Synthesis, antioxidant, and cytotoxic activities of n-azole substituted thiomorpholine Derivatives

Article abstract of DOI:10.1002/ardp.201300299

A new class of N-azole substituted thiomorpholine derivatives were prepared and their antioxidant and cytotoxic activities were studied. The methyl substituted oxazolyl thiomorpholine dioxide 9b exhibited radical scavenging activity greater than the standard ascorbic acid. On the other hand, the thiazolyl thiomorpholine 10c having a chloro substituent on the aromatic ring was identified as a remarkable lead molecule for cytotoxic activity against A549 and HeLa cells, with IC₅₀ values of 10.1 and 30.0 μM, respectively. A new series of N-azole substituted thiomorpholine derivatives were prepared to study their antioxidant and cytotoxic activities. The methyl substituted oxazolyl thiomorpholine dioxide 9b exhibited excellent radical scavenging activity greater than ascorbic acid. The thiazolyl thiomorpholine 10c showed noticeable cytotoxic activity against A549 and HeLa cells.

Full text of DOI:10.1002/ardp.201300299

Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
221
Full Paper
Synthesis, Antioxidant, and Cytotoxic Activities of N-Azole
Substituted Thiomorpholine Derivatives
Putta Ramachandra Reddy1, Guda Mallikarjuna Reddy1, Adivireddy Padmaja1,
Venkatapuram Padmavathi1, Paturu Kondaiah2, and Narra Siva Krishna2
1
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore,
2
Karnataka, India
A new class of N-azole substituted thiomorpholine derivatives were prepared and their antioxidant and
cytotoxic activities were studied. The methyl substituted oxazolyl thiomorpholine dioxide 9b exhibited
radical scavenging activity greater than the standard ascorbic acid. On the other hand, the thiazolyl
thiomorpholine 10c having a chloro substituent on the aromatic ring was identified as a remarkable
lead molecule for cytotoxic activity against A549 and HeLa cells, with IC50 values of 10.1 and 30.0 mM,
respectively.
Keywords: Antioxidant activity / Cytotoxicity / Imidazoles / Oxazoles / Thiazoles / Thiomorpholines
Received: August 10, 2013; Revised: September 17, 2013; Accepted: September 23, 2013
DOI 10.1002/ardp.201300299
Introduction
remarkable cytotoxic and antitumor properties [12, 13].
Imidazoles are common scaffolds in highly significant
biomolecules including biotin, histidine, and histamine.
Medicinal properties of imidazole containing compounds
include anticancer [14], antimicrobial [15–18], and antioxi-
dant [19]. Thiazoles are among the most frequently encoun-
tered heterocycles in compounds of biological interest. For
example, the aminothiazole ring system is a useful structural
element in medicinal chemistry and has found broad
application in drug development for the treatment of
allergies [20], hypertension [21], inflammation [22], bacterial
infection [23], and HIV [24]. In addition, these compounds
exhibited significant antimicrobial and anticancer activi-
ties [25–27]. Realizing the importance of thiomorpholines and
azoles, it is planned to conjugate these two ligands under one
construct and to study the cytotoxic activity.
Thiomorpholinone is a pharmacophore of great interest
because of its unique biological properties. Thiomorpholine,
and its S-oxide and S,S-dioxide derivatives are associated with
a variety of pharmacological activities including antibacteri-
al [1], antimicrobial [2], and anti-inflammatory [3]. This
nucleus is present in 1,4-benzothiazine calcium antagonist
semotiadil [4] as well as in pyrimidothiazine derivatives [5]
designed as an inhibitor of the glycinamide ribonucleotide
transformylase with potent cell growth inhibition. Thiomor-
pholines were synthesized by base-promoted aminoethylation
of ethyl thioglycolate with 2-oxazolidinones [6] and from
glycidic esters [7] besides the other methods like Ugi
reactions [8], microwave-assisted Smiles rearrangement [9],
and copper-catalyzed cascade reactions [10].
The azole derivatives are the prominent players in the
pharmaceutical research as they possess several biological Results and discussion
properties. In fact, simple oxazoles are common units in a
wide variety of polyoxazole marine natural products possess-
ing biological activity [11], while aminoalkyl oxazoles are
constituents of peptide-based alkaloids and antibiotics with
Chemistry
The N-azole substituted thiomorpholine derivatives were
prepared as outlined in Scheme 1. The compounds thio-
diglycolic acid chloride (1), sulfonyldiglycolic acid chloride
(2) [28], and the five-membered heterocyclic amines, 4-
aryloxazol-2-amine (3), 4-arylthiazol-2-amine (4) [29], and 4-
aryl-1H-imidazol-2-amine (5) [30], were prepared adopting the
literature precedent. The reaction of 1 with 3 in benzene
Correspondence: Dr. Venkatapuram Padmavathi, Department of Chem-
istry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh,
India.
