Synthesis and antioxidant evaluation of novel phenothiazine linked substitutedbenzylideneamino-1,2,4-triazole derivatives

Article abstract of DOI:10.4067/s0717-97072015000200012

A series of novel 5-((10H-phenothiazin-10yl)methyl)-4-(substitutedbenzylideneamino)-4H-1,2,4-triazole-3-thiol derivatives (6a-i) have been synthesized from compound (1) through a multi-step reaction. The key intermediate (5) afforded a series of title com

Full text of DOI:10.4067/s0717-97072015000200012

J. Chil. Chem. Soc., 60, Nº 2 (2015)
SYNTHESIS AND ANTIOXIDANT EVALUATION OF NOVEL PHENOTHIAZINE LINKED
SUBSTITUTEDBENZYLIDENEAMINO-1,2,4-TRIAZOLE DERIVATIVES
SURESH MADDILA1, MEHBUB MOMIN1, SRIDEVI GORLE3, LAVANYA PALAKONDU2* AND
SREEKANTH B JONNALAGADDA1
1School of Chemistry & Physics, University of KwaZulu-Natal, Westville Campus, Chiltern Hills, Private Bag 54001, Durban-4000, South Africa.
2Department of Chemistry, Annamacharya Institute of Technology & Sciences J.N.T.University, Tirupati -517 502, Andhra Pradesh, India.
3Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Chilten Hills, Durban-4000, South Africa.
ABSTRACT
A series of novel 5-((10H-phenothiazin-10yl)methyl)-4-(substitutedbenzylideneamino)-4H-1,2,4-triazole-3-thiol derivatives (6a-i) have been synthesized
from compound (1) through a multi-step reaction. The key intermediate (5) afforded a series of title compounds (6a–i) on condensation with various suitable
aldehydes in the presence of H2SO4. The structures of novel compounds were characterized based on their elemental analysis, IR, 1H-NMR, 13C-NMR and MS
spectral data. All these novel compounds were screened for their in vitro antioxidant activity by employing nitric oxide, hydrogen peroxide, and DPPH radical
scavenging assays. The compounds 6d, 6e and 6i demonstrated potent antioxidant activity as these contain the electron-releasing groups.
Keywords: Synthesis, Anti-oxidant activity, 1,2,4-Triazole, Phenothiazines.
synthesis of various triazole bearing phenothiazines derivatives. To mention a
few, such protocols employed microwave irradiation [44] and microwave and
catalyst [45]. These methods have some limitations and drawbacks, such as use
of toxic reagents, strong acidic or basic conditions, costly reagents and catalysts,
strict reaction conditions, tedious steps, low product yields and/or long reaction
times, which restrict their scope in practical applications. Therefore, a novel
protocol with good and inexpensive catalyst demanding less reaction times is
sought after. The method for the synthesis of triazole bearing phenothiazines
is described in Scheme 1. By adapting our previously described methods15, the
compound 10H-phenothiazine was preserved with chloroacetate 2 with K2CO3
catalyst present in acetone solvent medium to give ethyl 2-(10H-phenothiazin-
10-yl)acetate (3). Further, the compound was reacted with KOH as a basic
media to afford 2-(10H-phenothiazin-10-yl) acetic acid (4). The compound 4
was treated under solvent and catalyst free conditions with thiocarbohydrazide
to obtain the cyclic triazole compound (5). Next, substituted aldehydes were
treated with compound 5 in the presence of concentrated H2SO as a catalyst
in DMF solvent media to attain the proposed title (5-((10H-4phenothiazin-
10yl)methyl)-4-(substitutedbenzylideneamino)-4H-1,2,4-triazole-3-thiol (6a-
i) derivatives in good to high yields (Scheme 1). The characterization of the
synthesized title compounds was executed by FT-IR, 1H-NMR, 13C-NMR and
LC–MS spectral analysis.
INTRODUCTION
The heterocyclic compounds chemistry continues to be an emerging field
in the organic and pharmaceutical chemistry1. Heterocyclic compounds are
present in various drugs, several natural products, some vitamins, biomolecules,
and biologically active compounds such as anti-inflammatory, antitumour,
antimalarial, antidepressant, anti-HIV, antibiotic and antimicrobial agents
etc2,3. Nowadays, antioxidants provoke researcher’s interest in both medicinal
plants and synthetic compounds. Antioxidants (natural or synthetic) are the
molecules, which are able to neutralize the free radicals by acting at various
stages like interception, prevention and repair4,5. It is therefore necessary
to develop therapeutic agents with improved potential for treating broad-
spectrum of oxidant infections. Vast data suggests that the free radical-reactive
nitrogen and oxygen species influence on the damage of biomolecules, which
will further lead to etiology of many human diseases6. The high resistance
acquired by microbes against the antioxidant drugs existing in the market is of
a great challenge to the scientific organizations, involved in the development of
novel and effective drugs against human diseases7. Synthesis of nitrogen and
sulfur bearing heterocyclic derivatives has been the great interest of researchers
for the past few decades, due to their potential use in the pharmaceutical and
medicinal applications8,9.
