Improved synthesis of 1,3,4-thiadiazolium-2-phenylamines using microwave and ultrasound irradiation and investigation of their cytotoxic activity

Article abstract of DOI:10.1590/S0103-50532011000800014

Uma nova e eficiente si?ntese de oito derivados da classe 1,3,4-tiadiazolio-2-fenilaminas (1-8, sendo o derivado 8 ine?dito na literatura) foi realizada utilizando cloreto de tionila ou cloreto de trimetilsilano como catalisadores sob irradiac?a?o de micr

Full text of DOI:10.1590/S0103-50532011000800014

J. Braz. Chem. Soc., Vol. 22, No. 8, 1505-1510, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines using Microwave and
Ultrasound Irradiation and Investigation of their Cytotoxic Activity
Camilla Moretto dos Reis,a Juliana Echevarria-Lima,b Amanda Fraga Mirandaa and
Aurea Echevarria*,a
aDepartamento de Química, Instituto de Ciências Exatas,Universidade Federal Rural do
Rio de Janeiro, 23890-000 Seropédica-RJ, Brazil
bDepartamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do
Rio de Janeiro, 21941-590 Rio de Janeiro-RJ, Brazil
Uma nova e eficiente síntese de oito derivados da classe 1,3,4-tiadiazolio-2-fenilaminas (1-8,
sendo o derivado 8 inédito na literatura) foi realizada utilizando cloreto de tionila ou cloreto de
trimetilsilano como catalisadores sob irradiação de microondas ou ultra-som. Os compostos alvos
foram obtidos em bons rendimentos e em tempo extraordinariamente curtos, 5 min sob irradiação
de microondas e 10 min sob irradiação de ultra-som, quando comparados com a metodologia
tradicional (24 a 48 h em repouso a temperatura ambiente). Os melhores rendimentos foram
obtidos usando irradiação de microondas e, de maneira geral, usando o cloreto de tionila ao invés
de cloreto de trimetilsilano. A citotoxicidade foi avaliada frente à linhagem de leucemia humana
K562 e do linfoma Daudi, apresentando resultados promissores para o derivado cloreto de 4-fenil-
5-(4’-nitro-estiril)-1,3,4-tiadiazolio-2-fenilamina.
A new and efficient synthesis of eight 1,3,4-thiadiazolium-2-phenylamine derivatives (1-8,
where 8 is novel in the literature) was performed using thionyl chloride or trimethylsilyl chloride
as catalysts under microwave or ultrasound irradiation. The target compounds were obtained in
good yields and remarkably short times, 5 min under microwave irradiation and 10 min under
ultrasound irradiation, where compared to traditional methodology (24 to 48 h at room temperature
standing). The best yields were obtained using the microwave irradiation and, in general way, using
thionyl chloride instead trimethylsilyl chloride. The cytotoxicity against K562 human leukemia
and Daudi lymphoma lines was evaluated and showed promising results from the 4-phenyl-5-(4’-
nitro-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative.
Keywords: mesoionic heterocycle compounds, microwave, ultrasound, cytotoxic activity
Introduction
to cross biological membranes. Different classes of
mesoionic compounds have demonstrated a wide range
of biological activities such as anti-inflammatory and
analgesic,5 antiparasitic,6,7 antibacterial,8,9 antiplatelet,
fibrinolytic, thrombolytic and broncholytic effects,10 as
well as anticancer potency.11-17 The promising therapeutic
applications have led us to study these interesting
compounds in our laboratory. We have previously
described the survival enhancement of Ehrlich and
Sarcoma 180 tumor-bearing mice treated with 4-phenyl-
5-(4’-X-styryl)-1,3,5-thiadiazolium-2-phenylamine
chlorides.12 Respiratory chain inhibition, transmembrane
potential collapse, and ATPase activity in intact rat liver
mitochondria were observed when the 4-phenyl-5-(4’-
X-styryl)-1,3,5-thiadiazolium-2-phenylamine chlorides
Mesoionic compounds are a special class of
heterocycles with potential therapeutic applications
due to their unique chemical properties. They possess
a betaine-like character with a partial positive charge
on the heterocyclic ring that is balanced by a negative
charge located on an exocyclic atom or group.1,2 The
large separation between the charged regions leads to
large dipole moments of about 4-5 D.3,4 These properties
suggest the possibility of interacting with biomolecules
such as proteins and DNA. Additionally, the overall
neutral character of these compounds enables them
*e-mail: echevarr@ufrrj.br