E-mail: vkpuram2001@yahoo.com
Fax: þ91 877 2261825
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222
P. R. Reddy et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
Scheme 1. Synthesis of N-azole substituted thiomorpholines.
afforded 4-(4-aryloxazol-2-yl)thiomorpholine-3,5-dione (6) in
quantitative yield. Aiming at the construction of another
heterocyclic system, similar reaction of 1 was performed with
4, which resulted in 4-(4-arylthiazol-2-yl)thiomorpholine-3,5-
dione (7). Likewise, the compound 4-(4-aryl-1H-imidazol-2-yl)-
thiomorpholine-3,5-dione (8) was prepared by the reaction of 1
Biological results
Antioxidant activity
The compounds 6–11 were tested for antioxidant activity by
2,20-diphenyl-1-picrylhydrazyl (DPPH) [31, 32] and nitric oxide
(NO) [33, 34] methods. The results are presented in Tables 1
and 2 and Figs. 1 and 2. Among all the tested compounds, the
bis-heterocycles having thiomorpholinedioxides (9–11) dis-
played greater activity than the respective bis-heterocycles
having thiomorpholines (6–8). In fact, the compound 9b
displayed excellent radical scavenging activity greater than
the standard ascorbic acid. The compounds 9a, 10b, and 11b
also showed good radical scavenging activity. However, the
compounds oxazolyl thiomorpholines 6 and imidazolyl
thiomorpholines 8 displayed least activity. On the other
hand, the thiazolyl thiomorpholines showed no activity.
Thus, the results exemplified that thiomorpholinedioxide in
combination with oxazole 9 showed excellent activity, which
may be due to the presence of more oxygen atoms. The
presence of electron-donating methyl substituent on the
aromatic ring increases the activity when compared with
those having chloro substituent. It was also observed that
imidazolyl thiomorpholinedioxides 11 exhibited greater
activity than thiazolyl thiomorpholinedioxides 10. The free
radical scavenging activity of the compounds 9a, 9b, and 11b
was measured at different concentrations and mentioned the
change in absorbance at 10, 20, and 30 min in DPPH method
1
with 5 (Scheme 1). The H NMR spectrum of 6a displayed a
singlet for methylene protons of thiomorpholine at d 3.41 and
another singlet at 7.30 due to C5-H of oxazole. Similarly, the
compounds 7a and 8a exhibited two singlets at d 3.36, 7.24
and 3.38, 7.27 due to methylene protons and C5-H of thiazole/
imidazole rings. The compound 8a displayed a broad singlet
at 11.13 ppm due to NH, which disappeared on deuteration.
The compounds 1,1-dioxo-4-(4-aryloxazol-2-yl)thiomorpho-
line-3,5-dione (9), 1,1-dioxo-4-(4-arylthiazol-2-yl)thiomorpho-
line-3,5-dione (10), and 1,1-dioxo-4-(4-aryl-1H-imidazol-2-yl)-
thiomorpholine-3,5-dione (11) were obtained by the reaction
of 2 with 3, 4, and 5, respectively. The 1H NMR spectra of 9a,
10a, and 11a showed a singlet for methylene protons at
slightly downfield region than the thio derivatives at d 4.41,
4.35, and 4.37. In addition, another singlet was observed at
7.32, 7.23, and 7.30 due to C5-H of oxazole/thiazole/imidazole
rings. The compound 11a exhibited a broad singlet at
11.20 ppm for NH, which disappeared when D2O was added.
The structures of all the lead compounds were further
established by IR, 13C NMR, and elemental analyses.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
N-Azole Substituted Thiomorpholine Derivatives
223
Table 1. The in vitro antioxidant activity of 6–11 in the DPPH
method.
Table 2. The in vitro antioxidant activity of 6–11 in the nitric
oxide method.
Concentration (mg/mL)
Concentration (mg/mL)
Compound
50
75
100
Compound
50
75
100
6a
6b
6c
21.70 ꢁ 0.23
27.57 ꢁ 1.15
–
20.12 ꢁ 0.63
30.17 ꢁ 0.85
–
26.10 ꢁ 0.45
32.75 ꢁ 1.32
–
6a
6b
6c
20.25 ꢁ 0.25
29.95 ꢁ 1.23
–
22.27 ꢁ 0.49
31.81 ꢁ 0.96
–
25.11 ꢁ 0.65
34.87 ꢁ 1.10
–
7a
–
–
–
7a
–
–
–
7b
–
–
–
7b
–
–
–
7c
–
–
–
7c
–
–
–
8a
–
–
–
8a
–
–
–
8b
8c
22.37 ꢁ 0.35
–
25.93 ꢁ 0.48
–
27.56 ꢁ 0.61
–
8b
8c
