Microwave-assisted synthesis and antimicrobial activity of novel spiro 1,3,4-thiadiazolines from isatin derivatives

Article abstract of DOI:10.1002/jhet.4213

This work describes the synthesis of spiro 1,3,4-thiadiazolines from isatin-β-thiosemicarbazone acetylation, using microwave irradiation as a source of heating the reaction medium. N-substituted isatin derivatives were used as substrates to obtain thiosemicarbazones by adding thiosemicarbazide to the isatin ketone carbonyl. The final synthetic step was the reaction of thiosemicarbazones with acetic anhydride under microwave irradiation to get the spiro compounds. Reaction times ranged from 6 to 18 minutes resulting in yields of up to 90%. Biological assays have shown promising antibacterial and antifungal activity, especially spiro thiadiazolines derived from allylated isatins. All the proposed molecules proved to be potential drug candidates based on the results of the in silico investigation, with satisfactory drug-likeness and drug-score, respecting Lipinski's rule. The use of the microwave reactor was efficient for the synthesis of thiosemicarbazones and spiro compounds, resulting in a significant reduction in reaction times with conventional heating. Taking into account the threat of antimicrobial resistance, this work presents a series of bioactive molecules that are easily obtained via microwave reaction.

Full text of DOI:10.1002/jhet.4213

Microwave-assisted Synthesis and Antimicrobial Activity of
Novel Spiro 1,3,4-thiadiazolines From Isatin Derivatives
Daniel Pereira da Costaa, Aleff Cruz de Castroa, Girlyanderson Araújo da Silvaa,
Claudio Gabriel Lima-Junior*a, Francisco Patricio de Andrade Júniorb, Edeltrudes de
Oliveira Limac, Boniek Gontijo Vazd, Lidya Cardoso da Silvad
a
Department of Chemistry, Federal University of Paraíba, João Pessoa - PB, Brazil
b
Postgraduate Program in Natural and Synthetic Bioactive Products, Federal University of Paraíba, João Pessoa - PB,
Brazil
c
Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa - PB, Brazil
d
Institute of Chemistry, Federal University of Goiás, Goiânia - GO, Brazil
*
E-mail: claudio@quimica.ufpb.br
(Additional Supporting Information may be found in the online version of this article.)
Abstract. This work describes the synthesis of spiro 1,3,4- All the proposed molecules proved to be potential drug
thiadiazolines from isatin-β-thiosemicarbazone acetylation, candidates based on the results of the in silico investigation,
using microwave irradiation as a source of heating the with satisfactory drug-likeness and drug-score, respecting
reaction medium. N-substituted isatin derivatives were used Lipinski's Rule. The use of the microwave reactor was
as substrates to obtain thiosemicarbazones by adding efficient for the synthesis of thiosemicarbazones and spiro-
thiosemicarbazide to the isatin ketone carbonyl. The final compounds, resulting in a significant reduction in reaction
synthetic step was the reaction of thiosemicarbazones with times with conventional heating. Taking into account the
acetic anhydride under microwave irradiation to get the spiro threat of antimicrobial resistance, this work presents a
compounds. Reaction times ranged from 6 to 18 minutes series of bioactive molecules that are easily obtained via
resulting in yields of up to 90%. Biological assays have microwave reaction.
shown promising antibacterial and antifungal activity,
Keywords: isatin derivatives; spiro compounds; isatin-β-
especially spiro thiadiazolines derived from allylated isatins.
thiosemicarbazones; acetylation; biological activity.
The synthesis of thiadiazolinic compounds
represents a challenge for organic chemists. Several
Introduction
synthetic strategies are used for its execution, among
then oxidative condensation and cycloaddition
reactions from molecules with multiple carbon-
nitrogen bonds,[11] such as diazo compounds,[12]
isothiocyanates,[13] and nitrilimines.[14] However, the
main route of obtaining is based on the acetylation of
thiosemicarbazones under conventional heating,[15]
for example the incorporation of spiro 1,3,4-
thiadiazolines into α-pinene[16] and piperidine
derivatives[9] (Figure 1 a-b).
In general, these protocols are based on
conventional heating, whose main limitation is the
non-uniform temperature throughout the system,
which compromises the yield and reaction time.[17]
This difficulty has been gradually eliminated due to
the variety of new methodologies reported in recent
bibliographic reviews that show the application of
microwave irradiation as an indispensable tool for
synthesizing these compounds.[18–20]
Isatin is a heterocyclic compound widely used as a
building block in the synthesis and development of
new drug candidates. Its synthetic versatility has
allowed the synthesis of new compounds endowed
with biological activity, such as spirocyclic
derivatives.[1,2]
These polycyclic compounds have rings attached
through an ordinary atom, usually, a sp3 hybridized
carbon. Medicinal chemists worldwide have shown
enormous interest in spiro compounds, especially
molecules with 1,3,4-thiodiazolinic rings since this
characteristic is recurrent in many alkaloids of natural
origin widely studied due to their pharmaceutical
properties.[3,4]
Moreover, synthetic derivatives have obeying this
trend, showing up as promising anticancer,[5,6]
antifungals,[6] and especially antibacterial agents,[7–9]
whose biological activities are possibly associated
with the N=C–S portion of the 1,3,4-thiadiazolinic
ring.[10]
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Figure 1. α-pinene (a), piperidine (b), and isatin
derivatives (c) containing 1,3,4-thiadiazolinic moiety.
The literature reports few studies using microwave
radiation to obtain spiro 1,3,4-thiadiazolines.
However, the results published so far reinforce the
advantages mentioned above. Mamun et al.,[21] for
example, report the synthesis of spiro thiadiazolines
by acetylation of isatin-β-thiosemicarbazones (Figure
1 c) with reaction times of 3 to 4 minutes under
microwave irradiation. In turn, the reaction time can
reach 4 hours in protocols based on conventional
heating for similar molecules.[22]
Scheme 1. (i) MeOH, 100 ºC (microwave reactor), 30-45
min; (ii) Ac2O, 120 ºC (microwave reactor), 6-18 min.
