Design of new phenothiazine-thiadiazole hybrids via molecular hybridization approach for the development of potent antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2015.10.035

A new library of phenothiazine and 1,3,4-thiadiazole hybrid derivatives (5a-u) was designed based on the molecular hybridization approach and the molecules were synthesized in excellent yields using a facile single-step chloro-amine coupling reaction between 2-chloro-1-(10H-phenothiazin-10-yl)ethanones and 2-amino-5-subsituted-1,3,4-thiadiazoles. The compounds were evaluated for their in vitro inhibition activity against Mycobacterium tuberculosis H37Rv (MTB). Compounds 5g and 5n were emerged as the most active compounds of the series with MIC of 0.8 μg/mL (～1.9 μM). Also, compounds 5a, 5b, 5c, 5e, 5l and 5m (MIC = 1.6 μg/mL), and compounds 5j, 5k and 5o (MIC = 3.125 μg/mL) showed significant inhibition activity. The structure-activity relationship demonstrated that an alkyl (methyl/npropyl) or substituted (4-methyl/4-Cl/4-F) phenyl groups on the 1,3,4-thiadiazole ring enhance the inhibition activity of the compounds. The cytotoxicity study revealed that none of the active molecules are toxic to a normal Vero cell line thus proving the lack of general cellular toxicity. Further, the active molecules were subjected to molecular docking studies with target enzymes InhA and CYP121.

Full text of DOI:10.1016/j.ejmech.2015.10.035

Accepted Manuscript
Design of new phenothiazine-thiadiazole hybrids via molecular hybridization approach
for the development of potent antitubercular agents
Jurupula Ramprasad, Nagabhushana Nayak, Udayakumar Dalimba
PII:
S0223-5234(15)30317-2
10.1016/j.ejmech.2015.10.035
EJMECH 8170
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 August 2015
Revised Date: 10 October 2015
Accepted Date: 20 October 2015
Please cite this article as: J. Ramprasad, N. Nayak, U. Dalimba, Design of new phenothiazine-
thiadiazole hybrids via molecular hybridization approach for the development of potent antitubercular
agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.035.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design of new phenothiazine-thiadiazole hybrids via molecular
hybridization approach for the development of potent antitubercular
agents
a
a
a,*
Jurupula Ramprasad , Nagabhushana Nayak and Udayakumar Dalimba
a
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Srinivasanagar, Mangalore-575025, India.
*
Corresponding author email: udayaravi80@gmail.com, udayakumar@nitk.ac.in Phone:
+91-824-2473207. Fax: +91-824-2474033
Abstract
A new library of phenothiazine and 1,3,4-thiadiazole hybrid derivatives (5a-u) was
designed based on the molecular hybridization approach and the molecules were synthesized
in excellent yields using a facile single-step chloro-amine coupling reaction between 2-
chloro-1-(10H-phenothiazin-10-yl)ethanones and 2-amino-5-subsituted-1,3,4-thiadiazoles.
The compounds were evaluated for their in vitro inhibition activity against Mycobacterium
tuberculosis H Rv (MTB). Compounds 5g and 5n were emerged as the most active
37
compounds of the series with MIC of 0.8 ꢀg/mL (∼1.9 ꢀM). Also, compounds 5a, 5b, 5c, 5e,
l and 5m (MIC = 1.6 µg/mL), and compounds 5j, 5k and 5o (MIC = 3.125 µg/ml) showed
significant inhibition activity. The structure-activity relationship demonstrated that an alkyl
methyl/n-propyl) or substituted (4-methyl/4-Cl/4-F) phenyl groups on the 1,3,4-thiadiazole
5
(
ring enhance the inhibition activity of the compounds. The cytotoxicity study revealed that
none of the active molecules are toxic to a normal Vero cell line thus proving the lack of
general cellular toxicity. Further, the active molecules were subjected to molecular docking
studies with target enzymes InhA and CYP121.
Keywords: Mycobacterium tuberculosis; phenothiazine; 1,3,4-thiadiazole; antimycobacterial
activity; cytotoxicity; molecular docking
1

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1
. Introduction
Tuberculosis (TB) is a contagious disease caused by the infection with bacteria
Mycobacterium tuberculosis. It causes a massive amount of human deaths despite the
availability of more than 20 antiTB drugs and the Bacille Calmette Guerin (BCG) vaccine
[
1]. Further, the traditional anti-TB agents have limited efficacy against the new forms of TB,
extensively drug-resistant TB (XDRTB) and multidrug resistant TB (MDRTB). Hence there
is an emergent requirement for the development of newer fast acting antiTB drugs which
have a safer toxic profile. In this direction, several molecular design strategies are being
employed in order to indentify potent chemical entities [2]. The molecular hybridization
approach which involves the hybridization of two active pharmacophores into a single
molecular framework has become one of most promising approaches to develop potent
antiTB agents because many such hybrid derivatives possessed improved efficiency and
efficacy, when compared to the parent compounds [3-5]. In our previous studies on
imidazo[2,1-b][1,3,4]thiadazole (ITD) based hybrid molecules, we have incorporated
substituted benzimidazole or 1,2,3-triazole moiety at position-5 of the ITD ring and evaluated
the antitubercular activity of the hybrid molecules. Interestingly, most of the hybrid
compounds exhibited significant inhibition activity against Mtb H37Rv strain and a safe
toxicity profile towards a normal cell line [6, 7]. These results and some literature reports on
significant antitubercular activity of phenothiazine and 1,3,4-thiadiazole derivatives prompted
us to design new phenothiazine-thiadiazole hybrids for the development of potent molecules.
The in vitro antitubercular activity of phenothiazines is well-known for many years [8-10]. In
clinical trials, thioridazine (I) (figure 1) is being used in combination with Linezolid and
Moxifloxacin as front-line drug in combinatorial therapeutic approaches for the treatment of
Mtb infection [11]. Chlorpromazine (II) is effective against virulent Mtb strain H37Rv in
cultured human macrophage model of infection and it is described as synergistic with both
INH and RIF [12]. A recent report demonstrated the significant antitubercular activity of a
series of 4,5-dihydro-1H-phenothiazine containing pyrazolo[3,4-d]pyrimidines (III). One of
the derivatives was more potent (with MICMABA value of 0.025µg/mL) than the standard drug
Isoniazid [13]. Further, several N-substituted phenothiazine derivatives demonstrated
promising antitubercular activity [14-18]. On the other hand, 1,3,4-thiadiazole derivatives are
a important class of heterocyclic compounds in medicinal chemistry research [19, 20] which
exhibit wide variety of biological effects including anti-tubercular [21], anticancer [22],
antibacterial [23] and anti-fungal [24, 25] activities. Also, 1,3,4-thiadiazole ring is the core
2

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structural unit in several marketed drugs such as Acetazolamide, Methazolamide,
sulfamethizole, Cefazedone, Cefazolin, Ceftezole etc. In addition, a few literature reports
revealed promising antitubercular activity of 1,3,4-thiadiazoles [26]. For example, 3-
heteroarylthioquinoline derivatives [27] of 1,3,4-thiadiazole demonstrated MIC of ~3.5 ꢀM
against Mtb (IV) whereas a pyridinyl-thiadiazole derivative (V) exhibited MIC of 0.07 ꢀM
[
21] (figure 1). In view of these facts on promising antimycobacterial activity of 1,3,4-
thiadiazole and phenothiazine derivatives, we envisaged the incorporation of these two
molecular units in to a single molecular framework and synthesized a new series of hybrid
derivatives (5a-u).
(Figure 1 here)
2
2
. Results and discussion
.1. Chemistry
The target phenothiazine-thiadiazole hybrids were synthesized according to the
synthetic route presented in Scheme 1. 2-Subsituted-1-(2-chloro-10H-phenothiazin-10-yl)
ethanones (2a-c) were synthesized by the reaction of substituted phenothiazines (1a-c) with
chloroacetyl chloride under reflux conditions [15, 28]. 5-Methyl-2-amino-1,3,4-thiadiazole
(4a) was synthesized by reacting thiosemicarbazide with acetyl chloride (3a) using a reported
procedure [6]. Other 5-aryl-1,3,4-thiadiazole-2-amines (4b-g) were synthesized by treating
the corresponding aromatic acid (3b-g) with thiosemicarbazide in the presence of
phosphorous oxychloride. The target compounds, 1-(2-imino-1,3,4-thiadiazol-3(2H)-yl)-2-
(10H-phenothiazin-10-yl)ethanone derivatives (5a–u) were synthesized by the reaction
between compounds 4a-g and 2a-c in ethanol under reflux conditions. The plausible reaction
mechanism for the formation of final derivatives (5a-u) is shown in the Scheme 2. The
1
13
structure of the target molecules (5a-u) was confirmed by spectral ( H NMR, C NMR, ESI-
1
MS and FTIR) and elemental analysis. For instance, the H NMR spectrum of compound 5a
showed a broad singlet with one proton at δ 8.16 ppm due to the imine (C=NH) proton and
another singlet at δ 2.20 ppm due to methyl protons of the 1,3,4- thiadiazole ring. The singlet
at δ 4.74 ppm corresponds to the CH group. The broad NH at δ 8.16 ppm was disappeared
2
upon D O exchange. Also, its mass spectrum showed the molecular ion peak at m/z 355.1,
2
which corresponds to M+1 peak of the molecule and is in agreement with its molecular
formula C H N OS . The IR spectrum of compound 5a showed an absorption peak at 3314
1
7
14
4
2
3

