Synthesis of novel 1,3-thiazin-4-ones by acetylene diester cyclization and their anticancer activities

Article abstract of DOI:10.1080/10426507.2016.1209502

A novel series of 1,3-thiazin-4-one derivatives containing a piperidyl ring (7–16) were designed and synthesized efficiently by thioamide and acetylene diester cyclization reaction via aminolysis of the ester group and the elimination of an alcohol molecu

Full text of DOI:10.1080/10426507.2016.1209502

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Synthesis of novel 1,3–thiazin–4–ones by acetylene
diester cyclization and their anticancer activities
Selvam Athavan Alias Anand, Chandrasekaran Loganathan, Nisha
Susan Thomas, Kuppusamy Saravanan, Antony Therasa Alphonsa &
Senthamaraikannan Kabilan
To cite this article: Selvam Athavan Alias Anand, Chandrasekaran Loganathan, Nisha
Susan Thomas, Kuppusamy Saravanan, Antony Therasa Alphonsa & Senthamaraikannan
Kabilan (2016): Synthesis of novel 1,3–thiazin–4–ones by acetylene diester cyclization and
their anticancer activities, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:
10.1080/10426507.2016.1209502
To link to this article: http://dx.doi.org/10.1080/10426507.2016.1209502
Accepted author version posted online: 07
Jul 2016.
Published online: 07 Jul 2016.
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Date: 08 July 2016, At: 09:30

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Synthesis of novel 1,3–thiazin–4–ones by acetylene diester cyclization and their anticancer
activities
Selvam Athavan Alias Anand1, Chandrasekaran Loganathan1, Nisha Susan Thomas2,
Kuppusamy Saravanan1, Antony Therasa Alphonsa1 and Senthamaraikannan Kabilan1,*
1Drug Discovery Lab, Department of Chemistry, Annamalai University, Annamalainagar,
Tamilnadu, India.
2Department of Bio–Chemistry and Bio–Technology, Annamalai University, Annamalainagar,
Tamilnadu, India.
*Corresponding author: E-mail: profskabilanau@gmail.com; Tel: +91 94439 24629
Abstract
A novel series of 1,3–thiazin-4–one derivatives containing a piperidyl ring (7–16) were designed
and synthesized efficiently by thioamide and acetylene diester cyclization reaction via
aminolysis of the ester group and the elimination of an alcohol molecule. The structures of all the
1
novel compounds were established by their FTIR, Mass, H NMR and 13C NMR spectral
techniques. The newly synthesized compounds (7–16) were studied for their in vitro anticancer
activity against human liver cancer cell lines Hep G2 using MTT assay. The IC50 values of the
tested compounds were ranging in between 7.48 ± 0.71 and 56.57 ± 1.37 µM. Further, the
apoptosis evaluation by the mitochondrial membrane potential using JC–1 dye was carried out
and the results are in good agreement with the cytotoxicity studies.
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Keywords
1,3–Thiazin–4–one, Acetylene diesters, In vitro, Apoptosis, Cytotoxicity.
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1. Introduction
Hepatocellular carcinoma which develops through the malignant transformation of hepatocytes is
the second cancer causing more deaths worldwide. According to a recent survey, hepatoma is the
fifth most common cancer in men and the ninth in women.1 This hepatoma fatal malignant tumor
attributed due to the high frequency of hepatisis B and C viral infections and treated clinically by
chemotherapy, radiotherapy and surgery. Most of the anticancer drug candidates are heterocyclic
compounds with different functional groups and shows a significant antitumor property based on
the target. The heterocyclic system containing a piperidone nucleus has found broad application
in drug development. Modified piperidone derivatives have valuable medicinal chemistry
properties.2 For example, Risperdal was used for the treatment of schizophrenia3 and donepezil
for Alzheimer’s disease.4 Some investigations also reported that derivatives containing the
piperidyl ring produce remarkable effects against cancer cell lines.5,6 The substituted 1,3–
thiazin−4−ones was also an important analogues in medicinal chemistry and it is associated with
various biological activities such as anticancer,7 anticonvulsant,8 antifungal9 and antimalarial10
properties. From the above facts, we try to synthesis some new heterocyclic structures
incorporating 1,3−thiazin−4−one with N–substituted piperidyl ring for achieving potent
inhibitory activities against cancer cells. A new set of different modified 1,3–thiazine–4–ones
ring having a piperidyl nucleus was easily prepared via a cyclization reaction between the
electron-deficient alkyne diesters with varied piperidones thiosemicarbazones. The condensation
of substituted thiosemicarbazones and dimethyl/diethyl acetylene dicarboxylate (DMAD/DEAD)
was widely claimed for the formation of 1,3–thiazin–4–one derivatives11 which might be formed
from the thioamide cyclisation with the ester functionality, an alcohol molecule was eliminated.