30.61 ꢁ 0.24
–
33.27 ꢁ 0.71
–
35.68 ꢁ 1.02
–
9a
9b
9c
10a
10b
10c
11a
11b
62.34 ꢁ 0.99
79.92 ꢁ 0.76
55.27 ꢁ 1.14
39.46 ꢁ 1.23
50.17 ꢁ 0.76
31.37 ꢁ 0.89
49.49 ꢁ 0.95
62.37 ꢁ 0.33
39.73 ꢁ 0.98
78.16 ꢁ 0.36
–
65.27 ꢁ 0.75
82.79 ꢁ 1.21
56.98 ꢁ 0.16
40.71 ꢁ 0.45
52.34 ꢁ 0.37
32.98 ꢁ 0.19
51.70 ꢁ 0.68
65.96 ꢁ 0.51
41.89 ꢁ 0.23
80.98 ꢁ 1.12
–
66.41 ꢁ 0.09
85.56 ꢁ 0.55
58.36 ꢁ 1.09
43.38 ꢁ 0.36
54.95 ꢁ 0.83
35.37 ꢁ 0.67
53.35 ꢁ 0.41
67.65 ꢁ 0.52
43.37 ꢁ 0.51
83.82 ꢁ 0.83
–
9a
9b
9c
10a
10b
10c
11a
11b
65.53 ꢁ 0.15
88.75 ꢁ 0.57
58.54 ꢁ 0.92
47.75 ꢁ 1.20
54.59 ꢁ 1.04
40.73 ꢁ 0.84
50.57 ꢁ 0.78
64.61 ꢁ 0.56
44.37 ꢁ 0.48
86.02 ꢁ 0.73
–
66.98 ꢁ 0.78
89.96 ꢁ 0.76
60.20 ꢁ 0.35
49.12 ꢁ 0.82
56.90 ꢁ 0.16
43.75 ꢁ 0.30
51.21 ꢁ 0.28
66.33 ꢁ 0.51
46.58 ꢁ 0.40
89.46 ꢁ 0.80
–
67.24 ꢁ 0.36
92.79 ꢁ 0.86
61.39 ꢁ 1.08
50.65 ꢁ 0.87
57.37 ꢁ 1.25
45.21 ꢁ 1.13
53.56 ꢁ 0.45
67.75 ꢁ 1.37
48.12 ꢁ 1.31
91.53 ꢁ 0.71
–
11c
Ascorbic acid
Blank
11c
Ascorbic acid
Blank
(–) Showed no scavenging activity.
Values were the means of three replicates ꢁ SD.
(–) Showed no scavenging activity.
Values were the means of three replicates ꢁ SD.
(Table 3). It was observed that at these 10-min intervals the
values are very close and the results indicated that the
antioxidant activity is independent of time.
molecule that could be developed as therapeutic drug. Efforts
are in progress toward this goal.
Conclusion
Cytotoxicity
The newly synthesized compounds 6–11 are subjected to MTT
assay to determine growth-inhibitory/cytotoxic capability.
Only compound 10c showed noticeable cytotoxic activity on
A549 cells (IC50 ¼ 10.1mM) and HeLa cells (IC50 ¼ 30 mM).
However, all the other compounds did not show any
cytotoxicity when used up to 200 mM concentration. Figures 3
and 4 show the results of cytotoxicity of 10c using MTT assay
on A549 lung carcinoma cells and HeLa cells, respectively. The
cytotoxic activity observed with compound 10c is concentra-
tion dependent. Compound 10c at concentrations 12.5–
200 mM showed lowest viability, while viability more than
In conclusion, a new class of N-azole substituted thiomorpho-
line derivatives were prepared and their antioxidant and
cytotoxic activities were studied. The methyl substituted
oxazolyl thiomorpholine dioxide 9b exhibited radical scav-
enging activity greater than the standard ascorbic acid. On the
other hand, the thiazolyl thiomorpholine 10c having chloro
substituent on the aromatic ring was identified as a noticeable
lead molecule for cytotoxic activity against A549 cells and
HeLa cells with IC50 values of 10.1 and 30.0 mM, respectively,
which can be used as a lead compound in the future studies.
50% is observed when this compound is used at
a
Experimental
concentration below 12.50 mM on A549 cells and 75% cell
viability on HeLa cells at 12.50 mM concentration. This
suggests compound 10c as a noticeable lead molecule for
cytotoxic activity against tumor cells. It is of interest to note
that compound 10c is more potent on A549 cells compared to
HeLa cells. HeLa cells are cervical carcinoma cells that are
oncogenically driven by human papilloma viruses. On the
other hand, A549 cells have a mutated K-ras oncogene.
Further modifications to 10c may provide a more potent
Chemistry
Melting points were determined in open capillaries on a Mel
Temp apparatus and are uncorrected. The progress of the
reactions and the purity of the compounds were monitered by
thin layer chromatography (TLC; silica gel H, BDH, ethyl acetate/
hexane 0.5:2). The IR spectra were recorded on a Thermo Nicolet
IR 200 FT-IR spectrometer as KBr pellets, and the wave
numbers were given in cmꢀ1. The 1H NMR spectra were recorded
in DMSO-d6 on a Jeol JNM l-400 MHz. The 13C NMR spectra
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Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
Figure 1. The in vitro antioxidant activities of 6–11 in the DPPH method.
