Molecular hybridization approach for phenothiazine incorporated 1,2,3-triazole hybrids as promising antimicrobial agents: Design, synthesis, molecular docking and in silico ADME studies

Article abstract of DOI:10.1016/j.ejmech.2019.02.010

The objective of the current study is to synthesize a library consisting of four sets of phenothiazine incorporated 1,2,3-triazole compounds using molecular hybridization approach. In total, 36 new hybrid molecules were synthesized and screened for in vitro growth inhibition activity against Mycobacterium tuberculosis H37Rv strain (ATCC-27294). Among the tested compounds, nineteen compounds exhibited significant activity with MIC value 1.6 μg/mL, which is twofold higher than the MIC value of standard first-line TB drug Pyrazinamide. In addition, all these compounds are proved to be non-toxic (with selective index > 40) against VERO cell lines. However, these compounds did not inhibit significantly the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains: the activity profile is similar to that observed for standard anti-TB drugs (isoniazid and pyrazinamide), indicating the specificity of these compounds towards the Mycobacterium tuberculosis strain. Also, we report the molecular docking studies against two target enzymes (Inh A and CYP121) to further validate the antitubercular potency of these molecules. Furthermore, prediction of in silico-ADME and pharmacokinetic parameters indicated that these compounds have good oral bioavailability. The results suggest that these phenothiazine incorporated 1,2,3-triazole compounds are a promising class of molecular entities for the development of new antitubercular leads.

Full text of DOI:10.1016/j.ejmech.2019.02.010

Accepted Manuscript
Molecular hybridization approach for phenothiazine incorporated 1,2,3-triazole
hybrids as promising antimicrobial agents: Design, synthesis, molecular docking and
in silico ADME studies
Rajkumar Reddyrajula, Udayakumar Dalimba, S. Madan Kumar
PII:
S0223-5234(19)30120-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.010
EJMECH 11102
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 November 2018
Revised Date: 11 January 2019
Accepted Date: 3 February 2019
Please cite this article as: R. Reddyrajula, U. Dalimba, S. Madan Kumar, Molecular hybridization
approach for phenothiazine incorporated 1,2,3-triazole hybrids as promising antimicrobial agents:
Design, synthesis, molecular docking and in silico ADME studies, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.010.
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ACCEPTED MANUSCRIPT
MOLECULAR HYBRIDIZATION APPROACH FOR
PHENOTHIAZINE INCORPORATED 1,2,3-TRIAZOLE HYBRIDS AS
PROMISING ANTIMICROBIAL AGENTS : DESIGN, SYNTHESIS,
MOLECULAR DOCKING AND IN SILICO ADME STUDIES
a
a,*
b
Rajkumar Reddyrajula , Udayakumar Dalimba , Madan Kumar S
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
a
Karnataka, Surathkal, Srinivasanagar, Mangalore-575025, India.
b
DST-PURSE LAB, Mangalore University, Mangalagangotri 574199, India.
*
Corresponding author email: udayaravi80@gmail.com, udayakumar@nitk.ac.in
Graphical abstract

ACCEPTED MANUSCRIPT
MOLECULAR HYBRIDIZATION APPROACH FOR PHENOTHIAZINE
INCORPORATED 1,2,3-TRIAZOLE HYBRIDS AS PROMISING
ANTIMICROBIAL AGENTS : DESIGN, SYNTHESIS, MOLECULAR
DOCKING AND IN SILICO ADME STUDIES
a
a,*
b
Rajkumar Reddyrajula , Udayakumar Dalimba , Madan Kumar S
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
a
Karnataka, Surathkal, Srinivasanagar, Mangalore-575025, India.
b
DST-PURSE LAB, Mangalore University, Mangalagangotri 574199, India.
*
Corresponding author email: udayaravi80@gmail.com, udayakumar@nitk.ac.in
Abstract
The objective of the current study is to synthesize a library consisting of four sets of
phenothiazine incorporated 1,2,3-triazole compounds using molecular hybridization approach. In
total, 36 new hybrid molecules were synthesized and screened for in vitro growth inhibition
activity against Mycobacterium tuberculosis H37Rv strain (ATCC-27294). Among the tested
compounds, nineteen compounds exhibited significant activity with MIC value 1.6 µg/mL,
which is twofold higher than the MIC value of standard first-line TB drug Pyrazinamide. In
addition, all these compounds are proved to be non-toxic (with selective index > 40) against
VERO cell lines. However, these compounds did not inhibit significantly the growth of
Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains: the activity
profile is similar to that observed for standard anti-TB drugs (isoniazid and pyrazinamide),
indicating the specificity of these compounds towards the Mycobacterium tuberculosis strain.
Also, we report the molecular docking studies against two target enzymes (Inh A and CYP121)
to further validate the antitubercular potency of these molecules. Furthermore, prediction of in
silico-ADME and pharmacokinetic parameters indicated that these compounds have good oral
bioavailability. The results suggest that these phenothiazine incorporated 1,2,3-triazole
compounds are a promising class of molecular entities for the development of new antitubercular
leads.
1

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Keywords: Molecular hybridization, Phenothiazine, 1,2,3-triazole, Mycobacterium tuberculosis,
Molecular docking.
1
. Introduction
Mycobacterium tuberculosis (M.tb) is an airborne pathogen and the causative agent of
Tuberculosis (TB). TB is one of the ancient diseases but continues to be responsible for the
mortality of about one billion people worldwide for the last two centuries. According to statistics
of WHO 2017 [1] report, more than 10 million new TB cases noticed and 1.8 million people
died, including 0.4 million co-infected HIV cases. The currently recommended treatment for TB
employs a four-drug regimen includes isoniazid, rifampin, pyrazinamide and ethambutol for the
first two months and isoniazid and rifampin for further four months. Further, the traditional anti-
TB drugs have limited efficacy against the new forms of TB; MDR-TB (multidrug resistance
tuberculosis) is resistant to two first-line TB drugs isoniazid and rifampin [2], XDR-TB
(extensively drug-resistant tuberculosis) is additionally resistant to the second line TB drugs [3]
and totally drug drug-resistant tuberculosis (TDR-TB) is resistance to broad range of first-line
and second-line drugs [4]. Over the last 40 years only a few antitubercular drugs have either
clinically approved or now in advanced clinical trials [5]. Bedaquiline [6] (TMC207), a diaryl
quinoline-based molecule, has been approved by the US-FDA and EMA (European Medicines
Agency) recently to treat pulmonary MDR-TB in adults [7]. Similarly, delamanid [8] (OPC-
6
7683), a nitro dihydro imidazole derivative received approval by the EMA to treat the
pulmonary MDR-TB in adults [9]. Diamine analogue [10] (SQ-109), Pretomanid [11] a
nitroimidazole derivative (PA-8240), and salicylic acid-1,2,3-triazolyl acetamide analogue (I-
A09) are other represents, which are under clinical trial. However, bedaquiline displayed toxic
side-effects such as cardiac arrhythmias, which led to significant death rates in clinical testing
[12]. Hence, there is an emergent need to address these challenges and to develop novel safe and
fast acting TB drugs. In this direction, various design strategies are being conformed to identify
potent chemical entities. Molecular hybridization is a powerful tool in drug design, which
involves the hybridization of two active pharmacophores of either similar bioactive substances or
different bioactive substances into a single molecular framework. It is being widely employed
because of the fact that many such hybrid derivatives possessed improved efficiency and safer
toxicity profile, when compared to the parent compounds [13]. In view of this, several
2

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pharmacologically active structural units, mostly heterocyclic moieties are being explored to
identify novel lead molecules.
Phenothiazines are well known over a hundred years for their in vitro antitubercular
activity against M.tb [14]. Few of the phenothiazine derivatives such as chlorpromazine,
thioridazine, methdilazine, and trifluoperazine (Figure 1) which are effective in treating
neurodegenerative disorders, also possess effective inhibition activity against M. tuberculosis
[15]. Example, thioridazine accelerated the chemotherapeutic activity against the in vitro latent
M.tb bacilli, when it is used as supportive drug along with isoniazid or rifampicin (>1 log CFU
reduction) [16]. Also it is being used as a front-line drug in combination therapy along with
Moxifloxacin and Linezolid for the treatment of M.tb and XDR-TB [17]. One of the other
derivatives, chlorpromazine is effective against replicating M.tb strain in cultured normal human
macrophage model and it is found to act in synergy with M.tb susceptible to primary drugs
isoniazid and rifampicin [18]. Interestingly, phenothiazines are known to have a specific
mechanism of action, inhibits the type II NADH dehydrogenase of M.tb [19,20]. Type II NADH
dehydrogenase is a key enzyme, which plays a vital role in the metabolic transition of M.tb to
dormant state. Hence, phenothiazine analogs may be effective in preventing the transition of
M.tb from latent stage to non-replicating stage. Additionally, a few recent reports demonstrated
the promising antitubercular activity of phenothiazines (Figure 2). For instance, a series of novel
pyrazolo[3,4-d]pyrimidine derivatives containing a 4,5-dihydro-1H-phenothiazine (I) exhibited
significant antitubercular activity against M.tb H37Rv [21]. Ramprasad et al. synthesized a set
of phenothiazine and 1,3,4-thiadiazole hybrid derivatives using the molecular hybridization
approach and among these, compounds 1-(2-chloro-10H-phenothiazin-10-yl)-2-(2-imino-5-(4-
propylphenyl)-1,3,4-thiadiazol-3(2H)-yl)ethanone(II)
and
2-(2-imino-5-(p-tolyl)-1,3,4-
thiadiazol-3(2H)-yl)-1-(10H-phenothiazin-10-yl)ethanone (III) exhibited excellent antitubercular
activity with MIC of 0.8 µg/mL [22]. Further, several literatures demonstrated the promising
antitubercular activity of N-substituted phenothiazine derivatives [23–25].
Over the past few decades, 1,2,3-triazoles have been an important class of nitrogen-based
heterocyclic compounds because of their intriguing pharmacological and biological applications
such as anti-cancer [26], anti-tubercular [27], antibacterial [28], anti-fungal [29], anti-HIV [30],
and Src-kinase inhibition [31] activities. It is quite interesting that the triazole ring owns some
favorable properties like modest dipole moment, the capability of hydrogen bond formation,
3