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After spectral characterization, the synthesized thiazinone derivatives were screened for their
inhibitory activities against Hep G2 cells due to the presence of biologically significant
functional groups.
2. Results and Discussion
2.1. Chemistry
In the present work, the reaction between acetylene diesters and N-substituted piperidin–4–one
thiosemicarbazones has been investigated and summarized in Scheme 1. The N-tosylpiperidin-4-
one and N–Bocpiperidin–4–one were used for the target molecule synthesis. The N-
tosylpiperidin-4-one was synthesized by the reaction between toluenesulphonyl chloride and
piperidone hydrochloride in water and chloroform (1:1) biphasic mixture medium as reported
earlier.12 The thiosemicarbazone derivatives were synthesized by treating thiosemicarbazide with
N-substituted piperidones in acidic methanol medium under reflux condition.13 To synthesize the
title compounds, the substituted thiosemicarbazones and acetylene diesters were stirred in
methanol for 1 h at 60°C. The yellow precipitate obtained was filtered and washed with warm
methanol. A possible reaction mechanism is displayed in Figure 1. The first step of the reaction
mechanism involves the conjugate addition of sulphur to the triple bond of acetylene diester and
further the intramolecular N−acylation leads to the final product.14,15 Further, the ester moiety
attached to the thiazinone ring was converted to acid using lithium hydroxide as base in
tetrahydrofuran and methanol mixture stirred for 1 h at 50°C.
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2.2 Spectral Characterization and Analysis
1
The structures of newly synthesized compounds were analyzed by FT–IR, Mass, H NMR and
1
13C NMR spectral techniques. The H NMR splitting patterns were noted as singlet (s), broad
singlet (bs), doublet (d), triplet (t), quartet (q) and multiplet (m). In order to assign the proton and
carbon signals of NMR spectra, compound 7 has been chosen as a representative compound.
2.2.1 Analysis of FTIR and Mass spectra
The FT–IR spectrum of compound 7 shows sharp intense bands at 1717, 1636 and 1593 cm-1
confirm the presence of ester carbonyl, amido carbonyl and imino group. The asymmetric and
symmetric stretching of –SO2– of compounds 7–10 give intense bands around 1320 and 1150
cm-1 respectively. For compounds 11–16 the –Boc carbonyl appeared between 1732 and 1719
cm-1. From the peak values observed in the mass spectra, the product formation was confirmed
by its molecular mass. FT–IR and Mass spectra of all compounds are given in the supplementary
data file.
