),
12.41 (s, 1H, NOH); 13C NMR (125 MHz, DMSO-d6) d
8.26 (CH3), 44.62 (CH2), 73.65 (CH), 110.60 (d, J 7.5 Hz,
CH), 112.12 (d, J 25.0 Hz, CH), 115.52 (d, J 23.8 Hz, CH),
133.93 (d, J 7.5 Hz, C), 139.41 (d, J 1.25 Hz, C), 155.20
(C), 158.33 (d, J 235.0 Hz, C), 178.73 (C), 195.65 (C);
13C NMR DEPT-135 (125 MHz, DMSO-d6) d 8.22 (CH3),
44.58 (CH2), 110.56 (d, J 7.5 Hz, CH), 112.08 (d, J 25.0 Hz,
CH), 115.48 (d, J 23.8 Hz, CH); IR (KBr) νmax / cm-1 3325,
3224, 1716, 1682, 1493, 1188, 1032, 822; HRMS (ESI)
calcd. for C12H11FN2NaO4+ [M + Na]+: 289.06006, found:
289.05928, error (ppm): 2.69; calcd. for C24H22F2N4NaO8+
[2M + Na]+: 555.13035, found: 555.12926, error (ppm): 1.96.
+
for C24H22Br2N4NaO8 [2M + Na]+: 676.96712, found:
678.96611, error (ppm): 2.95.
5’-Chloro-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5c)
1
Brown solid; yield 57%; mp 155-159 ºC; H NMR
(500 MHz, DMSO-d6) d 1.65 (s, 3H, H-11), 3.34 (d,
J 20.0 Hz, 1H, H-8a), 3.60 (d, J 20.0 Hz, 4H, H-8b)*,
6.16 (s, 1H, OH), 6.80 (d, J 10.0 Hz, 1H, H-7), 7.21 (dd,

8
A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity J. Braz. Chem. Soc.
5’-Methyl-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
2.12; calcd. for C24H20Cl4N4NaO8+ [2M + Na]+: 654.99074,
found: 656.98925, error (ppm): 3.03.
imino-butane (5i)
1
Brown solid; yield 36%; mp 156-158 ºC; H NMR
(400 MHz, DMSO-d6) d 1.64 (s, 3H, H-12), 2.20 (s, 3H,
H-11), 3.26 (d, J 16.0 Hz, 1H, H-8a), 3.77 (d, J 16.0 Hz,
1H, H-8a), 5.96 (s, 1H, OH), 6.67 (d, J 8.0 Hz, 1H, H-7),
6.96 (d, J 8.0 Hz, 1H, H-6), 7.02 (s,1H, H-4), 10.12 (s, 1H,
NH), 12.40 (s, 1H, NOH); 13C NMR (100 MHz, DMSO-d6)
d 8.18 (CH3), 21.10 (CH3), 44.65 (CH2), 73.41 (CH),
109.58 (CH), 124.76 (CH), 129.53 (CH), 130.34 (CH),
132.05 (C), 140.71 (C), 155.18 (C), 178.67 (C), 195.56
(C); 13C NMR DEPT-135 (100 MHz, DMSO-d6) d 8.18
(CH3), 21.11 (CH3), 44.61 (CH2), 109.57 (CH), 124.76
(CH), 129.53 (CH); IR (KBr) νmax / cm-1 3346, 3230,
2926, 1709, 1678, 1497, 1041, 1020, 731; HRMS (ESI)
C13H14N2NaO4+ [M + Na]+: 285.08513, found: 285.08434,
error (ppm): 2.77; calcd. for C26H28N4NaO8+ [2M + Na]+:
547.18049, found: 547.17939, error (ppm): 2.0.
5’,7’-Dibromo-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5f)
Light brown solid; yield 65%; mp 181-184 ºC;
1H NMR (400 MHz, DMSO-d6) d 1.68 (s, 3H, H-11),
3.43 (d, J 16.0 Hz, 1H, H-8a), 3.84 (d, J 16.0 Hz, 1H,
H-8b), 6.33 (s, 1H, OH), 7.48 (s, 1H, H-6), 7.63 (s, 1H,
H-4), 10.75 (s, 1H, NH), 12.49 (s, 1H, NOH); 13C NMR
(100 MHz, DMSO-d6) d 8.90 (CH3), 44.76 (C), 73.89 (C),
114.68 (C), 123.26 (CH), 126.30 (C), 128.69 (CH),
135.64 (C), 140.16 (C), 155.02 (C), 178.31 (C), 195.74 (C);
13C NMR DEPT-135 (100 MHz, DMSO-d6) d 8.19 (CH3),
44.76 (CH2), 123.25 (CH), 128.68 (CH); IR (KBr)
νmax / cm-1 3485, 3199, 1713, 1697, 1618, 1375, 1182,
650; HRMS (ESI) calcd. for C12H10Br2N2NaO4+ [M + Na]+:
428.88646, found: 428.88804, error (ppm): 3.68; calcd.