1506
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
were used.13,14 In addition, Senff-Ribeiro et al.15 showed
that 4-phenyl-5-(4’-nitro-styryl)-1,3,5-thiadiazolium-2-
phenylamine chloride displays an important antitumor
activity against murine melanoma B16F10. This
compound was cytotoxic in vitro, decreasing cell
viability and proliferation, and was able to inhibit
tumor growth by ca. 85% in vivo as well. Additionally,
it decreased the viability and proliferation of cell lines
of human melanoma (MEL-85, SK-MEL, A2058 and
MEWO).15,16 To contribute to the understanding of the
molecular pathways involved in the pharmacological
activities of mesoionic compounds, we have recently
described the effects of 4-phenyl-5-(4’-nitro-styryl)-
1,3,5-thiadiazolium-2-phenylamine chloride on lipid
peroxidation in rat liver mitochondria, submitochondrial
particles and phosphatidylcholine, as well as its ability
to scavenge radicals.17
In addition, our group has investigated new,
more efficient and cleaner synthetic methods for the
preparation of these mesoionic compounds,18 especially
due to important environmental concerns with the
current synthesis.19,20 Therefore, the synthesis of the
1,3,4-thiadiazolium-2-phenylamine class of compounds
was revisited using alternative energy sources to obtain
better yields and lower reaction times.
Towards that end, this work presents the synthesis
of 4-phenyl-5-(4’-X-phenyl)-1,3,5-thiadiazolium-2-
phenylamine chlorides, where X = H (1), 4’−OCH3 (2),
4’−NO2 (3) and 3’,4’−OCH2O (4) (referred to as the
benzaldehyde series), and 4-phenyl-5-(4’-X-styryl)-
1,3,5-thiadiazolium-2-phenylamine chlorides, where
X = H (5), 2’−OCH3 (6), 4’−NO2 (7) and 4’−N(CH3)2
(8) (the cinnamaldehyde series), by two new alternative
methods: using microwave irradiation and an ultrasound
bath. Furthermore, derivatives of 1, 3 and 7 were assayed
against K562 human leukemia and Daudi lymphoma lines.
General procedure for preparation of mesoionic chlorides
(1-8)
Method A
Using microwave irradiation. The aldehyde (0.35 mmol)
and 1,4-diphenyl-thiosemicarbazide (0.35 mmol) were
mixed in the presence of a Lewis acid (TMS-Cl or SOCl2)
and a few drops of 1,4-dioxane. The resulting mixture
was submitted to microwave irradiation for full 5 min in
open vessel. After this continuous time, the mixture was
added to 1,4-dioxane and left to stand overnight at room
temperature. The obtained solid was filtered, washed with
ice-cold water and 1,4-dioxane, and recrystallized from
chloroform:ethanol (60:40, v/v).
Method B
Using ultrasound bath irradiation. The aldehyde
(0.35 mmol) and 1,4-diphenyl-thiosemicarbazide
(0.35 mmol) were mixed in the presence of a Lewis
acid (TMS-Cl or SOCl2) and 1,4 dioxane as solvent.
The resulting mixture was submitted to ultrasound bath
irradiation for 10 min. The solution containing the reaction
mixture was left to stand overnight. The formed precipitate
was treated in a similar way as described above.
Table 1 shows the yields obtained for 1-8 using the
methodA and method B, and the characterization data6,12,21
for all compounds are in Supplementary Information.
Cell culture
The human erythroleukemia K562 and Burkett
lymphoma Daudi cell lines were maintained in culture
medium RPMI 1640 (Sigma) supplemented with
β-mercaptoethanol 50 µmol L−1, HEPES 25 mmol L−1
(Sigma), penicillin 60 mg L−1 (Sigma), streptomycin
100 mg L−1 (Sigma) and 10% (v/v) fetal calf serum
(Gibco, Life Technologies). They were incubated at 37 °C
in a humidified atmosphere of air and 5% CO2. The cell
line was passaged twice a week at a concentration of
1 × 104 cell mL−1.
Experimental
Chemistry
Melting points were determined with a Meltemp
II apparatus and were uncorrected. Infrared spectra
(KBr pellets) were recorded on a Perkin-Elmer 1605
Cytotoxic assays
1 × 104 cell mL−1 were seeded on a 96-well cell culture
plate. The cells were incubated with or without a mesoionic
compound 1, 3 or 7 at a concentration of 3.125-50 µmol L−1
each. They were incubated for 24 h, 48 h, 72 h, 96 h and
7 days at 37 °C with 5% CO2. After, cell viability was
assessed by adding 20 µL cell−1 of MTT (5 mg mL−1;
3-[4,5-dimethylthiazol--2-yl]-2,5-diphenyl tetrazolium
bromide, Sigma) as described earlier.22 The plates were
1
FT-IR spectrophotometer. H and 13C NMR spectra
were obtained on a Bruker AC200 (200 and 50.3 MHz)
spectrometer with TMS as the internal reference and
DMSO-d6 as the solvent. The microwave and ultrasound
bath assisted organic reactions were performed in a
Panasonic Piccilo-NN-S45BH (a domestic oven) and
an Ultra-cleaner 700, respectively.