2.2.2 Analysis of NMR Spectra
1
In H NMR spectrum of compound 7, a singlet at 2.42 ppm with integral value three was
assigned for methyl protons attached to the aromatic ring. The four triplets appeared at 2.62 (J =
6.0 Hz), 2.91 (J = 6.0 Hz), 3.18 (J = 6.0 Hz) and 3.25 (J = 6.0 Hz) ppm were assigned for the
methylene protons present in the piperidyl ring. Moreover, the N–CH3 protons present in the
thiazinone ring resonate as a sharp singlet at 3.34 ppm with three protons integral. Similarly, the
methyl protons of the ester group were resonated as a singlet at 3.83 ppm with three protons
integral. The signal at 6.86 ppm with one proton integral corresponds to methine proton present
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in the thiazinone ring. The two doublets with two protons integral each at 7.32 (J = 8.0 Hz) ppm
and 7.66 (J = 8.4 Hz) ppm was assigned to the protons of aromatic ring. Whereas for compounds
11, 12, 13, 14, 15 and 16, the tert–butyl methyl protons peak appeared around 1.4 to 1.5 ppm as a
singlet with nine protons integral value.
In 13C NMR of compound 7, the signal appeared at 21.5 ppm was assigned to the methyl carbon
attached to the aromatic ring. The signals at 27.9, 29.6, 45.8 and 46.8 ppm corresponds to the
methylene carbons which are present in the piperidyl ring. The carbon signal at 34.1 ppm was
assigned to N–CH3 carbon present in the thiazinone ring. Further, the methyl carbon of ester
group gives signal at 52.5 ppm and the methine carbon present in the thiazinone ring gives signal
at 116.0 ppm. The signals at the deshielded regions 127.6 and 129.8 ppm corresponds to the
methine carbons of the aromatic ring and the signals at 133.0 and 143.9 ppm were assignable to
the carbons attached to the methyl and sulphonyl groups of the same aromatic ring. The signals
at 159.0 and 166.9 ppm were assigned to the imino carbons present in the thiazinone and
piperidyl rings respectively. The carbons of amido carbonyl and ester carbonyl were assigned at
165.0 and 166.3 ppm respectively.
2.3. Anticancer studies
2.3.1 Cytotoxicity
The cytotoxicity of all novel compounds (7–16) were tested against human liver cancer cell line
(Hep G2) at various concentrations, viz., 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM,
80 μM, 90 μM and 100 μM through MTT (3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyl
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tetrazolium bromide, a yellow tetrazole) assay. The results are described in the Supplemental
Materials (Figure S 42, Table S 1)
3. Experimental
3.1. Instrumentation
All the reactions were carried out using analytical grade solvents without further purification.
The reactions were monitored by thin layer chromatography using 2x5 cm silica gel precoated
aluminium sheets of Merck TLC 60 F254 and visualized in ultra–violet light (254 nm) and iodine
chamber. The melting points of the newly synthesized compounds were determined in open
capillary tubes and are uncorrected. Fourier Transform Infra–red spectra were recorded using
1
KBr pellet on Thermo Nicolet FT–IR model iS5 spectrophotometer. The H and 13C NMR
spectra were recorded on a Bruker instrument (400 MHz) using Tetramethylsilane (TMS) as an
internal standard. Deuterated chloroform/deuterated dimethyl sulfoxide was used to record all
NMR spectra. The chemical shifts are expressed in δ (parts per million) units relative to the
standard and the coupling constants reported in Hertz (Hz). High resolution mass spectra were
recorded on Waters Synapt–G2 high definition mass spectrometer. The Supplemental Materials
contains sample 1H and 13C NMR and HR-MS spectra of the products (Figures S 1 – S 40)
3.2. Synthetic Procedures
3.2.1 Synthesis of 1-Tosylpiperidin-4-one (1)
To the completely dissolved solution of 4-piperidone hydrochloride monohydrate (3.0 g, 22
mmol) in 20mL of biphasic system of CHCl3−H2O (1:1), K2CO3 (9.13 g, 66 mmol) was added
followed by the addition of toluenesulphonyl chloride (5.48 g, 26 mmol). The above reaction
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mixture was allowed to stir at 50°C for 1 hour. After completion of reaction, the organic layer
was separated and dried over sodium sulphate. The excess solvent was removed using rota
evaporator and the crude product was washed with diethyl ether to afford the pure compound.