+
for C24H20Br4N4NaO8 [2M + Na]+: 834.78815, found:
General procedure for the preparation of (2’-oxo-3’-hydroxy-
834.78816, error (ppm): 1.20.
3’-indolyl)-3-oxo-4-hydroxy-imino-butane (5e-g)
5’-Nitro-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
imino-butane (5g)
To a round bottom flask under magnetic stirring coupled
to a condenser 2 mmol of 3-(hydroxyimino)butan-2-one
(4), 20 mL of dry tetrahydrofuran and 1 mL of distilled
triethylamine were added.After the mixture was kept under
stirring for 15 min, 1 mmol of the desired isatin was added.
The reaction remained under reflux and was monitored
by thin layer chromatography. After consuming all the
starting material, liquid-liquid extraction was performed
with ethyl acetate (3 × 20 mL), the organic phase was
dried over anhydrous sodium sulfate, filtered into a flask
and concentrated in vacuum to give a brown solid that was
purified by silica gel column chromatography using 1:1
hexane:ethyl acetate as the eluent.
Light brown solid; yield 70%; mp 209-211 ºC; 1H NMR
(500 MHz, DMSO-d6) d 1.61 (s, 3H, H-11), 3.41 (d,
J 20.0 Hz, 1H, H-8a), 3.94 (d, J 20.0 Hz, 1H, H-8b), 6.36
(s, 1H, OH), 6.98 (d, J 10.0 Hz, 1H, H-7), 8.12 (d, 1H,
J 10.0 Hz, H-6), 8.12 (dd, J 10 and 5.0 Hz, 1H, H-4), 10.98
(s, 1H, NH), 12.47 (s, 1H, NOH); 13C NMR (125 MHz,
DMSO-d6) d 8.27 (CH3), 44.74 (CH2), 72.94 (CH),
110.15 (CH), 120.03 (CH), 126.99 (CH), 133.30 (C),
142.47 (C), 150.00 (C), 155.10 (C), 179.14 (C), 195.89 (C);
13C NMR DEPT-135 (125 MHz, DMSO-d6) d 8.23 (CH3),
44.70 (CH2), 110.10 (CH), 119.99 (CH), 126.95 (CH);
IR (KBr) νmax / cm-1 3458, 3250, 1724, 1689, 1628,
1325, 1016, 648; HRMS (ESI) calcd. for C12H11N3NaO6+
[M + Na]+: 316.05456, found: 316.05371, error (ppm):
5’,7’-Dichloro-(2’-oxo-3’-hydroxy-3’-indolyl)-3-oxo-4-hydroxy-
+
imino-butane (5e)
2.69; calcd. for C24H22N6NaO12 [2M + Na]+: 609.11935,
1
Brown solid; yield 45%; mp 212-215 ºC; H NMR
found: 609.118227, error (ppm): 1.77.
(400 MHz, DMSO-d6) d 1.66 (s, 3H, H-11), 3.43 (d,
J 16.0 Hz, 1H, H-8a), 3.84 (d, J 16.0 Hz, 1H, H-8b), 6.31
(s, 1H, OH), 7.33 (d, J 8.0 Hz, 1H, H-6), 7.41 (d, J 8.0 Hz,
1H, H-4), 10.85 (s, 1H, NH), 12.47 (s, 1H, NOH); 13C NMR
(100 MHz, DMSO-d6) d 8.19 (CH3), 44.6 (CH2), 73.89 (C),
114.68 (C), 123.26 (CH), 126.30 (C), 128.69 (CH),
135.64 (C), 140.16 (C), 155.02 (C), 178.31 (C), 195.74 (C);
13C NMR DEPT-135 (100 MHz, DMSO-d6) d 8.19 (CH3),
44.76 (CH2), 123.25 (CH), 128.68 (CH); IR (KBr)
νmax / cm-1 3294, 3082, 1720, 1689, 1618, 1460, 1389,
1115, 1034, 708; HMRS (ESI) calcd. for C12H10Cl2N2NaO4+
[M + Na]+: 338.99146, found: 338.99074, error (ppm):
General procedure for the preparation of 4-methyl-
spiro[(5-aza-4-eno-3-one-cyclohexane)-1,3’-(1H-indol-one)
(6a-i)
In a round bottom flask coupled to a condenser, 1 mmol
of the aldol adduct (6a-i) and 5 mL of trifluoroacetic acid
(TFA) were added. The reaction medium was maintained
under reflux under magnetic stirring for 40 min. The
consumption of the starting material was checked by thin
layer chromatography. Isolation was done with the addition
of saturated sodium carbonate solution (Na2CO3) to pH 9.0

Vol. 00, No. 00, 2018
Santos et al.