Vol. 22, No. 8, 2011
Reis et al.
1507
read on a microplate reader (Sunrise-Basic TECAN) at a
wavelength of 490 nm. The results are represented as the
mean SD of 5 determinations.
Results and Discussion
Chemistry
Analysis of the cell cycle
Eight mesoionic chloride salts were synthesized by two
different methods, microwave irradiation and an ultrasound
bath, aimedatreductionofthereactiontimeandadaptationto
the principles of Green Chemistry.23 The convergent strategy
formesoionicchloridesaltssynthesisincludedthereactionof
1,4-diphenyl-thiosemicarbazidewiththeappropriatealdehyde
in the presence of a Lewis acid and 1,4-dioxane as solvent. In
methods, two different Lewis acids, SOCl2 and trimethylsilyl
chloride (TMS-Cl), were investigated separately (Scheme 1).
When compared to the traditional method,7,24 the use of an
aldehyde and a Lewis acid enables the elimination of a step
involving a carboxylic acid and the subsequent acid chloride
preparation, as well as a reduction of the time required to
obtain the target compounds from 24-48 h standing at room
temperature, after 8 h of stirring, to 5 min under microwave
irradiation or 10 min under ultrasound irradiation.
1 × 104 cell mL−1 were seeded on a 96-well cell culture
plate. The cells were incubated with or without mesoionic
compound 7 at a concentration of 3.125-50 µmol L−1 each.
They were incubated for 24 h at 37 °C with 5% CO2. After,
the cells were collected and washed in Hank’s buffer.The cell
cycle was assessed by adding 200 µL of cell cycle solution
(5mgmL−1).Thecellswerethenanalyzedbyflowcytometry.
The samples were excited at 488 nm with an argon laser;
fluorescence emission was detected at 585 nm (FL-2). Ten
thousand cells were acquired. All flow cytometry analyses
were accomplished with WinMDI, version 2.9.
Statistical analysis
The values are given as a mean SD. The statistical
significance was calculated by one-way analysis of variance
(ANOVA) followed by Bonferroni’s t-test using Prism
5 software. P values of less than 0.05 were considered
significant.
The first method consisted of submitting a mixture of
1,4-diphenyl-thiosemicarbazide, the appropriate aldehyde,
SOCl2 (three equivalents) or TMS-Cl (two equiv.) and a few
drops of 1,4-dioxane to microwave irradiation (MW) for
5 min. Subsequently, an organic solvent was added, and the
Entry A (saturated series):
16
17
a
15
14
Cl–
O
H
N
H
N
9
8
N
N
Ph
+
11
2
5
10
H
Ph
N
H
12
13
6
N
b
S
7
S
X
H
11'
12'
X
X = H (1); 4'-OCH3 (2); 4'-NO2 (3); 3',4'-OCH2O (4)
Entry B (unsaturated series):
16
17
a
15
14
Cl–
O
H
N
H
N
9
8
N
N
Ph
+
α
11
2
5
H
Ph
N
H
12
13
6
N
b
S
10
7
S
β
X
H
11'
12'
X
X = H (5); 2'-OCH3 (6); 4'-NO2 (7); 4'-N(CH3)2 (8)
Scheme 1. Reagents and conditions: 1,4-dioxane, Lewis acid (SOCl2 or TMS-Cl), MW for 5 min (a) or ))) for 10 min (b).