All the analysed compounds gave satisfactory analyses for the anticipated
structures, which were established by their spectral data. The 1H-NMR spectra
of the compound (3) demonstrated distinctive peaks in the range of triplet at δ
1.32 ppm for CH2CH3, quartet at δ 4.24 ppm for CH2CH3 and singlet at δ 4.84
ppm due to CH2CO. The IR spectra of the compound (3) exhibited absorption
bands at 1576, 1609, 1672, 2832 and 3378 cm—1 corresponding to the stretching
frequencies of, C=N, C=C, C=O, CH and COOH groups, respectively. The
1H-NMR spectra of the compound (4) exhibited a singlet for the two protons
of the CH CO group at δ = 4.35 ppm and a broad singlet at δ 11.49 ppm due to
COOH pr2oton. IR spectra of compound (5) showed characteristic absorption
bands at 1571, 1658, 2762, 2894, and 3458 cm—1for the C=N, C=O, SH, CH
Phenothiazines are heterocyclic molecules with two benzene rings
associated to the tricyclic system via nitrogen, sulfur atoms. Phenothiazine
11
occurs in various antipsychotic and antihistaminic drugs10,
. These
phenothiazine moieties are widely employed as antibacterial12, antiviral13,
anti-inflammatory14,15, anticancer16,17, tranquilizers agents18,19, antitubercular20,
anticonvulsant21, antiproliferative22 and as anti-HIV agents23. Triazoles
and their derivatives established a main class of organic molecules in a
broad spectrum of biological activities, various industrial, agricultural and
pharmaceutical fields24-26. Triazole ring derivatization lies on the ‘bioisosterism
phenomenon’ in which oxygen of oxadiazole nucleus is replaced with the
nitrogen of triazole analogue27. Triazole derivatives are the therapeutically
fascinating drug candidates including antifungal28, antioxidant29, antibacterial30,
insecticidal31, anti-inflammatory15, antineoplastic32, antiviral33, sedatives34, anti-
convulsant35,36, anti-histaminic 27,32, antitumor37, and CNS stimulants38 etc.
In continuation of our research in the domain of biologically active
molecules15,27-31, 39-43, the novel phenothiazine and triazole derivatives, in
combination, were synthesized with the hope that these derivatives may exhibit
a synergistic effect for the antioxidant activity.
1
and NH2, stretching vibrations, respectively. Its H-NMR spectra showed a
broad singlet for the one proton of the SH group at δ 12.53 ppm, while the NH2
group appeared as a singlet for the two protons at δ 5.67 ppm and singlet at 5.30
ppm due to the NCH group respectively.
The title derivati2ves (6a–i) were assigned on the basis of spectral analysis.
The IR spectra of the title compounds exhibited very similar features and
showed the absorption bands range at 2888–2898 cm—1, 2758–2770 cm—1
,
1650–1658 cm—1 and 1570–1578 cm—1 for the CH, SH, C=O and C=N
stretching vibrations. The physical data, 1H-NMR, 13C-NMR, LC–MS and
elemental spectral data for all the synthesized compounds are reported in
experimental protocols.
RESULTS AND DISCUSSION
Chemistry
Antioxidant testing
The synthesized novel compounds 6a-i were evaluated for in vitro
antioxidant activity by NO, H2O2, DPPH methods and the results are tabulated
A few procedures have been reported in the literature for the synthesis
of triazole bearing phenothiazines derivatives and their antimicrobial activity.