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stability and rigidity, which might be responsible for their enhanced pharmacological activities
[
32]. Moreover, 1,2,3-triazole derivatives exhibit a promising antitubercular activity profile
Figure 2). For example, dibenzo[b,d]thiophene-1,2,3-triazole derivatives (IV) [33] and 4-
(
substituted N-phenyl-1,2,3-triazole derivatives inhibit the growth of M.tb H37Rv. Among them,
E)-N-[(1-aryl)-1H-1,2,3-triazole-4-yl)methylene]isonicotinoyl hydrazides (V) displayed
excellent activity with MIC values ranging from 2.5 to 0.62 µg/mL [34]. Further, [1,1'-biphenyl]-
-yloxy incorporated 1H-1,2,3-triazole analogs (VI) exhibited significant anti-tubercular activity
35]. Recently, benzofuran salicylic acid containing 1,2,3-triazolyl acetamide I-A09 (VII) has
(
4
[
emerged as the promising antitubercular drug, which is under clinical trial [36]. In addition, a
library of novel 2-(trifluoromethyl) phenothiazine-1,2,3-triazole hybrids (VIII) was developed
through molecular hybridization approch by Addla et al and three of these compounds exhibited
promising MIC value (6.25 µg/mL) against M.tb H37Rv strain [37]. In view of these literature
reports on the promising antitubercular activity of phenothiazines and 1,2,3-triazole derivatives
we designed and synthesized phenothiazine-1,2,3-triazole hybrids by incorporation of these two
molecular entities in to a single framework, in which the 1,2,3-triazole fragment was attached to
phenothiazine through different linkers as represented in Figure 3.
2
2
. Results and discussion
.1 Chemistry
The synthesis of phenothiazine incorporated 1,2,3-triazole-based compounds 6a-g, 9a-g,
1a-h and 14a-n was achieved through efficient and versatile synthetic routes from
1
commercially available starting materials as summarized in schemes 1-4. The general synthetic
strategy for 3-(((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10-methyl-10H-phenothiazine
derivatives (6a-g), involves the Huisgen 1,3-dipolar cycloaddition reaction (click reaction) in
which 10-methyl-3-((prop-2-yn-1-yloxy)methyl)-10H-phenothiazine intermediate (5) was treated
with diversely substituted aryl bromides in presence of sodium azide, sodium ascorbate and
copper sulphate as catalyst (Scheme 1). In the first step, commercially available phenothiazine
was alkylated with methyl iodide using sodium hydride to afford 10-methyl-10H-phenothiazine
(2). Intermediate, 10-methyl-10H-phenothiazine-3-carbaldehyde (3) was synthesized via
Vilsmeier-Haack formylation of compound 2 with DMF and phosphorous oxychloride.
Reduction of the phenothiazine aldehyde (3) was carried out by using NaBH to yield (10-
4
4

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methyl-10H-phenothiazin-3-yl)methanol (4). Then O-alkylation of compound 4 with propargyl
bromide resulted the intermediate 5.
In order to investigate the effect of replacement of the ether linker in 6a-g by an oxime
linker on the antitubercular activity of the compounds, we have synthesized the oxime analogs
9
a-g (Scheme 2). The key intermediate 10-methyl-10H-phenothiazine-3-carbaldehyde oxime (7)
was synthesized by the condensation of compound 3 with hydroxylamine hydrochloride. The
oxime derivative (7) was then propargylated and the propargyl derivative (8) was subjected to
Huisgen 1,3-dipolar cycloaddition reaction with different aryl bromides to get the target oxime
analogs 9a-g. In the third series, 1,2,3-triazole unit is linked to core phenothiazine ring via the
ring nitrogen of later and N-substituted phenothiazine-1,2,3-triazole hybrid derivatives (11a-h)
were synthesized according to the synthetic route given in Scheme 3. The key intermediate 10-
(prop-2-yn-1-yl)-10H-phenothiazine (10) was prepared in good yield by treating phenothiazine
with propargyl bromide and sodium hydride. Huisgen 1,3-dipolar cycloaddition reaction of
compound 10 with a series of benzyl bromides afforded the target molecules 11a-h. The
synthetic strategy developed for the synthesis of a final set of analogs 14a-n is outlined in
Scheme 4. These molecules are designed by the molecular hybridization of N-substituted
phenothiazine-1,2,3-triazole skeleton with some of the anti-TB agents such as isoniazid,
pyrazine-2-carbohydrazide and nicotinohydrazide etc. Synthesis of 4-((4-((3-formyl-10H-
phenothiazin-10-yl) methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile derivatives 12 and 13 was
achieved by the Vilsmeier-Haack formylation of compounds 11e and 11h respectively.
Intermediates 12 and 13 were then condensed with differently substituted aromatic hydrazides to
yield
(E)-N'-((10-((1-(4-cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
yl)methylene) hydrazide derivatives (14a-n).
The chemical structure of all the synthesized compounds was confirmed by elemental
1
13
1
analysis, mass and NMR ( H and C) spectral analysis. The H NMR spectrum of compound 3
displayed a singlet at δ 9.81 ppm corresponding to the aldehyde hydrogen, which confirmed the
formylation of compound 2. The key intermediate 7 displayed two singlet signals at δ 11.34 and
1
8
.45 ppm corresponding to the oxime proton (N=OH) and proton of CH=N- respectively in H
1
NMR spectrum. The H NMR spectrum of final compounds (6a-g, 9a-g) exhibited distinct
singlet signals at around δ 8.10-7.43 ppm due to triazole-CH proton, δ 5.80-5.51 ppm due to
methylene (CH
2
) protons attached to the triazole, δ 4.56-4.45 ppm due to bridge methylene(–O-
5

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CH
2
3
–) protons attached to the triazole, and δ 3.37-3.18 due to methyl (N-CH ) protons. In
addition, compounds 6a-g displayed a singlet signal at around δ 4.74-4.61 ppm, for bridge
methylene (-CH -O-) protons attached to the phenothiazine ring whereas compounds 9a-g
2
displayed a singlet signal at around δ 8.19-8.24 ppm, for oxime (CH=N-) proton. Further, these
1
3
findings were confirmed by respective C NMR spectra of all the compounds. For instance, the
1
3
C NMR spectrum of 11c showed all the characteristic peaks according to its molecular
structure: the two methylene carbons appeared at δ 47.7 and δ 45.0 ppm. The most prominent
carbon signals observed at δ 161.5 and δ 123.9 ppm accounted for aromatic carbons carrying
flouro group and triazole (CH) ring respectively. The mass spectrum of all the compounds
showed their molecular ion peaks, corresponds to M+1 peak of the respective molecules and is in
agreement with their molecular formula. For example, The ESI mass spectra of compounds 6d,
+
9
b, 11b and 14a exhibited [M+H] peaks at m/z 415.2, 473.3, 416.1 and 543.1 which are in
agreement with their molecular formula C H N OS, C H N O S, C H N O S and
2
4
22
4
24 20
6
3
22 17
5
2
30 22 8
C H N OS respectively. The three dimensional structure of final compound 9c was confirmed
by single crystal XRD study (Figure 4) and the crystal data is given in Table 1. The structural
characterization data of all the synthesized compounds is presented in the experimental part and
ESI.
2
.2 In vitro antitubercular activity
The in vitro antimycobacterial screening of target compounds 6a-g, 9a-g, 11a-h and 14a-
n at the minimum inhibitory concentration (MIC) was carried out using MABA method against
the M.tb H37Rv (ATCC No- 27294) strain [38]. Pyrazinamide, Ciprofloxacin and Streptomycin
were used as the reference drugs. Initially, we designed, synthesized, and evaluated various
phenothiazine-1,2,3-triazole hybrids 6a-g bearing ether linker to probe the SAR exploration of
the 1,2,3-triazole region. It is noteworthy that the presence of lipophilic groups such as fluorine
in compounds 6c and 6g or the CN in compound 6e, increases the inhibition activity against M.tb
H37Rv, with the MIC of 12.5 µg/mL (Table 2). Replacement of the ether linker with oxime
linker resulted in no notable enhancement in the activity of compounds (9a-g). In fact, the
introduction of oxime linker led to a loss of activity in compounds 9c and 9g (MIC of 25 µg/mL)
in comparison with the activity of their ether analogs 6c and 6g (MIC of 12.5 µg/mL). In order to
further explore SAR of these hybrid derivatives, we have designed another set of molecules
(11a-h) in which the 1,2,3-triazole residue is attached to the N-atom of the phenothiazine ring.
6