9
and liquid-liquid extraction with ethyl acetate, washing the
organic phase with brine, drying the organic phase with
anhydrous sodium sulfate, filtration, and evaporation of the
solvent, resulting in a dark brown solid that was purified
by silica gel column chromatography using hexane: ethyl
acetate in concentrations of 10 to 100% as eluent.
79.35 (C), 112.41 (CH), 126.19 (CH), 127.12 (C), 127.72 (C),
131.49 (CH), 143.16 (C), 143.34 (C), 172.84 (C), 195.83 (C);
13C NMR DEPT-135 (125 MHz, DMSO-d6) d 8.09 (CH3),
42.05 (CH2), 112.37 (CH), 126.15 (CH), 131.45 (CH);
IR (KBr) νmax / cm-1 3209, 1738, 1713, 1562, 1477, 1358,
+
1174, 823; HRMS (ESI) calcd. for C12H9ClN2NaO3
[M + Na]+: 287.0194, found: 287.0202, error (ppm): 2.70;
4-Methyl-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6a)
GC-MS (70 eV) m/z 264 [M]+, 266 [M + 2]+.
Light brown solid; yield 46%; mp 196-199 ºC; 1H NMR
(500 MHz, DMSO-d6) d 1.95 (s, 3H, H-11), 3.20 (d,
J 18.2 Hz, 1H, H-8a), 3.15 (d, J 18.2 Hz,1H, H-8b), 6.93
(d, J 10.0 Hz, 1H, H-7), 7.04 (t, J 7.5 Hz, 1H, H-6), 7.35 (t,
J 7.5 Hz, 1H, H-5), 7.49 (d, J 10.0 Hz, 1H, H-4), 11.03 (s,
1H, NH); 13C NMR (125 MHz, DMSO-d6) d 8.10 (CH3),
42.24 (CH2), 79.54 (C), 110.99 (CH), 123.13 (CH), 125.67
(CH), 125.77 (C), 131.71 (CH), 142.92 (C), 144.38 (C),
173.02 (C), 196.07 (C); IR νmax / cm-1 3410, 3157, 3097,
1728, 1716, 1560, 1475, 1101, 746; HRMS (ESI) calcd.
for C12H10N2NaO3+ [M + Na]+: 253.0583, found: 253.0585,
error (ppm): 0.40; calcd. for C24H20N4NaO6+ [2M + Na]+:
483.1275, found: 483.1279, error (ppm): 0.80; GC-MS (gas
chromatography-mass spectrometry) (70 eV) m/z 230 [M]+.
4-Methyl-5’-iodo-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6d)
Dark brown solid; yield 30%; mp 215-217 ºC; 1H NMR
(400 MHz, DMSO-d6) d 1.94 (s, 3H, H-11), 3.10 (d,
J 20.0 Hz, 1H, H-8a), 3.20 (d, J 20.0 Hz, 1H, H-8b), 6.79
(d, J 8.0 Hz, 1H, H-7), 7.70 (d, J 8.0 Hz, 1H, H-6), 7.96
(s, 1H, H-4), 11.12 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d 8.05 (CH3), 42.06 (CH2), 79.03 (C), 85.78 (C),
113.22 (CH), 128.21 (C), 134.12 (CH), 140.00 (CH),
143.06 (C), 144.12 (C), 172.49 (C), 195.72 (C); IR (KBr)
ν
max / cm-1 3294, 2922, 2850, 1751, 1707, 1614, 1562, 1346,
823; HRMS (ESI) calcd. for C12H9IN2NaO3+ [M + Na]+:
378.9550, found: 378.9549, error (ppm): 0.26; calcd. for
C24H18I2N4NaO6+ [2M + Na]+: 734.9208, found: 734.9212,
error (ppm): 0.54; GC-MS (70 eV) m/z 356 [M]+.