Literature survey shows quite many reaction methods were reported for
e-mail: gajulapalillavanya@gmail.com
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J. Chil. Chem. Soc., 60, Nº 2 (2015)
in Table 1, 2 and 3 respectively. Among all the compounds screened for
antioxidant activity, the compounds 6d, 6e, 6i showed potential antioxidant
activity as compared to the ascorbic acid (Reference drug) in all three methods
employed. This potential activity by compounds 6d, 6e, 6i can be ascribed
to the presence of mild electron releasing groups (methoxy, dimethoxy and
hydroxyl) appended to the aromatic benzene rings. However, the compounds
6a, 6b, 6c, 6f, 6g and 6h revealed moderate antioxidant activity. Results
suggest that methoxy, dimethoxy and hydroxyl substituted compounds (6d, 6e,
6i) offered better antioxidant activity when compared to that of the compounds
6a, 6b, 6c, 6f, 6g and 6h with electron-withdrawing groups (halo). The IC50
value of the standard ascorbic acid using the NO method was found to be 15.70
μg/mL at 25 μg/mL whereas the IC50 values of the compounds 6e, 6i and 6d
were found to be 16.98, 17.54 and 17.91 μg/mL, respectively. More, the tables
1, 2 and 3 designates that the antioxidant activity in NO, DPPH and H2O2
methods which increased with concentration.
Reagents & Conditions: (a) K2CO3, 78 °C, reflux, 3-4 h; (b) KOH, R.T., 12 h; (c) thiocarbohydrazide, heat, 4-5 h; (d) substituted aldehydes, Con
H2SO4, DMF, 25 °C, 5−6 h.
Table 1: In vitro antioxidant activity of compounds (6a-i) determined by NO method.
Compound
Concentration (μg/mL)
IC50
25
50
75
100
6a
68.41 ± 0.90
60.97 ± 1.41
62.73 ± 1.17
75.02 ± 0.22
78.18 ± 0.30
65.37 ± 1.17
73.90 ± 0.86
54.64 ± 1.38
71.26 ± 0.84
85.76± 0.13
–
72.59 ± 1.37
63.37 ± 1.30
67.48 ± 1.24
78.14 ± 0.45
83.25 ± 0.49
70.22 ± 1.57
75.06 ± 0.94
57.49 ± 1.24
74.84 ± 1.05
84.92 ± 0.38
–
74.18 ± 0.94
67.65 ± 1.24
69.94 ± 0.88
85.45 ± 0.61
86.60 ± 0.71
75.69 ± 1.41
79.25 ± 1.06
61.03 ± 0.71
79.14 ± 1.40
87.32± 0.55
–
82.40 ± 1.23
72.35 ± 0.80
73.16 ± 0.95
84.88 ± 0.76
86.15 ± 0.78
80.53 ± 0.71
83.36 ± 1.41
65.28 ± 1.08
84.93 ± 1.26
91.42 ± 0.68
–
19.52 ± 1.04
20.49 ± 0.95
21.01 ± 0.70
17.91 ± 0.86
16.98 ± 0.69
20.41 ± 1.24
18.14 ± 0.57
24.30 ± 1.08
17.54 ± 0.93
15.70 ± 0.54
–
6b
6c
6d
6e
6f
6g
6h
6i
Ascorbic acid*
Control
(–) Showed no scavenging activity. Values were the means of three replicates ± SD.
* = Standard
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J. Chil. Chem. Soc., 60, Nº 2 (2015)
Table 2: In vitro antioxidant activity of compounds (6a-i) determined by H2O2 method.
Compound
Concentration (μg/mL)
75
IC50
25
50
100
6a
61.27 ± 1.07
52.36 ± 1.17
55.51 ± 1.37
64.88 ± 0.31
67.29 ± 1.14
54.09 ± 0.89
57.68 ± 1.14
47.08 ± 0.87
63.07 ± 0.81
77.42 ± 0.17
–
64.49 ± 1.28
55.03 ± 0.87
58.31 ± 1.22
66.50 ± 0.47
70.52 ± 1.30
57.60 ± 1.38
61.48 ± 1.19
49.37 ± 1.16
66.84 ± 1.58
79.61 ± 0.34
–
68.84 ± 1.58
59.12 ± 0.96
61.11 ± 0.71
69.97 ± 0.66
72.68 ± 0.61
60.95 ± 0.79
68.12 ± 1.47
52.44 ± 1.26
70.22 ± 1.06
83.39 ± 0.63
–
71.93 ± 0.83
64.72 ± 0.63
64.27 ± 1.08
73.52 ± 0.77
75.19 ± 0.71
65.20 ± 1.05
70.93 ± 1.54
55.62 ± 1.38
72.59 ± 1.37
87.59 ± 0.70
–
21.74 ± 0.53
25.33 ± 1.05
26.10 ± 0.97
20.57 ± 1.25
19.85 ± 0.61
24.54 ± 0.41
21.33 ± 1.07
27.76 ± 0.64
20.47 ± 1.24
17.35 ± 0.28
–
6b
6c
6d
6e
6f
6g
6h
6i
Ascorbic acid*
Control
(–) Showed no scavenging activity. Values were the means of three replicates ± SD.