Based on this context, this work aimed to evaluate
the use of microwave irradiation in the synthesis of
new spiro 1,3,4-thiadiazolines derived from isatin.
Paying attention to antimicrobial resistance as one
of our most serious health threats, as well as the wide
exploitation of spirooxindoles as antimicrobial
agents,[23] we also propose an in silico study of the
pharmacokinetics and toxicological properties of the
proposed molecules and in vitro investigation of their
antimicrobial and antifungal activity.
Table 1. Reaction times and yields for obtaining
thiosemicarbazones 2a-2l.
Time Yield
Compound
R1
R2
R3
(min)
(%)
67
51
63
53
57
75
64
87
77
88
73
81
H
H
H
Cl
Cl
Cl
Cl
H
H
H
Br
Br
Br
Br
H
Cl
Cl
Cl
Cl
H
H
H
H
H
45
45
30
30
30
30
30
30
30
45
45
45
2a
2b
2c
2d
2e
2f
2g
2h
2i
CH3
C3H5
C7H7
CH3
C3H5
C7H7
H
CH3
C3H5
C7H7
Results and Discussion
Synthesis of isatin-derived thiosemicarbazones
(2a-2l)
Thiosemicarbazones (2a-2l) were obtained via
condensation reactions involving thiosemicarbazide
and ketone carbonyl present in isatin derivatives (1a-
1l) using methanol solvent under microwave
irradiation at 100 °C, as shown in Scheme 1.
2j
2k
2l
H
H
Acid catalysis was not used to obtain the products,
considering that some authors claim that acid is a
dispensable condition in obtaining these molecules.
¹H NMR spectra (see supporting information)
indicate the formation of the desired products due to
the appearance of the three characteristic peaks of
thiosemicarbazone: i) a singlet around 12.0 ppm,
corresponding to thioamides hydrogen; ii) two
diastereotopic hydrogens singlets between near to 9.3
and 8.7 ppm, corresponding to the -NH2 portion of
thiosemicarbazone due to the restricted rotation of the
bond C-NH2 by resonance effects.[27,28] We also
highlight the presence of peaks of thioamides carbons
in the most unshielded region of the 13C NMR
spectrum (about 179.0 ppm) that attest to the
formation of compounds 2a-2l.
[24–26]
Reaction conditions and yields for getting
thiosemicarbazones can be seen in Table 1.
It can be observed that the parameters used to
obtain thiosemicarbazones were satisfactory,
providing moderate yields (51-88%) with reaction
times between 30 and 45 minutes, with no formation
of co-products. These yields can be associated with
the use of methanol as a solvent since its high
polarity can result in solubilization and consequent
loss of part of the product by filtration.
Synthesis of isatin-derived 1,3,4-thiadiazolines
(3a-3l)
2
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Few synthetic protocols are described in the literature
for obtaining spiro 1,3,4-thiadiazolines derived from
isatin. It is commonly a reaction that uses
thiosemicarbazone derived from isatin and excess of
acetic anhydride. Reaction times vary from 1 to 4
hours under conventional heating[22,29,30] and from 3
to 4 minutes using an adapted domestic microwave
oven.[21]
Being aware of this information, we submitted the
first three reactions for the preparation of spiro 1,3,4-
thiadiazoline (3a) to microwave irradiation, varying
the temperature and evaluating by TLC the time
required for total consumption of thiosemicarbazone
(Table 2). All synthetic protocols described in the
literature use an excess of acetic anhydride in the
cyclization reactions, leading us to use a fixed
amount of this reagent in the reactions carried out in
this study (3.5 mL).
C3H5
C7H7
CH3
C3H5
C7H7
H
CH3
C3H5
C7H7
Cl
Cl
H
H
H
Br
Br
Br
Br
Cl
Cl
H
H
H
H
H
H
H
6
6
6
6
6
81
65
62
70
90
25
57
61
65
3d
3e
3f
3g
3h
3i
3j
3k
3l
6
18
18
18
a Isolated yield.
¹H NMR spectroscopic data confirm the successful
formation of the desired thiadiazolines, evidenced by
the disappearance of the two singlets mentioned
above, characteristic of the hydrogens of the -NH2
portion of the precursor thiosemicarbazones. The
appearance of two singlets around 2.0 ppm is also
noted, which are related to α-amidic hydrogens
resulting from the acetylation of thiosemicarbazone,
Table 2. Investigation of the influence of temperature on
the synthesis of 3a.
[28]
Entry a
Temp. (°C) Time (min) Yield (%)b
consistent with the literature data for similar
compounds previously described.[21,22]
80
100
120
18
12
6
85
87
82
1
2
3
Regarding the ¹³C NMR spectra obtained for the
synthesized 1,3,4-thiadiazolines, there is an increase
in the number of carbon signals compared to
thiosemicarbazones. Two of these signals are
attributed to the methyl carbons introduced by
acetylation and are located close to 22.0 ppm. Based
on the spectroscopic associations available in the
literature for similar compounds, we highlight the
signals' appearance in the most unshielded region of
the APT spectra (~170.0 ppm), referring to the
carbonyl carbons resulting from acetylation. The
presence of a discrete signal around 74.0 ppm,
attributed by Somogyi[22] to the carbon spiro,
confirms the formation of the desired compounds.
Based on the twin drug proposal, widely explored
in the literature, we also performed the synthesis of
two dimeric thiadiazolines derived from isatin.
a Reactions using 0.5 mmol of thiosemicarbazone (2a) and
3.5 mL of acetic anhydride; b isolated yield.
Based on the results obtained in the preparation of the
spiro 1,3,4-thiadiazoline 3a, we can observe that the
increase in temperature in the microwave reactor
promotes a gradual decrease in reaction time. It was
also observed the total conversion of the starting
material and the formation of the same products,
regardless of the temperature used.
Considering that reference used does not detail
synthetic aspects such as the possible formation of
co-products and subsequent purification of the
desired products, we proceeded to synthesize the
other spiro compounds using microwave irradiation
at 120 °C. These results are shown in Table 3.