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Interestingly, these compounds 11a-g exhibited superior activity compared to their ether and
oxime analogs 6a-g and 9a-g. Remarkably, four compounds namely 11b, 11c, 11e and 11f
showed potent anti-tubercular activity with MIC of 1.6 µg/mL (Figure 5) whereas compound 11g
exhibited moderate anti-tubercular activity with MIC of 3.12 µg/mL. It is evident from figure 5
2
that the nature of the substituent on 1,2,3-triazole (R ) rings affect the activity of the compounds.
2
The presence of electron-withdrawing groups (such as NO , F and CN) as well as electron
donating groups (-OCH ), enhanced the activity of these compounds. At this point, it was
3
encouraging to synthesize an analog compound 11h with a triflouromethyl group at position-2 of
the phenothiazine ring and to compare its anti-TB activity with the unsubstituted analog 11e.
However, no change in the activity was observed by the introduction of a triflouromethyl group;
both 11e and 11h exhibited similar activity profile (MIC = 1.6 µg/mL) which makes them
interesting lead compounds for further structural optimization. Finally, different hydrazine
derivatives (including Isoniazid, a standard drug for TB) were hybridized with the most potent
molecules (11e and 11h) to obtain compounds 14a-n. Surprisingly, we observed that such a
molecular hybridization did not change the activity of the molecules; instead all the compounds
(14a-n) retained the activity level at a MIC of 1.6 µg/mL(Table 3). Comparative study of the
antitubercular activity of the final compounds and the reference drugs indicated that among 36
compounds, nineteen compounds showed significant inhibition activity against M.tb H Rv strain
3
7
(MIC of 1.6 µg/mL) which is twofold and fourfold superior to the activity of standard first-line
TB drug Pyrazinamide (MIC of 3.125 µg/mL) and the second-line TB drugs (Ciprofloxacin and
Streptomycin) respectively.
2
.3 In vitro antibacterial activity
All the final compounds were screened for their in vitro antibacterial activity against
Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains using the agar
dilution method by measuring the minimum inhibitory concentration [39]. Ciprofloxacin, the
commercial antibiotic was taken as the standard drug for comparison. Most of the tested
compounds exhibited better inhibition activity against Staphylococcus aureus strain than the
other tested strains (Table 4). Three compounds 11e, 14i, and 14m showed significant activity
with MIC of 6.25 µg/mL whereas other eight compounds 6e, 9c, 9e, 9g, 11c, 11g, 14c and 14h
exhibited moderate activity with MIC of 12.5 µg/mL. It is noteworthy that all these active
7

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molecules (except 14c and 14h) carry an electron withdrawing substitution. Interestingly, all the
fluoro substituted analogs showed excellent inhibition activity which reveals the significant
contribution of the fluoro substituent towards the antibacterial activity of these compounds.
However, it can be seen from the MIC values that some of the molecules show superior
inhibitory activity against M.tb H37Rv in comparison to their activity against the other tested
bacterial strains.
2
.4 Cytotoxicity
Compounds with MIC of 1.6 µg/mL against M.tb were further evaluated for their in vitro
cytotoxicity using MTT assay [40] by measuring the concentration of compounds resulting in
0% inhibition (IC ) of a VERO cell line. The graphical representation of IC values of the
5
5
0
50
compounds is represented in Figure 6. Among the tested compounds 11c, 11f, 11h, 14c, 14d,
4f, 14g and 14n exhibited very less growth inhibition with 3.3, 7.57, 5.0, 5.13, 7.32, 7.14, 7.57
and 6.28% respectively. Selectivity Index (SI = IC against VERO cell / MIC against M.tb) of
1
50
all the potent molecules is > 40 (Table 5) which indicate that none of these compounds are toxic
to the normal cells and hence their anti-TB activity was not due to some general cytotoxicity.
2
.5 Molecular docking studies
Molecular docking studies are generally employed to investigate the binding energy and
to validate molecular mechanisms for ligands at the active site of a protein. To understand the
binding mode of the phenothiazine-1,2,3-triazole hybrids, all the title compounds were subjected
to molecular docking studies against selected enzymes viz. InhA and CYP 121 of M.tb using
Auto-dock Vina 1.1.2 software [41]. InhA is an NADH-dependent enoyl-acyl carrier protein
reductase, essential for cell viability of the bacteria and is endorsed as an effective anti-TB target
[42,43]. Further, isoniazid and ethionamide (anti-TB drugs) are predicted as the Inh A inhibitors.
Another target, CYP 121 enzyme also was selected for the docking studies based on the recent
literature on some triazole derivatives effectively binding with this target enzyme [44]. The
crystal structures of enzymes, Inh A in complex with 641 (PDB entry: 4TZK) and CYP 121
(PDB entry: 4KTF) in complex with 1TM was retrieved from the PDB (protein data bank) and
used as the template in molecular docking calculations.
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Docking study with the enoyl acyl carrier protein reductase (Inh A) of M. tuberculosis
H37Rv
The calculated binding energies for the studied 36 molecules are substantially high and
are in the range of −9.2 to −12.2 kcal/mol owing to the fact that the target compounds have
formed one or more hydrogen bonds with amino acid residues in the active pocket of Inh A
(
Figure 7). Tyr 158 is the crucial amino acid residue, which interacts with the long chain fatty
acyl substrates that are required for mycolic acid synthesis in mycobacteria [45,46]. Almost all
the active compounds showed hydrogen bond formation and pi-pi interaction with hydrophobic
residue Tyr 158, which were the key binding interactions of the original ligand as well with
enzyme Inh A (4TZK). In addition, few of them showed hydrogen bond formation with Lys 165
residue as well as key pi-pi stacking interaction with Phe 149 residue. One of the active
compounds 11g with the docking score (-9.9 kcal/mol) demonstrated substantial specific binding
as illustrated in Figure 8, where the nitrogen atom (N ) of triazole ring is hydrogen bonded to the
3
Lys 165 residue with a bond distance of 2.45 Å and nitrogen atom of the phenothiazine ring is
bonded with the Tyr 158 (2.74 Å) residue. Also, prominent pi-pi stacking interactions with the
Tyr 158 (3.9 Å) residue and additional van der Waals interactions with Phe 149, Met 147, Asp
1
48, Ala 191, Pro 193, Ile 195 and Thr 196 residues are the other binding modes observed for the
molecule. Whereas its oxime derivative 9g (which was moderately active against M.tb MIC 25
µg/mL) didn’t exhibit any interaction with Tyr 158 and Phe 149 residues which could be the
reason for the higher activity of compounds 11a-g than their oxime analogs. In the best docked
pose of 14c, three crucial hydrogen bonds are formed: the first between nitrogen of isoniazid ring
and Gln 100 (2.33 Å) residue; the second hydrogen bonding interaction was observed between
amide hydrogen and Met 91 (1.98 Å) residue and the third between nitrogen (N ) of triazole ring
3
and Tyr 158 (2.24 Å) residue. Additionally, a pi-pi interaction is seen between triazole ring and
Phe 149 residue. These interactions resulted a docking score of -11.7 kcal/mol for the molecule.
Docking study with the cytochrome P450 monooxygenase (CYP 121) of M. tuberculosis
H37Rv
The calculated binding energies for the target molecules are in the range of −9.6 to −11.7
kcal/mol. The binding sites of CYP 121 involved in these interactions are Gln 385, Arg 386, Phe
2
80, Gly 232, Val 228, Trp 182, Phe 168, Phe 161, Asn 85, Val 83, Leu 76, Asn 74, Leu 73, Arg
9