3.2.2 General procedure for the synthesis of compounds (3–6)
In a 50 mL RB flask of N-substituted piperidone (1.0 mmol) was dissolved in of methanol (20
mL). To this solution, thiosemicarbazide/N-methyl thiosemicarbazide (1.0 mmol) and
concentrated hydrochloric acid (0.2 mL) were added with stirring. The reaction mixture was
allowed to reflux for 1 h. After the completion of reaction was confirmed by thin layer
chromatography, the reaction mixture was cooled to room temperature and the solid separated
was filtered. The crude product was recrystallised from methanol.
3.2.3 General procedure for the synthesis of compounds 7−14
To a warm solution of N–substituted piperidin−4−one thiosemicarbazones (1.0 mmol) in
methanol, methanolic solution of DMAD/DEAD (1.0 mmol) was added drop wise with
stirring. The reaction mixture was stirred at 60°C for 1 h. The completion of reaction was
confirmed by thin layer chromatography and the reaction mixture was cooled to room
temperature. The yellow solid obtained was filtered and washed with warm methanol. The
crude product was recrystallised from chloroform–methanol mixture.
(E)-methyl-3-methyl-4-oxo-2-((1-tosylpiperidin-4-ylidene)hydrazono)-3,4-dihydro-2H-1,3-
thiazine-6-carboxylate (7) Yield: 79%; Chemical formula: C19H22N4O5S2; Yellow solid; mp:
1
162–164°C; H NMR (400 MHz, CDCl3, δ ppm): 7.66 (d, J =8.4 Hz, 2H), 7.32 (d, J = 8.0 Hz,
2H), 6.86 (s, 1H), 3.83 (s, 3H), 3.34 (s, 3H), 3.25 (t, J = 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H),
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2.91(t, J = 6.0 Hz, 2H), 2.62 (t, J = 6.0 Hz, 2H), 2.42 (s, 3H); C NMR (100 MHz, CDCl3 ): δ
166.8, 166.3, 165.0, 159.0, 143.9, 141.6, 133.0, 129.8, 127.6, 116.0, 52.5, 46.8, 45.8, 34.1, 29.6,
27.9, 21.5; FTIR (KBr, cm-1): νmax 3427, 1717, 1698, 1636, 1593, 1323 and 1159; HRMS:
(ESI/(M+H)+) calculated 451.1111 found 450.9696.
(E)-ethyl-3-methyl-4-oxo-2-((1-tosylpiperidin-4-ylidene)hydrazono)-3,4-dihydro-2H-1,3-
thiazine-6-carboxylate (8) Yield: 74%; Chemical formula: C20H24N4O5S2; Yellow solid; mp:
1
187–189°C; H NMR (400 MHz, CDCl3, δ ppm): 7.66 (d, J = 7.2 Hz, 2H), 7.32 (d, J = 7.6 Hz,
2H), 6.86 (s,1H), 4.29 (q, J = 7.2 Hz, 2H), 3.34 (s, 3H), 3.25 (bs, 2H), 3.18 (bs, 2H), 2.91 (d, J =
5.2 Hz, 2H), 2.61 (bs, 2H), 2.42 (s, 3H), 1.33 (t, J = 4.8 Hz, 3H); 13C NMR (100 MHz, CDCl3 ):
δ 166.6, 165.9, 165.1, 159.2, 143.9, 141.3, 133.0, 129.8, 127.6, 116.5, 61.6, 46.8, 45.8, 34.1,
29.5, 27.9, 21.5, 14.2; FTIR (KBr, cm-1): νmax 3410, 1715, 1695, 1630, 1594, 1339 and 1159;
HRMS: (ESI/(M+H)+) calculated 465.1267 found 465.1920.