The dimeric compounds (1m and 1n) were
submitted
to
addition
to generate
reactions
with
thiosemicarbazide
homodimeric
The values in Table 3 reveal that two compounds
(3b and 3i) showed significantly lower yields than the
other synthesized compounds due to the formation of
thiosemicarbazones (2m and 2n) and their subsequent
conversion to homodimeric 1,3,4-thiadiazolines (3m
and 3n), according to the synthesis protocols adopted
in this research. Scheme 2 shows the synthetic route
for obtaining these homodimers.
co-products
which
made
purification
via
chromatographic column difficult. These compounds
come from isatin that was not subjected to
substitution reactions in the NH portion of the indole
nucleus (R1=H), an essential factor for the low yields
obtained, since this problem was not verified for the
N-substituted derivatives. Compound 3a was an
exception, which yielded 82% even though it
belonged to the class of compounds 3b and 3i.
Table 3. Reaction times and yields for obtaining 1,3,4-
thiadiazolines 3a-3l.
Time Yield
(min) (%)a
Compound
R1
R2
R3
H
H
CH3
H
Cl
Cl
H
Cl
Cl
6
6
6
82
36
88
3a
3b
3c
3
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being only excreted. To better understand this
behavior in spiro 1,3,4-thiadiazolines, the prediction
of the compounds' physical-chemical properties was
made.
Values of physicochemical properties, molecular
weight, number of acceptors (HBA), and hydrogen
donors (HBD), Log P and molar refractivity were
calculated using the SwissADME software.[34]
The results given in Table 4 show that the
monomeric spiro 1,3,4-thiadiazolines evaluated do
not violate any parameter of Lipinski's Rule. All
compounds have a molecular mass below 500 g mol-1,
indicating good solubility and cross cell membranes
without difficulty. On the other hand, the studied
homodimers (3m and 3n) violated this rule,
presenting respectively molar mass values of 648.71
and 662.74 g mol-1.
The increase in the number of possibilities for
hydrogen bonds, represented as a function of HBA
and HBD, contributes to increasing the compound's
polarity. A high number of hydrogen bonds hinders
the drug's penetration through the lipid bilayer of cell
membranes. These bonds need to be broken as the
molecule passes through the hydrophilic to
hydrophobic medium.[33]
Scheme 2. (i) MeOH, 100 ºC (microwave reactor), 30 min;
(ii) Ac2O, 120 ºC (microwave reactor), 30 min.
Lipophilicity of a compound is expressed as Log P.
Compounds with cLog P > 5 are poorly soluble in
aqueous solutions; therefore, they cannot access the
membrane surface.[35] In an optimal situation for
effective gastrointestinal absorption after oral
administration of the drug, the Log P value must be
between 0 and 3.[36] All synthesized molecules obey
this parameter, except for the dimeric molecule 3n.
This compound showed cLog P = 3.48 so it did not
violate the limit established by Lipinski's Rule (cLog
P > 5).
The synthesis of dimeric compounds demanded a
longer reaction time concerning monomeric
thiadiazolines. The total consumption of the starting
thiosemicarbazones (2m and 2n) occurred after 30
min under heating in a microwave reactor, resulting
in yields of 52 and 27% for 3m and 3n, respectively.
These compounds were fully characterized,
1
presenting behavior similar to monomers at H and
13C NMR spectra.
The main objective of homodimer synthesis is to
obtain more selective and potent drugs than the
monomeric units that compose it, exhibiting a
different selectivity profile and pharmacokinetic
properties.[31]
Molar refractivity is
a
constitutive-additive
physical-chemical property related to the adjustment
of the molecule to its receptor site. Positive values
indicate the presence of dispersive forces in the active
center that allow the ligand-bioreceptor interaction. In
contrast, negative values represent the ligand's ability
to distort the bioreceptor conformation, causing a
stereochemical impediment that makes molecular
interaction difficult.[37] All compounds analyzed,
except homodimers 3m and 3n, showed values within
the reference standard established (40 ≤ molar
refractivity ≤ 130),[38] indicating an excellent ligand-
bioreceptor interaction.
In silico study
The in silico study has become an excellent strategy
to assist medicinal chemists in discovering new
possible drugs. This study is based on a theoretical
investigation
pharmacodynamic properties and toxicological
effects (ADMET Absorption, Distribution,
of
the
pharmacokinetic
and
-
Metabolization, Excretion, and Toxicity) of
compounds with potential biological activity, making
it possible to reduce the number of compounds
Studies for
a
possible prediction of the
toxicological properties (mutagenic, tumorigenic,
irritant, and reproductive effects) of the proposed
compounds were also carried out using the Osiris
Property Explorer.[39] Among all the synthesized
prepared
with
undesirable
physicochemical
properties.[32]
One of the most important theoretical approaches
to pharmacokinetics in silico study is the Lipinski’s
Rule of Five.[33] This rule is based on the physical-
chemical criteria that the compounds must obey to be
absorbed and distributed by the human organism.
These parameters also indicate that the drugs can
present metabolic stability, remaining in the body for
the time necessary to act appropriately instead of
1,3,4-thiadiazolines,
only
compounds
3b-3e
presented a single risk group: irritation with a
medium level of risk (Table 5).
The drug-likeness parameter assesses the presence
of functional groups and physical properties similar
to most known drugs. Negative values represent
minimal similarities, and positive values represent a
more remarkable similarity between the molecules
4
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studied and the drugs in use.[40] On the other hand, the
drug-score combines values of some parameters into
a single numerical value that is useful for judging the
potential of the compound to become a possible
drug.[39] All spiro compounds provided positive
values that indicate substantial similarities with
pharmacophoric subunits and properties present in
already known drugs.
We emphasize here that in silico studies are
essential, but not decisive for searching for new drug
candidates. These results guide the development of
new structures, but the biological activity and toxicity
of
the compounds must
be
considered.
Table 4. Physicochemical properties are predicted via SwissADME.