* = Standard
Table 3: In vitro antioxidant activity of compounds (6a-i) determined by DPPH method.
Compound
Concentration (μg/mL)
IC50
25
50
75
100
6a
64.87 ± 0.71
61.27 ± 1.07
54.75 ± 1.74
71.30 ± 1.06
78.43± 0.28
63.04 ± 1.40
65.70 ± 1.44
49.65 ± 0.60
76.46 ± 0.30
84.68 ± 0.18
–
68.93 ± 1.32
64.49 ± 1.28
60.25 ± 1.05
75.56 ± 0.88
81.64 ± 0.48
67.83 ± 1.56
69.41 ± 1.21
53.86 ± 1.24
73.83 ± 0.66
83.52 ± 0.32
–
72.48 ± 1.29
68.84 ± 1.58
63.37 ± 0.92
75.87 ±1.30
85.28 ± 0.62
71.92 ± 0.78
74.84 ±1.56
57.63 ± 0.55
74.36 ± 0.76
88.52 ± 0.43
–
77.64 ± 1.34
71.93 ± 0.83
67.81 ± 1.58
82.28 ±1.00
84.76 ± 0.78
74.89 ± 1.07
78.53 ± 0.96
62.93 ± 0.82
89.13 ± 0.80
86.22 ± 0.54
–
20.57 ± 1.23
21.74 ± 0.53
25.70 ± 1.47
18.48 ± 1.04
17.88 ± 0.28
21.14 ± 0.96
20.30 ± 1.12
26.69 ± 1.10
18.08 ± 0.51
16.10 ± 0.45
–
6b
6c
6d
6e
6f
6g
6h
6i
Ascorbic acid*
Control
(–) Showed no scavenging activity. Values were the means of three replicates ± SD.
* = Standard
IR spectrometer at the 400-4000 cm-1 area. LCMS spectral data was recorded
on a MASPEC low resolution mass spectrometer operated at 70 eV. Purity of
all the reaction products was confirmed by TLC using aluminum plates coated
with silica gel (Merck Kieselgel 60 F254).
CONCLUSION
In the current study, we have developed the synthesis of 9 new derivatives
of novel phenothiazine linked substitutedbenzylideneamino-1,2,4-triazole
derivatives (6a-i). All have been characterized by spectral studies. In vitro,
their antioxidant activity was also investigated by employing nitric oxide,
hydrogen peroxide, and DPPH radical scavenging assays. The evaluation of
antioxidant screening data reveals that among the 9 compounds screened,
compounds 6d, 6e & 6i showed potent antioxidant activity almost equivalent
to that of standards.
Synthesis of ethyl 2-(10H-phenothiazin-10-yl)acetate (3)
To a solution of 10H-phenothiazine 1 (1 mmol) in acetone (20 mL) was
added K2CO (3 mmol) at 25 °C followed by the addition of ethyl chloroacetate
2 (1.2 mmol3) with reflux at 78 °C for 3-4 h. The completion of reaction was
monitored by TLC. Next, the reaction mixture was cooled, poured into ice-
water and was extracted using ethyl acetate solution. Further, the organic layer
was evaporated and recrystalized to obtain ethyl 2-(10H-phenothiazin-10-yl)
acetate as solid.
EXPERIMENTAL
Synthesis of 2-(10H-phenothiazin-10-yl)acetic acid (4)
The solutions of compound 3 (1 mmol), dissolved in ethanol (10 mL)
and potassium hydroxide (3 mmol) in water (5 mL) were combined and the
mixture was left for stirring for 12 h at 25 °C. The reaction was monitored
by TLC for completion. The reaction mixture was then transferred into cold
water and acidified with HCl to obtain a solid product by filtration. Finally,
the crude was recrystallized with ethyl acetate to afford a pure compound
2-(10H-phenothiazin-10-yl)acetic acid.