1508
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
Table 1. Mesoionic chloride yields obtained under microwave irradiation
and ultrasound bath, using TMS-Cl and SOCl2 as catalys
mixture was allowed to stand overnight, after which time
the precipitate was filtered and washed with ice-cold water
to afford the target compounds. In the second method, using
similar conditions as above, the reagents were dissolved
in 1,4-dioxane and put in the ultrasound bath for 10 min,
and the mixture was allowed to stand overnight. The
isolation of the target compounds was performed in the
same manner as for the microwave experiment. Scheme 1
shows the synthetic route of the two methods used in this
work. In both methods, the mesoionic salts were obtained
in good yields and high purity (Table 1). When microwave
irradiation was employed, the yields were in the range of
41-95% when TMS-Cl was used and 90-98% when SOCl2
was used. Using the ultrasound bath method, the respective
yields for TMS-Cl and SOCl2 were 66-98% and 72-87%.
Thus, the best results were observed using SOCl2 as the
catalyst under microwave irradiation conditions.
Microwaveb
Yield (%)
Ultrasoundc
Yield (%)
Mesoionic Traditionala
chlorides
Yield (%)
TMS-Cl
SOCl2
92
TMS-Cl
SOCl2
83
1
2
3
4
5
6
7
8
87
52
78
68
58
73
89
nod
68
41
95
74
87
87
92
93
89
66
98
87
73
83
90
68
96
74
98
83
92
87
90
86
95
84
93
91
91
72
a24-48 h at room temperature standing; b5 min of microwave irradiation
c
and stand overnight; 10 min on ultrasound bath and stand overnight;
d no product obtained.
The mesoionic chloride structures 1-8 were fully
characterized by 1H and 13C NMR and IR spectroscopies.
The 1H and 13C chemical shifts (d) were assigned based on
literature data6,12,21,25 and were consistent with the structures
proposed.
Cytotoxicity activity
InBrazil, leukemiarepresentsabout2%ofthetotalcancer
cases and affects adults, the elderly and young individuals.26
Figure 1. Effects of mesoionic compound 7 on Daudi and K562 cell viability. 1 × 104 cell mL−1 were incubated with or without 7 at 37 °C and CO2 5%.
After 24, 48, 72, 96 h and 7 days, Daudi cell line (A and B) or K562 cell line (C and D) were incubated with MTT for 3 h at the same conditions. The
optical density was acquired at 490 nm wavelength. The graphs above represent mean standard deviation values of the optical density. A p < 0.05 was
considered significant when compared to control (*), or to control and 6.25 µmol L−1 (**), or when compared to control, 12.5 and 25 µmol L−1 (#).