Lipinski’s parameter
Compound
Molar Refractivity
MW
HBA
HBD
cLog P
nViol
304.32
373.21
387.24
413.28
463.34
318.35
344.38
394.45
383.22
397.25
423.28
473.34
648.71
662.74
4
4
4
4
4
4
4
4
4
4
4
4
8
8
2
2
1
1
1
1
1
1
2
1
1
1
2
2
1.42
1.95
2.37
2.54
2.92
1.90
2.22
2.49
1.80
2.28
2.75
2.92
2.87
3.48
0
0
0
0
0
0
0
0
0
0
0
0
1
1
88.15
98.17
3a
3b
3c
3d
3e
3f
103.07
112.21
127.56
93.05
102.19
117.54
95.85
3g
3h
3i
100.75
109.89
125.24
188.80
193.61
3j
3k
3l
3m
3n
MW: molecular weight; HBA: hydrogen bond acceptor; HBD: hydrogen bond donor; MW: cLog P: octanol/water partition
coefficient; nViol: number of violations;
Table 5. Physicochemical properties predicted via Osiris Property Explorer.
Mutagenic
effects
NR
Irritating
effects
NR
Tumorigenic Reproductive
Compound
Drug-likeness
Drug-score
effects
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
effects
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
7.04
6.74
7.27
5.02
7.96
7.57
5.31
8.24
4.91
5.44
3.19
6.13
8.30
7.15
0.81
0.51
0.50
0.43
0.34
0.83
0.76
0.63
0.71
0.69
0.60
0.47
0.30
0.28
3a
3b
3c
3d
3e
3f
NR
MR
MR
MR
MR
NR
NR
NR
NR
NR
NR
NR
3g
3h
3i
NR
NR
NR
NR
NR
NR
3j
NR
NR
3k
3l
NR
NR
NR
NR
3m
3n
NR
NR
NR: no risk; MR: medium risk.
In vitro biological evaluation
Strains of bacteria (Staphylococcus aureus, ATCC-
13150; Escherichia coli, ATCC-18739; Pseudomonas
5
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aeruginosa, ATCC-9027) and fungi (Candida
albicans, ATCC-90028; Candida tropicalis, ATCC-
13803; Cryptococcus neoformans, FCF-119) were
used to evaluate the biological activity of the
synthesized spiro compounds.
Biological activity of the compounds was based on
the values of minimum inhibitory concentration
(MIC) obtained in vitro, and their activity was
considered according to the following criteria: intense
activity (MIC < 600 μg mL-1); moderate exercise
(600 ≤ MIC ≤ 1500 μg mL-1); inadequate training or
inactive product (MIC > 1500 μg mL-1).[41–43]
Negative control was carried out in parallel with the
antimicrobials gentamicin (64 μg mL-1) inhibiting
bacteria and amphotericin B (32 μg mL-1) inhibiting
fungi. Table 6 shows the results of the antimicrobial
activity of the tested compounds.
the lowest MIC values (137.5 µg mL-1) for all
microorganisms tested. The only exception concerns
the performance of 3g against P. aureuginosa, whose
MIC was 550.0 µg mL-1.
Compound 3l showed activity on all trial strains,
whose MIC values varied between 125.0 and 500.0
µg mL-1. An opposite behavior was observed for the
compound N-methylated 3f, which showed only
antifungal activity with MIC ranging between 137.5
and 550.0 µg mL-1. Compound 3h was active only
against P. aureuginosa with MIC of 275 µg mL-1.
Despite recent reports regarding the use of
dimerization as a viable alternative to improve the
biological activity of monomeric precursors,[44–47] the
evaluated dimers (3m and 3n) did not show physical
activity against the tested microorganisms. These
results reinforce the need for studies involving aryl-
substituted isatin derivatives, because of the
promising results of some thiadiazolines derived from
chlorinated and brominated isatins.
As observed in Table 6, only five compounds were
active against at least one of the studied
microorganisms. Among the tested molecules, it was
found that the N-allylated compounds 3d and 3g had
Table 6. Minimum inhibitory concentration (µg mL-1) of the 1,3,4-thiadiazolines against bacterial and fungal strains.
Bacteria
Fungi
Compound
S. aureus
E. coli
P. aeruginosa
C. albicans
C. tropicalis
C. neoformans
ATCC-13150
ATCC–18739
ATCC-9027
ATCC-90028
ATCC-13803
FCF-119
+
+
+
+
+
+
3b
+
+
+
+
+
+
3c
137.5
137.5
137.5
137.5
137.5
137.5
3d
+
+
+
+
+
+
3e
+
+
+
550.0
137.5
137.5
3f
137.5
137.5
550.0
137.5
137.5
137.5
3g
+
+
+
+
275.0
+
+
+
+
+
+
+
3h
3j
+
+
+
+
+
+
+
+
+
3k
3l
250.0
125.0
500.0
+
+
+
+
+
+
+
+
+
+
+
+
3m
3n
Culture media
Microorganism
Amphotericin B
Gentamicine
-
+
NT
-
-
+
NT
-
-
+
NT
-
-
+
-
-
+
-
-
+
-
NT
NT
NT
MIC: minimum inhibitory concentration; ATCC: American Type Culture Collection; (+): the presence of microbial growth; (-): no
microbial growth; NT: Not tested.
potential of these thiadiazolines for drug candidates.
The 3d, 3f, 3g, 3h and 3l compounds showed intense
Conclusion
antimicrobial activity, especially 3d and 3g. These N-
allylated derivatives showed activity at
a
concentration of 137.5 µg mL-1 in practically all tests
with microorganisms. Finally, the synthesized dimers
(3m and 3n) did not show activity against the
microorganisms evaluated. This behavior reinforces
the importance of further studies extending the series
of dimeric compounds with substituent groups in the
aromatic ring.
The microwave radiation synthesis of was
successfully applied, resulting in spiro compounds
with yields between 25 and 90%. The use of the
microwave reactor proved to be efficient for the
synthesis of thiosemicarbazones and compound spiro,
resulting in a significant reduction in reaction times
with conventional heating. In silico studies have
shown that the spiro compounds synthesized
respected Lipinski's parameters, indicating a good
oral availability. In addition, the results obtained for
toxicity, drug-likeness and drug-score reinforce the
Experimental Section
6
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General
reduced pressure, solid red products were obtained
with yields of 62% (1d), 79% (1g) e 71% (1k).