Figure 2. The in vitro antioxidant activities of 6–11 in the nitric oxide method.
were recorded in DMSO-d6 on a Jeol JNM spectrometer operating
at 100 MHz. All chemical shifts are reported in d (ppm) using TMS
as an internal standard. The microanalyses were performed on
Perkin-Elmer 240C elemental analyzer. High-resolution mass
spectra were recorded on Micromass Q-TOF micromass spectro-
meter using electrospray ionization. The compounds thiodigly-
colic acid chloride (1), sulfonyldiglycolic acid chloride (2) [28],
4-aryloxazol-2-amine (3), 4-arylthiazol-2-amine (4) [29], and 4-aryl-
1H-imidazol-2-amine (5) [30] were prepared as per the literature
Table 3. Antioxidant activities of the compounds 9a, 9b, and 11b
at 10-min intervals by the DPPH scavenging method.
Compound
10 min
20 min
30 min
9a
9b
11b
66.24
85.40
67.38
66.35
85.61
67.50
66.48
85.95
67.67
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Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
N-Azole Substituted Thiomorpholine Derivatives
225
d (ppm): 3.41 (s, 4H, CH2), 7.30 (s, 1H, C5-H), 7.45–7.67 (m, 5H,
Ar–H); 13C NMR (DMSO-d6) d (ppm): 38.9 (CH2), 127.5, 127.9, 128.7,
133.8 (Ar–C), 142.1 (C-5), 143.2 (C-4), 153.2 (C-2), 164.0 (CO); HRMS
[MþNa] (m/z): 297.2840. Anal. calcd. for C13H10N2O3S: C 56.92,
H 3.67, N 10.21; Found: C 56.83; H 3.61; N 10.29%.
4-(4-p-Tolyloxazol-2-yl)thiomorpholine-3,5-dione (6b)
Brown solid in 75% (0.22 g) yield; m.p.: 162–164°C; IR (KBr) nmax
1
(cmꢀ1): 1568 (C N), 1620 (C C), 1627 (C O); H NMR (DMSO-d )
–
–
–
–
–
–
6
d (ppm): 2.23 (s, 3H, Ar–CH3), 3.38 (s, 4H, CH2), 7.26 (s, 1H, C5-H),
7.40–7.58 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 25.5
(Ar–CH3), 38.4 (CH2), 127.1, 127.4, 128.2, 133.2 (Ar–C), 141.7 (C-5),
142.9 (C-4), 152.8 (C-2), 163.6 (CO); HRMS [MþNa] (m/z): 311.3118;
Anal. calcd. for C14H12N2O3S: C 58.32, H 4.19, N 9.71; Found: C
58.45, H 4.14, N 9.82%.
4-(4-(4-Chlorophenyl)oxazol-2-yl)thiomorpholine-3,5-
Figure 3. The dose–response curve of 10c measured by MTT
assay on A549 lung carcinoma cells. X-axis shows the concentra-
tion of the compound, and Y-axis, the cell viability.
dione (6c)
Brown solid in 74% (0.23 g) yield; m.p.: 177–179°C; IR (KBr) nmax
1
(cmꢀ1): 1576 (C N), 1629 (C C), 1635 (C O); H NMR (DMSO-d )
–
–
–
–
–
–
6
d (ppm): 3.45 (s, 4H, CH2), 7.34 (s, 1H, C5-H), 7.51–7.71 (m, 4H,
Ar–H); 13C NMR (DMSO-d6) d (ppm): 39.2 (CH2), 127.9, 128.3, 129.1,
134.1 (Ar–C), 142.5 (C-5), 143.7 (C-4), 153.6 (C-2), 164.8 (CO); HRMS
[MþNa] (m/z): 331.7293. Anal. calcd. for C13H9ClN2O3S: C 50.57,
H 2.93, N 9.07; Found: C 50.65, H 2.88, N 8.96%.
4-(4-Phenylthiazol-2-yl)thiomorpholine-3,5-dione (7a)
Brown solid in 69% (0.20 g) yield; m.p.: 157–159°C; IR (KBr) nmax
1
(cmꢀ1): 1570 (C N), 1623 (C C), 1630 (C O); H NMR (DMSO-d )
–
–
–
–
–
–
6
d (ppm): 3.36 (s, 4H, CH2), 7.24 (s, 1H, C5-H), 7.38–7.60 (m, 5H,
Ar–H); 13C NMR (DMSO-d6) d (ppm): 36.7 (CH2), 103.2 (C-5), 127.0,
127.6, 128.4, 133.5 (Ar–C), 138.5 (C-4), 163.8 (CO), 169.7 (C-2);
HRMS [MþNa] (m/z): 313.3502. Anal. calcd. for C13H10N2O2S2: C
53.77, H 3.47, N 9.64; Found: C 53.87, H 3.39, N 9.78%.
4-(4-p-Tolylthiazol-2-yl)thiomorpholine-3,5-dione (7b)
Brown solid in 74% (0.22 g) yield; m.p.: 143–145°C; IR (KBr) nmax
(cmꢀ1): 1565 (C N), 1619 (C C), 1628 (C O) 1H NMR (DMSO-d6)
–
–
–
–
–
–
;
d (ppm): 2.20 (s, 3H, Ar–CH3), 3.33 (s, 4H, CH2), 7.18 (s, 1H, C5-H),
7.32–7.54 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 24.8
(Ar–CH3), 36.2 (CH2), 102.8 (C-5), 126.6, 127.1, 128.0, 132.9 (Ar–C),
138.1 (C-4), 163.4 (CO), 169.1 (C-2); HRMS [MþNa] (m/z): 327.3766.