All chemicals and reagents required for the reaction were of analytical
grade and were used without any further purification. Melting points were
recorded on a Buchi Melting Point B-545 apparatus. Bruker AMX 400 MHz
NMR spectrometer was used to record the 1H NMR and 13C NMR (400 MHz)
spectral values. The CDCl3/DMSO−d solution was utilized for this while TMS
served as the internal standard. TMS6was further used as an internal standard
for reporting the all chemical shifts in δ (ppm). The FT-IR spectrum for the
samples was established using a Perkin Elmer Perkin Elmer Precisely 100 FT-
General procedure for the synthesis of 5-((10H-phenothiazin-10-yl)
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J. Chil. Chem. Soc., 60, Nº 2 (2015)
methyl)-4-amino-4H-1,2,4-triazole-3-thiol (5)
5-((10H-Phenothiazin-10yl)methyl)-4-(2,4-dichlorobenzylideneamino)-
4H-1,2,4-triazole-3-thiol (6f)
A mixture of compound 4 (1 mmol) and thiocarbohydrazide (1 mmol)
was heated under solvent-free conditions for 4-5 h and the reaction end-point
was examined by TLC. The reaction mixture was left to cool for 1 h and
subsequently excess acid compound was removed using sodium bicarbonate
solution. Thereafter, the above mixture was filtered, dried and finally
recrystallized with DMF to achieve compound 5.
Yield 75%; m.p. 230-232 ºC; IR (KBr)ν (cm–1): 2893 (CH), 2768 (SH),
1
1650 (C=O), 1574 (C=N) 682 (C-Cl); H NMR (DMSO–d6, 400 MHz) δ:
13.12 (s, 1H, SH), 9.18 (s, 1H), 7.92 (d, Ar–H, J = 7.98 Hz), 7.72 (s, Ar-H),
7.38 (d, Ar–H, J = 8.10 Hz), 7.20 (m, 4H, Ar-H), 7.10 (dd, 2H, Ar–H, J =
7.96, 2.38 Hz) 6.95(dd, 2H, J = 7.88, 2.15 Hz ), 5.24 (s, 2H, NCH2); 13C NMR
(DMSO–d6, 100 MHz) δ: 163.15, 158.30, 154.24, 144.88 (2C), 131.40, 131.10,
129.00, 129.60, 128.50 (2C), 128.10, 127.50 (2C), 127.10, 123.14 (2C), 122.40
(2C), 115.20 (2C), 55.18; LC—MS (70 eV): m/z = 483.01. Anal. Calcd. for
C22H15Cl2N5S : C, 54.55; H, 3.12; N, 14.46. Found: C, 54.57; H, 3.15; N, 14.48.
5-((10H-2Phenothiazin-10yl)methyl)-4-(2,5-dibromobenzylideneamino)-
4H-1,2,4-triazole-3-thiol (6g)
Synthesis
of
5-((10H-Phenothiazin-10yl)methyl)-4-
(substitutedbenzylideneamino)-4H-1,2,4-triazole-3-thiol (6a-i)
An equimolar ratio of compound 5 and substituted aldehydes (1x1), a
few drops of concentrated H SO4 in DMF (10 mL) solvent were stirred at 25
°C for 5−6 h. After this, the 2reaction mixture was poured into ice-cold water,
filtered to collect the solid product and was dried. The resultant crude was then
recrystallized with ethyl acetate to accomplish the title compounds as excellent
yields (6a-i).
Yield 81%; m.p. 228-230 ºC; IR (KBr)ν (cm–1): 2892 (CH), 2761 (SH),
1654 (C=O), 1570 (C=N) 726 (C-Br); 1H NMR (DMSO–d6, 400 MHz) δ: 12.50
(s, 1H, SH), 9.30 (s, 1H), 7.92 (s, Ar–H), 7.60 (d, J = 8.05, Ar–H), 7.55 (d,
J = 8.10, Ar–H ), 7.22 (dd, J= 8.20, 2.40, 2H, Ar-H), 7.20(d, J = 8.10, 2H,
Ar–H ), 7.12 (dd, 2H, Ar–H, J = 7.98, 2.36 Hz), 7.00 (dd, 2H, J = 7.80, 2.30
Hz ), 5.22 (s, 2H, NCH2); 13C NMR (DMSO–d6, 100 MHz) δ: 165.10, 158.18,
154.20, 144.12 (2C), 134.00, 133.20, 132.90, 130.30, 128.80 (2C), 127.60
(2C), 125.10, 124.16, 123.22 (2C), 122.10(2C), 115.66 (2C), 55.14; LC—MS
(70 eV): m/z = 572.91. Anal. Calcd. for C H16Br2N5S2: C, 46.09; H, 2.64; N,
12.22. Found: C, 46.07; H, 2.61; N, 12.25. 22
5-((10H-Phenothiazin-10yl)methyl)-4-(4-bromobenzylideneamino)-4H-
1,2,4-triazole-3-thiol (6a)
Yield 80%; m.p. 222-224 ºC; IR (KBr)ν (cm–1): 2892 (CH), 2761 (SH),
1654 (C=O), 1570 (C=N) 720 (C-Br); 1H NMR (DMSO–d6, 400 MHz) δ: 12.50
(s, 1H, SH), 9.30 (s, 1H), 7.71 (dd, 2H, Ar–H, J = 8.01, 2.51 Hz), 7.59 (dd, 2H,
J = 8.05, 2.49, Ar–H), 7.28 (m, 4H, Ar-H), 7.11 (dd, 2H, Ar–H, J = 7.95, 2.36
Hz), 7.05(dd, 2H, J = 7.80, 2.10 Hz ), 5.20 (s, 2H, NCH ); 13C NMR (DMSO–
d , 100 MHz) δ: 165.10, 158.18, 154.20, 144.12 (2C),2131.20 (2C), 130.30,
1628.90 (2C), 128.10 (2), 127.62 (2C), 125.32, 123.24 (2C), 122 (2C), 115.67
(2C), 55.14; LC—MS (70 eV): m/z = 495. Anal. Calcd. for C22H16BrN5S2: C,
53.44; H, 3.26; N, 14.16. Found: C, 53.47; H, 3.29; N, 14.19.