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−
1
due to N-H stretching (C=NH). Further, the signals at 1696 and 1601cm correspond to C=O
and C=N stretching respectively. The spectral and elemental analysis data of all target
compounds are given in the experimental part and some representative spectra are given the
supplementary information. The physical data of compounds (5a-u) are tabulated in Table 1.
(Scheme 1 here)
(Table 1 here)
2
.2. In vitro antimycobacterial activity
All the target derivatives (5a-u) were screened against Mtb H37Rv (ATCC27294)
using MABA method [29] and their antimycobacterial activity was evaluated in terms of
minimum inhibitory concentration (MIC) values. The MIC values in ꢀg/mL of 5a-u along
with those of standard drugs for comparison are presented in figure 2. The MIC values of the
compounds are in the range of 0.8-50 ꢀg/mL. Interestingly, eleven compounds of the series
showed significant inhibitory activity (MIC ≤ 3.125 ꢀg/mL) among which eight compounds
exhibited MIC ≤ 1.6 ꢀg/mL (figure 3). The MIC values of these compounds are comparable
with those of the standard drugs ethambutol (EMB) and ciprofloxacin (INN). Compounds 5g
and 5n which contain 4-methylphenyl and n-propyl groups respectively on the thiadiazole
ring emerged as the most potent leads with a MIC of 0.8 ꢀg/mL and are more potent than
1
standard drugs EMB and INN. The nature of the substituents on the phenothiazine (R ) and
2
1
,3,4-thiadiazole (R ) rings affected the activity of the molecules. The presence of a methyl
(compounds 5a-c) or n-propyl (compounds 5m-o) group on the 1,3,4-thiadiazole ring
substantially increased the antitubercular activity regardless of the nature of the substituent at
1
R (H, Cl or CF ). Among derivatives which contain an unsubstituted phenothiazine ring
3
1
2
(
R =H), those with a 4-methylphenyl (5g) or 4-fluorophenyl (5j) substituent at R displayed
1
substantial activity. In the case of chloro substituted (R =Cl) derivatives, a 4-chlorophenyl
2
(
5e) or 4-fulorophenyl (5k) substituent at R enhanced the activity. Among trifluoromethyl
2
substituted analogs, only one compound (5l) with a 4-fluoro phenyl substituent at R showed
promising activity. The presence of electron rich groups such as 3,4-dimethoxy phenyl or
2
thienyl at R failed to enhance the inhibition activity of the molecules (5p-u). This is evident
from the observation that except compound 5p which showed a MIC of 12.5 ꢀg/mL, all other
molecules (5q-u) were only moderately active. Further, an alkyl/substituted (4-methyl/4-
Cl/4-F) phenyl group on the 1,3,4-thiadiazole ring and a chlorosubstituted/unsubstituted
phenothiazine ring serve to enhance the antitubercular activity of the hybrid derivatives. The
4

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structure activity relationship suggest that the phenothiazine-thiadiazole core could be
considered as a promising structural unit for the development of new antiTB agents.
(
Figure 2 here)
Figure 3 here)
(
2
.3. Antibacterial activity
The in vitro antibacterial activity of synthesized compounds 5a-u was tested using the
disc diffusion method [30]. All the compounds were screened against three bacterial strains
viz. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli using
ciprofloxacin as the standard drug. The compounds were dissolved in DMSO with two
concentrations (75 µg/mL and 50 µg/mL). Compounds 5a, 5g and 5p demonstrated
significant inhibition activity (Table 2) against all three bacterial strains at both
concentrations. It is interesting to note that all three derivatives contain an unsubstituted
1
2
phenothiazine ring (R =H) and an electron rich substitution (R ) on the 1,3,4-thiadiazole ring.
(Table 2 here)
2
.4. In vitro cytotoxicity studies
The cytotoxicity of the potent compounds was evaluated against VERO (African
Green Monkey Kidney) cell lines using the MTT assay [31]. The graphical representation of
the IC value and selectivity index (SI) of the compounds is shown in figure 4. The high IC
5
0
50
value (>215 ꢀg/mL) indicates the nontoxicity of the compounds. Further, the compounds
exhibited high selectivity index (SI = IC against Vero cells/MIC against M. tuberculosis) in
5
0
the range 76 - 473. The compounds with SI values greater than 10 against Vero cells are
considered as nontoxic [32]. Hence, these molecules which possess significant antiTB
activity with a low toxicity profile are suitable for further development as new antiTB agents.
(Figure 4 here)
2
.5. Molecular docking studies
In order to further understand the mechanism of action of the new phenothiazine-
,3,4-thiadiazole hybrids, the active molecules were subjected to molecular docking studies
1
against two enzymes, enoyl-acyl carrier protein reductase (InhA) and cytochrome P450
monooxygenase (CYP121) of M. tuberculosis, which have been validated as effective antiTB
targets. The molecules were docked with in the active sites of InhA (PDB code: 1P44) and
5

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CYP121 (PDB code: 4KTF) using Glide 6.6 (Schrodinger, 2015-1) package. The ligands
from the crystal structure of the enzyme-ligand complexes were rebuilt and redocked to
validate the docking procedure. The RMSD values of the docking pose from the original
orientation of the ligands were found to be 0.6106 and 1.1467 Å respectively for 1P44 and
4
KTF. The docking poses of molecules 5g, 5n, 5l and 5o are shown in figure 5. The docking
score of all the active molecules and details of interacting amino acid residues are given in
the supplementary data. The target molecules, as they contain 4 or 5 aromatic rings, showed
strong pi-pi stacking interactions in addition to hydrogen bonding interaction with the target
enzymes. Most of the active compounds interacted with Phe 149 residue of 1P44 and Phe 168
residue of CYP121. For instance, both 5g and 5n (the most active compounds of the series)
showed pi-pi stacking interaction with these amino acid residues. Compound 5g with a
docking score of -8.98 displayed additional interactions with 1P44 viz a pi-pi stacking
interaction with Tyr 158 and an H-bonding interaction with Gly 104. It is interesting to note
that the two phenyl rings of 5g interacted simultaneously with both Phe 149 and Tyr 158
residues (figure 5a) which ensures the stronger binding of the compound with the target
enzyme. The docking pose of 5g with target enzyme 4KTF (figure 5d) reveals the additional
pi-pi stacking interaction with Trp 182 residue. Compound 5l, which showed an MIC of 1.6
µg/mL, displayed the highest docking scores with both target enzymes (-9.37 with 1P44 and -
9
.1 with 4KTF). The pi-pi stacking interaction of the molecule with the target enzymes is
evident from the docking poses presented in figures 5b and 5e. These results suggest that the
strong pi-pi stacking interaction of the active molecules with the target enzymes could be
responsible for their significant inhibition activity against the MTB strain. The presence of a
pi-pi stacking interaction was observed also in the case of isoniazid (INH), which is a first
line antiTB drug. It is known that INH acts by inhibiting InhA and shows a pi-pi stacking
interaction with Phe 149 residue of the enzyme. Hence, the MTB inhibition and docking
studies signifies that the active phenothiazine-thiadiazole hybrids are suitable for the
development new antiTB agents.
(Figure 5 here)
3
. Conclusions
We have designed a series of 2-(2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-
phenothiazin-10-yl)ethanone derivatives (5a-u) following the molecular hybridization
6