4-Methyl-5’-bromo-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6b)
4-Methyl-5’,7’-dichloro-spiro[(5-aza-4-eno-3-one-
cyclohexane)-1,3’-(1H-indol-one) (6e)
1
Brown solid; yield 50%; mp 198-200 ºC; H NMR
(400 MHz, DMSO-d6) d 1.96 (s, 3H, H-11), 3.12 (d,
J 16.0 Hz, 1H, H-8a), 3.22 (d, J 16.0 Hz, 1H, H-8b), 6.91
(d, J 8.0 Hz, 1H, H-7), 7.56 (d, J 8.0 Hz, 1H, H-6), 7.87
(s, 1H, H-4), 11.17 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d 8.05 (CH3), 42.03 (CH2), 79.22 (C),
112.82 (CH), 114.64 (C), 128.01 (C), 128.78 (CH),
134.25 (CH), 143.09 (C), 143.69 (C), 172.66 (C),
195.70 (C); 13C NMR DEPT-135 (100 MHz, DMSO-d6)
d 8.05(CH3), 42.03 (CH2), 112.82 (CH), 128.78 (CH),
134.25 (CH); IR (KBr) νmax / cm-1 3207, 2981, 2846,
1747, 1616, 1562, 1390, 1105, 814; HRMS (ESI) calcd.
Light brown solid; yield 36%; mp 220-223 ºC; 1H NMR
(400 MHz, DMSO-d6) d 1.97 (s, 3H, H-11), 3.16 (d,
J 16.0 Hz, 1H, H-8a), 3.24 (d, J 16.0 Hz, 1H, H-8b), 7.65
(d, J 8.0 Hz, 1H, H-6), 7.79 (d, J 8.0 Hz, 1H, H-4), 11.68
(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d 8.08 (CH3),
42.13 (CH2), 79.73 (C), 115.66 (C), 125.03 (CH), 127.59 (C),
128.88 (C), 130.89 (CH), 141.45 (C), 143.13 (C), 172.80 (C),
195.51 (C); 13C NMR DEPT-135 (100 MHz, DMSO-d6)
d 8.07 (CH3), 42.13 (CH2), 125.03 (CH), 130.89 (CH);
IR (KBr) νmax / cm-1 3167, 3066, 2922, 2850, 1753,
1720, 1570, 1470, 1163, 889; HRMS (ESI) calcd. for
C12H8Cl2N2NaO3+ [M + Na]+: 320.98097, found: 320.98034,
error (ppm): 1.96; calcd. for C24H16Cl4N4NaO6+ [2M + Na]+:
618.97217, found: 620.96882, error (ppm): 3.22; GC-MS
(70 eV) m/z 298 [M]+, 300 [M + 2]+, 302 [M + 4]+.
+
for C12H10BrN2NaO3 [M + H]+: 308.98748, found:
308.98698, error (ppm): 1.62; calcd. for C24H18Br2N4NaO6+
[M + Na]+: 638.94907, found: 640.94646, error (ppm):
3.11; GC-MS (70 eV) m/z 308 [M]+, 310 [M + 2]+.
4-Methyl-5’-chloro-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6c)
4-Methyl-5’,7’-dibromo-spiro[(5-aza-4-eno-3-one-
cyclohexane)-1,3’-(1H-indol-one) (6f)
Light brown solid; yield 58%; mp 214-217 ºC; 1H NMR
(500 MHz, DMSO-d6) d 1.92 (s, 3H, H-11), 3.09 (d,
J 20.0 Hz, 1H, H-8a), 3.18 (d, J 20.0 Hz, 1H, H-8b), 6.92
(d, J 10.0 Hz, 1H, H-7), 7.39 (dd, J 10.0 and 5.0 Hz, 1H,
H-6), 7.71 (d, J 10,0 Hz, 1H, H-4), 11.13 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) d 8.30 (CH3), 42.08 (CH2),
Yellow solid; yield 71%; mp 225-227 ºC; H NMR
1
(400 MHz, acetone-d6 + TMS) d 2.00 (s, 3H, H-11), 3.23
(d, J 20.0 Hz, 1H, H-8a), 3.30 (d, J 20.0 Hz, 1H, H-8b),
7.80 (d, J 8.0 Hz, 1H, H-6), 7.84 (d, J 8.0 Hz, 1H, H-4);
13C NMR (100 MHz, CD3OD-d4 + DMSO-d6) d 7.70 (CH3),
79.76 (C), 103.62 (C), 115.07 (C), 127.83 (CH), 129.00 (C),

10
A Facile Synthesis of Novel Isatinspirooxazine Derivatives and Potential in vitro Anti-Proliferative Activity J. Braz. Chem. Soc.