Anal. calcd. for C14H12N2O2S2: C 55.24, H 3.97, N 9.20; Found:
C 55.17, H 4.03, N 9.32%.
Figure 4. The dose–response curve of 10c measured by MTT
assay on HeLa cervical carcinoma cells. X-axis shows the
concentration of the compound, and Y-axis, the cell viability.
procedure. All chemicals and solvents were purchased from
Merck and used without further purification.
4-(4-(4-Chlorophenyl)thiazol-2-yl)thiomorpholine-3,5-
dione (7c)
General procedure for the synthesis of 4-(4-aryloxazol-2-
yl)thiomorpholine-3,5-dione (6a–c)/4-(4-arylthiazol-2-yl)-
thiomorpholine-3,5-dione (7a–c)/4-(4-aryl-1H-imidazol-2-
Brown solid in 72% (0.23 g) yield; m.p.: 169–171°C; IR (KBr) nmax
(cmꢀ1): 1574 (C N), 1627 (C C), 1634 (C O); H NMR (DMSO-d )
1
–
–
–
–
–
–
6
d (ppm): 3.45 (s, 4H, CH2), 7.29 (s, 1H, C5-H), 7.42–7.66 (m, 4H,
Ar–H); 13C NMR (DMSO-d6) d (ppm): 37.1 (CH2), 103.7 (C-5), 127.3,
128.1, 128.9, 133.9 (Ar–C), 139.0 (C-4), 164.1 (CO), 170.0 (C-2);
HRMS [MþNa] (m/z): 347.7959. Anal. calcd. for C13H9ClN2O2S2: C
48.07, H 2.79, N 8.62; Found: C 47.95, H 2.64, N 8.73%.
yl)thiomorpholine-3,5-dione (8a–c)
A mixture of 3/4/5 (1 mmol), thiodiglycolic acid chloride (1.1 mmol)
and benzene (10 mL) was heated to reflux for 3–4h and the solvent
was removed under reduced pressure. The brown colored solid
obtained was recrystallized from aqueous methanol (1:1).
4-(4-Phenyl-1H-imidazol-2-yl)thiomorpholine-3,5-
4-(4-Phenyloxazol-2-yl)thiomorpholine-3,5-dione (6a)
dione (8a)
Brown solid in 72% (0.28 g) yield; m.p.: 145–147°C; IR (KBr) nmax
Brown solid in 73% (0.20 g) yield; m.p.: 235–237°C; IR (KBr) nmax
1
(cmꢀ1): 1572 (C N), 1625 (C C), 1631 (C O); H NMR (DMSO-d )
–
–
–
–
–
–
(cmꢀ1): 1567 (C N), 1624 (C C), 1632 (C O), 3227 (NH); H NMR
1
–
–
–
–
–
–
6
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(DMSO-d6) d (ppm): 3.38 (s, 4H, CH2), 7.27 (s, 1H, C5-H), 7.43–7.62
(m, 5H, Ar–H), 11.13 (bs, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 37.6
(CH2), 122.7 (C-5), 127.7, 128.4, 129.5, 134.6 (Ar–C), 140.3 (C-2),
142.7 (C-4), 163.7 (CO); HRMS [MþNa] (m/z): 296.3008. Anal. calcd.
for C13H11N3O2S: C 57.12, H 4.05, N 15.37; Found: C 57.23, H 4.13,
N 15.50%.
4-(4-(4-Chlorophenyl)oxazol-2-yl)-1,1-
dioxothiomorpholine-3,5-dione (9c)
Yellow solid in 86% (0.29 g) yield; m.p.: 197–199°C; IR (KBr) nmax
(cmꢀ1): 1153, 1354 (SO ), 1580 (C N), 1629 (C C), 1637 (C O);
–
–
–
–
–
–
2
1H NMR (DMSO-d6) d (ppm): 4.45 (s, 4H, CH2), 7.35 (s, 1H, C5-H),
7.57–7.85 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 58.7 (CH2),
128.2, 128.7, 129.7, 134.6 (Ar–C), 143.0 (C-5), 144.1 (C-4), 154.0
(C-2), 165.2 (CO); HRMS [MþNa] (m/z): 363.7287; Anal. calcd. for
4-(4-p-Tolyl-1H-imidazol-2-yl)thiomorpholine-3,5-
C
13H9ClN2O5S: C 45.82, H 2.61, N 8.22; Found: C 45.89, H 2.61, N
dione (8b)
8.33%.
Brown solid in 68% (0.19 g) yield; m.p.: 217–219°C; IR (KBr) nmax
1
(cmꢀ1): 1570 (C N), 1621 (C C), 1629 (C O), 3221 (NH); H NMR
–
–
–
–
–
–
(DMSO-d6) d (ppm): 2.18 (s, 3H, Ar–CH3), 3.34 (s, 4H, CH2), 7.23
(s, 1H, C5-H), 7.39–7.57 (m, 4H, Ar–H), 11.11 (bs, 1H, NH); 13C NMR
(DMSO-d6) d (ppm): 24.3 (Ar–CH3), 37.1 (CH2), 122.3 (C-5), 127.1,
128.0, 129.2, 134.1 (Ar–C), 139.8 (C-2), 142.2 (C-4), 163.1 (CO);
HRMS [MþNa] (m/z): 310.3264. Anal. calcd. for C14H13N3O2S: C
58.52, H 4.56, N 14.62; Found: C58.64, H 4.50, N 14.49%.