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approach. The antiTB assay against Mtb H37Rv revealed the significant inhibition activity of
the molecules. Interestingly, eleven compounds of the series showed remarkable inhibitory
activity with MIC ≤ 3.125 ꢀg/mL, among which derivatives 5g and 5n emerged as the most
potent leads with MIC of 0.8 ꢀg/mL which suggests that the phenothiazine-thiadiazole core
could be considered as a new active structural unit for the development of antiTB leads. The
structure-activity relationship revealed that alkyl (methyl/n-propyl) or substituted (4-
methyl/4-Cl/4-F) phenyl groups on the 1,3,4-thiadiazole ring enhance the inhibition activity
of the compounds. Also, compounds containing chloro/unsubstituted phenothiazine ring
exhibited remarkable antiTB activity. Further, the cytotoxicity study revealed the nontoxic
nature of the active molecules to a normal Vero cell line. The molecular docking study
demonstrated that strong pi-pi stacking interaction of the active molecules with the target
enzymes could be responsible for their significant inhibition activity against MTB strain.
These results suggest that the molecules are promising leads for further studies and
development of new antiTB agents.
4
4
4
. Experimental part
.1. Synthesis
.1.1. 2-chloro-1-(10H-phenothiazin-10-yl)ethanone (2a): To the solution of 10H-
phenothiazine 1 (5.0 g, 25.11 mmol) in toluene (75 mL) was cooled to 0 °C. To the above
solution chloroacetyl chloride (3.0 mL, 37.6 mmol) was added and reaction mixture was
heated at 80 °C for 12 h. The reaction mixture was allowed cool to room temperature,
concentrated under reduced pressure (< 45 °C) and to the crude material water (50 mL) was
added and extracted with dichloromethane (2 × 50 mL). Organic layer was dried over
anhydrous Na SO and the solvent was removed to get compound 2a as off white solid.
2
4
°
−1
Yield: 6.56 g, 95 %; m.p: 114-115 C; FTIR (ATR, cm ): 2998, 2950, 1670, 1586, 1476,
1
1
459, 1442, 1401, 1339, 1275, 1248, 1193, 1170, 1123, 782, 751, 647, 615; H NMR (400
MHz, CDCl ) δ (ppm):7.52 (s, 2H, Ar-H), 7.40 (s, 2H, Ar-H), 7.29 (d, J = 5.7 Hz, 2H, Ar-H),
3
13
7
.21 (d, J = 6.3 Hz, 2H, Ar-H), 4.12 (s, 2H, CH ); C NMR (100 MHz, CDCl ) δ (ppm):
2 3
1
65.5, 137.9, 133.1, 128.1, 127.4, 127.3, 126.5, 41.8; ESI-MS (m/z) = 276.1 (M + 1);
calculated for C H ClNOS; C, 60.98; H, 3.66; N, 5.08; S, 11.63. Found: C, 60.87; H, 3.68;
1
4
10
N, 5.10; S, 11.65.
4
.1.2 2-chloro-1-(2-chloro-10H-phenothiazin-10-yl)ethanone (2b): White solid. Yield: 6.0
°
−1
g, 90 %; m.p: 118-119 C; FTIR (ATR, cm ): 3057, 2993, 2943, 1673, 1574, 1451, 1398,
7

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1
1
328, 1268, 1162, 1125, 1090, 801, 755, 732, 689, 654; H NMR (400 MHz, CDCl ) δ
3
(ppm): 7.58 (s, 1H, Ar-H), 7.46 (s, 1H, Ar-H), 7.39 (s, 1H, Ar-H), 7.31 (d, J = 3.4 Hz, 2H,
Ar-H), 7.21 (d, J = 14.9 Hz, 2H, Ar-H), 4.16 (d, J = 12.5 Hz, 1H, CH), 4.08 (d, J = 12.5 Hz,
1
3
CH); C NMR (100 MHz, DMSO-d ) δ (ppm):165.4, 139.2, 137.5, 132.4, 132.3, 131.9,
6
1
29.7, 128.6, 128.3, 128.2, 127.9, 127.4, 127.3, 43.2; ESI-MS (m/z) = 310.1 (M + 1);
calculated for C H Cl NOS; C, 54.21; H, 2.92; N, 4.52; S, 10.34. Found: C, 54.18; H, 2.93;
1
4
9
2
N, 4.55; S, 10.35.
.1.3 2-chloro-1-(2-(trifluoromethyl)-10H-phenothiazin-10-yl)ethanone (2c): White solid.
4
°
−1
Yield: 6.16 g, 96 %; m.p: 110-111 C; FTIR (ATR, cm ): 3002, 2947, 1683, 1607, 1465,
1
1
324, 1298, 1274, 1242, 1163, 1128, 1083, 823, 745, 639.63; H NMR (400 MHz, CDCl ) δ
3
(ppm): 7.84 (s, 1H, Ar-H), 7.54 – 7.38 (m, 4H, Ar-H), 7.33 (s, 1H, Ar-H), 7.27 (s, 1H, Ar-H),
13
4
.18 (d, J = 12.5 Hz, 1H, CH), 4.06 (d, J = 12.5 Hz, 1H, CH); C NMR (100 MHz, CDCl ) δ
3
(ppm): 165.4, 138.0, 137.1, 128.9, 128.3, 128.2, 127.9, 126.1, 125.2, 124.1, 123.9, 41.5; ESI-
MS (m/z) = 344.1 (M + 1); calculated for C H ClF NOS; C, 52.41; H, 2.64; N, 4.07; S, 9.33.
1
5
9
3
Found: C, 52.38; H, 2.63; N, 4.05; S, 9.35.
.2.1. 5-amino-2-methyl-1, 3, 4-thiadiazole (4a): To the thiosemicarbazide (7.5 g, 82.29
4
mmol), acetyl chloride (14.27 mL, 164.58 mmol) was added slowly and the mixture was
stirred for 4 h at RT. To this reaction mixture, ice cold water was added and the solid
obtained was filtered off. The solid was then taken in ice cold water and to this slurry, a
solution of 50% NaOH was added till the pH of the solution becomes basic. The solid
obtained was filtered off and dried under vacuum to get the compound (4a) as white solid.
°
−1
1
Yield: 7.2 g, 76 %; m.p: 269–270 C; FTIR (ATR, cm ): 3288, 3105, 2925, 1614, 689; H
13
NMR (400 MHz, DMSO-d ) δ (ppm): 13.05 (br s, 2H, NH ), 2.18 (s, 3H, CH ); C NMR
6
2
3
(
100 MHz, DMSO-d ) δ (ppm): 165.7, 148.8, 10.9; ESI-MS (m/z) = 116.02 (M + 1);
6
calculated for C H N S; C, 31.9; H, 4.38; N, 36.49; S, 27.84. Found: C, 31.89; H, 4.41; N,
3
5
3
3
6.42; S, 27.44.
4
.2.2. General procedure for the synthesis of 5-subsituted-1,3,4-thiadiazole-2-yl amine (4b-
g): A mixture of appropriate aromatic carboxylic acid (5.0 g, 1 eq), thiosemicarbazide (1 eq)
°
and POCl (1.85 eq) was heated to 75 C and maintained at the same temperature for 30 min
3
under stirring. The reaction mixture was then cooled to room temperature, water (11 v) was
added and the mixture was refluxed for 4 h. After cooling, the mixture was basified with 50%
8