All commercially available reagents were purchased
from Aldrich and used without further purification.
N- benzyl isatin derivatives (1e, 1h and 1l)
Reactions
were
monitored
by
thin-layer
5.0 mmol of 1a, 1b or 1i were solubilized in 5 mL of
DMF previously dried, followed by the addition of
K2CO3 (1,2 equiv.) under magnetic stirring. Then
benzyl bromide (1.0 equiv.) was added and the
reaction was carried out at room temperature under
an inert argon atmosphere and monitored by CCDA.
After 2 hours of reaction liquid-liquid extraction was
performed using ethyl acetate and distilled water. The
organic phase was treated with anhydrous Na2SO4.
After filtration and removal of the solvent under
reduced pressure, solid red products were obtained
with yields of 78% (1e), 87% (1h) e 82% (1l).
chromatography (TLC) using Silica gel 60 UV254
Macherey-Nagel pre-coated silica gel plates. Flash
column chromatography was performed on a silica
gel (300–400 mesh) using an ethyl acetate-hexane
mixture as eluent. Organic phases were dried using
anhydrous Na2SO4 prior to evaporation on a rotary
evaporator. NMR spectra were recorded in DMSO-d6
with a Varian Mercury Spectra AC 20 or Bruker
Advance II (400 MHz to ¹H NMR and 100 MHz to
¹³C NMR). Chemical shift values are expressed as
parts per million (ppm) downfield from TMS and J
values are in hertz. Splitting patterns are indicated as
singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), double doublet (dd), doublet of
doublets of doublets(ddd) and broad peak (br).
General procedure for synthesis of homodimeric
derivatives of isatin (1m and 1n)
2 mmol of 1a was solubilized in 2 mL of DMF
previously dried, followed by the addition of K2CO3
(1,2 equiv.) under magnetic stirring. Then 1 mmol of
Synthesis of the isatin derivatives (1b-1l)
5,7-Dichloroisatin (1b)
the
corresponding
dibromoalkane
(1,3-
Isatin (1a) (10.0 mmol, 1 equiv.) was added to a
solution of trichloroisocyanuric acid (10.0 mmol, 1
equiv.) in 6 mL of H2SO4 at 0 ºC. This mixture was
stirring under ambient conditions for 30 min and
accompanied by TLC. After this time, crushed ice
was added to the reaction flask and the precipitate
was filtered under reduced pressure and washed with
ice water resulting in a solid yellow product (1b) with
89% yield.
dibromopropane for 1m or 1,4-dibromobutane for 1n)
was added and the reaction was carried out at room
temperature under an inert argon atmosphere and
monitored by TLC. After 24 hours, ice water was
added to the reaction flask and the precipitate was
filtered under reduced pressure and washed with
ethanol resulting in a solid orange product (1m and
1n).
General procedure for synthesis of isatin
thiosemicarbazones (2a-2n)
N-methyl isatin derivatives (1c, 1f and 1j)
5.0 mmol of isatin (1a) or its derivatives 5,7-
dichloroisatin (1b) and 5-bromoisatin (1i) were
solubilized in 5 mL of DMF previously dried,
followed by the addition of K2CO3 (1,2 equiv.) under
magnetic stirring. Then methyl iodide (1.2 equiv.)
was added and the reaction was carried out at room
temperature under an inert argon atmosphere and
monitored by TLC. After 2 hours of reaction liquid-
liquid extraction was performed using ethyl acetate
and distilled water. The organic phase was treated
with anhydrous Na2SO4. After filtration and removal
of the solvent under reduced pressure, solid red
products were obtained with yields of 81% (1c), 93%
(1f) and 87% (1j).
1 mmol of isatin (1a) or its derivatives (1b-1n) were
added to 4 mL of methanol in a glass tube specific for
a
microwave
reactor
(10
mL).
Then
thiosemicarbazide was add to the system under
magnetic stirring, obeying 1 equiv. for monomeric
(1b-1l) and 2 equiv. for dimeric derivatives (1m-1n).
The reaction was carried out at 100 °C under
microwave irradiation with closed vessel conditions
and monitored by TLC every 15 minutes complete
reagent consumption. At the end of the reaction, the
mixture was cooled to room temperature in order to
form a precipitate which was isolated by vacuum
filtration, using ethanol for washing of the obtained
solid.
N-allyl isatin derivatives (1d, 1g and 1k)
Compound 2a: Yellow solid (67% yield), mp 220-
5.0 mmol of 1a, 1b or 1i were solubilized in 5 mL of
DMF previously dried, followed by the addition of
K2CO3 (1,2 equiv.) under magnetic stirring. Then
allyl bromide (1.2 equiv.) was added and the reaction
was carried out at room temperature under an inert
argon atmosphere and monitored by TLC. After 2
hours of reaction liquid-liquid extraction was
performed using ethyl acetate and distilled water. The
organic phase was treated with anhydrous Na2SO4.
After filtration and removal of the solvent under
1
225 ºC. H NMR (400 MHz, DMSO-d6) δ 12.48 (s,
1H), 11.19 (s, 1H), 9.03 (s, 1H), 8.67 (s, 1H), 7.64 (d,
1H), 7.33 (t, 1H), 7,06 (t, 1H), 6.91 (d, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 179.17, 163.10, 142.81,
132.52, 131.73, 122.84, 121.43, 120.43, 111.50.
Compound 2b: Orange solid (51% yield), mp
1
240-245 ºC. H NMR (400 MHz, DMSO-d6) δ 12.24
(s, 1H), 11.73 (s, 1H), 9.15 (s, 1H), 8.84 (s, 1H), 7.70
(d, J = 2.0 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 179.24, 162.91,
7
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138.99, 130.67, 129.88, 127.38, 123.77, 119.81,
116.28.
Hz, 1H), 3.34 (s, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 179.21, 160.81, 143.02, 133.46, 130.17, 123.55,
121.92, 115.24, 112.18, 26.28.