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7
2, Thr 65, and Met 62 residues which seemed to play important roles in the drug binding
through hydrogen bond formation. As shown in Figure 9, inhibitor 11b, with a binding energy of
10.3 kcal/mol forms three hydrogen bonds with Thr 65 (2.6 Å), Arg 72 (2.3Å) and Gln 385(2.4
Å), among which two bonds originate through the nitro group. ompounds 14a and 14c exhibited
-
C
binding energies of -11.2 and -11.4 kcal/mol, respectively, with two hydrogen bonding
interactions with Leu 73 and Val 228 (Figure 9). Compound 14d which exhibited the highest
docking score (-11.7 kcal/mol) interacted with Val 228 (2.54 Å) whereas compound 14f
interacted with Asn 74 (2.2 Å), Val 228 (2.64 Å) and Asp 281 (2.09 Å) with a binding energy of
-11.0 kcal/mol. Hence, the docking study further validates the activity profile observed in the in
vitro studies of active analogs.
2
.6 In silico ADME studies
In silico ADME properties (adsorption, distribution, metabolism and excretion), and
another ten pharmacokinetics properties viz. partition coefficient of n-octanol and water, aqua
solubility, polar surface area, number of rotatable bonds, number of hydrogen bond
donors/acceptors, caco-2 cell permeability, human serum albumin binding, blood/brain partition
co-efficient and human oral absorption of the 36 compounds were computed by using the
QikProp program. The results of these computations are summarized in Table 6. Estimation of
octanol-water partition coefficient (lipophilicity) is examined by QPlogPo/w and higher
QPlogPo/w value was related to poor absorption. It is evident from the Table 6 that the most
potent compounds 11b, 11c, 11e, 11f and 11h follow the Lipinski’s rule and display favorable
pharmacokinetic profiles for all descriptors expect QPlogPo/w value. Polar surface area (PSA),
which should not be >140 Å, is another key property linked to drug bioavailability and the values
for the target molecules are in the range of 34.649 – 80.821 Å. A noteworthy difference was
observed in the Caco-2 cell permeability parameter (QPPcaco) and the aqua solubility parameter
(QPlog S) of three different sets of molecules. The most potent compounds 11b, 11c, 11e, 11f
and 11h displayed higher permeability and aqua solubility values than the respective ether and
oxime linked analogs. Blood/Brain partition co-efficient (QPlogBB) value which is a measure of
the ability of a drug to cross the blood-brain barrier, is in the acceptable range for these target
molecules. Further, Human serum albumin binding co-efficient (QPlogKhsa) also is one of the
key factors and predicted values for the compounds are in the range of 0.788 – 1.071. The
1
0

ACCEPTED MANUSCRIPT
predicted values of human oral absorption are in the range of 91-100 %, which indicates the
possibility of higher oral bioavailability of the active compounds. It is worthwhile reveling that
the library of most active compounds (14a-n) falls into low QPlog S aqua solubility, QPPcaco
permeability and oral bioavailability. In summary, these in silico ADME prediction of the active
compounds may help in further development of new drug candidates with favorable oral
bioavailability.
3
. Conclusions
In conclusion, we have designed and synthesized four series of phenothiazine
incorporated 1,2,3-triazole hybrids through molecular hybridization method by hybridizing two
active pharmacophores, phenothiazine and 1,2,3-triazole. All the newly synthesized compounds
6
a-g, 9a-g, 11a-h and 14a-n were evaluated for in vitro growth inhibition activity against M.tb
H37Rv strain (ATCC-27294). Three compounds bearing ether linker (6c, 6e and 6g) showed
moderate antitubercular activity with MIC 12.5 µg /mL. Modification of the ether linker by an
oxime linker in compounds 6a-g did not affect significantly the activity profile of the oxime
analogs (9a-g). All the phenothiazine-N-substituted 1,2,3-triazole derivatives 11a-g exhibited
superior activity compared to their respective ether (6a-g) and oxime (9a-g) analogs. Four
compounds 11b, 11c, 11e and 11f showed potent anti-tubercular activity with MIC of 1.6 µg/mL,
which is twofold higher than the activity of standard first-line TB drug Pyrazinamide (MIC of
3
.125 µg/mL) and one of the other compound 11g showed significant activity with MIC of 3.12
µg/mL. Introduction of a triflouromethyl substituent at position-2 of the phenothiazine ring in
1e did not alter the activity profile of the molecule. Hybridization of TB-active pharmacophores
1
with active compounds 11e and 11h caused no further enhancement in the anti-TB activity of
hybrid derivatives. However, the molecular hybridization resulted a library of potent molecules;
all the 14 compounds (14a-n) exhibited MIC of 1.6 µg/mL. The cytotoxicity studies of the active
compounds (MIC= 1.6 µg/mL) revealed that these compounds are non-toxic to the normal
VERO cell lines. Further, compounds 11e, 14i, and 14m showed significant antibacterial activity
against all the tested bacterial strains. The SAR studies revealed that the electron withdrawing
groups such as NO , F and CN on phenyl ring attached to 1,2,3-triazole nucleus play a vital role
2
1
1

ACCEPTED MANUSCRIPT
in enhancing the pharmacological activities of the final compounds. These results were further
supported by the molecular docking studies; the potent molecules displayed stronger binding
affinity towards the target enzymes through multiple hydrogen bonds and pi-pi stacking
interactions. In-silico ADME studies predicts these molecules to exhibit good oral
bioavailability. Hence, the phenothiazine incorporated 1,2,3-triazole hybrids described here
could be promising lead compounds for the identification and development of novel anti-TB
drugs.
4
4
4
. Experimental section
.1 Synthesis
.1.1. 10-Methyl-10H-phenothiazine (2).
Phenothiazine 1 (5 g, 25.09 mmol) was taken in anhydrous N, N-dimethyl formamide (DMF) (50
mL) in a dry round bottom flask. Sodium hydride (2 g, 50.18 mmol) was added under nitrogen
condition at 0 °C and stirred for 30 minutes at room temperature. Methyl iodide (3.4 mL, 50.18
mmol) was added and continued the stirring for 8h. After that, the reaction mixture (monitored
by TLC) was quenched with ice cold water (150 mL). The separated solid was filtered off, dried
1
and used as such for the next step. White solid. Yield: 4.8 g, 89%; m.p: 105-106 °C; H NMR
(
CDCl , 400 MHz, δ in ppm): 7.262-7.160 (m, 3H, Ar-H), 7.141-6.822 (m, 5H, Ar-H), 3.495 (s,
3
1
3
3
H, CH
3
3
); C NMR (100 MHz, CDCl ) δ (ppm): 151.2, 129.3, 128.1, 124.9, 123.3, 115.8, 37.2;
+
ESI-MS (m/z) = 214.3 (M+H) ; calculated for C H NS; C, 73.20; H, 5.20; N, 6.57; S, 15.03.
13
11
Found: C, 73.18; H, 5.21; N, 6.57; S, 15.04.
.1.2. 10-Methyl-10H-phenothiazine-3-carbaldehyde (3).
Phosphorous oxychloride (POCl ) (3.5 mL, 37.50 mmol) was added drop wise to DMF (2.9 mL,
4
3
3
7.50 mmol) in RB flask under the inert condition at 0-5 °C. After the formation of the
Vilsmeier-Haack salt, a solution of 2 (4 g, 18.75 mmol) in DMF was added and continued the
stirring at 60 °C for about 4h. The reaction mixture was cooled to RT and poured into ice water
slowly. The separated solid was filtered off, dried and washed with an excess of cold water and
then purified by column chromatography with pet ether/ethyl acetate (9:1) system to give
1
compound 3. Yellow crystalline solid. Yield: 3.8g, 83.9%; m.p: 110-111 °C; H NMR (CDCl
3
,
1
2