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NaOH to pH 8 by the drop wise addition under stirring. The obtained solid was filtered and
recrystallized from ethanol to give the target compounds 4b-g.
4
.2.2.1.
5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-amine (4b): White solid. Yield: 5.20
°
−1
1
g, 77 %; m.p: 210–211 C; FTIR (ATR, cm ): 3258, 3093, 1633, 751, 684; H NMR (400
MHz, DMSO-d ) δ (ppm): 7.47 (s, 2H), 7.52–7.54 (d, J = 6.55 Hz, 2H, Ar-H), 7.76–7.78 (d, J
6
1
3
=
6.24 Hz, 2H, Ar-H); C NMR (100 MHz, DMSO-d ) δ (ppm):128.38, 129.63, 130.34,
6
1
34.43, 155.59, 168.30; ESI-MS (m/z) = 212.6 (M + 1); calculated for C H ClN S; C, 45.39;
8 6 3
H, 2.86; N, 19.85; S, 15.15. Found: C, 45.30; H, 2.88; N, 19.90; S, 15.14.
4
.2.2.2.
5-(4-Methylphenyl)-1,3,4-thiadiazol-2-amine (4c): White solid; yield: 6.31 g,
°
−1
1
9
0 %; m.p: 213–214 C; FTIR (ATR, cm ): 3278, 3103, 2955, 1611, 690; H NMR (400
MHz, DMSO-d ) δ (ppm): 2.33 (s, 3H), 7.25–7.27 (d, J = 8.00 Hz, 2H, Ar-H), 7.36 (s, 2H),
6
1
3
7
.63–7.65 (d, J = 8.12 Hz, 2H, Ar-H); C NMR (100 MHz, DMSO-d ) δ (ppm): 21.35,
6
1
26.72, 128.80, 130.11, 139.76, 156.95, 168.65; ESI-MS (m/z) = 192.2 (M + 1); Calculated
for C H N S; C, 56.22; H, 4.74; N, 21.97; S, 16.77. Found: C, 56.20; H, 4.74; N, 21.90; S,
9
9
3
1
6.74.
4
.2.2.3.
5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-amine (4d): Colorless solid. Yield:
°
−1
1
5
.69 g, 82 %; m.p: 230-231 C; FTIR (ATR, cm ): 3346, 3251, 2934, 1633, 1063, 683; H
NMR (400 MHz, DMSO-d ) δ (ppm): 7.29–7.33 (t, J = 8.77 Hz, 2H, Ar-H), 7.41 (s, 2H),
6
1
3
7
.79–7.83 (dd, J = 5.46 Hz, 8.59 Hz, 2H, Ar-H); C NMR (100 MHz, DMSO-d ) δ (ppm):
6
1
18.13, 129.43, 130.21, 154.32, 157.28, 168.10; ESI-MS (m/z) = 196.4 (M + 1); calculated
for C H FN S; C, 49.22; H, 3.10; N, 21.52; S, 16.43. Found: C, 49.20; H, 3.11; N, 21.52; S,
8
6
3
1
6.44.
4
.2.2.4.
5-Propyl-1,3,4-thiadiazol-2-amine (4e): White solid. Yield: 6.33 g, 78 %;
°
−1
m.p: 209-210 C; FTIR (ATR, cm ): 3228, 3075, 2956, 2928, 1634, 1520, 1497, 1459, 1327,
1
1
2
187, 1039, 686; H NMR (400 MHz, DMSO-d ) δ (ppm): 6.82 (br s, 2H), 2.61 (t, J = 7.6 Hz,
6
1
3
H), 1.61 (sext, J = 7.6 Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H); C NMR (100 MHz, DMSO-d ) δ
6
(ppm): 165.7, 148.8, 31.4, 21.7, 13.3; ESI-MS (m/z) = 144.1 (M + 1); calculated for
C H N S; C, 41.93; H, 6.33; N, 29.34; S, 22.39. Found: C, 41.89; H, 6.35; N, 29.22; S, 22.44.
5
9
3
4
.2.2.5.
5-(3,4-Dimethoxyphenyl)-1,3,4-thiadiazol-2-amine (4f): White solid. Yield:
°
−1
5
.53 g, 85 %; m.p: 213-214 C; FTIR (ATR, cm ): 3251, 3081, 3004, 2949, 1619, 1589,
9

ACCEPTED MANUSCRIPT
1
1
517, 1468, 1419, 1248, 1058, 1044, 1022, 861, 797, 758, 704, 662; H NMR (400 MHz,
DMSO-d ) δ (ppm): 8.65–8.69 (m, 3H, Ar-H), 7.61 (br s, 2H), 3.71 (s, 3H, OCH ), 3.70 (s,
6
3
1
3
3
1
H, OCH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 168.7, 154.1, 144.1, 132.8, 125.5,
3 6
18.4, 115.8, 108.1, 55.8, 55.2; ESI-MS (m/z) = 238.1 (M + 1); calculated for C H N O S;
1
0
11
3
2
C, 50.62; H, 4.67; N, 17.71; S, 13.51. Found: C, 50.72; H, 4.66; N, 17.75; S, 13.54.
4
9
1
7
.2.2.6.
5-(Thiophen-2-yl)-1,3,4-thiadiazol-2-amine (4g): White solid. Yield: 6.57 g,
°
−1
2 %; m.p: 175-176 C; FTIR (ATR, cm ): 3271, 3097, 1618, 1510, 1463, 1419, 1328,
1
257, 1229, 1134, 1068, 1044, 914, 844, 775, 703; H NMR (400 MHz, DMSO-d ) δ (ppm):
6
13
.70 – 7.64 (m, 2H, Ar-H), 7.63 – 7.57 (m, 1H, Ar-H), 7.34 (br s, 2H); C NMR (100 MHz,
DMSO-d ) δ (ppm): 169.3, 154.3, 134.9, 131.6, 133.7, 127.4; ESI-MS (m/z) = 183.9 (M + 1);
6
calculated for C H N S ; C, 39.32; H, 2.75; N, 22.93; S, 35.00. Found: C, 39.29; H, 2.74; N,
6
5
3 2
2
2.89; S, 35.03.
4
.2.3. General Procedure for the Synthesis of derivatives (5a-u): A mixture of 5-subsituted 2-
amino-1,3,4-thiadiazole (4a-g) (0.2 g, 1.0 mmol) and 2-chloro-1-(2-substituted)-10H-
phenothiazin-10-yl)ethanone (2a-c) (1.0 mmol) in ethanol was stirred at 80-85 °C for 17 h.
After the completion of reaction (as confirmed by TLC), the reaction mass was cooled to
room temperature and then concentrated under reduced pressure. The obtained crude product
was basified with aqueous 10% Na CO solution. To the above solution ethyl acetate (20 mL)
2
3
was added, the organic layer was separated, washed with brine solution (2 × 10 mL), dried
over anhydrous Na S O and concentrated under reduced pressure. The crude residue was
2
2
4
purified over silica gel column chromatography (100-200 mesh) eluted with methanol and
dichloromethane (0.5 : 9.5) to give pure products 5a-u.
4
.2.3.1.
2-(2-imino-5-methyl-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-10-
−
1
yl)ethanone (5a): White solid. Yield: 0.647 g; FTIR (ATR, cm ): 3314, 1696, 1601, 1460,
1
1
259, 1157, 948, 751, 704; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.16 (br s, 1H, NH, D O
6
2
exchangeable proton), 7.68 (s, 2H, Ar-H), 7.56 (d, J = 7.3 Hz, 2H, Ar-H), 7.40 (t, J = 7.2 Hz,
1
2
H, Ar-H), 7.32 (t, J = 7.3 Hz, 2H, Ar-H), 4.74 (s, 2H), 2.20 (s, 3H, CH ); H NMR (400
3
MHz, D O) δ 7.61 (s, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.3 Hz, 2H), 7.29 (t, J = 7.4
2
1
3
Hz, 2H), 4.68 (s, 2H), 2.15 (s, 3H). C NMR (100 MHz, DMSO-d ) δ (ppm): 165.9, 137.9,
6
1
32.5, 128.4, 127.9, 127.8, 127.5, 124.7, 49.3, 17.08; ESI-MS (m/z) = 355.1 (M + 1); anal.
1
0