Compound 2c: Orange solid (63% yield), mp 230-
1
235 ºC. H NMR (400 MHz, DMSO-d6) δ 12.17 (s,
Compound 2k: Yellow solid (73% yield), mp
1
1H), 9.21 (s, 1H), 8.93 (s, 1H), 7.84 (d, J = 2.1 Hz,
205-210 ºC. H NMR (400 MHz, DMSO-d6) δ 12.18
13
1H), 7.57 (d,J = 2.1 Hz, 1H), 3.49 (s, 3H). C NMR
(s, 1H), 9.15 (s, 1H), 8.86 (s, 1H), 7.92 (d, J = 2.0 Hz,
1H), 7.55 (dd, J = 8.4, 2.1 Hz, 1H), 7.01 (d, J = 8.4
Hz, 1H), 5.89-5.80 (m, 1H), 5.19 (ddd, J = 11.3, 9.2,
1.3 Hz, 2H), 4.36 (d,2H). 13C NMR (100 MHz,
DMSO-d6) δ 179.22, 160.56, 142.12, 133.43, 131.70,
130.03, 123.75, 122.15, 117.93, 115.32, 112.73,
41.86.
(100 MHz, DMSO-d6) δ 179.24, 161.27, 138.34,
131.56, 129.34, 127.85, 124.36, 119.85, 116.62,
29.12.
Compound 2d: Orange solid (53% yield), mp
188-193 ºC.1H NMR (400 MHz, DMSO-d6) δ 12.12
(s, 1H), 9.22 (s, 1H), 8.95 (s, 1H), 7.87 (s, 1H), 7.54
(s, 1H), 6.01-5.92 (m, 1H), 5.14 (dd, J = 10.5. 1H),
5.07 (dd, J = 17.3, 1H), 4.63-4.61 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 179.23, 161.12, 137.41,
133.37, 131.66, 129.03, 128.07, 124.59, 120.00,
116.50, 116.38, 43.11.
Compound 2l: Yellow solid (81% yield), mp 235-
1
240 ºC. H NMR (400 MHz, DMSO-d6) δ 12.22 (s,
1H), 9.18 (s, 1H), 8.89 (s, 1H), 7.94 (d, J = 2.1 Hz,
1H), 7.50 (dd, J = 8.4, 2.1 Hz, 1H), 7.37-7.24 (m, 5H),
6.95 (d, 1H), 4.96 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 179.23, 160.90, 142.00, 135.91, 133.43,
130.00, 129.18, 128.12, 127.85, 123.81, 122.28,
115.47, 112.73, 43.05.
Compound 2e: Brown solid (57% yield), mp 223-
1
228 ºC. H NMR (400 MHz, DMSO-d6) δ 12.14 (s,
1H), 9.28 (s, 1H), 9.01 (s, 1H), 7.91 (d, J = 2.1 Hz,
1H), 7.48 (d, J = 2.1 Hz, 1H), 7.34-7.23 (m, 5H), 5.25
(s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 179.23,
161.53, 137.34, 137.31, 131.57, 129.05, 128.91,
128.24, 127.66, 126.60, 124.79, 120.03, 116.37,
44.39;
Compound 2m: Orange solid (80% yield), mp
190-195 ºC.1H NMR (400 MHz, DMSO-d6) δ 12.33
(s, 2H), 9.09 (s, 2H), 8.70 (s, 2H) 7.65 (d, 2H), 7.36 (t,
2H), 7.16 (m, 2 H), 7.10 (m, 2H), 3.84 (t, 4H), 2.04
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 178.45,
160.58, 142.41, 137.78, 130.89, 122.67, 120. 57,
119.27, 109.84, 36.96, 24.72.
Compound 2f: Yellow solid (75% yield), mp 220-
1
225 ºC. H NMR (400 MHz, DMSO-d6) δ 12.38 (s,
1H), 9.07 (s, 1H), 8.70 (s, 1H), 7.66 (d, J = 6.8 Hz,
1H), 7.42 (td, J = 7.7 Hz, 1.3 Hz, 1H), 7.15-7.09 (m,
Compound 2n: Orange solid (78% yield), mp
1
240-245 ºC. H NMR (400 MHz, DMSO-d6) δ 12.37
13
2H), 3.36 (s, 3H). C NMR (100 MHz, DMSO-d6) δ
(s, 2H). 9.05 (s, 2H). 8.67 (s, 2H). 7.64 (d, 2H). 7.39
(t, 2H). 7.37 (m, 2H). 7.09 (m, 2H). 3.76 (m, 4H).
1.68 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ
178.71, 160.89, 142.71, 131.09, 131.05, 122.86,
120.80, 119.39, 110.04, 38.79, 24.20.
179.16, 161.21, 144.03, 131.64, 131.60, 123.33,
121.03, 119.70, 110.23, 26.14.
Compound 2g: Yellow solid (64% yield), mp 148-
1
153 ºC. H NMR (400 MHz, DMSO-d6) δ 12.32 (s,
1H), 9.02 (s, 1H), 8.60 (s, 1H), 7.75 (d, 1H), 7.48 (t,
1H), 7.18 (t, 1H), 7.02 (d, 1H), 5.57 (m, 1H), 5.50 (t.
2H), 4.45 (d, 2H). 13C NMR (100 MHz, DMSO-d6) δ
178.72, 160.46, 142.64, 131.42, 131.04, 130.97,
122.89, 120.73, 119.36, 117.39, 110.27, 41.32.
General procedure for synthesis of the spiro
thiadiazolines (3a-3n)
0.5 mmol of isatin thiosemicarbazones (2a-2n) were
added under magnetic stirring to 3.5 mL of acetic
anhydride in a glass tube specific for a microwave
reactor (10 mL). The reaction was carried out at
120 °C under microwave irradiation with closed
vessel conditions and monitored by TLC every 6
minutes until complete reagent consumption. At the
end of the reaction the mixture was cooled to room
temperature and diluted in ethyl acetate and treated
with saturated solution of NaHCO3 to neutralize the
remaining acetic acid. washed with in order to form a
precipitate which was isolated by vacuum filtration,
using ethanol for washing of the obtained solid. After
removal of the solvent under reduced pressure, the
products (3a-3n) were purified by flash
chromatography using silica gel and ethyl
acetate/hexane as eluent, resulting in yellow solids.