Vol. 22, No. 8, 2011
Reis et al.
1509
Cases of leukemia are classified as acute or chronic and,
for lymphoid or myeloid, according to the origin of the cell
type. The treatment of leukemia is performed using different
combinations of chemotherapy drugs; however, the index of
remission remains unsatisfactory. The lymphoma cell line
Daudi and myeloid cell line K562 were used for studying the
effects of the synthesized mesoionic compounds on leukemic
cells. The cytotoxic activities of the 1, 3 and 7 were assayed
in vitro using MTT.22 Compounds 1 and 3 did not alter the
viability of the lymphoma cell line Daudi in 72 h of culture.
The positive results obtained with 7 (the unsaturated
series compound with an NO2 moiety), led us to assay it in
24 h, 48 h, 72 h, 96 h and 7 days incubations with Daudi
cell cultures. The compound showed significant cytotoxic
activity after 24 h. Moreover, this mesoionic derivative
decreased the cellular viability in a concentration-dependent
manner (Figure 1A and B). Similar results were obtained in
the human erythroleukemia cell line K562. We observed that
the mesoionic compound 7 reduced the cellular viability in a
concentration-dependentmannerforthiscelllineaswell.The
compound showed significant cytotoxic activity beginning at
24 h and continuously growing over time (Figure 1C and D).
The IC50 values were determined in µmol L−1 in at least
three independent experiments using 7 at concentrations in
the range of 5-25 µmol L−1. The compound 7 was incubated
for 24 h, 48 h, 72 h, 96 h, and 7 days with cultures of Daudi
(lymphoid origin) and K562 cells (myeloid cells). When
the results with the different cell lines were compared, they
showed similar sensitivities for the first 72 h; however, the
myeloid cells demonstrated more sensitivity after a long
exposure time (7 days), as shown in Table 2.
Figure 2. Mesoionic compound 7 induces apoptosis on Daudi and K562.
1 × 104 cell mL−1 of Daudi (A) and K562 (B) were incubated with or
without mesoionic compound 7 at 37 °C and CO2 5%. After 24 h, cells
were incubated with cell cycle solution at room temperature for 15 min.
Following, cells were acquired by flow cytometry and analyzed on
WinMDI 2.9. The graphs above represent mean values of hyplodiploid
cells percentage.
(Figure 2). This suggests that the mesoionic 7 induces cell
death by apoptosis.
Table 2. IC50 (µmol L−1) values of compound 7 against Daudi lymphoma
and K562 leukemia cells
Conclusions
In summary, the new synthetic methods using
microwave irradiation and an ultrasound bath provided the
products 1-8, where 8 is novel in the literature, in shorter
reaction times and higher yields when compared to the
traditional method. SOCl2 proved to be a better Lewis acid
than TMS-Cl, and microwave irradiation led to a more
efficient reaction when compared to the ultrasound bath.
In addition to improving the synthetic route, these methods
adhere to the principles of Green Chemistry. Furthermore,
the 4’-nitro-styryl derivative has shown to be cytotoxic
against Daudi lymphoma and K562 leukemic myeloid cells
in preliminary in vitro studies of cytotoxicity.
time of culture
Daudi IC50
K562 IC50
(µmol L−1 SDa)
(µmol L−1 SD)
24 h
10.76 0.09
8.62 0.07
7.93 0.08
10.17 0.11
11.55 0.07
12.12 0.10
7.63 0.06
7.08 0.08
6.03 0.07
8.76 0.09
48 h
72 h
96 h
7 days
aSD: standard deviation.
Tounderstandthemechanismofthecytotoxicityexhibited
by the mesoionic compound 7 on the Daudi and K562 cells,
they were incubated for 24 h with 7 at a concentration of
3.25-50 µmol L−1. The capacity of this compound to induce
apoptosis in Daudi and K562 cells was analyzed with a cell
cycle assay. The results showed an increase in hypodiploid
cells, which was correlated with the cytotoxicity results
Supplementary Information
Supplementary information with characterization data
1
for all compounds (IR, H and 13C NMR data) and the