ACCEPTED MANUSCRIPT
calcd (%) for C H N OS : C, 57.61; H, 3.98; N, 15.81; S, 18.09. Found: C, 57.42; H, 4.02;
1
7
14
4
2
N, 15.78; S, 18.12.
4
.2.3.2. 1-(2-chloro-10H-phenothiazin-10-yl) -2- (2-imino-5-methyl-1, 3, 4-thiadiazol
−
1
-
3(2H)yl)ethanone (5b): Off-white solid. Yield: 0.573 g; FTIR (ATR, cm ): 3313, 2919,
1
1
694, 1598, 1456, 1242, 1172, 1131, 1092, 948, 808, 744, 700; H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.95 (br s, 1H, NH), 7.78 (s, 1H, Ar-H), 7.70 (d, J = 7.5 Hz, 1H, Ar-H), 7.57 (d,
J = 8.3 Hz, 2H, Ar-H), 7.45 – 7.37 (m, 2H, Ar-H), 7.34 (t, J = 7.6 Hz, 1H, Ar-H), 4.84 (s, 1H,
13
CH), 4.62 (s, 1H, CH), 2.18 (s, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 168.3,
3
6
1
4
3
41.6, 140.3, 138.2, 137.7, 134.5, 129.3, 128.3, 127.6, 125.7, 125.1, 124.4, 122.2, 121.2,
9.7, 17.1; ESI-MS (m/z) = 389.1 (M + 1); anal. calcd (%) for C H ClN OS : C, 52.50; H,
1
7
13
4
2
.37; N, 14.41; S, 16.49. Found: C, 52.48; H, 3.40; N, 14.38; S, 16.52.
4
.2.3.3. 2-(2-imino-5-methyl-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)-10H-
−
1
phenothiazin-10-yl) ethanone (5c): Light brown solid. Yield: 0.645 g, FTIR (ATR, cm ):
3
9
7
341, 2893, 1692, 1594, 1467, 1414, 1362, 1326, 1296, 1245, 1166, 1122, 1085, 1029, 984,
1
51, 885, 814, 744, 695, 656; H NMR (400 MHz, DMSO-d ) δ (ppm): 7.98 (br s, 1H, NH),
6
.84 (d, J = 8.0 Hz, 1H, Ar-H), 7.68 (dd, J = 22.2, 7.5 Hz, 3H, Ar-H), 7.52 – 7.35 (m, 3H,
1
3
Ar-H), 4.74 (s, 2H, CH ), 2.46 (s, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm):
2
3
6
1
67.4, 143.9, 141.81, 138.87, 137.73, 129.9, 128.33, 127.6, 127.04, 125.77, 125.12, 124.84,
1
22.29, 116.02, 52.79, 17.6; ESI-MS (m/z) = 423.1 (M + 1); anal. calcd (%) for
C H F N OS : C, 51.18; H, 3.10; N, 13.26; S, 15.18. Found: C, 51.22; H, 3.12; N, 13.28; S,
18
13
3
4
2
1
5.22.
4
.2.3.4.
2-(5-(4-chlorophenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-
−
1
phenothiazin-10-yl )ethanone (5d): White solid. Yield: 0.375 g, FTIR (ATR, cm ): 3314,
1
3
056, 1690, 1459, 1343, 1262, 1236, , 1182, 1129, 827, 757, 659; H NMR (400 MHz,
DMSO-d ) δ (ppm): 8.01 (br s, 1H, NH), 7.78 (d, J = 7.5 Hz, 4H, Ar-H), 7.61 (d, J = 7.7 Hz,
6
1
3
4
H, Ar-H), 7.46 (s, 2H, Ar-H), 7.38 (s, 2H, Ar-H), 5.27 (s, 2H); C NMR (100 MHz,
DMSO-d ) δ (ppm): 164.1, 137.4, 137.3, 137.0, 136.9, 130.0, 129.6, 128.6, 128.1, 127.3,
6
5
2.8; ESI-MS (m/z) = 451.1 (M + 1); anal. calcd (%) for C H ClN OS : C, 58.59; H, 3.35;
22 15 4 2
N, 12.42; S, 14.22. Found: C, 58.52; H, 3.32; N, 12.48; S, 14.25.
4
.2.3.5.
1-(2-chloro-10H-phenothiazin-10-yl)-2-(5-(4-chlorophenyl)-2-imino-1,3,4-
−
1
thiadiazol-3(2H)-yl) ethanone (5e): Light brown solid. Yield: 0.412 g; FTIR (ATR, cm ):
1
1

ACCEPTED MANUSCRIPT
3
7
303, 2976, 1689, 1634, 1572, 1458, 1401, 1366, 1287, 1245, 1177, 1091, 996, 903, 821,
1
49, 690; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.02 (br s, 1NH), 7.82-7.75 (m, 4H), 7.60
6
13
(
d, J = 7.8 Hz, 4H), 7.45 (m, 3H), 5.28 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ
2 6
(
ppm): 163.5, 146.8, 146.7, 145.8, 144.7, 142.3, 141.5, 137.6, 137.5, 137.2, 130.1, 129.0,
26.5, 53.9; ESI-MS (m/z) = 484.9 (M + 1); anal. calcd (%) for C H Cl N OS : C, 54.44;
1
2
2
14
2
4
2
H, 2.91; N, 11.54; S, 13.21. Found: C, 54.48; H, 2.91; N, 11.56; S, 13.23.
4
.2.3.6.
2-(5-(4-chlorophenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)-
1
0H-phenothiazin-10-yl)ethanone (5f): Light brown solid. Yield: 0.451 g; FTIR (ATR,
−
1
cm ): 3340, 1690, 1604, 1418, 1400, 1370, 1327, 1299, 1248, 1169, 1126, 1087, 1014, 985,
1
9
1
7
1
1
55, 934, 905, 886, 827,745, 652; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.08 (br s,
6
NH), 7.79 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.60-7.54 (m, 5H), 7.45 (d, J = 7.4 Hz, 2H),
13
.40 (s, 1H, Ar-H), 4.83 (s, 1H), 4.70 (s, 1H). C NMR (100 MHz, DMSO-d ) δ (ppm):
6
66.15, 138.2, 135.1, 129.7, 129.4, 129.3, 128.7 128.5, 128.3, 127.5, 124.4, 124.3, 124.2,
22.9, 49.84; ESI-MS (m/z) = 519.1 (M + 1); anal. calcd (%) for C H ClF N OS : C, 53.23;
2
3
14
3
4
2
H, 2.72; N, 10.80; S, 12.36. Found: C, 53.33; H, 2.71; N, 10.79; S, 12.40.
4
.2.3.7. 2-(2-imino-5-p-tolyl-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-10-yl)
−
1
ethanone (5g): Off-white solid. Yield: 0.382 g; FTIR (ATR, cm ): 3330, 2949, 1690, 1593,
1
1
458, 1362, 1256, 1178, 1146, 979.76, 750, 724, 696; H NMR (400 MHz, DMSO-d ) δ
6
(ppm): 8.78 (s, 1H), 7.73 (s, 2H), 7.58 (d, J = 6.5 Hz, 2H), 7.48 (d, J = 6.7 Hz, 2H), 7.41 (s,
13
2
H), 7.33 (s, 2H), 7.28 (d, J = 6.4 Hz, 2H), 4.96 (s, 2H), 2.33 (s, 3H); C NMR (100 MHz,
DMSO-d ) δ (ppm): 165.5, 141.0, 137.8, 132.5, 130.2, 128.5, 127.9, 127.5, 127.3, 126.07,
6
5
0.50, 21.42; ESI-MS (m/z) = 431.1 (M + 1); anal. calcd (%) for C H N OS : C, 64.16; H,
23 18 4 2
4
.21; N, 13.01; S, 14.90. Found: C, 64.20; H, 4.19; N, 13.10; S, 14.88.
4
.2.3.8. 1-(2-chloro-10H-phenothiazin-10-yl)-2-(2-imino-5-(p-tolyl)-1,3,4-thiadiazol-3(2H)
−
1
yl) ethanone (5h): Light yellow solid. Yield: 0.426 g; FTIR (ATR, cm ): 3316, 2920, 1694,
1
8
586, 1503, 1455, 1395, 1356, 1277, 1242, 1206, 1175, 1129, 1093, 1026, 981, 949, 901,
1
12, 743, 693; H NMR (400 MHz, DMSO) δ 8.45 (br s, NH), 7.82 (s, 1H, Ar-H), 7.73-7.69
(m, 2H, Ar-H), 7.60 (d, J = 7.4 Hz, 2H), 7.48 (d, J = 7.3 Hz, 2H), 7.41 (s, 2H), 7.25 – 7.17
1
3
(
m, 2H 96 (s, 2H), 2.38 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm): 165.7, 141.6,
6
1
1
39.1, 136.8, 135.4, 136.7, 135.5, 130.0, 129.3, 129.0, 128.4, 127.9, 126.3, 125.8, 125.4,
24.6, 122.6, 121.2, 51.2, 21.33; ESI-MS (m/z) = 464.1 (M + 1); anal. Calcd (%) for
1
2