136.02 (CH), 143.15 (C), 143.37 (C), 172.46 (C), 195.34 (C);
13C NMR DEPT-135 (100 MHz, CD3OD-d4 + DMSO-d6)
d 7.71 (CH3), 127.82 (CH), 136.02 (CH); IR (KBr) νmax / cm-1
3413, 3174, 2921, 1712, 1613, 1575, 1458, 1307, 1156,
864; HRMS (ESI) calcd. for C12H8Br2N2NaO3+ [M + Na]+:
410.87789, found: 410.87736, error (ppm): 1.29; calcd.
H-12), 3.07 (d, J 20.0 Hz, 1H, H-8a), 3.12 (d, J 20.0 Hz,
1H, H-8b), 6.79 (d, J 10.0 Hz, 1H, H-7), 7.13 (d, J 10.0 Hz,
1H, H-6), 7.30 (s, 1H, H-4), 10.89 (s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6) d 8.00 (CH3), 21.01 (CH3),
42.18 (CH2), 79.54 (CH), 110.63 (CH), 125.77 (C),
126.02 (CH), 131.77 (CH), 132.16 (C), 141.77 (C),
172.90 (C), 195.94 (C); 13C NMR DEPT-135 (125 MHz,
DMSO-d6) d 8.00 (CH3), 21.01 (CH3), 42.18 (CH2), 110.63
(CH), 126.02 (CH), 131.77 (CH); IR (KBr) νmax / cm-1
3411, 3224, 3027, 2921, 1724, 1627, 1560, 1353, 815;
+
for C24H16Br4N4NaO6 [2M + Na]+: 798.76601, found:
798.76636, error (ppm): 0.44; GC-MS (70 eV) m/z 386 [M]+,
388 [M + 2]+, 391 [M + 4]+.
+
4-Methyl-5’-nitro-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6g)
HRMS (ESI) calcd. for C13H12N2NaO3 [M + Na]+:
267.0740, found: 267.0744, error (ppm): 1.50; calcd. for
C26H24N4NaO6+ [2M + Na]+: 511.1588, found: 511.1593,
error (ppm): 0.90; GC-MS (70 eV) m/z 244 [M]+.
Dark brown solid; yield 32%; mp 197-200 ºC; 1H NMR
(400 MHz, DMSO-d6) d 1.98 (s, 3H, H-11), 3.16 (d,
J 20.0 Hz, 1H, H-8a), 3.31 (s, J 20.0 Hz, 4H, H-8b), 7.15
(d, J 8.0 Hz, 1H, H-7), 8.32 (dd, J 8.0 and 4.0 Hz, 1H, H-6),
8.68 (d, J 4.0 Hz, 1H, H-4), 11.74 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d 8.10 (CH3), 41.97 (CH2), 78.90 (C),
111.17 (CH), 122.28 (CH), 126.76 (C), 128.56 (CH),
143.34 (C), 150.75 (C), 173.56 (C), 195.58 (C); 13C NMR
DEPT-135 (100 MHz, DMSO-d6) d8.10 (CH3), 41.97 (CH2),
111.17 (CH), 122.28 (CH), 128.55 (CH); IR (KBr)
νmax / cm-1 3385, 3123, 1739, 1630, 1339, 833; HRMS
(ESI) calcd. for C24H18N6NaO10+ [2M + Na]+: 573.09821,
found: 573.09804, error (ppm): 0.29.
MTT assay-cytotoxic assay
Compounds (0.15-30.0 μM) were tested for cytotoxic
activity against MCF-7 (human mammary epithelial
adenocarcinoma,ATCC No. HTB-22),A-549 (human lung
adenocarcinoma, ATCC No. CCL-185), MDA-MB231
(human mammary epitelial adenocarcinoma triple
negative, ATCC No. HTB-26), freshly prepared human
blood leukocytes and erythrocytes. All cell lines were
maintained in Dulbecco’s Modifed Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum,
2.0 mM glutamine, 100 U mL-1 penicillin and 100 mg mL-1
streptomycin at 37 °C with 5% CO2. Each compound
was dissolved with DMSO and diluted with cell culture
medium to obtain a concentration of 100.0 μM. They were
incubated with the cells for 48 h. The negative control
received the same amount of DMSO (0.005% in the highest
concentration). Doxorubicin was used as a positive control.