1,1-Dioxo-4-(4-phenylthiazol-2-yl)thiomorpholine-3,5-
dione (10a)
Yellow solid in 83% (0.27 g) yield; m.p.: 163–165°C; IR (KBr) nmax
(cmꢀ1): 1148, 1342 (SO ), 1574 (C N), 1627 (C C), 1632 (C O);
–
–
–
–
–
–
2
1H NMR (DMSO-d6) d (ppm): 4.35 (s, 4H, CH2), 7.23 (s, 1H, C5-H),
7.50–7.72 (m, 5H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 57.7 (CH2),
103.6 (C-5), 127.6, 128.1, 128.9, 133.9 (Ar–C), 139.1 (C-4), 163.8
(CO), 170.2 (C-2); HRMS [MþNa] (m/z): 345.3499. Anal. calcd. for
C13H10N2O4S2: C 48.44, H 3.13, N 8.69; Found: C 48.54, H 3.19,
N 8.24%.
4-(4-(4-Chlorophenyl)-1H-imidazol-2-yl)thiomorpholine-
3,5-dione (8c)
Brown solid in 67% (0.20 g) yield; m.p.: 262–264°C; IR (KBr) nmax
1
(cmꢀ1): 1578 (C N), 1628 (C C), 1636 (C O), 3230 (NH); H NMR
–
–
–
–
–
–
(DMSO-d6) d (ppm): 3.41 (s, 4H, CH2), 7.31 (s, 1H, C5-H), 7.46–7.66
(m, 4H, Ar–H), 11.15 (bs, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 38.2
(CH2), 123.4 (C-5), 128.2, 128.9, 129.9, 135.1 (Ar–C), 140.8 (C-2),
143.2 (C-4), 164.2 (CO); HRMS [MþNa] (m/z): 330.7449. Anal. calcd.
for C13H10ClN3O2S: C 50.73, H 3.27, N 13.65; Found: C 50.61, H
3.19, N 13.77%.
1,1-Dioxo-4-(4-p-tolylthiazol-2-yl)thiomorpholine-3,5-
dione (10b)
Yellow solid in 80% (0.27 g) yield; m.p.: 154–156°C; IR (KBr) nmax
(cmꢀ1): 1145, 1338 (SO ), 1567 (C N), 1624 (C C), 1628 (C O);
–
–
–
–
–
–
2
1H NMR (DMSO-d6) d (ppm): 2.24 (s, 3H, Ar–CH3), 4.33 (s, 4H, CH2),
7.20 (s, 1H, C5-H), 7.48–7.69 (m, 4H, Ar–H); 13C NMR (DMSO-d6)
d (ppm): 25.2 (Ar–CH3), 57.1 (CH2), 103.3 (C-5), 127.4, 127.7, 128.3,
133.4 (Ar–C), 138.8 (C-4), 164.0 (CO), 169.9 (C-2); HRMS [MþNa]
(m/z): 359.3758. Anal. calcd. for C14H12N2O4S2: C 49.99, H 3.59, N
8.33, Found: C 50.10, H 3.67, N 8.24%.
General procedure for the synthesis of 1,1-dioxo-4-(4-
aryloxazol-2-yl)-thiomorpholine-3,5-dione (9a–c)/1,1-
dioxo-4-(4-arylthiazol-2-yl)thiomorpholine-3,5-dione
(10a–c)/1,1-dioxo-4-(4-aryl-1H-imidazol-2-yl)-
thiomorpholine-3,5-dione (11a–c)
4-(4-(4-Chlorophenyl)thiazol-2-yl)-1,1-
A mixture of 3/4/5 (1 mmol), sulfonyldiglycolic acid chloride
(1.1 mmol) and benzene (10 mL) was heated to reflux for 3–4 h and
solvent was removed under vacuum. The yellow colored solid
obtained was recrystallized from aqueous methanol (1:1).
dioxothiomorpholine-3,5-dione (10c)
Yellow solid in 87% (0.31 g) yield; m.p.: 171–174°C; IR (KBr) nmax
(cmꢀ1): 1156, 1346 (SO ), 1579 (C N), 1630 (C C), 1638 (C O);
–
–
–
–
–
–
2
1H NMR (DMSO-d6) d (ppm): 4.38 (s, 4H, CH2), 7.27 (s, 1H, C5-H),
7.54–7.76 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 58.1 (CH2),
104.0 (C-5), 128.0, 128.6, 129.4, 134.1 (Ar–C), 139.4 (C-4), 164.7
(CO), 170.5 (C-2); HRMS [MþNa] (m/z): 379.7941. Anal. calcd. for
C13H9ClN2O4S2: C 43.76, H 2.54, N 7.85; Found: C 43.88, H 2.62,
N 7.98%.