ACCEPTED MANUSCRIPT
4
00 MHz, δ in ppm): 9.812 (s, 1H, -CHO), 7.681-7.656 (m, 1H, Ar-H), 7.615-7.610 (m, 1H, Ar-
H), 7.196-7.145 (m, 2H, Ar-H), 7.129-6.974 (m, 1H, Ar-H), 6.878-6.843 (m, 2H, Ar-H), 3.440
1
3
(
s, 3H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 191.4, 151.6, 145.3, 131.2, 130.7, 129.8,
3
3
+
1
28.5, 127.5, 124.6, 123.9, 122.7, 116.4, 114.1, 37.5; ESI-MS (m/z) = 242.3 (M+H) ; calculated
for C H NOS; C, 69.68; H, 4.59; N, 5.80; S, 13.29. Found: C, 69.66; H, 4.59; N, 5.73; S,
1
4
11
1
3.26.
4
.1.3. (10-Methyl-10H-phenothiazin-3-yl)methanol (4).
Intermediate 3 (3 g, 12.43 mmol) was taken in methanol (30 ml) to which NaBH (0.705 g, 18.64
4
°
mmol) was added in portions at 0 C under inert condition. Then the reaction was stirred for 4h.
After that, the reaction mixture (Monitored by TLC) was quenched with ice cold water. The solid
obtained was filtered under suction. The residue was washed with cold methanol, dried and
recrystallized from ethanol to get desired product 4 as white solid. Yield: 2.6 g, 89 %; m.p: 143-
o
1
1
44 C; H NMR (CDCl , 400 MHz, δ in ppm): 7.415-7.341 (m, 2H, Ar-H), 7.072-7.011 (m, 2H,
3
2 3
Ar-H), 6.714-6.598 (m, 3H, Ar-H), 4.914 (s, 2H, CH ), 3.845 (s, 1H, OH), 3.438 (s, 3H, CH );
1
3
C NMR (100 MHz, CDCl ) δ (ppm): 149.1, 146.3, 135.1, 129.8, 128.7, 128.2, 127.6, 125.2,
3
+
1
24.6, 122.2, 116.4, 115.3, 65.8, 37.3; ESI-MS (m/z) = 244.3 (M+H) ; calculated for
C H NOS; C, 69.11; H, 5.39; N, 5.76; S, 13.18. Found: C, 69.02; H, 5.41; N, 5.81; S, 13.07.
14
13
4
.1.4. 10-Methyl-3-((prop-2-yn-1-yloxy)methyl)-10H-phenothiazine (5).
The above procedure (synthesis of 2) was adopted to synthesize the intermediate 5 by alkylation
of compound 4 (2 g, 8.2 mmol) with propargyl bromide(1.24 ml, 16.43 mmol) in presence of
1
sodium hydride (0.657 g , 16.43 mmol). Brown solid. Yield: 1.9 g, 82%; H NMR (CDCl
3
, 400
MHz, δ in ppm): 7.193-7.128 (m, 4H, Ar-H), 6.951-6.911 (m, 1H, Ar-H), 6.826-6.773 (m, 2H,
Ar-H), 4.507 (s, 2H, CH ), 4.129-4.122 (d, 2H, J = 2.8 Hz, CH ), 3.374 (s, 3H, CH ), 2.469-
2
2
3
1
3
2
1
3
1
4
1
.457 (t, 1H, J = 4.8, 2.0 Hz, Acetylene CH); C NMR (100 MHz, CDCl
3
) δ (ppm): 149.3,
46.4, 131.4, 129.7, 128.8, 128.2, 127.9, 125.1, 124.7, 123.1, 116.7, 114.8, 79.7, 77.4, 74.5, 61.2,
+
7.4; ESI-MS (m/z) = 282.1 (M+H) ; calculated for C17
H15NOS; C, 72.57; H, 5.37; N, 4.98; S,
1.40. Found: C, 72.49; H, 5.40; N, 4.98; S, 11.41.
.1.5. General Procedure for synthesis of 3-(((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy) methyl)-
0-methyl-10H-phenothiazine derivatives (6a-g): To a mixture of compound 5 (1.0 mmol),
substituted benzyl bromide (1.0 mmol) and NaN (1.0 mmol) in 1:1 mixture of t-BuOH and
3
water (4 mL), sodium ascorbate (10 mol %) and copper (II) sulfate (5 mol %) were added
1
3

ACCEPTED MANUSCRIPT
consecutively and the reaction mixture was stirred for 24 h. The reaction mixture was diluted
with ice cold water (15 mL) once the starting material consumed completely (Monitored by
TLC) and extracted with ethyl acetate. The solvent was removed under vacuum and the residue
was purified by column chromatography with pet ether/ethyl acetate system to give compounds
6
a-g.
4
.1.5.1.
10-Methyl-3-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10H-
1
phenothiazine (6a). Brown solid. Yield: 83%, m.p: 145-146 °C; H NMR (CDCl , 400 MHz, δ in
3
ppm): 7.902-7.882 (d, J = 8.0 Hz, 2H, Ar-H), 7.697 (s, 1H, Traizole-CH), 7.341-7.321 (d, J = 8.0
Hz, 2H, Ar-H), 7.174-7.116 (m, 4H, Ar-H), 6.934-6.915 (t, J = 7.6 Hz, 1H, Ar-H), 6.812-6.766
(
t, J = 18.4 Hz, 8.0Hz, 2H, Ar-H), 5.799 (s, 2H, CH ), 4.696 (s, 2H, CH ), 4.505 (s, 2H, CH ),
2 2 2
1
3
3
1
1
3 3 3
.367 (s, 3H, CH ), 2.449 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 149.4, 146.5,
43.7, 136.8, 134.8, 130.1, 129.6, 129.1, 128.5, 127.9, 127.6, 127.3, 125.2, 124.8, 123.6, 122.9,
+
16.8, 114.9, 73.7, 70.4, 58.5, 37.6, 22.8; ESI-MS (m/z) = 429.1 (M+H) ; calculated for
C H N OS; C, 70.07; H, 5.64; N, 13.07; S, 7.48. Found: C, 70.06; H, 5.57; N, 13.12; S, 7.49.
25
24
4
4
.1.5.2.
10-Methyl-3-(((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10H-
1
phenothiazine (6b). Pale yellow solid. Yield: 86%; m.p: 176-177 °C; H NMR (CDCl
3
, 400
MHz, δ in ppm): 8.238-8.216 (d, J = 8.8 Hz, 2H, Ar-H), 7.503 (s, 1H, Traizole-CH), 7.407-7.385
(d, J = 8.8 Hz, 2H, Ar-H), 7.170-7.117 (m, 4H, Ar-H), 6.949-6.910 (m, 1H, Ar-H), 6.820-6.753
(
m, 2H, Ar-H), 5.619 (s, 2H, CH ), 4.638 (s, 2H, CH ), 4.491 (s, 2H, CH ), 3.363 (s, 3H, CH );
2
2
2
3
1
3
C NMR (100 MHz, CDCl ) δ (ppm): 149.7, 147.8, 146.6, 144.4, 143.8, 130.2, 129.7, 129.3,
3
1
28.6, 127.8, 127.4, 125.4, 124.9, 124.1, 123.7, 122.5, 116.9, 114.8, 73.7, 70.5, 58.6, 37.6; ESI-
+
MS (m/z) = 460.1 (M+H) ; calculated for C H N O S; C, 62.73; H, 4.61; N, 15.24; S, 6.98.
2
4
21
5
3
Found: C, 62.80; H, 4.66; N, 15.29; S, 6.82.
4
.1.5.3.
3-(((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10-methyl-10H-
1
phenothiazine (6c). Grey solid. Yield: 78%; m.p: 118-119 °C; H NMR (CDCl
3
, 400 MHz, δ in
ppm): 8.154-8.134 (m, 2H, Ar-H), 7.512 (s, 1H, traizole-CH), 7.425-7.391 (m, 3H, Ar-H), 7.051-
.015 (m, 3H, Ar-H), 6.912-6.895 (m, 1H, Ar-H), 6.789-6.545 (m, 2H, Ar-H), 5.626 (s, 2H,
7
13
CH ), 4.647 (s, 2H, CH ), 4.454 (s, 2H, CH ), 3.362 (s, 3H, CH ); C NMR (100 MHz, CDCl )
2
2
2
3
3
δ (ppm): 162.7, 149.6, 147.9, 144.5, 132.5, 130.1, 129.9, 129.1, 128.7, 127.9, 127.6, 126.1,
25.5, 125.1, 124.7, 123.8, 122.4, 118.4, 117.1, 114.9, 73.8, 70.5, 50.2, 37.7; ESI-MS (m/z) =
1
1
4

ACCEPTED MANUSCRIPT
+
4
6
33.51 (M+H) ; calculated for C24
6.53; H, 4.94; N, 12.98; S, 7.46.
4
H21FN OS; C, 66.65; H, 4.89; N, 12.95; S, 7.41 Found: C,
4
.1.5.4. 3-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10-methyl-10H-phenothiazine (6d).
1
Grey solid. Yield: 82%; m.p: 124-125 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.433 (s, 1H,
3
Triazole-CH), 7.377-7.360 (m, 3H, Ar-H), 7.280-7.274 (d, J = 2.4 Hz, 2H, Ar-H), 7.186-7.117
(m, 4H, Ar-H), 6.944-6.907 (t, J = 14.8 Hz, 7.2 Hz, 1H, Ar-H), 6.816-6.749 (m, 2H, Ar-H),
1
3
5
.507 (s, 2H, CH ), 4.608 (s, 2H, CH ), 4.471 (s, 2H, CH ), 3.361 (s, 3H, N-CH ); C NMR
2 2 2 3
3
(100 MHz, CDCl ) δ (ppm): 149.4, 146.7, 143.6, 135.5, 130.2, 129.7, 129.0, 128.6, 128.2, 127.8,
1
4
6
27.4, 126.7, 125.3, 124.7, 123.7, 122.9, 116.9, 114.8, 73.8, 70.7, 58.7, 37.5; ESI-MS (m/z) =
+
15.20 (M+H) ; calculated for C H N OS; C, 69.54; H, 5.35; N, 13.52; S, 7.74 Found: C,
24
22
4
9.64; H, 5.43; N, 13.51; S, 7.81.
4
.1.5.5.
4-((4-(((10-Methyl-10H-phenothiazin-3-yl)methoxy)methyl)-1H-1,2,3-triazol-1-yl)
1
methyl)benzonitrile (6e). Pale green solid. Yield: 91%; m.p: 125-126 °C; H NMR (CDCl , 400
3
MHz, δ in ppm): 8.187-8.167 (d, J = 8.0 Hz, 2H, Ar-H), 7.509 (s, 1H, Traizole-CH), 7.414-7.393
(d, J = 8.4 Hz, 2H, Ar-H), 7.154-7.102 (m, 4H, Ar-H), 6.927-6.889 (m, 1H, Ar-H), 6.802-6.734
(
m, 2H, Ar-H), 5.604 (s, 2H, CH ), 4.622 (s, 2H, CH ), 4.47 (s, 2H, CH ), 3.358 (s, 3H, CH );
2
2
2
3
1
3
C NMR (100 MHz, CDCl ) δ (ppm): 149.6, 147.8, 144.5, 141.7, 134.8, 131.5, 130.1, 129.8,
3
1
28.7, 127.9, 127.5, 125.3, 124.9, 123.8, 122.6, 119.7, 116.9, 114.7, 110.8, 73.8, 70.6, 58.5,
+
3
7.7; ESI-MS (m/z) = 440.20 (M+H) ; calculated for C H N OS; C, 68.32; H, 4.82; N, 15.93;
2
5
21
5
S, 7.30 Found: C, 68.60; H, 4.87; N, 15.86; S, 7.33.
4
.1.5.6.
3-(((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10-methyl-10H-
1
phenothiazine (6f). Pale yellow solid. Yield: 88%; m.p: 121-122 °C; H NMR (CDCl
3
, 400 MHz,
δ in ppm): 7.990-7.968 (m, 2H, Ar-H), 7.708 (s, 1H, Traizole-CH), 7.184-7.122 (m, 4H, Ar-H),
7
5
.011-6.989 (d, J = 8.8 Hz, 2H, Ar-H), 6.940-6.818 (m, 1H, Ar-H), 6.797-6.770 (m, 2H, Ar-H),
.782 (s, 2H, CH ), 4.700 (s, 2H, CH ), 4.508 (s, 2H, CH ), 3.904 (s, 3H, O-CH ), 3.369 (s, 3H,
2
2
2
3
13
CH ); C NMR (100 MHz, CDCl ) δ (ppm): 158.7, 149.6, 146.7, 143.8, 131.9, 131.2, 130.3,
3
3
1
29.4, 129.0, 128.6, 127.9, 125.1, 124.9, 123.7, 122.9, 116.7, 115.6, 114.9, 73.8, 70.5, 58.6, 57.6,
+
3
7.5; ESI-MS (m/z) = 445.40 (M+H) ; calculated for C H N O S; C, 67.54; H, 5.44; N, 12.60;
25
24
4
2
S, 7.21 Found: C, 67.52; H, 5.48; N, 12.55; S, 7.23.
1
5