1510
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
corresponding spectra for compound 8 are available free
of charge at http://jbcs.sbq.org.br as PDF file.
11. Schinzato, T. O.; Grynberg, N. F.; Gomes, R. M.; Echevarria,
A.; Miller, J.; J. Med. Sci. Res. 1989, 17, 865.
12. Grynberg, N.; Santos, A. C. S.; Echevarria, A.; Anti-Cancer
Drugs 1997, 8, 88.
Acknowledgments
13. Cadena, S. M. S. C.; Carnieri, E. G. S.; Echevarria,A.; Oliveira,
M. B. M.; FEBS Lett. 1998, 440, 46.
The authors thank CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico) and CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior) for financial support and fellowships.
14. Cadena, S. M. S. C.; Carnieri, E. G. S.; Echevarria,A.; Oliveira,
M. B. M.; Cell Biochem. Funct. 2002, 20, 31.
15. Senff-Ribeiro, A.; Echevarria, A.; Silva, E. F.; Veiga, S. S.;
Oliveira, M. B. M.; Melanoma Res. 2003, 13, 465.
16. Senff-Ribeiro, A.; Echevarria, A.; Silva, E. F.; Franco, C. R.;
Viegas, S. S.; Oliveira, M. B. M.; Br. J. Cancer 2004, 91, 297.
17. Mendez-Sanches, S. C.; Martinez, G. R.; Romão, S.; Echevarria,
A.; Silva, E. F.; Rocha, M. E. M.; Chem.-Biol. Interact. 2009,
179, 125.
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Submitted: November 6, 2010
Published online: May 3, 2011

J. Braz. Chem. Soc., Vol. 22, No. 8, S1-S6, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Supplementary Information
SI
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines Using Microwave and
Ultrasound Irradiation and Investigation of their Cytotoxic Activity
Camilla Moretto dos Reis,a Juliana Echevarria-Lima,b Amanda Fraga Mirandaa and
Aurea Echevarria*,a
aDepartamento de Química, Instituto de Ciências Exatas,Universidade Federal Rural do
Rio de Janeiro, 23890-000 Seropédica-RJ, Brazil
bDepartamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do
Rio de Janeiro, 21941-590 Rio de Janeiro-RJ, Brazil
Characterization data
(d, H-11, H-11’), 7.63-7.08 (m, Harom), 3.86 (s, CH3O);
13C NMR (DMSO-d6) d 163.00 (C-5), 159.48 (C-2),
138.34 (C-14), 137.74 (C-6), 131.89 (C-10), 131.07
(C-17), 129.40 (C-12), 129.73 (C-16), 125.79 (C-8),