1,1-Dioxo-4-(4-phenyl-oxazol-2-yl)thiomorpholine-3,5-
dione (9a)
Yellow solid in 85% (0.26 g) yield; m.p.: 157–159°C; IR (KBr) nmax
1
(cmꢀ1): 1150, 1350 (SO ), 1576 (C N), 1626 (C C), 1633 (C O); H
–
–
–
–
–
–
2
NMR (DMSO-d6) d (ppm): 4.41 (s, 4H, CH2), 7.32 (s, 1H, C5-H), 7.52–
7.81 (m, 5H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 58.3 (CH2), 127.9,
128.3, 129.2, 134.1 (Ar–C), 142.5 (C-5), 143.6 (C-4), 153.7 (C-2), 164.5
(CO); HRMS [MþNa] (m/z): 329.2840. Anal. calcd. for C13H10N2O5S:
C 50.98, H 3.29, N 9.14; Found: C 50.86, H 3.22, N 9.03%.
1,1-Dioxo-4-(4-phenyl-1H-imidazol-2-yl)thiomorpholine-
3,5-dione (11a)
Yellow solid in 79% (0.24 g) yield; m.p.: 242–244°C; IR (KBr) nmax
(cmꢀ1): 1150, 1349 (SO ), 1577 (C N), 1628 (C C), 1633 (C O),
–
–
–
–
–
–
2
3230 (NH); 1H NMR (DMSO-d6) d (ppm): 4.37 (s, 4H, CH2), 7.30 (s, 1H,
C5-H), 7.50–7.79 (m, 5H, Ar–H), 11.20 (bs, 1H, NH); 13C NMR (DMSO-
d6) d (ppm): 57.9 (CH2), 123.6 (C-5), 128.5, 129.1, 129.9, 135.2
(Ar–C), 140.9 (C-2), 143.3 (C-4), 164.1 (CO); HRMS [MþNa] (m/z):
305.3096; Anal. calcd. for C13H11N3O4S: C 51.14, H 3.63, N 13.76;
Found: C 51.07, H 3.58, N 13.85%.
1,1-Dioxo-4-(4-p-tolyl-oxazol-2-yl)thiomorpholine-3,5-
dione (9b)
Yellow solid in 82% (0.26 g) yield; m.p.: 186–188°C; IR (KBr) nmax
(cmꢀ1): 1145, 1345 (SO ), 1569 (C N), 1622 (C C), 1631 (C O)
–
–
–
–
–
–
2
cmꢀ1; 1H NMR (DMSO-d6) d (ppm): 2.28 (s, 3H, Ar–CH3), 4.38 (s, 4H,
CH2), 7.30 (s, 1H, C5-H), 7.50–7.76 (m, 4H, Ar–H); 13C NMR (DMSO-
d6) d (ppm): 25.8 (Ar–CH3), 57.8 (CH2), 127.4, 128.0, 128.7, 133.7
(Ar–C), 142.1 (C-5), 143.3 (C-4), 153.3 (C-2), 163.7 (CO); HRMS
[MþNa] (m/z): 343.3107. Anal. calcd. for C14H12N2O5S: C 52.49, H
3.77, N 8.74; Found: C 52.61, H 3.85, N 8.88%.
1,1-Dioxo-4-(4-p-tolyl-1H-imidazol-2-yl)thiomorpholine-
3,5-dione (11b)
Yellow solid in 80% (0.25 g); m.p.: 234–236°C; IR (KBr) nmax (cmꢀ1):
–
–
–
1146, 1344 (SO ), 1573 (C N), 1622 (C C), 1630 (C O), 3225 (NH);
–
–
–
2
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 221–228
N-Azole Substituted Thiomorpholine Derivatives
227
1H NMR (DMSO-d6) d (ppm): 2.67 (s, 3H, Ar–CH3), 4.35 (s, 4H, CH2),
7.27 (s, 1H, C5-H), 7.48–7.74 (m, 4H, Ar–H), 11.17 (bs, 1H, NH);
13C NMR (DMSO-d6) d (ppm): 24.7 (Ar–CH3), 57.4 (CH2), 123.2 (C-5),
128.1, 128.9, 129.4, 134.8 (Ar–C), 140.5 (C-2), 143.0 (C-4), 163.8
(CO); HRMS [MþNa] (m/z): 319.3351. Anal. calcd. for C14H13N3O4S:
C 52.65, H 4.10, N 13.16; Found: C 52.52, H 3.99, N 13.06%.
where Acontrol is the absorbance of the control reaction
(containing all reagents and ascorbic acid), Asample is the
absorbance of the test compound (containing all reagents and
test compound), and Ablank is the absorbance of the blank
(containing only reagents). Tests were carried out in triplicate.
Cytotoxic activity
Compounds 6–11 were dissolved in DMSO at different concen-
trations of 12.5–200 mM.