Methyl-4-oxo-2-(2-(1-tosylpiperidin-4-ylidene)hydrazinyl)-4H-1,3-thiazine-6-carboxylate
(9)
1
Yield: 81%; Chemical formula: C18H20N4O5S2; Yellow solid; mp: 171–173°C; H NMR (400
MHz, CDCl3, δ ppm): 7.66 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.78 (s, 1H), 3.84 (s,
3H), 3.26 (t, J = 5.6 Hz, 2H), 3.17 (t, J = 6.0 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 5.6
Hz, 2H), 2.44 (s, 3H); 13C NMR (100 MHz, CDCl3 ): δ 167.4, 166.9, 159.3, 145.1, 143.8, 133.6,
130.8, 128.3, 116.2, 53.2, 47.6, 46.7, 34.8, 28.7, 22.1; FTIR (KBr, cm-1): νmax 3438, 1729, 1700,
1642, 1601, 1340 and 1152; HRMS: (ESI/(M+H)+) calculated 437.0954 found 437.1516.
Ethyl-4-oxo-2-(2-(1-tosylpiperidin-4-ylidene)hydrazinyl)-4H-1,3-thiazine-6-carboxylate
(10)
1
Yield: 77%; Chemical formula: C19H22N4O5S2; Yellow solid; mp: 192–198°C; H NMR (400
MHz, CDCl3, δ ppm): 7.62 (d, J = 6.8 Hz, 2H), 7.33 (d, J = 7.6, 2H), 6.72 (s, 1H), 4.26 (m, 2H),
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3.21 (bs, 2H), 3.12 (bs, 2H), 2.84 (m, 4H), 2.42 (s, 3H), 1.31 (m, 3H); C NMR (100 MHz,
CDCl3 ): δ 167.0, 166.6, 166.0, 144.7, 143.6, 133.3, 130.7, 128.2, 116.2, 62.2, 47.5, 46.6, 34.8,
28.7, 22.3, 15.0; FTIR (KBr, cm-1): νmax 3446, 1733, 1695, 1637, 1600, 1328 and 1152; HRMS:
(ESI/(M+H)+) calculated 451.1111 found 451.1651.
Methyl-2-(2-(1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazinyl)-4-oxo-4H-1,3-thiazine-6-
carboxylate (11) Yield: 83%; Chemical formula: C16H22N4O5S; Yellow solid; mp: 185–187°C;
1H NMR (400 MHz, CDCl3, δ ppm): 6.85 (s, 1H), 3.85 (s, 3H), 3.62 (m, 4H), 2.74 (bs, 2H), 2.52
13
(t, J = 6.0 Hz, 2H), 1.50 (s, 9H); C NMR (100 MHz, CDCl3 ): δ 168.5, 166.2, 165.2, 154.7,
142.1, 116.1, 96.1, 80.4, 52.4, 44.1, 42.8, 34.4, 28.9, 28.4; FTIR (KBr, cm-1): νmax 3438, 1727,
1698, 1643, 1607, 1335 and 1173; HRMS: (ESI/(M+H)+) calculated 383.1390 found 383.2100.
Ethyl-2-(2-(1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazinyl)-4-oxo-4H-1,3-thiazine-6-
carboxylate (12) Yield: 72%; Chemical formula: C17H24N4O5S; Yellow solid; mp: 211–213°C;
1H NMR (400 MHz, CDCl3, δ ppm): 6.84 (s, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.62 (t, J = 5.6 Hz,
2H), 3.57 (bs, 2H), 2.73 (bs, 2H), 2.51 (t, J = 5.6 Hz, 2H), 1.49 (s, 9H), 1.34 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3 ): δ 169.5, 168.4, 165.8, 165.4, 157.6, 154.7, 116.6, 80.4, 61.6, 34.4,
30.9, 28.9, 28.4, 14.2; FTIR (KBr, cm-1): νmax 3438, 1729, 1694, 1642, 1604, 1334 and 1181;
HRMS: (ESI/(M+H)+) calculated 397.1546 found 397.2265.