126.10 (C-9, C-13), 123.64 (C-11), 118.18 (C-15), 114.69
(C-7), 55.54 (OCH3).
16
17
15
14
Cl–
9
8
N
N
11
2
5
10
12
13
6
4-Phenyl-5-(4’-nitrophenyl)-1,3,4-thiadiazolium-2-
phenylamine chloride (3)
N
S
7
H
11'
Orange solid; mp 138-139 °C (lit.21 mp 138-139 °C);
IR (KBr) nmax/cm-1 3434, 3043, 2721, 1573, 1537, 1454,
1348, 854, 746, 694; 1H NMR (CDCl3) d 12.75 (s, N-H),
8.21 (d, H-12,H-12’), 8.16 (d, H-11, H-11’), 7.71-7.00 (m,
Harom); 13C NMR (DMSO-d6) d 163.84 (C-5), 159.20 (C-2),
137.95 (C-13), 136.95 (C-10), 131.43 (C-14), 131.25 (C-6),
129.46 (C-17), 128.85 (C-16), 126.00 (C-9), 125.67 (C-8),
124.90 (C-12), 123.79 (C-11), 123.33 (C-15), 118.24 (C-7).
12'
X
X = H (1), 4’-OCH3 (2), 4’-NO2 (3), 3’,4’-OCH2O (4)
4,5-Diphenyl-1,3,4-thiadiazolium-2-phenylamine chloride
(1)
Pale yellow solid; mp 356-358 °C (lit.21 mp 358 °C);
IR (KBr) nmax/cm-1 3419, 3047, 2723, 1569, 1542, 1494,
1444, 1319, 755, 690; 1H NMR (CDCl3) d 12.50 (s, N-H),
7.08-7.72 (m, Harom); 13C NMR (DMSO-d6 d 163.84 (C-5),
160.48 (C-2), 138.56 (C-14), 138.28 (C-10), 138.00 (C-6),
137.46 (C-17), 135.80 (C-13), 134.07 (C-11), 132.98
(C-16), 131.19 (C-8), 130.19 (C-12), 129.88 (C-15), 129.61
(C-9), 118.33 (C-7).
4-Phenyl-5-(3’,4’-methylenedioxyphenyl)-1,3,4-
thiadiazolium-2-phenylamine chloride (4)
Pale yellow solid; mp 290-293 °C (lit.6 mp 286-287 °C);
IR (KBr) nmax/cm-1 3438, 3050, 2902, 2642, 1604, 1540,
1450, 1567, 1311, 1243, 1105, 1037, 754, 694; 1H NMR
(CDCl3) d 11.76 (s, N-H), 7.60-7.20 (m, Harom), 7.17 (d,
H-11’), 7.10 (d, H-12’), 7.02 (d, H-11), 6.08 (s, OCH2O);
13C NMR (DMSO-d6) d 163.05 (C-5), 160.05 (C-2), 150.70
(C-12), 148.00 (C-13), 140.50 (C-14), 138.80 (C-6), 133.45
(C-17), 130.95 (C-8), 129.75 (C-16), 126.13 (C-11’),
127.57 (C-15), 127.42 (C-9), 117.56 (C-7), 115.12 (C-10),
115.46 (C-11), 110.42 (C-12’), 103.86 (OCH2O).
4-Phenyl-5-(4’-methoxy-phenyl)-1,3,4-thiadiazolium-2-
phenylamine chloride (2)
Yellow solid; mp 242-243 °C (lit.21 mp 238 °C); IR
(KBr) nmax/cm-1 3425, 3048, 2935, 2665, 1602, 1542,
1
1448, 1567, 1309, 1175, 1024, 827, 752, 692; H NMR
(CDCl3) d 12.90 (s, N-H), 7.70 (d, H-12,H-12’), 6.90
*e-mail: echevarr@ufrrj.br