ACCEPTED MANUSCRIPT
C H ClN OS : C, 59.41; H, 3.69; N, 12.05; S, 13.79. Found: C, 59.43; H, 3.70; N, 12.08; S,
23
17
4
2
1
3.80.
4
.2.3.9.
2-(2-imino-5-(p-tolyl)-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)-10H-
−
1
phenothiazin-10-yl) ethanone (5i): Yellow solid. Yield: 0.474 g; FTIR (ATR, cm ): 3339,
3
9
7
2
021, 2893, 1693, 1592, 1466, 1414, 1362, 1326, 1296, 1245, 1166, 1122, 1085, 1029, 984,
1
51, 886, 816, 746, 694, 656; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.38 (br s, NH, 1H),
6
.92 (s, 1H, Ar-H), 7.71 (t, J = 6.1 Hz, 2H), 7.66 – 7.58 (m, 4H, Ar-H), 7.40 (d, J = 7.4 Hz,
H, Ar-H), 7.31 (dd, J = 14.23, 7.4 Hz, 2H, Ar-H), 4.83 (s, 1H), 4.70 (s, 1H), 2.35 (s, 1H);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 167.7, 143.9, 142.81, 140.1, 137.7, 136.4, 131.0,
6
1
29.9, 129.0, 128.3, 127.6, 127.0, 126.2, 125.7, 125.1 , 124.8, 122.2, 116.0, 52.7, 21.3; ESI-
MS (m/z) = 499.1 (M + 1); anal. calcd (%) for C H F N OS : C, 57.82; H, 3.44; N, 11.24;
2
4
17
3
4
2
S, 12.86. Found: C, 57.78; H, 3.48; N, 11.22; S, 12.90.
4
.2.3.10. 2-(5-(4-fluorophenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-10-
−
1
yl) ethanone (5j): Light brown solid. Yield: 0.409 g; FTIR (ATR, cm ): 3305, 3055, 2355,
1
8
7
1
1
4
687, 1593, 1501, 1459, 1395, 1357, 1300, 1255, 1228, 1177, 1157, 1127, 1030, 987, 948,
1
35, 811, 756, 692, 660, 630; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.35 (br s, 1H, NH),
6
.71 (s, 2H, Ar-H), 7.63 – 7.54 (m, 4H, Ar-H), 7.40 (t, J = 7.6 Hz, 2H, Ar-H), 7.31 (dd, J =
13
6.3, 7.8 Hz, 4H, Ar-H), 4.88 (s, 2H); C NMR (100 MHz, DMSO-d ) δ (ppm): 166.1,
6
62.8, 156.4, 137.7, 129.3, 128.4, 128.2, 127.6, 125.86, 122.3, 114.6, 50.21; ESI-MS (m/z):=
35.1 (M + 1); anal. calcd (%) for C H FN OS : C, 60.81; H, 3.48; N, 12.89; S, 14.76.
2
2
15
4
2
Found: C, 60.77; H, 3.50; N, 12.88; S, 14.70.
4
.2.3.11.
1-(2-chloro-10H-phenothiazin-10-yl)-2-(5-(4-fluorophenyl)-2-imino-1,3,4-
−
1
thiadiazol-3(2H)-yl)ethanone (5k): Brown solid. Yield: 0.456 g; FTIR (ATR, cm ): 3313,
3
9
066, 1691, 1592, 1501, 1456, 1396, 1356, 1322, 1279, 1232, 1175, 1158, 1131, 1094, 987,
1
50, 835, 811, 744, 694; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.35 (br s, 1H, NH), 7.82
6
(s, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.64 – 7.53 (m, 3H), 7.46-7.35 (m , 3H, Ar-H), 7.31 (dd, J
1
3
=
15.7, 7.0 Hz, 3H), 5.01 (s, 1H), 4.77 (s, 1H); C NMR (100 MHz, DMSO-d ) δ (ppm):
6
1
1
66.0, 164.67, 159.91, 159.24, 158.90, 149.25, 139.1, 132.2, 129.57, 128.65, 127.9, 127.5,
27.46, 117.3, 116.7, 49.8; ESI-MS (m/z) = 469.1 (M + 1); anal. calcd (%) for
C H ClFN OS : C, 56.35; H, 3.01; N, 11.95; S, 13.68. Found: C, 56.28; H, 3.00; N, 11.93;
22
14
4
2
S, 13.70.
1
3

ACCEPTED MANUSCRIPT
4
1
3
9
.2.3.12.
2-(5-(4-fluorophenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)-
−
1
0H-phenothiazin-10-yl) ethanone (5l): Yellow solid. Yield: 0.453 g; FTIR (ATR, cm ):
328, 2921, 1694, 1594, 1501, 1466, 1414, 1359, 1326, 1296, 1239, 1165, 1122, 1086, 1029,
1
87, 950, 889, 833, 751, 694, 629; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.33 (br s, 1H,
6
NH), 8.03 (s, 1H, Ar-H), 7.78 (d, J = 8.2 Hz, 2H, Ar-H), 7.65 (d, J = 6.9 Hz, 1H, Ar-H), 7.60
dd, J = 8.9, 5.3 Hz, 3H, Ar-H), 7.45 (dd, J = 14.2, 7.6 Hz, 1H, Ar-H), 7.38 (dd, J = 13.0, 6.3
(
13
Hz, 1H), 7.34 – 7.26 (m, 2H, Ar-H), 5.06 (s, 1H), 4.75 (s, 1H); C NMR (100 MHz, DMSO-
d6) δ (ppm): 166.1, 162.22, 138.3, 137.1, 129.3, 128.76, 128.5, 128.3, 128.2, 128.15, 124.3,
1
16.8, 116.6, 49.8; ESI-MS (m/z) = 503.1 (M + 1); anal. calcd (%) for C H F N OS : C,
23 14 4 4 2
5
4.97; H, 2.81; N, 11.15; S, 12.76. Found: C, 54.88; H, 2.81; N, 11.16; S, 12.66.
4
.2.3.13. 2-(2-imino-5-propyl-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-10-yl)
−
1
ethanone (5m): Off-white solid. Yield: 0.432 g; FTIR (ATR, cm ): 3370, 2992, 2925, 2871,
2
1
784, 1687, 1636, 1577, 1550, 1480, 1460, 1416, 1363, 1302, 1283, 1262, 1240, 1182, 1163,
1
128, 1077, 1026, 974, 766, 656; H NMR (400 MHz, DMSO-d ) δ (ppm): 7.68 (s, 2H, Ar-
6
H), 7.63 (d, J = 6.9 Hz, 2H, Ar-H), 7.45 (s, 2H, Ar-H), 7.38 (s, 2H, Ar-H), 5.84 (s, 1H), 4.92
1
3
(
s, 1H), 2.80 (t, J = 7.2 Hz, 2H), 1.61 (sextet, J = 7.2 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H); C
NMR (100 MHz, DMSO-d ) δ (ppm):169.01, 163.5, 158.3, 158.1, 128.7, 128.6, 128.5, 128.3,
6
1
28.2, 53.3, 31.5, 21.7, 13.4; ESI-MS (m/z) = 383.1 (M + 1); anal. calcd (%) for
C H N OS : C, 56.85; H, 3.34; N, 13.26; S, 22.77. Found: C, 56.94; H, 3.36; N, 13.24; S,
20
14
4
3
2
2.69.
4
3
2
7
7
4
.2.3.14.
1-(2-chloro-10H-phenothiazin-10-yl)-2-(2-imino-5-propyl-1,3,4-thiadiazol-
−
1
(2H)yl)ethanone (5n): Light yellow solid. Yield: 0.504 g; FTIR (ATR, cm ): 3321, 2957,
926, 2869, 1690, 1589, 1561, 1478, 1455, 1391, 1277, 1231, 1174, 1122, 1091, 1028, 916,
1
94, 739; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.21 (br s, 1H, NH), 7.79 (s, 1H, Ar-H),
6
.70 (d, J = 7.6 Hz, 2H), 7.63 – 7.54 (m, 2H, Ar-H), 7.19 (d, J = 7.6 Hz, 2H), 5.08 (s, 1H),
.85 (s, 1H), 2.77 (t, J = 7.8 Hz, 2H), 1.54 (sextet, J = 7.8 Hz, 2H), 0.89 (t, J = 7.8 Hz, 3H);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 168.2, 163.8, 159.3, 141.6, 138.5, 134.5, 130.2,
6
1
28.3, 127.9, 126.7, 125.4, 124.5, 122.31, 121.23, 49.5, 31.3, 21.4, 13.2; ESI-MS (m/z) =
4
17.1 (M + 1); anal. calcd (%) for C H ClN OS : C, 54.73; H, 4.11; N, 13.44; S, 15.38.
1
9
17
4
2
Found: C, 54.62; H, 4.14; N, 13.39; S, 15.44. ESI-MS (m/z) = 417.1 (M + 1);
1
4