ACCEPTED MANUSCRIPT
4
.1.5.7
3-(((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-10-methyl-10H-
1
phenothiazine (6g). Yellow solid. Yield: 80%; m.p: 119-120 °C; H NMR (CDCl , 400 MHz, δ
3
in ppm): 8.014-7.993 (m, 2H, Ar-H), 7.561 (s, 1H, Traizole-CH), 7.345-7.284 (m, 4H, Ar-H),
7
.181-7.160 (d, J = 8.4 Hz, 2H, Ar-H), 6.994-6.872 (m, 1H, Ar-H), 6.810-6.785 (m, 2H, Ar-H),
13
5
.764 (s, 2H, CH ), 4.714 (s, 2H, CH ), 4.491 (s, 2H, CH ), 3.357 (s, 3H, CH ); C NMR (100
2 2 2 3
3
MHz, CDCl ) δ (ppm): 161.4, 149.8, 147.8, 144.3, 133.1, 130.4, 129.8, 129.2, 128.6, 127.5,
1
4
6
26.4, 125.6, 124.8, 123.8, 122.3, 118.5, 117.2, 114.9, 73.9, 70.6, 58.4, 37.5; ESI-MS (m/z) =
+
33.1 (M+H) ; calculated for C H FN OS; C, 66.65; H, 4.89; N, 12.95; S, 7.41 Found: C,
2
4
21
4
6.53; H, 4.94; N, 12.98; S, 7.46.
4
.1.6. N-[(E)-(10-Methyl-10H-phenothiazin-3-yl)methylidene]hydroxylamine (7). To a solution
of 3 (2 g, 8.26 mmol) in a minimum amount of anhydrous ethanol in a dry RB, hydroxylamine
hydrochloride (684 mg, 9.92 mmol) and concentrated sulphuric acid (catalytic amount) were
added and refluxed at 80 °C for 1 hour. After that, the reaction mixture (monitored by TLC) was
quenched with ice cold water (150 mL). The separated solid was filtered off, dried, weighed and
1
used as such for the next step. Pale yellow solid. Yield: 1.65 g, 78%; H NMR (DMSO-d , 400
6
MHz, δ in ppm): 11.341 (s, 1H, N-OH), 8.451 (s, 1H, -CH=N), 7.673-7.612 (m, 1H, Ar-H),
7
.505-7.410 (m, 1H, Ar-H), 7.269-7.155 (m, 2H, Ar-H), 6.960-6.921 (m, 1H, Ar-H), 6.857-6.814
1
3
(
3 3
m, 2H, Ar-H), 3.41 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 149.3, 146.1, 144.5,
1
35.6, 129.1, 127.9, 127.5, 126.4, 124.9, 122.3, 120.7, 115.8, 113.5, 37.5; ESI-MS (m/z) = 257.3
+
(
M+H) ; calculated for C H N OS; C, 65.60; H, 4.72; N, 10.93; S, 12.51. Found: C, 65.50; H,
14
12
2
4
.75; N, 10.96; S, 12.47.
4
.1.7. (E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(prop-2-yn-1-yl)oxy]methanimine (8). The
above procedure (synthesis of 2) was adopted to synthesize the intermediate 8 by O-alkylation of
compound 7 (1 g, 3.9 mmol) with propargyl bromide (0.59 ml, 7.81 mmol) in presence of
1
sodium hydride (1.07 g, 7.81 mmol). Brown solid. Yield: 0.98 g, 85%; H NMR (DMSO-d
6
, 400
MHz, δ in ppm): 8.405 (s, 1H, -CH=N), 7.994-7.101 (m, 4H, Ar-H), 6.985-6.919 (m, 1H, Ar-H),
6
4
1
.870-6.791 (m, 2H, Ar-H), 4.289 (d, J = 2.4 Hz, 2H, CH ), 3.394 (s, 3H, CH ), 3.186 (t, 1H, J =
2 3
1
3
.4, 2.0 Hz, 1H, Acetylene-CH); C NMR (100 MHz, CDCl ) δ (ppm): 150.8, 146.2, 144.8,
3
35.3, 129.2, 128.1, 127.4, 126.9, 124.9, 122.4, 122.2, 115.7, 112.6, 89.2, 79.1, 61.1, 37.5; ESI-
1
6

ACCEPTED MANUSCRIPT
+
14 2
MS (m/z) = 295.2 (M+H) ; calculated for C17H N OS; C, 69.36; H, 4.79; N, 9.52; S, 10.89.
Found: C, 69.35; H, 4.81; N, 9.46; S, 10.88.
4
.1.8. General procedure for the synthesis of (E)-1-(10-methyl-10H-phenothiazin-3-yl)-N-[(1-
benzyl-1H-1,2,3-triazol-4-yl)methoxy]methanimine derivatives (9a-g). The above procedure
synthesis of 6a-g) was adopted to synthesize the compounds 9a-g by click reaction between
substituted benzyl bromides (1 mmol) and Intermediate 8 (1 mmol) in presence of sodium azide
1 mmol), sodium ascorbate (10 mol %) and copper (II) sulfate (5 mol %).
(
(
4
.1.8.1.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(4-methylbenzyl-1H-1,2,3-triazol-4-yl)
1
methoxy]methanimine (9a). Pale brown solid. Yield: 79%; m.p: 207-208 °C; H NMR (CDCl ,
3
4
00 MHz, δ in ppm): 8.210 (s, 1H, -CH=N), 7.926-7.891 (m, 2H, Ar-H), 7.729 (s, 1H, Triazole-
H), 7.392-7.280 (m, 2H, Ar-H), 7.208-7.164 (m, 4H, Ar-H), 6.987-6.862 (m, 1H, Ar-H), 6.821-
6
3
1
3
1
.745 (m, 2H, Ar-H), 5.714 (s, 2H, CH ), 4.558 (s, 2H, CH ), 3.282 (s, 3H, N-CH ), 2.412 (s,
2 2 3
13
3 3
H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 155.5, 150.3, 146.4, 144.8, 137.1, 135.3, 134.5,
31.9, 129.8, 129.3, 128.2, 127.8, 127.2, 126.0, 125.4, 125.2, 123.8, 123.1, 117.9, 68.2, 58.3,
+
7.3, 22.4; ESI-MS (m/z) = 442.51 (M+H) ; calculated for C H N OS; C, 68.00; H, 5.25; N,
2
5
23
5
5.86; S, 7.26 Found: C, 68.03; H, 5.25; N, 15.90; S, 7.21.
4
.1.8.2.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(4-nitrobenzyl-1H-1,2,3-triazol-4-yl)
1
methoxy]methanimine (9b). Brick red solid. Yield: 81%; m.p. 235-236 °C; H NMR (CDCl
3
, 400
MHz, δ in ppm): 8.191 (s, 1H, -CH=N), 8.109-7.968 (m, 2H, Ar-H), 7.712-7.652 (s, 2H, Ar-H),
.538-7.593 (m, 2H, Ar-H), 7.202-7.146 (m, 3H, Ar-H), 6.928-6.876 (m, 1H, Ar-H), 6.812-6.774
7
1
3
(
m, 2H, Ar-H), 5.697 (s, 2H, CH ), 4.495 (s, 2H, CH ), 3.291 (s, 3H, N-CH ); C NMR (100
2 2 3
MHz, CDCl ) δ (ppm): 155.3, 150.2, 146.2, 145.9, 144.7, 144.6, 135.2, 131.8, 129.8, 129.2,
3
1
4
6
28.2, 127.1, 126.0, 125.2, 125.1, 124.9, 123.7, 123.0, 117.9, 68.4, 58.4, 37.4; ESI-MS (m/z) =
+
73.30 (M+H) ; calculated for C H N O S; C, 61.00; H, 4.27; N, 17.79; S, 6.79 Found: C,
2
4
20
6
3
0.99; H, 4.28; N, 17.81; S, 6.80.
4
.1.8.3.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(2-flourobenzyl-1H-1,2,3-triazol-4-yl)
1
methoxy]methanimine (9c). Brown solid. Yield: 74%; m.p. 198-199 °C; H NMR (CDCl
3
, 400
MHz, δ in ppm): 8.189 (s, 1H, -CH=N), 8.104 -7.956 (m, 2H, Ar-H), 7.703-7.645 (s, 2H, Ar-H),
.581-7.523 (m, 2H, Ar-H), 7.209-7.134 (m, 3H, Ar-H), 6.912-6.857 (m, 1H, Ar-H), 6.801-6.767
7
1
3
(
m, 2H, Ar-H), 5.669 (s, 2H, CH ), 4.449 (s, 2H, CH ), 3.289 (s, 3H, N-CH ); C NMR (100
2 2 3
1
7