The cell viability was determined by reduction of the
yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) to a blue formazan product
after 3 h incubation as described by Denizot and Lang.37
4-Methyl-5’-fluoro-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6h)
Dark brown solid; yield 51%; mp 198-203 ºC; 1H NMR
(500 MHz, DMSO-d6) d 1.92 (s, 3H, H-11), 3.10 (d,
J 20.0 Hz, 1H, H-8a), 3.16 (d, J 20.0 Hz, 1H, H-8b), 6.90-
6.92 (m, 1H, H-7), 7.17 (td, J 10.0 and 5.0 Hz, 1H, H-6),
7.52 (dd, J 10.0 and 5.0 Hz, 1H, H-4), 11.02 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) d 8.03 (CH3), 42.06 (CH2),
79.55 (C), 111.84 (d, J 7.5 Hz, CH), 113.75 (d, J 25.0 Hz,
CH), 118.00 (d, J 23.75 Hz, CH), 127.25 (d, J 8.75 Hz, C),
140.57 (d, J 2.5 Hz, C), 142.99 (C), 158.70 (d, J 236.25 Hz,
C), 172.94 (C), 195.79 (C); 13C NMR DEPT-135 (125 MHz,
DMSO-d6) d 8.04 (CH3), 42.06 (CH2), 111.84 (d, J 7.5 Hz,
CH), 113.75 (d, J 25.0 Hz, CH), 118.00 (d, J 23.75 Hz,
CH); IR (KBr) νmax / cm-1 3429, 3210, 1720, 1630, 1488,
820; HRMS (ESI) calcd. for C12H9FN2NaO3+ [M + Na]+:
271.04949, found: 271.04884, error (ppm): 2.40; calcd.
Erythrocytes hemolysis
The test was performed as described by Campos et al.38
in 96-well plates using a 2% human erythrocyte suspension
in 0.85% NaCl containing 10 mM CaCl2. The compounds
diluted as mentioned above were tested at concentration of
100 μM. After incubation at room temperature for 30 min
and centrifugation, the supernatant was removed and the
liberated hemoglobin was measured spectrophotometrically
at 540 nm. DMSO was used as a negative control and Triton
X-100 (1%) was used as positive control.
+
for C24H18F2N4NaO6 [2M + Na]+: 519.10921, found:
519.10853, error (ppm): 1.30; GC-MS (70 eV) m/z 248
[M]+.
4-Methyl-5’-methyl-spiro[(5-aza-4-eno-3-one-cyclohexane)-
1,3’-(1H-indol-one) (6i)
Light brown solid; yield 80%; mp 215-217 ºC; 1H NMR
(500 MHz, DMSO-d6) d 1.91 (s, 3H, H-11), 2.21 (s, 3H,
Compounds (0.15-30.0 μM) were tested for cytotoxic
activity against MCF-7 (human mammary epithelial

Vol. 00, No. 00, 2018
Santos et al.
11
adenocarcinoma,ATCC No. HTB-22),A-549 (human lung
adenocarcinoma, ATCC No. CCL-185), MDA-MB435
(human melanoma,ATCC No. HTB-129), freshly prepared
human blood leukocytes and erythrocytes. All cell lines
were maintained in DMEM medium supplemented with
10% fetal bovine serum, 2.0 mM glutamine, 100 U mL-1
penicillin and 100 mg mL-1 streptomycin at 37 °C with
5% CO2. Each compound was dissolved with DMSO and
diluted with cell culture medium to obtain a concentration
of 100.0 μM. They were incubated with the cells for 48 h.
The negative control received the same amount of DMSO
(0.005% in the highest concentration). Doxorubicin was
used as a positive control. The cell viability was determined
by reduction of the yellow dye MTT to a blue formazan
product after 3 h incubation as described by Denizot and
Lang.37
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Supplementary Information
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Supplementary information (spectroscopic data,
1H NMR and 13C NMR spectra and mass spectra (EI)) is
available free of charge at http://jbcs.sbq.org.br as PDF file.
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Acknowledgments
The authors thank the Brazilian agencies National
Counsel for Technological and Scientific Development
(CNPq), State of Rio de Janeiro Research Foundation
(FAPERJ) and Coordination for the Improvement of Higher
Education Personnel (CAPES).
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Submitted: June 15, 2018
Published online: August 15, 2018
This is an open-access article distributed under the terms of the Creative Commons Attribution License.