ACCEPTED MANUSCRIPT
4
.2.3.15.
2-(2-imino-5-propyl-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)-10H-
−
1
phenothiazin-10-yl)ethanone (5o): Light brown solid. Yield: 0.560 g; FTIR (ATR, cm ):
3
1
310, 2961, 2932, 2873, 1693, 1603, 1553, 1464, 1433, 1390, 1326, 1299, 1229, 1162, 1113,
1
084, 1029, 952, 929, 869, 810, 743, 688, 649; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.21
6
(s, 1H, Ar-H), 7.89 – 7.73 (m, 2H, Ar-H), 7.73 – 7.54 (m, 3H, Ar-H), 7.52 – 7.29 (m, 2H, Ar-
H), 4.80 (s, 2H, CH ), 2.79 (t, J = 7.3 Hz, 2H), 1.57 (sextet, J = 7.2 Hz, 2H), 0.89 (t, J = 7.4
2
13
Hz, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm): 168.3, 163.4, 159.4, 144.9, 138.5, 137.7,
6
1
36.82, 133.5, 132.3, 125.5, 125.0, 124.3, 122.2, 116.0, 50.8, 31.2, 22.1, 13.5; ESI-MS (m/z)
451.1 (M + 1); anal. calcd (%) for C H F N OS : C, 53.32; H, 3.80; N, 12.44; S, 14.24.
=
2
0
17
3
4
2
Found: C, 53.38; H, 3.81; N, 12.45; S, 14.26.
4
.2.3.16.
2-(5-(3,4-dimethoxyphenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-
−
1
phenothiazin-10-yl) ethanone (5p): White solid. Yield: 0.317 g; FTIR (ATR, cm ): 3319,
2
8
7
7
3
1
4
1
932, 2834, 1689, 1587, 1505, 1459, 1417, 1356, 1301, 1248, 1173, 1131, 1019, 947, 859,
1
36, 805, 759, 735, 696, 660, 634; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.20 (s, 1H),
6
.69 (d, J = 17.4 Hz, 2H, Ar-H), 7.60 – 7.53 (m, 2H, Ar-H), 7.40 (t, J = 7.0 Hz, 2H, Ar-H),
.32 (t, J = 7.1 Hz, 2H, Ar-H), 7.12 (s, 1H, Ar-H), 7.06 – 6.97 (m, 2H, Ar-H), 4.86 (s, 2H),
13
.78 (s, 6H, dimethoxy); C NMR (100 MHz, DMSO-d ) δ (ppm): 169.2, 159.0, 158.7,
6
49.4, 145.3, 141.9, 136.7, 132.7, 132.6, 128.4, 127.8, 127.5, 119.7, 112.2, 108.0, 56.1, 56.0,
9.8; ESI-MS (m/z) = 477.2 (M + 1); anal. calcd (%) for C H N O S : C, 60.49; H, 4.23; N,
2
4
20
4
3 2
1.76; S, 13.46. Found: C, 60.52; H, 4.23; N, 11.78; S, 13.50.
.2.3.17. 1-(2-chloro-10H-phenothiazin-10-yl)-2-(5-(3,4-dimethoxyphenyl)-2-imino-
4
1
3
8
1
–
1
−
1
,3,4-thiadiazol-3(2H)-yl)ethanone (5q): Off-white solid. Yield: 0.348 g; FTIR (ATR, cm ):
318, 2934, 2835, 1687, 1586, 1503, 1456, 1414, 1354, 1301, 1248, 1173, 1131, 1019, 947,
1
59, 836, 811, 759, 735, 696, 660, 634; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.19 (s,
6
H), 7.98 (s, 1H, Ar-H), 7.74 (d, J = 7.4 Hz, 2H, Ar-H), 7.64 (d, J = 7.2 Hz, 1H, Ar-H), 7.57
7.51 (m, 1H, Ar-H), 7.46 (t, J = 7.6 Hz, 1H, Ar-H), 7.32 (t, J = 7.3 Hz, 1H, Ar-H), 7.21 (s,
H, Ar-H), 7.04 – 6.95 (m, 2H, Ar-H), 4.98 (s, 1H), 4.69 (s, 1H), 3.74 (s, 6H, dimethoxy);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 169.6, 161.8, 155.8, 153.1, 150.6, 141.6, 137.7,
6
1
34.5, 129.3, 128.2, 127.6, 125.7, 125.1, 124.4, 122.2, 121.2, 118.4, 115.8, 107.9, 56.8, 56.5,
5
0.3; ESI-MS (m/z) = 511.1 (M + 1); anal. Calcd (%) for C H ClN O S : C, 56.41; H, 3.75;
2
4
19
4
3 2
N, 10.96; S, 12.55. Found: C, 56.38; H, 3.74; N, 10.97; S, 12.49.
1
5

ACCEPTED MANUSCRIPT
4
.2.3.18.
2-(5-(3,4-dimethoxyphenyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)-1-(2-
(trifluoromethyl)-10H-phenothiazin-10-yl)ethanone (5r): Brown solid. Yield: 0.390 g; FTIR
−
1
(
ATR, cm ): 3302, 2933, 1693, 1590, 1505, 1462, 1414, 1358, 1326, 1244, 1166, 1121,
1
1
084, 1019, 949, 887, 831, 756, 701, 634; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.25 (br
6
s, 1H, NH), 8.05 (s, 1H, Ar-H), 7.79 (d, J = 8.2 Hz, 2H, Ar-H), 7.66 (d, J = 7.1 Hz, 1H, Ar-
H), 7.64 – 7.57 (m, 1H, Ar-H), 7.46 (t, J = 7.0 Hz, 1H, Ar-H), 7.37 (t, J = 7.3 Hz, 1H, Ar-H),
7
3
1
1
.15 – 7.08 (m, 1H, Ar-H), 7.04 – 6.97 (m, 2H, Ar-H), 5.07 (s, 1H, CH), 4.71 (s, 1H, CH),
13
.78 (s, 6H, dimethoxy); C NMR (100 MHz, DMSO-d ) δ (ppm): 169.7, 159.8, 155.84 (s),
6
53.1, 150.6, 143.9, 137.7, 137.7, 133.8, 131.0, 125.9, 125.5, 125.03, 124.04, 122.2, 118.4,
16.0, 115.8, 107.9, 56.2, 55.9, 49.9; ESI-MS (m/z) = 545.1 (M + 1); anal. calcd (%) for
C H F N O S : C, 55.14; H, 3.52; N, 10.29; S, 11.78. Found: C, 55.10; H, 3.56; N, 10.22;
25
19
3
4
3
2
S, 11.80.
4
1
2
1
7
.2.3.19.
2-(2-imino-5-(thiophen-2-yl)-1,3,4-thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-
−
1
0-yl) ethanone (5s): Light brown solid. Yield: 0.405 g; FTIR (ATR, cm ): 3298, 3061,
933, 1688, 1588, 1477, 1459, 1427, 1392, 1357, 1300, 1279, 1254, 1177, 1126, 1080, 1051,
1
030, 946, 897, 840, 757, 700; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.33 (br s, 1H, NH),
6
.72 (s, 2H, Ar-H), 7.68 (dd, J = 5.1, 1.1 Hz, 1H, Ar-H), 7.57 (d, J = 7.7 Hz, 2H, Ar-H), 7.39
(dd, J = 10.9, 4.4 Hz, 2H, Ar-H), 7.32 (dd, J = 8.4, 4.6 Hz, 3H, Ar-H), 7.11 (dd, J = 5.1, 3.7
1
3
Hz, 1H, Ar-H), 4.84 (s, 2H); C NMR (100 MHz, DMSO-d ) δ (ppm): 165.9, 137.9, 129.0,
6
1
4
28.9, 128.7, 128.5, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5,127.4, 49.7; ESI-MS (m/z) =
23.1 (M + 1); anal. calcd (%) for C H N OS : C, 56.85; H, 3.34; N, 13.26; S, 22.77.
2
0
14
4
3
Found: C, 56.94; H, 3.36; N, 13.24; S, 22.69.
4
.2.3.20.
1-(2-chloro-10H-phenothiazin-10-yl)-2-(2-imino-5-(thiophen-2-yl)-1,3,4-
−
1
thiadiazol-3(2H)-yl) ethanone (5t): Off-white solid. Yield: 0.443 g; FTIR (ATR, cm ): 3317,
3
8
7
066, 1692, 1588, 1456, 1428, 1395, 1357, 1279, 1243, 1175, 1129, 1092, 1052, 947, 917,
1
76, 841, 811, 765, 742, 701; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.31 (br s, 1H, NH),
6
.82 (s, 1H, Ar-H), 7.79 (d, J = 6.4 Hz, 2H, Ar-H), 7.68 – 7.64 (m, 2H, Ar-H), 7.62 – 7.56
(m, 2H, Ar-H), 7.33 (d, J = 6.4 Hz, 1H, Ar-H), 7.16 (t, J = 5.4 Hz, 2H), 5.03 (s, 1H, CH),
1
3
4
1
1
2
.82 (s, 1H, CH); C NMR (100 MHz, DMSO-d ) δ (ppm): 166.2, 148.3, 145.8, 141.6,
6
37.7, 134.5, 133.5, 130.0, 129.3, 128.3, 127.6, 127.4, 127.2, 125.7, 125.1, 124.4, 122.2,
21.2, 50.7; ESI-MS (m/z) = 456.9 (M + 1); anal. calcd (%) for C H ClN OS : C, 52.56; H,
2
0
13
4
3
.87; N, 12.26; S, 21.05. Found: C, 52.63; H, 2.88; N, 12.30; S, 20.98.
1
6