5-((10H-Phenothiazin-10yl)methyl)-4-(3,5-difluorobenzylideneamino)-
4H-1,2,4-triazole-3-thiol (6h)
Yield 68%; m.p. 150-152 ºC; IR (KBr)ν (cm–1): 2892 (CH), 2766 (SH),
1
5-((10H-Phenothiazin-10yl)methyl)-4-(4-chlorobenzylideneamino)-4H-
1,2,4-triazole-3-thiol (6b)
1656 (C=O), 1572 (C=N) 1190 (C-F); H NMR (DMSO–d , 400 MHz) δ:
12.80 (s, 1H, SH), 9.08 (s, 1H), 7.50 (d, 2H, Ar–H, J = 7.560 Hz), 7.20 (m,
4H, Ar-H), 7.10 (dd, 2H, Ar–H, J = 7.98, 2.30 Hz), 7.00(dd, 2H, J = 7.88,
2.22 Hz ), 6.95 (t, J=7.5, Ar-H), 5.14 (s, 2H, NCH2); 13C NMR (DMSO–d ,
100 MHz) δ: 163.80 (d, J = 246.80, 2C-F), 161.10, 154.40, 147.28, 144.660
(2C), 134.70, 128.20(2C), 127.15 (2C), 123.10 (2C), 122.60 (2C), 115.90 (2C),
110.50 (d, 2C), 104.50, 54.16; LC—MS (70 eV): m/z = 451.07. Anal. Calcd.
for C H16F2N5S2: C, 58.52; H, 3.35; N, 15.51. Found: C, 58.54; H, 3.37; N,
15.542.2
Yield 70%; m.p. 208-210 ºC; IR (KBr)ν (cm–1): 2893 (CH), 2768 (SH),
1650 (C=O), 1574 (C=N) 680 (C-Cl); 1H NMR (DMSO–d6, 400 MHz) δ: 12.40
(s, 1H, SH), 9.35 (s, 1H), 7.77 (dd, 2H, Ar–H, J = 8.05, 2.55 Hz), 7.54 (dd, 2H,
J = 8.10, 2.48, Ar–H), 7.22 (m, 4H, Ar-H), 7.15 (dd, 2H, Ar–H, J = 7.95, 2.36
Hz), 7.01(dd, 2H, J = 7.85, 2.10 Hz ), 5.10 (s, 2H, NCH2); 13C NMR (DMSO–
d6, 100 MHz) δ: 165.15, 158.22, 154.28, 144.25 (2C), 135.40, 131.20, 130.80
(2C), 128.70 (2C), 128.12 (2), 127.23 (2C), 123.22 (2C), 122.50 (2C), 115.60
(2C), 55.14; LC—MS (70 eV): m/z = 449.05. Anal. Calcd. for C22H16ClN5S2:
C, 58.72; H, 3.58; N, 15.56. Found: C, 58.75; H, 3.56; N, 15.59.