Compound 2h: Yellow solid (87% yield), mp
1
250-255 ºC. H NMR (400 MHz, DMSO-d6) δ 12.41
(s, 1H), 9.11 (s, 1H), 8.76 (s, 1H), 7.71 (dd, J = 6.8
Hz, 1H), 7.39-7.30 (m, 5H), 7.26 (tt, 1H), 7.12 (td, J
= 7.6 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 4.97 (s, 2H).
13C NMR (100 MHz, DMSO-d6) δ 179.16, 161.29,
143.01, 136.20, 131.51, 131.42, 129.18, 128.08,
127.91, 123.50, 121.28, 119.97, 110.81, 42.98.
Compound 2i: Yellow solid (77% yield), mp 245-
1
250 ºC. H NMR (400 MHz, DMSO-d6) δ 12.28 (s,
1H), 11.28 (s, 1H), 9.10 (s, 1H), 8.81 (s, 1H), 7.86 (d,
J = 2.0 Hz, 1H), 7.48 (dd, J = 8.3, 2.1 Hz, 1H), 6.86
(d, J = 8.3 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 179.22, 162.70, 141.80, 133.64, 131.10, 123.92,
122.77, 114.63, 113.40.
Compound 3a: Yellow solid (82% yield), mp >
320 ºC. 1H NMR (400 MHz, DMSO-d6) δ 7.40 (d, J =
7.2 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.6
Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 2.19 (s, 3H), 2.12
(s, 3H).
Compound 2j: Orange solid (88% yield), mp 250-
1
255 ºC. H NMR (400 MHz, DMSO-d6) δ 12.18 (s,
1H), 9.13 (s, 1H), 8.82 (s, 1H), 7.86 (d, J = 2.0 Hz,
1H), 7.55 (dd, J = 8.4, 2.1 Hz, 1H), 7.06 (d, J = 8.4
8
This article is protected by copyright. All rights reserved.

Compound 3b: Yellow solid (36% yield), the
crystals became black without melting. 1H NMR (400
MHz, DMSO-d6) δ 7.49 (d, J = 2.0 Hz, 1H), 7.45 (d,
7.8 Hz, 1H), 5.02 (d, J = 16.2 Hz, 1H), 4.83 (d, J =
16.2 Hz, 1H), 2.19 (s, 3H), 2.09 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 172.49, 170.55, 167.52,
143.31, 141.86, 136.21, 130.53, 129.03, 128.40,
127.82, 127.46, 124.17, 123.79, 110.14, 74.81, 43.85,
22.77, 22.56.
13
J = 2.0 Hz, 1H), 2.16 (s, 3H), 2.09 (s, 3H). C NMR
(100 MHz, DMSO-d6) δ 173.60, 170.51, 167.57,
143.43, 138.57, 132.03, 129.75, 127.32, 123.34,
115.42, 75.11, 22.65, 22.40. HRMS (FTMS + pESI)
m/z, calcd. for C13H10Cl2N4O3S: 371.9851; found:
370.9783 (M-1).
Compound 3i: Yellow solid (25% yield), the
crystals became black without melting. 1H NMR (400
MHz, DMSO-d6) δ 12.01 (s, 1H), 10.86 (s, 1H), 7.52
(d, J = 2.0 Hz, 1H), 7.43 (dd, J = 8.3, 2.1 Hz, 1H),
6.80 (d, J = 8.3 Hz, 1H), 2.15 (s, 1H), 2.08 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ 173.45, 170.50,
167.50, 143.23, 140.88, 133.24, 131.24, 127.13,
114.42, 112.61, 74.66, 22.73, 22.55.
Compound 3c: Yellow solid (88% yield), mp 277-
1
282 ºC. H NMR (400 MHz, DMSO-d6) δ 12.00 (s,
1H), 7.51 (q, J = 2.1 Hz, 2H), 3.44 (s, 3H), 2.15 (s,
13
3H), 2.09 (s, 3H). C NMR (100 MHz, DMSO-d6) δ
172.68, 170.52, 167.55, 143.65, 138.17, 132.93,
131.49, 127.88, 123.51, 115.74, 74.02, 30.31, 22.65,
22.37. HRMS (FTMS + pESI) m/z, calcd. for
C14H12Cl2N4O3S: 386.0007; found: 384.9900 (M-1).
Compound 3j: Yellow solid (57% yield), mp 280-
1
285 ºC. H NMR (400 MHz, DMSO-d6) δ 12.03 (s,
1H), 7.58 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 8.3, 2.0
Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 3.13 (s, 3H), 2.14
(s, 3H), 2.08 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 171.98, 170.53, 167.48, 143.47, 142.47, 133.27,
130.55, 126.84, 115.11, 111.54, 74.23, 27.23, 22.73,
22.53. HRMS (FTMS + pESI) m/z, calcd. for
C14H13BrN4O3S: 395.9892; found: 396.2807 (M+1).
Compound 3d: Yellow solid (81% yield), mp
1
218-223 ºC. H NMR (400 MHz, DMSO-d6) δ 12.09
(s, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 2.1 Hz,
1H), 5.97-5.88 (m, 1H), 5.25 (dd, J = 17.4. 1H), 5.12
(dd, J = 10.7. 1H), 4.58-4.57 (m, 1H), 2.18 (s, 2H),
2.09 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ
172.66, 170.65, 167.64, 143.54, 137.26, 133.34,
133.05, 131.66, 128.11, 123.61, 116.06, 115.65,
73.99, 44.19, 22.67, 22.40. HRMS (FTMS + pESI)
m/z, calcd. for C16H14Cl2N4O3S: 412.0164; found:
411.0100 (M-1).