(E)-methyl-2-((1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazono)-3-methyl-4-oxo-3,4-
dihydro-2H-1,3-thiazine-6-carboxylate (13) Yield: 76%; Chemical formula: C17H24N4O5S;
1
Yellow solid; mp: 189–191°C; H NMR (400 MHz, CDCl3, δ ppm): 6.88 (s, 1H), 3.85 (s, 3H),
3.62 (t, J = 5.6 Hz, 2H), 3.56 (bs, 2H), 3.38 (s, 3H), 2.80 (t, J = 5.6 Hz, 2H), 2.52 (bs, 2H), 1.49
13
(s, 9H); C NMR (100 MHz, CDCl3 ): δ 168.9, 166.3, 165.1, 158.2, 154.6, 141.8, 115.8, 80.1,
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52.4, 34.5, 29.6, 28.7, 28.4; FTIR (KBr, cm-1): νmax 3424, 1719, 1689, 1641, 1595, 1428 and
1182; HRMS: (ESI/(M+H)+) calculated 397.1546 found 397.2048.
(E)-ethyl-2-((1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazono)-3-methyl-4-oxo-3,4-
dihydro-2H-1,3-thiazine-6-carboxylate (14) Yield: 69%; Chemical formula: C18H26N4O5S;
Yellow solid; mp: 193–195°C; 1H NMR (400 MHz, CDCl3, δ ppm): 6.87 (s, 1H), 4.30 (q, J = 7.2
Hz, 2H), 3.62 (t, J = 5.6 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.38 (s, 3H), 2.79 (t, J = 5.6 Hz, 2H),
2.51 (t, J = 5.6 Hz, 2H), 1.49 (s, 9H), 1.34 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3 ): δ
168.8, 165.9, 165.1, 158.4, 154.6, 141.5, 116.3, 80.1, 61.6, 34.5, 29.6, 28.7, 28.4, 14.2; FTIR
(KBr, cm-1): νmax 3424, 1720, 1688, 1639, 1596, 1429 and 1190; HRMS: (ESI/(M+H)+)
calculated 411.1703 found 411.2157.
3.2.4 General procedure for the synthesis of compounds 15 and 16
The ester (11 or 13) compound (1 mmol) was dissolved in tetrahydrofuran (3 mL), methanol (2
mL) mixture and the lithium hydroxide (2mmol, in 2 mL water) was added to the solution. The
above mixture was stirred at 50 °C for 1 hour, then cooled to room temperature and neutralized
with dilute hydrochloric acid. The crude product obtained was filtered and recrystallised in
aqueous dimethylformamide.
2-(2-(1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazinyl)-4-oxo-4H-1,3-thiazine-6-
carboxylic acid (15) Yield: 73%; Chemical formula: C15H20N4O5S; Yellow solid; mp: 202–
204°C; 1H NMR (400 MHz, DMSO–d6, δ ppm): 12.72 (bs, 1H), 6.58 (s, 1H), 3.51 (t, J = 5.6 Hz,
13
2H), 3.46 (t, J = 5.6 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.43 (t, J = 6.0 Hz, 2H), 1.42 (s,9H); C
NMR (100 MHz, DMSO-d6): δ 167.6, 166.6, 165.7, 158.4, 153.7, 142.2, 115.6, 79.0, 33.8, 28.3,
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28.0; FTIR (KBr, cm-1): νmax 3449, 1732, 1692, 1647, 1425 and 1168; HRMS: (ESI/(M+H)+)
calculated 369.1233 found 368.9700.
(E)-2-((1-(tert-butoxycarbonyl)piperidin-4-ylidene)hydrazono)-3-methyl-4-oxo-3,4-dihydro-2H-
1,3-thiazine-6-carboxylic acid (16) Yield: 71%; Chemical formula: C16H22N4O5S; Yellow solid;
mp: 232–234°C; 1H NMR (400 MHz, CDCl3, δ ppm): 6.86 (s, 1H), 3.61 (bs, 2H), 3.55 (bs, 2H),
13
3.37 (s, 3H), 2.80 (bs, 2H), 2.51 (bs, 2H), 1.48 (s, 9H); C NMR (100 MHz, CDCl3 ): δ 168.4,
167.3, 165.2, 158.5, 154.5, 141.0, 117.3, 79.9, 34.4, 29.4, 28.6, 28.3; FTIR (KBr, cm-1): νmax
3424, 1720, 1693, 1638, 1429 and 1184; HRMS: (ESI/(M+H)+) calculated 383.1390 found
383.1797.