5-((10H-Phenothiazin-10yl)methyl)-4-(4-fluorobenzylideneamino)-4H-
1,2,4-triazole-3-thiol (6c)
5 - ( ( 1 0 H - P h e n o t h i a z i n - 1 0 y l ) m e t h y l ) - 4 - ( 3 , 5 -
dimethoxybenzylideneamino)-4H-1,2,4-triazole-3-thiol (6i)
Yield 70%; m.p. 239-240 ºC; IR (KBr)ν (cm–1): 2898 (CH), 2770 (SH),
1
1658 (C=O), 1578 (C=N); H NMR (DMSO–d6, 400 MHz) δ: 13.48 (s, 1H,
Yield 72%; m.p. 160-162 ºC; IR (KBr)ν (cm–1): 2890 (CH), 2760 (SH),
SH), 9.26 (s, 1H), 7.22 (dd, 2H, Ar–H, J = 7.90, 2.40 Hz), 7.20 (dd, 2H, J =
8.10, 2.44, Ar–H), 7.14 (d, J=8.00, 2H, Ar-H), 7.00 (dd, 2H, Ar–H, J = 8.18,
2.46 Hz), 7.00(dd, 2H, J = 7.84, 2.10 Hz ), 6.90 (s, Ar-H), 5.20 (s, 2H, NCH2),
3.84 (s, 6H, CH3); 13C NMR (DMSO–d6, 100 MHz) δ: 164.15 (2C), 160.15,
154.24, 148.50, 144.20 (2C), 134.40, 127.80 (2C), 127.40 (2C), 123.22 (2C),
122.44 (2C), 115.48 (2C), 104.20 (2C), 103.50, 55.40 (2CH3), 55.14; LC—MS
(70 eV): m/z = 475.11. Anal. Calcd. for C24H21N5O2S2: C, 60.61; H, 4.45; N,
14.73. Found: C, 60.63; H, 4.42; N, 14.75.
1
1658 (C=O), 1572 (C=N) 1195 (C-F); H NMR (DMSO–d , 400 MHz) δ:
12.42 (s, 1H, SH), 9.10 (s, 1H), 7.67 (dd, 2H, Ar–H, J = 8.06, 2.40 Hz), 7.23
(dd, 2H, J = 8.06, 2.40, Ar-H), 7.20 (dd, 2H, J = 8.0, 2.40, Ar-H), 7.14 (dd, 2H,
Ar–H, J = 7.94, 2.38 Hz), 7.08 (t, 2H, J = 8.50, Ar–H), 7.04(dd, 2H, J = 7.84,
2.12 Hz ), 5.12 (s, 2H, NCH ); 13C NMR (DMSO–d , 100 MHz) δ: 165.50 (d,
J = 248.20), 161.20, 154.202, 147.28, 144.30 (2C), 6130.70 (2C), 128.10(2C),
127.90, 127.15 (2C), 123.30 (2C), 121.60 (2C), 116.90 (2C), 115.50 (d, J=18
Hz, 2C), 54.14; LC—MS (70 eV): m/z = 433.08. Anal. Calcd. for C22H16FN5S2:
C, 60.95; H, 3.72; N, 16.15. Found: C, 60.98; H, 3.75; N, 16.17.
PHARMACOLOGICAL SCREENING
Antioxidant screening in vitro
5-((10H-Phenothiazin-10yl)methyl)-4-(4-methoxybenzylideneamino)-
4H-1,2,4-triazole-3-thiol (6d)
The compounds 6a-i were evaluated for antioxidant activity using the Nitric
oxide (NO), Hydrogen peroxide (H2O2) and 1,1-Diphenyl-1-picrylhydrazyl
(DPPH) methods.
Yield 68%; m.p. 218-220 ºC; IR (KBr)ν (cm–1): 2893 (CH), 2768 (SH),
1
1650 (C=O), 1574 (C=N); H NMR (DMSO–d6, 400 MHz) δ: 13.10 (s, 1H,
NO scavenging activity
SH), 9.16 (s, 1H), 7.90 (dd, 2H, Ar–H, J = 7.98, 2.45 Hz), 7.51 (dd, 2H, J =
8.00, 2.44, Ar–H), 7.24 (m, 4H, Ar-H), 7.14 (dd, 2H, Ar–H, J = 7.96, 2.42
Hz), 7.00(dd, 2H, J = 7.84, 2.10 Hz ), 5.24 (s, 2H, NCH2), 3.82 (s, 3H, CH3);
13C NMR (DMSO–d6, 100 MHz) δ: 165.15, 160.10, 158.20, 154.24, 144.38
(2C), 130.40 (2C), 127.70 (2C), 127.23 (2C), 125.12, 123.18 (2C), 122.46
(2C), 115.40 (2C), 114.20 (2C), 55.20 (CH3), 55.14; LC—MS (70 eV): m/z =
445.10. Anal. Calcd. for C22H16ClN5S2: C, 62.00; H, 4.30; N, 15.72. Found: C,
61.98; H, 4.33; N, 15.70.