ACCEPTED MANUSCRIPT
MHz, CDCl
3
) δ (ppm): 162.7, 155.6, 150.4, 146.6, 144.9, 135.6, 132.3, 131.2, 129.4, 128.4,
1
27.9, 127.1, 126.4, 126.0, 125.7, 125.3, 125.3, 123.7, 123.1, 117.8, 116.7, 68.5, 50.4, 37.6;
+
ESI-MS (m/z) = 446.10 (M+H) ; calculated for C H FN OS ; C, 64.70; H, 4.52; N, 15.72; S,
24
20
5
7
.20. Found: C, 64.72; H, 4.54; N, 15.71; S, 7.19.
4
.1.8.4.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(1-benzyl-1H-1,2,3-triazol-4-yl)
1
methoxy]methanimine (9d). Grey solid. Yield: 79%; m.p. 214-215 °C; H NMR (CDCl
3
, 400
MHz, δ in ppm): 8.221 (s, 1H, -CH=N), 7.912-7.872 (m, 3H, Ar-H), 7.732 (s, 1H, Triazole-H),
.389-7.278 (m, 2H, Ar-H), 7.198-7.152 (m, 4H, Ar-H), 6.978-6.856 (m, 1H, Ar-H), 6.811-6.734
7
1
3
(m, 2H, Ar-H), 5.701 (s, 2H, CH
2 2 3
), 4.545 (s, 2H, CH ), 3.278 (s, 3H, N-CH ); C NMR (100
MHz, CDCl ) δ (ppm): 155.1, 150.0, 146.3, 144.9, 135.8, 135.4, 131.9, 129.5, 129.4, 128.7,
3
1
4
6
4
27.7, 127.1, 126.8, 125.9, 125.2, 125.0, 123.9, 123.2, 117.9, 68.1, 58.2, 37.1; ESI-MS (m/z) =
+
28.51 (M+H) ; calculated for C H N OS; C, 67.43; H, 4.95; N, 16.38; S, 7.50. Found: C,
2
4
21
5
7.45; H, 4.93; N, 16.43; S, 7.47.
.1.8.5. (E)-4-((4-(((((10-Methyl-10H-phenothiazin-3-yl)methylene)amino)oxy)methyl)-1H-1,2,3-
1
triazol-1-yl)methyl)benzonitrile (9e). Pale yellow solid. Yield: 85%; m.p. 217-218 °C. H NMR
(
CDCl
3
, 400 MHz, δ in ppm): 8.235 (s, 1H, -CH=N), 8.191-7.976 (m, 2H, Ar-H), 7.724-7.665 (s,
2
H, Ar-H), 7.210- 7.154 (m, 3H, Ar-H), 7.601-7.543 (m, 2H, Ar-H), 6.932-6.884 (m, 1H, Ar-H),
1
3
6
.821-6.787 (m, 2H, Ar-H), 5.715 (s, 2H, CH ), 4.512 (s, 2H, CH ), 3.297 (s, 3H, N-CH ); C
2 2 3
3
NMR (100 MHz, CDCl ) δ (ppm): 155.7, 150.5, 146.7, 144.9, 141.7, 135.8, 133.4, 132.0, 130.6,
1
29.5, 127.9, 127.3, 126.2, 125.5, 125.3, 123.9, 123.2, 119.9, 117.8, 111.0, 68.1, 58.1, 37.4; ESI-
+
20 6
MS (m/z) = 453.10 (M+H) ; calculated for C25H N OS; C, 66.35; H, 4.45; N, 18.57; S, 7.09.
Found: C, 66.31; H, 4.43; N, 18.59; S, 7.01.
4
.1.8.6.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(1-methoxybenzyl-1H-1,2,3-triazol-4-
1
yl)methoxy]methanimine (9f). Brown solid. Yield: 83%; m.p. 222-223 °C; H NMR (CDCl , 400
3
MHz, δ in ppm): 8.196 (s, 1H, -CH=N), 7.951-7.915 (m, 2H, Ar-H), 7.179-7.072 (m, 3H, Ar-H),
7
3
1
1
.514 (s, 1H, Triazole-H), 6.987-6.817 (m, 6H, Ar-H), 5.724 (s, 2H, CH
2
), 4.508 (s, 2H, CH
2
),
13
.912 (s, 3H, O-CH ), 3.314 (s, 3H, N-CH ); C NMR (100 MHz, CDCl ) δ (ppm): 159.1,
3
3
3
55.4, 150.2, 146.4, 144.9, 135.2, 131.9, 131.1, 130.2, 129.5, 127.7, 127.3, 126.1, 125.3, 125.3,
+
23.9, 123.1, 117.7, 115.3, 68.5, 58.6, 57.0, 37.6; ESI-MS (m/z) = 458.40 (M+H) ; calculated
for C H N O S; C, 65.63; H, 5.07; N, 15.31; S, 7.01. Found: C, 65.61; H, 5.07; N, 15.33; S,
2
5
23
5
2
7
.00.
1
8

ACCEPTED MANUSCRIPT
4
.1.8.7.
(E)-1-(10-Methyl-10H-phenothiazin-3-yl)-N-[(4-methoxybenzyl-1H-1,2,3-triazol-4-
1
yl)methoxy]methanimine (9g). Brown solid. Yield: 80%; m.p. 200-201 °C; H NMR (CDCl , 400
3
MHz, δ in ppm): 8.214 (s, 1H, -CH=N), 8.182-7.965 (m, 2H, Ar-H), 7.692-7.634 (s, 2H, Ar-H),
7
.588-7.529 (m, 2H, Ar-H), 7.187-7.133 (m, 3H, Ar-H), 6.897-6.849 (m, 1H, Ar-H), 6.801-6.754
1
3
(
m, 2H, Ar-H), 5.704 (s, 2H, CH ), 4.498 (s, 2H, CH ), 3.321 (s, 3H, N-CH ); C NMR (100
2 2 3
3
MHz, CDCl ) δ (ppm): 161.2, 155.7, 150.4, 146.5, 144.8, 135.4, 133.0, 132.4, 129.7, 129.4,
1
4
6
27.9, 127.2, 126.1, 125.5, 125.2, 123.9, 123.0, 118.1, 116.8, 68.1, 58.2, 37.3; ESI-MS (m/z) =
+
46.10 (M+H) ; calculated for C H FN OS; C, 64.70; H, 4.52; N, 15.72; S, 7.20. Found: C,
2
4
20
5
4.67; H, 4.50; N, 15.70; S, 7.21.
4
.1.9. General procedure for synthesis of 10-(prop-2-yn-1-yl)-10H-phenothiazine derivatives (10
and 10a). The similar procedure (synthesis of 2) was adopted to synthesize the intermediates (10
and 10a) by N-alkylation of Phenothiazine derivatives (1 and 1a) (1 mmol) with propargyl
bromide (1.5 mmol) and sodium hydride (2 mmol).
1
4
.1.9.1 10-(Prop-2-yn-1-yl)-10H-phenothiazine (10). Brown solid. Yield: 89%; H NMR
3
(CDCl , 400 MHz, δ in ppm): 7.193-7.154 (m, 4H, Ar-H), 6.867-6.752 (m, 2H, Ar-H), 6.801-
6
1
1
4
4
.747 (m, 2H, Ar-H), 4.749-4.743 (d, J = 2.4 Hz, 2H, CH ), 2.783-2.771 (t, J = 4.8 Hz, 2.4 Hz,
2
1
3
H, Acetylene-CH); C NMR (100 MHz, CDCl ) δ (ppm): 145.3, 130.2, 129.4, 125.1, 122.8,
3
+
17.7, 99.7, 75.4, 39.1; ESI-MS (m/z) = 238.21 (M+H) ; calculated for C15
H11NS; C, 75.91; H,
.67; N, 5.90; S, 13.51. Found: C, 75.88; H, 4.65; N, 5.92; S, 13.53.
.1.9.2. 10-(Prop-2-yn-1-yl)-2-(trifluoromethyl)-10H-phenothiazine (10a). Pale brown solid.
1
Yield: 88%; H NMR (CDCl , 400 MHz, δ in ppm): 7.181-7.145 (m, 2H, Ar-H), 6.815-6.645 (m,
3
3
H, Ar-H), 6.514 (s, 1H, Ar-H), 6.491-6.464 (m, 1H, Ar-H), 4.757-4.750 (d, J = 2.8 Hz, 2H,
1
3
CH ), 2.794-2.781 (t, J = 5.2 Hz, 2.8 Hz, 1H, Acetylene-CH); C NMR (100 MHz, CDCl ) δ
2
3
(ppm): 147.5, 145.4, 132.7, 130.4, 129.2, 128.4, 125.3, 125.1, 124.5, 123.6, 122.8, 117.6, 111.2,
+
9
9.8, 75.5, 39.2; ESI-MS (m/z) = 306.4 (M+H) ; calculated for C16
H
10
F
3
NS; C, 62.94; H, 3.30;
N, 4.59; S, 10.50. Found: C, 62.98; H, 3.25; N, 4.58; S, 10.56.
.1.10. General procedure for synthesis of 10-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-10H-
4
phenothiazine derivatives (11a-h). The above procedure (synthesis of 6a-g) was adopted to
synthesize the compounds 11a-h by click reaction between substituted benzyl bromides (1
mmol) and respective 10-(prop-2-yn-1-yl)-10H-phenothiazine derivatives(10 and 10a) (1 mmol)
1
9