3.3. In vitro studies
3.3.1 Cytotoxicity
The synthesized compounds (7–16) were evaluated for their cytotoxic effect on human hepatoma
cell line Hep G2 through MTT assay.16 The cell line was obtained from the National Center for
Cell Sciences, Pune, India. Dimethyl sulfoxide (DMSO, sigma, USA) was used to dissolve the
compounds quantitatively and diluted to make the stock solution. Hep G2 were seeded in 96 well
plates at a density of 5×103 cells per well. After 24 h, the cancer cells were treated with various
concentrations of the tested compounds in the range of 10 to 100 μM and incubated for 24 h and
48 h. Further, the cells were assigned by the addition of 20 μL of MTT (5 mg/mL in PBS-
phosphate buffered saline) per well and incubated again in the dark at 37°C for 3 h. Later, the
purple formazan crystals formed were dissolved in 100 μL of DMSO after aspirating the MTT
and incubated for further 10 min. The conversion of MTT to blue formazan product due to the
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mitochondrial dehydrogenase in viable cells was measured at 570 nm (measurement) and 630
nm (reference) using a 96 well plates reader (Bio-Rad, Hercules, CA, USA). The means were
collected from the six independent experiments and the percentage of inhibition was calculated
from the data using the following formula
 Mean absorbance of treated cells/Mean absorbance of sham control cells  100




3.3.2 Cell Morphology assessment
Hep G2 cells at a density of 5×105 cells per well were grown on glass cover slips (22×22mm)
placed in six well plates and incubated for 24 h before treatment with the IC50 (50% inhibitory
concentration after treatment) concentrations of compounds (7–16). The medium was
subsequently removed from each well of the sham control and the cells were treated with the
compounds after 24 h and 48 h incubation. The cover slips with treated cells were then inverted
and placed over micro slides. The gross morphological changes in the sham control and
compound treated cells were observed and calculated.
3.3.3 Assessment of mitochondrial membrane potential
Hep G2 cells were grown on glass cover slip, placed in six well plates and treated with 24 h IC50
concentrations of the selected compounds 7, 8, 13, 14 and 15. Since loss of mitochondrial
membrane potential is an early apoptotic marker, the cells were stained with 5,5´,6,6´–
tetrachloro–1,1´,3,3´–tetraethyl benzimidazol–carbocyanine iodide (JC-1) dye 24 h exposure.
The mitochondrial depolarization pattern of the cells were observed using fluorescent
microscope (Carl Zeiss, Jena, Germany) fitted with a 377–355 nm filter., at 40× magnification.
Mitochondrial depolarization is indicated by a decrease in the red fluorescence intensity.
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4. Conclusion
In this study, a convenient and effective synthesis of substituted 1,3-thiazin-4-one derivatives in
good yields by the reaction of acetylene diesters and N-substituted piperidone
thiosemicarbazones were reported. The novel compounds were tested for their cytotoxicity
against human hepatoma cell line Hep G2 through MTT assay. Compounds 7, 8, 13, 14 and 15
were displayed better activity against Hep G2 cell line among the tested compounds. The
apoptotic induction of the compounds was tested by the mitochondrial membrane potential using
JC–1 dye. The results of apoptotic evaluation were found to be identical with the cytotoxicity
results.
Acknowledgements
The authors gratefully acknowledge the funding support from University Grants
Commission (UGC) Grant No. 39–689–2010 SR, New Delhi, India.
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Scheme 1. The reaction between N-substituted piperidin–4–one thiosemicarbazones and
DMAD/DEAD.
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Figure 1 A plausible mechanism for thioamide and DMAD cyclization reaction.
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