NO biological activity was determined using the methods by Green et al.
and Marcocci et al. with slight modification46,47. Briefly, NO radicals were first
created with the sodium nitroprusside. Sodium nitroprusside (10 mmol, 1 mL),
phosphate buffer saline (PBS) (0.25 mmol, 1.5 mL, pH 7.5) were added to
the various amounts of experimental compounds (25, 50, 75 and 100 μg/mL)
and incubated at 25 ˚C for 2.5 h. Next, the reaction mixtures (1 mL) were
preserved with 1 mL of Griess reagent. All experiments were performed in
triplicates. The absorbance of the samples was measured at 546 nm using the
chromatophore and NO activity was calculated by the following formula
Scavenging activity (%) = [(A control˗A sample)/A control] X 100
A control = Absorbance of the control reaction (all reagents without the
test compounds)
5-((10H-Phenothiazin-10yl)methyl)-4-(4-hydroxybenzylideneamino)-
4H-1,2,4-triazole-3-thiol (6e)
Yield 75%; m.p. 214-216 ºC; IR (KBr)ν (cm–1): 3320 (O-H), 2888 (CH),
2758 (SH), 1656 (C=O), 1578 (C=N); 1H NMR (DMSO–d , 400 MHz) δ: 13.12
(s, 1H, SH), 9.90 (s, O-H), 9.16 (s, 1H), 7.80 (dd, J = 7.98,62.45 Hz, 2H, Ar–H),
7.22 (dd, 2H, Ar-H, J = 8.10, 2.44 Hz), 7.20 (dd, 2H, Ar–H, J = 7.94, 2.46
Hz), 7.10(dd, 2H, J = 7.82, 2.20 Hz ), 6.90 (m, 4H, Ar-H), 5.24 (s, 2H, NCH2);
13C NMR (DMSO–d6, 100 MHz) δ: 165.00, 161.10, 158.20, 154.24, 144.98
(2C), 130.30 (2C), 127.80 (2C), 126.90 (2C), 124.92, 123.16 (2C), 122.26
(2C), 116.10 (2C), 115.00 (2C), 53.80; LC—MS (70 eV): m/z = 431.01. Anal.
Calcd. for C22H16ClN5S2: C, 61.23; H, 3.97; N, 16.23. Found: C, 61.21; H, 4.00;
N, 16.25.
A sample = Absorbance of the test compound.
H2O2 scavenging activity
The compounds H2O2 biological activity was determined using the previous
method48. A solution of H2O2 (10 mmol) in PBS (0.25 mmol) was prepared and
various amounts of test compounds as described in above section were added
to the PBS containing H2O2 (4 mL). The absorbance of the compounds was
recorded at 230 nm, percent of H2O2 scavenging activity was calculated by
using the above mentioned formula. All the tests were carried out in triplicate.
2922

J. Chil. Chem. Soc., 60, Nº 2 (2015)
DPPH scavenging activity
24. S. Maddila, P. Ramakanth, S. B. Jonnalagadda, Lett. Org. Chem. 10(10),
693, (2013).
The compounds ability to donate electrons or hydrogen atoms was
evaluated by the DPPH colorimetric method based on the decolorization of
purple methanol solution49. This assay produces the DPPH stable radicals.
Various concentrations of compounds (refer to above sections) in methanol
(1 mL) were added to the 4 mL of DPPH methanol solution (0.004% (w/v)
and incubated for 0.5 h at 25 °C. After this incubation period, the absorbance
was measured at 517 nm. The percentage of DPPH scavenging activity was
calculated using the above equation.
28. S. Maddila, S. B. Jonnagadda, Pharma. Chem. J. 46(11), 1, (2013).
29. S. Maddila, A. S. Kumar, S. Gorle, M. Singh, P. Lavanya, S. B.
Jonnalagadda, Lett. Drug Desig. & Discov. 10(2), 186, (2013).
30. S. Maddila, S. B. Jonnalagadda, Lett. Drug Desig. & Discov. 9(7), 687,
(2012).
31. S. Maddila, P. Ramakanth, S. B. Jonnalagadda, J. Heter. Chem. (MS ID:
JHET-13-0053.R1-In Press) (2013).
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ACKNOWLEDGEMENT
33. Y. A. Al-Soud, M. N. Al-Dweri, N. A. Al-Masoudi, IL Farmaco. 59, 775,
(2004).
The authors acknowledge to the National Research Foundation of South
Africa, University of KwaZulu-Natal, Durban, South Africa and Department
of Chemistry, Annamacharya Institute of Technology & Sciences, Tirupati,
India for the facilities.
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