4-(4-(4-Chlorophenyl)-1H-imidazol-2-yl)-1,1-
dioxothiomorpholine-3,5-dione (11c)
Yellow solid in 76% (0.26 g) yield; m.p.: 273–275°C; IR (KBr) nmax
(cmꢀ1): 1154, 1356 (SO ), 1582 (C N), 1630 (C C), 1640 (C O),
–
–
–
–
–
–
2
Cells
1
3236 (NH); H NMR (DMSO-d6) d (ppm): 4.40 (s, 4H, CH2), 7.33 (s,
1H, C5-H), 7.53–7.83 (m, 4H, Ar–H), 11.24 (bs, 1H, NH); 13C NMR
(DMSO-d6) d (ppm): 58.1 (CH2), 124.1 (C-5), 129.0, 129.7, 130.1,
135.7 (Ar–C), 141.2 (C-2), 143.8 (C-4), 164.7 (CO); HRMS [MþNa]
(m/z): 339.7547. Anal. calcd. for C13H10ClN3O4S: C 45.95, H 2.97, N
12.37; Found: C 45.87, H 2.92, N 12.49%.
A549 lung adenocarcinoma cells and HeLa cervical carcinoma
cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) medium substituted with 10% fetal bovine serum and 1%
penicillin and streptomycin. The cells were plated in T-25 tissue
culture flask and incubated at 37°C in a 5% CO2 atmosphere with
90% humidity.
Biological assays
Antioxidant activity
MTT assay for cell viability
The cytotoxicity of the compounds was tested using A549 lung
carcinoma cells and HeLa cervical carcinoma cells. 5 ꢂ 104 cells
were plated in each well of a 96-well tissue culture cluster (Nunc,
Inc., Germany) and incubated at 37°C in a medium containing
DMEM, 10% fetal bovine serum, and antibiotics (Invitrogen, USA),
in 5% CO2 atmosphere [35, 36].Compounds were dissolved in
DMSO. Serial dilutions were made for the active compound 10c
from stock solution 10 mg/mL (43800 mM) in DMSO. Compound
10c stock solution 6.39 mL was added to 1393.61 mL (final volume
1400 mL) of DMEM medium substituted with 10% fetal bovine
serum and 1% penicillin and streptomycin to make concentration
200 mM, which was further used for serial dilutions. After
attachment of the cells (usually 3–4 h), different concentrations of
dilutions were added to cells in 96-well plate and incubated for
20 h. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide) solution (20 mL of 5 mg/mL) was added to each well
and the incubation continued for additional 3 h. The dark blue
formazan crystals formed within the healthy cells were solubi-
lized with DMSO, and the absorbance was estimated in ELISA
plate reader (7520 Micro plate reader, Cambridge Technologies,
Inc.) at 550 nm and the absorbance was correlated with the cell
number. Experiments were performed in triplicates, and the
values are the average of three (n ¼ 3) independent experiments.
The inhibitory concentration (IC50) of the compound was assessed
by Graph Pad Prism software.
The compounds 6–11 are tested for antioxidant activity by DPPH
and NO methods.
DPPH radical scavenging activity
The hydrogen atom or electron-donating ability of the com-
pounds was measured from the bleaching of the purple colored
methanol solution of DPPH. The spectrophotometric assay uses
the stable radical DPPH as a reagent. To 4 mL of 0.004% w/v
methanol solution of DPPH, 1 mL of various concentrations of the
test compounds (50, 75, and 100 mg/mL) in methanol were added.
After a 30-min incubation period at room temperature, the
absorbance was read against blank at 517 nm. Ascorbic acid was
used as the standard. The percent of inhibition (I%) of free radical
production from DPPH was calculated by the following equation
ꢀ
ꢁ
Acontrol ꢀ Asample
I% ¼
ꢂ 100
Ablank
where Acontrol is the absorbance of the control reaction
(containing methanolic DPPH and ascorbic acid), Asample is the
absorbance of the test compound (containing methanolic DPPH
and test compound), and Ablank is the absorbance of the blank
(containing only methanolic DPPH). Tests were carried out in
triplicate.
Nitric oxide-scavenging activity
The authors are grateful to Council of Scientific and Industrial Research
(CSIR), New Delhi for financial assistance under major research project.
One of the authors, P. Ramachandra Reddy, is thankful to Council of
Scientific and Industrial Research (CSIR), New Delhi, India for the
sanction of Senior Research Fellowship (SRF).
NO-scavenging activity was measured by slightly modified
methods of Green et al. [33] and Marcocci et al. [34]. NO radicals
were generated from sodium nitroprusside. One milliliter of
sodium nitroprusside (10 mM) and 1.5 mL of phosphate buffer
saline (0.2 M, pH 7.4) were added to different concentrations
(50, 75, and 100 mg/mL) of the test compounds and incubated for
150 min at 25°C. After incubation, 1 mL of the reaction mixture
was treated with 1 mL of Griess reagent (1% sulfanilamide, 2%
H3PO4, and 0.1% naphthylethylenediamine dihydrochloride). The
absorbance of the chromatophore was measured at 546 nm.
Ascorbic acid was used as standard. NO scavenging activity was
calculated by the following equation
The authors have declared no conflict of interest.
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