ACCEPTED MANUSCRIPT
4
.2.3.21.
2-(2-imino-5-(thiophen-2-yl)-1,3,4-thiadiazol-3(2H)-yl)-1-(2-(trifluoromethyl)
-10H-phenothiazin-10-yl)ethanone (5u): Off-white solid. Yield: 0.465 g, 87 %; m.p: 108-
°
−1
1
09 C; FTIR (ATR, cm ): 3321, 3075, 2936, 1693, 1591, 1486, 1465, 1417, 1358, 1326,
1
1
298, 1245, 1166, 1120, 1084, 980, 947, 922, 880, 841, 825, 754, 738, 702, 634, 618; H
NMR (400 MHz, DMSO-d ) δ (ppm): 8.31 (br s, 1H, NH), 8.04 (s, 1H, Ar-H), 7.79 (d, J =
6
8
3
=
.2 Hz, 2H, Ar-H), 7.70 – 7.64 (m, 2H, Ar-H), 7.63 – 7.57 (m, 1H, Ar-H), 7.45 (dd, J = 11.5,
.8 Hz, 1H, Ar-H), 7.37 (t, J = 7.1 Hz, 1H, Ar-H), 7.31 (d, J = 2.7 Hz, 1H, Ar-H), 7.11 (dd, J
1
3
5.0, 3.7 Hz, 1H, Ar-H), 5.02 (s, 1H), 4.75 (s, 1H); C NMR (100 MHz, DMSO-d ) δ
6
(
ppm): 167.1, 148.3, 146.8, 143.9, 137.7, 133.4, 130.9, 128.4, 127.6, 127.4, 127.1, 125.7,
25.1, 124.8, 122.2, 116.02, 50.7; ESI-MS (m/z) = 491.1 (M + 1); anal. calcd (%) for
C H F N OS : C, 51.42; H, 2.67; N, 11.42; S, 19.61. Found: C, 51.50; H, 2.63; N, 11.42; S,
1
21
13
3
4
3
1
9.62.
Acknowledgements
Authors are thankful to NITK, Surathkal for providing the research facilities. We
thank Dr. Reddy’s institute of life sciences, Central University of Hyderabad for providing
NMR and mass spectral analysis and Dr. K. G. Bhat, Maratha Mandal’s Dental College,
Hospital and Research Centre, Belgaum, India, for providing the facility for biological
screening.
1
7

ACCEPTED MANUSCRIPT
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List of figures and tables
Figure 1. Representative phenothiazine and 1,3,4-thiadiazole based antitubercular agents.
Figure 2. Antitubercular activity of 5a-u against Mtb H Rv (INH: Isoniazid; INN:
37
ciprofloxacin; EMB: Ethambutol; STM: Streptomycin).
Figure 3. The structure and MIC value against Mtb H Rv of the most active phenothiazine-
3
7
thiadiazole derivatives.
Figure 4. In vitro cytotoxicity (IC ) and selectivity index (SI) of the active compounds
5
0
against Vero cells. (SI = IC against Vero cells/MIC against M. tuberculosis).
5
0
Figure 5. Three dimensional docking pose showing the interaction of some active antiTB
compounds (5g, 5l, 5n and 5o) with the target enzymes, InhA and CP121 (a) 5g
with InhA; (b) 5l with InhA; (c) 5n with InhA; (d) 5g with CP121; (e) 5l with
CP121; (f) 5o with CP121.
Scheme 1. Synthesis of 1-(2-imino-1,3,4-thiadiazol-3(2H)-yl)-2-(10H-phenothiazin-10-yl)
ethanone derivatives (5a-u). Reagents and conditions: (a) toulene, reflux, 6 h;
(b) Acetyl chloride (3a), 0 °C - RT, 3 h; (c) Substituted aromatic carboxylic acid
(
3b-3g), POCl , 75 °C, 30 min; (d) Ethanol, 80-85 °C, 17h.
3
Scheme 2. Plausible reaction mechanism for the formation of target derivatives (5a-u).
Table 1. Physical properties and structural features of target compounds (5a-u).
Table 2. Antibacterial activity of target compounds (5a-u) against Staphylococcus aureus,
Pseudomonas aeruginosa and Escherichia coli.
2
2

ACCEPTED MANUSCRIPT
Table 1. Physical properties and structural features of target compounds (5a-u).
1
2
Entry Product
R
R
Yield (%) m.p (°C)
1
2
3
4
5
5a
5b
5c
5d
5e
H
Cl
CH3
CH3
CH3
82
85
88
88
90
184-185
167-168
203-204
219-220
180-181
CF3
H
4-Cl C H
6
4
4
4
Cl
4-Cl C H
6
6
7
5f
5g
CF3
H
4-Cl C H
92
85
150-151
189-190
6
4-CH C H
3 6
4
4
4
8
9
5h
5i
Cl
CF3
H
4-CH C H
88
91
92
95
160-161
175-176
161-162
120-121
3
6
4-CH C H
3
6
1
0
5j
4-F C H
6
4
4
4
1
1
5k
Cl
4-F C H
6
1
1
2
3
5l
5m
CF3
H
4-F C H
88
82
118-119
210-211
6
CH (CH )
3
2 2
14
15
16
17
18
19
20
21
5n
5o
5p
5q
5r
Cl
CF3
H
CH (CH )
88
89
79
81
85
88
89
87
150-151
135-136
105-106
110-111
106-107
110-111
109-110
108-109
3
2 2
CH (CH )
3
2 2
3,4-diOMe C H
6
3
3
3
Cl 3,4-diOMe C H
6
CF3 3,4-diOMe C H
6
5s
5t
H
Cl
2-Thienyl
2-Thienyl
2-Thienyl
5u
CF3
2
3

ACCEPTED MANUSCRIPT
Table 2. Antibacterial activity of target compounds (5a-u) against Staphylococcus aureus,
Pseudomonas aeruginosa and Escherichia coli.
Zone of inhibition in mm; Mean±SD
Escherichia
Coli
Staphylococcus
aureus
Pseudomonas
Aeruginosa
Compounds
Cocn. in
µg/ml
7
5
50
75
50
75
50
5
a
25±0.1
16±0.2
20±0.1
10±0.2
15±0.4
12±0.1
27±0.3
09±0.3
13±0.4
11±0.1
12±0.4
11±0.1
14±0.4
12±0.1
09±0.4
16±0.3
08±0.1
20±0.4
14±0.3
15±0.1
08±0.2
12±0.4
11±0.2
24±0.2
07±0.4
11±0.4
08±0.2
07±0.2
09±0.4
11±0.2
08±0.4
06±0.3
13±0.2
04±0.2
26±0.2
13±0.3
17±0.1
09±0.3
17±0.2
13±0.1
21±0.2
12±0.3
11±0.1
08±0.2
09±0.1
10±0.4
16±0.1
10±0.3
11±0.4
21±0.1
08±0.2
22±0.3
10±0.3
14±0.2
07±0.3
15±0.1
11±0.2
16±0.2
10±0.1
09±0.2
05±0.3
05±0.3
07±0.2
12±0.1
08±0.2
07±0.2
18±0.3
06±0.2
25±0.4
13±0.2
15±0.1
10±0.2
20±0.3
14±0.2
11±0.1
07±0.2
09±0.1
12±0.2
10±0.1
13±0.2
15±0.3
12±0.2
07±0.3
25±0.1
06±0.1
20±0.1
09±0.4
12±0.1
07±0.4
15±0.1
09±0.2
09±0.2
04±0.1
06±0.2
08±0.2
07±0.1
07±0.3
11±0.1
10±0.2
03±0.1
20±0.4
04±0.2
5
b
5
c
5
d
5
5
5
e
f
g
5
h
5
5
i
j
5
k
5
l
5
m
5
n
5
o
p
q
5
5
5
r
10±0.2
08±0.1
07±0.1
04±0.1
09±0.2
06±0.1
5
5
s
t
07±0.3
08±0.1
10±0.2
00
06±0.2
04±0.2
08±0.1
00
−
−
−
−
04±0.2
11±0.2
12±0.1
00
02±0.2
07±0.1
08±0.3
00
5
u
08±0.1
00
26±0.1
05±0.1
00
21±0.2
Control
INN
32±0.2
27±0.2
21±0.2
18±0.1
INN: Ciprofloxacin (anti-bacterial standard); −: inhibition not detected; control: DMSO
2
4

ACCEPTED MANUSCRIPT
Figure 1.
Figure 2.
2
5

ACCEPTED MANUSCRIPT
Figure 3.
Figure 4.
2
6

ACCEPTED MANUSCRIPT
Figure 5.
Scheme 1
2
7

ACCEPTED MANUSCRIPT
Scheme 2.
2
8