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Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
IR (KBr) nmax/cm-1 3432, 3048, 2721, 1616, 1571, 1519,
1450, 1342, 956, 840, 746, 688; 1H NMR (CDCl3) d 12.74
(s, N-H), 8.33 (d, H-12, H-12’), 8.25 (d, Ha), 8.04 (d,
H-11, H-11’), 7.96-7.39 (m, Harom), 7.33 (d, Hb); 13C NMR
(DMSO-d6) d 165.10 (C-5), 162.38 (C-2), 151.00 (C-13),
147.55 (C-a), 142.99 (C-10), 140.20 (C-14), 139.05 (C-6),
132.92 (C-17), 131.98 (C-16), 130.50 (C-8), 129.68
(C-11), 127.20 (C-15), 126.33 (C-9), 125.25 (C-12),
117.71 (C-7), 114.81 (C-b).
16
17
15
14
Cl–
9
8
N
N
α
11
2
5
12
13
6
N
S
10
7
β
H
11'
12'
X
X = H (5), 2’-OCH3 (6), 4’-NO2 (7), 4’-N(CH3)2 (8)
4-Phenyl-5-(4’-dimethylamino-styryl)-1,3,4-
thiadiazolium-2-phenylamine chloride (8)
4-Phenyl-5-styryl-1,3,4-thiadiazolium-2-phenylamine
chloride (5)
Purple solid; mp 201-203 °C; IR (KBr) nmax/cm-1:
3425, 3062, 2923, 2852, 1612, 1565, 1577, 1525, 1444,
1378, 1268, 941, 808, 750, 692; 1H NMR (CDCl3) d 12.39
(s, N-H), 7.85 (d, Ha), 7.81 (d, H-12, H-12’), 7.73 (d,
H-11, H-11’), 7.71- 6.71 (m, Harom), 6.70 (d, Hb), 3.02
(m, N(CH3)2); 13C NMR (DMSO-d6) d 163.27 (C-13),
157.14 (C-5), 153.01 (C-2), 149.47 (C-a), 138.81 (C-14),
137.15 (C-6), 131.76 (C-11, 11’), 131.20 (C-17), 130.12
(C-16), 129.33 (C-8), 126.16 (C-15), 123.66 (C-9), 121.08
(C-10), 118.30 (C-7), 111.88 (C-12, 12’), 103.81 (C-b),
39.75 (N(CH3)2); Elemental analysis: Found: C, 66.38;
H, 5.15; N, 12.94. Calc. for C24H23N4SCl: C, 66.27; H,
5.33; N, 12.88%.
Yellow solid; mp 264-265 °C (lit.12 mp 266-267 °C);
IR (KBr) nmax/cm-1 3432, 3056, 2670, 1604, 1567, 1538,
1
1498, 1448, 1330, 954, 746, 690; H NMR (CDCl3) d
12.58 (s, N-H), 7.95 (d, Ha), 7.19-7.83 (m, Harom), 7.06 (d,
Hb); 13C NMR (DMSO-d6) d 164.90 (C-5), 160.30 (C-2),
149.84 (C-a), 138.00 (C-14), 137.82 (C-6), 135.35 (C-10),
135.90 (C-17), 134.89 (C-13), 132.30 (C-11), 131.95
(C-16), 133.64 (C-8), 130.56 (C-12), 129.20 (C-15),
127.28 (C-9), 120.79 (C-7), 115.67 (C-b).
4-Phenyl-5-(2’-methoxy-styryl)-1,3,4-thiadiazolium-2-
phenylamine chloride (6)
Yellow solid; mp 198-200 °C (lit.12 mp 195-196 °C);
IR (KBr) nmax/cm-1 3423, 3054, 2925, 2786, 1604, 1594,
1540, 1444, 1565, 1295, 1249, 1110, 1031, 966, 836,
References
1
754, 690; H NMR (CDCl3) d 11.90 (s, N-H), 7.92 (d,
Same as cited in the Article
Ha), 7.84-6.90 (m, Harom), 6.97 (d, Hb), 3.81 (s, CH3O);
13C NMR (DMSO-d6) d 162.29 (C-11), 159.15 (C-5),
158.57 (C-2), 144.58 (C-a), 138.26 (C-14), 136.87 (C-6),
133.94 (C-11’), 131.70 (C-13), 130.51 (C-17), 130.07
(C-16), 128.89 (C-8), 125.56 (C-15), 124.04 (C-9), 122.11
(C-10), 118.89 (C-7), 111.40 (C-b), 55.46 (OCH3).
6. Ferreira, W. S.; Lima, L. F.; Saraiva,V. B.; Silva, F.A.; Previato,
L. M.; Previato, J. O.; Echevarria, A.; Lima, M. E. F.; Bioorg.
Med. Chem. 2008, 16, 2984.
12. Grynberg, N.; Santos, A. C. S.; Echevarria, A.; Anti-Cancer
Drugs 1997, 8, 88.
21. Maciel, M. A. M.; Echevarria, A.; Rumjanek, V. M.; Quim.
Nova 1998, 21, 569.
4-Phenyl-5-(4’-nitro-styryl)-1,3,4-thiadiazolium-2-
phenylamine chloride (7)
25. Santos,A. C. S.; Echevarria,A.; Magn. Reson. Chem. 2001, 39,
182.
Orange solid; mp 234-236 °C (lit.12 mp 232-233 °C);

Vol. 22, No. 8, 2011
Reis et al.
S3
IR, 13C NMR, DEPT-Q and1H, spectra of compound 8
Figure S1. IR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4- thiadiazolium-2-phenylamine chloride (8).
Figure S2. 13C NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.

S4
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
Figure S3. Expansion of 13C NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.
Figure S4. DEPT-Q spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.

Vol. 22, No. 8, 2011
Reis et al.
S5
Figure S5. Expansion of DEPT-Q spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.
Figure S6. 1H NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.

S6
Improved Synthesis of 1,3,4-Thiadiazolium-2-phenylamines
J. Braz. Chem. Soc.
Figure S7. Expansion of 1H NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in DMSO-d6.
Figure S8. 13C NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in MeOH-d4.
Figure S9. Expansion of 13C NMR spectrum of 4-phenyl-5-(4’-dimethylamino-styryl)-1,3,4-thiadiazolium-2-phenylamine chloride (8) in MeOH-d4.