Compound 3k: Yellow solid (61% yield), mp
1
225-230 ºC. H NMR (400 MHz, DMSO-d6) δ 7.61
(d, J = 2.0 Hz, 1H), 7.51 (dd, J = 8.4, 2.1 Hz, 1H),
6.91 (d, J = 8.4 Hz, 1H), 5.85-5.76 (m. 1H), 5.28 (dd,
J = 17.3, 1.4 Hz, 1H), 5.14 (dd, J = 10.5, 1.4 Hz, 1H),
4.47-4.21 (m, 1H), 2.16 (s, 1H), 2.09 (s, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 171.82, 170.58,
167.56, 143.35, 141.39, 133.17, 131.45, 130.53,
126.95, 117.26, 115.21, 112.16, 74.27, 42.65, 22.73,
22.50. HRMS (FTMS + pESI) m/z, calcd. for
C16H15BrN4O3S: 422.0048; found: 420.9978 (M-1).
Compound 3e: Yellow solid (65% yield), mp 248-
1
253 ºC. H NMR (400 MHz, DMSO-d6) δ 12.07 (s,
1H), 7.59 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 2.1 Hz,
1H), 7.34-7.22 (m, 5H), 5.21 (q, 2H), 2.21 (s, 3H),
2.10 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
173.11, 170.62, 167.76, 143.51, 137.75, 137.28,
133.09, 131.61, 128.87, 128.28, 127.35, 126.28,
123.59, 115.65, 74.14, 45.63, 22.65, 22.38; HRMS
(FTMS + pESI) m/z, calcd. for C20H16Cl2N4O3S:
462.0320; found: 461.0238 (M-1).
Compound 3l: Yellow solid (65% yield), mp 270-
1
275 ºC. H NMR (400 MHz, DMSO-d6) δ 12.09 (s,
1H), 7.64 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 8.4, 2.1
Hz, 1H), 7.41-7.21 (m, 5H), 6.74 (d, J = 8.4 Hz, 1H),
5.04 (d, J = 16.3 Hz, 1H), 4.80 (d, J = 16.3 Hz, 1H),
2.21 (s, 3H), 2.10 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 172.18, 170.61, 167.67, 143.38, 141.23,
135.85, 133.18, 130.61, 129.05, 127.88, 127.42,
127.02, 115.40, 112.15, 74.40, 43.94, 22.73, 22.51.
Compound 3f: Yellow solid (62% yield), mp 245-
1
250 ºC. H NMR (400 MHz, DMSO-d6) δ 12.03 (s,
1H), 7.37-7.33 (m, 2H), 7.09-7.02 (m, 2H), 3.14 (s,
13
3H), 2.13 (s, 3H), 2.08 (s, 3H). C NMR (100 MHz,
DMSO-d6) δ 172.30, 170.48, 167.33, 143.37, 143.07,
130.67, 128.37, 124.01, 123.61, 109.47, 74.62, 27.10,
22.76, 22.58.
HRMS (FTMS
+
pESI) m/z, calcd. for
C20H17BrN4O3S: 472.0205; found: 471.0100 (M-1).
Compound 3g: Yellow solid (70% yield), mp 220-
Compound 3m: Yellow solid (52% yield), mp
1
1
225 ºC. H NMR (400 MHz, DMSO-d6) δ 12.03 (s,
220-225 ºC. H NMR (400 MHz, DMSO-d6) δ 12.03
1H), 7.39 (dd, J = 7.5, 0.8 Hz, 1H), 7.32 (td, J = 7.8,
1.2 Hz, 1H), 7.07 (td. J = 7.6, 0.8 Hz, 1H), 6.93 (d, J
= 7.8 Hz, 1H), 5.87-5.78 (m, 1H), 5.30 (dd, J = 17.3,
1H), 5.14 (dd, J = 10.5, 1H), 4.40-4.22 (m, 2H), 2.15
(s, 3H), 2.09 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 172.13, 170.50, 167.39, 143.27, 142.02, 131.73,
130.53, 128.32, 124.12, 123.64, 117.18, 110.10,
74.68, 42.55, 22.76, 22.55.
(s, 2H), 7.39 (d, 2H), 7.31(ddd, J = 7.7, 1.2 Hz, 2H),
7.16-7,06 (m, 4H), 3.78 (m, 4H), 2.16 (s, 6H) 2.09 (s,
6H), 1.93 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
172.24, 170.51, 167.49, 142.29, 130.69, 128.33,
124.35, 123.63, 109.60, 75.03, 38.39, 38.35, 22.38.
HRMS (FTMS + pESI) m/z, calcd. for C29H28N8O6S2:
648.1573; found: 647.1487 (M-1).
Compound 3n: Yellow solid (27% yield), mp
1
Compound 3h: Yellow solid (90% yield), mp
245-250 ºC. H NMR (400 MHz, DMSO-d6) δ 12.02
1
225-230 ºC. H NMR (400 MHz, DMSO-d6) δ 12.06
(s, 2H), 7.39-7.31 (m, 4H), 7.11-7.15 (m, 4H), 3.72
13
(s, 1H), 7.40 (tt, 3H), 7.34-7.30 (m, 2H), 7.27-7.21
(br, 4H), 2.16 (s, 6H), 2.10 (s, 6H), 1.68 (br, 4H). C
(m, 2H), 7.05 (td, J = 7.6, 0.9 Hz, 1H), 6.78 (d, J =
NMR (100 MHz, DMSO-d6) δ 171.96, 170.08,
9
This article is protected by copyright. All rights reserved.

166.94, 142.86, 141.81, 130.26, 128.03, 123.74,
123.10, 109.42, 74.26, 39.52, 23.84, 22.36, 22.17.
HRMS (FTMS + pESI) m/z, calcd. for C30H30N8O6S2:
662.1730; found: 661.1658 (M-1).
M. K. Islyaikin, Russ. J. Gen. Chem. 2008, 78,
480–484.
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the Brazilian agencies Conselho Nacional de
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and Coordenação de Aperfeiçoamento de Pessoal de
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Conflict of Interest
[19] V. V Konovalova, A. N. Maslivets, Mini. Rev. Org.
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The authors declare no conflict of interest, financial
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