ACCEPTED MANUSCRIPT
in presence of sodium azide(1mmol), sodium ascorbate (10 mol %) and copper (II) sulfate (5 mol
%).
4
.1.10.1. 10-((1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazine (11a). Pale
1
yellow solid. Yield: 85%; m.p. 148-149 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.836-7.816
3
(d, J = 8.0 Hz, 2H, Ar-H), 7.477 (s, 1H, Triazole-H), 7.311-7.292 (d, J = 7.6 Hz, 2H, Ar-H),
7
5
.121-7.051 (m, 4H, Ar-H), 6.915-6.878 (m, 2H, Ar-H), 6.837-6.816 (d, J = 8.4 Hz, 2H, Ar-H),
1
3
.734 (s, 2H, CH ), 5.263 (s, 2H, CH ), 2.436 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ
2
2
3
3
(ppm): 145.4, 139.3, 137.6, 134.5, 130.7, 130.4, 130.2, 129.1, 128.6, 125.9, 122.8, 117.6, 51.6,
+
4
1
4
4.7, 22.3; ESI-MS (m/z) = 385.40 (M+H) ; calculated for C H N S; C, 71.85; H, 5.24; N,
2
3
20
4
4.57; S, 8.34. Found: C, 71.89; H, 5.21; N, 14.57; S, 8.33.
.1.10.2. 10-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazine (11b). Yellow
1
solid. Yield: 91%; m.p. 174-175 °C. H NMR (CDCl , 400 MHz, δ in ppm): 8.149-8.127 (d, J =
3
8
.8 Hz, 2H, Ar-H), 7.344 (s, 1H, Triazole-H), 7.195-7.173 (d, J = 8.8 Hz, 2H, ArH), 7.137-7.040
(
m, 4H, Ar-H), 6.936-6.915 (m, 2H, ArH), 6.899-6.759 (m, 2H, ArH), 5.560 (s, 2H, CH ), 5.239
2
1
3
(
s, 2H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 147.9, 144.2, 141.7, 128.3, 128.0, 127.3,
2
3
+
1
24.3, 124.2, 122.9, 115.4, 53.1, 44.7; ESI-MS (m/z) = 416.10 (M+H) ; calculated for
C H N O S; C, 63.60; H, 4.12; N, 16.86; S, 7.72. Found: C, 63.59, H, 4.10; N, 16.85; S, 7.71.
22
17
5
2
4
.1.10.3. 10-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl) methyl)-10H-phenothiazine (11c). Grey
1
solid. Yield: 80%; m.p. 134-135 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.351 (s, 1H,
3
Triazole-H), 7.310-7.292 (d, J = 7.2 Hz, 1H, Ar-H), 7.125-7.103 (m, 2H, ArH), 7.082-7.024 (m,
5
H, Ar-H), 6.915-6.876 (m, 2H, Ar-H), 6.774-6.753 (m, 2H, Ar-H), 5.510 (s, 2H, CH ), 5.207
2
1
3
(
s, 2H, CH2); C NMR (100 MHz, CDCl ) δ (ppm): 162.6, 145.3, 144.2, 130.7, 130.6, 129.9,
3
1
27.3, 124.7, 124.0, 122.8, 122.0, 121.9, 115.6, 115.3, 47.7, 45.0; ESI-MS (m/z) = 389.10
+
(
M+H) ; calculated for C H FN S ; C, 68.02; H, 4.41; N, 14.42; S, 8.25. Found: C, 68.09; H,
2
2
17
4
4
4
.40; N, 14.39; S, 8.25.
.1.10.4. 10-((1-Benzyl-1H-1,2,3-triazol-4-yl) methyl)-10H-phenothiazine (11d). Grey solid.
1
Yield: 84%; m.p. 148-149 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.113-7.013 (m, 6H, Ar-
3
H), 6.907-6.867 (m, 2H, Ar-H), 6.767-6.747 (m, 2H, Ar-H), 7.296-7.273 (m, 4H, Ar-H), 5.450
1
3
(
s, 2H, CH ), 5.199 (s, 2H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 145.6, 139.4, 137.4,
2 2 3
1
30.8, 130.9, 129.7, 129.4, 128.6, 128.2, 125.6, 123.1, 117.9, 51.7, 44.6; ESI-MS (m/z) = 371.10
2
0

ACCEPTED MANUSCRIPT
+
(
18 4
M+H) ; calculated for C22H N S; C, 71.32; H, 4.90; N, 15.12; S, 8.66. Found: C, 71.30; H,
4
.93; N, 15.17; S, 8.67.
4
.1.10.5.
4-((4-((10H-Phenothiazin-10-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile
1
(
11e). Colourless solid. Yield: 98%; m.p. 184-185 °C; H NMR (CDCl
3
, 400 MHz, δ in ppm):
7
7
5
1
.581-7.560 (d, J = 8.4 Hz, 2H, Ar-H), 7.259 (s, 1H, Triazole-H), 7.136-7.117 (m, 4H, Ar-H),
.113-7.031 (m, 2H, Ar-H), 6.932-6.891 (m, 2H, Ar-H), 6.771-6.752 (d, J = 7.6 Hz, 2H, Ar-H),
13
.508 (s, 2H, CH ), 5.234 (s, 2H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 145.6, 142.8,
2
2
3
39.4, 133.7, 131.9, 130.2, 129.4, 128.7, 125.1, 122.9, 120.2, 117.6, 113.5, 51.3, 44.7; ESI-MS
+
(
m/z) = 396.10 (M+H) ; calculated for C23
H
17
5
N S; C, 69.85; H, 4.33; N, 17.71; S, 8.11. Found:
C, 69.89; H, 4.32; N, 17.63; S, 8.13.
4
.1.10.6. 10-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl) methyl)-10H-phenothiazine (11f). Pale
1
grey solid. Yield: 89%; m.p. 144-145 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.843-7.821
3
(m, 2H, ArH), 7.497 (s, 1H, Triazole-H), 7.324-7.276 (m, 2H, ArH), 7.132-7.107 (m, 4H, ArH),
6
3
1
.924-6.891 (m, 2H, ArH), 6.843-6.821 (m, 2H, ArH), 5.285 (s, 2H, CH
2
), 5.792 (s, 2H, CH
2
),
13
.914 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 160.1, 145.5, 139.0, 130.5, 130.1,
3
3
29.2, 128.6, 127.5, 125.0, 122.8, 117.4, 115.6, 57.5, 51.2, 43.6; ESI-MS (m/z) = 401.40
+
(
M+H) ; calculated for C H N OS; C, 68.98; H, 5.03; N, 13.99; S, 8.01. Found: C, 69.02; H,
2
3
20
4
5
.04; N, 13.97; S, 7.95.
4
.1.10.7. 10-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazine (11g). Grey
1
solid. Yield: 84%; m.p. 136-137 °C; H NMR (CDCl , 400 MHz, δ in ppm): 7.568-7.546 (d, J =
3
8
.8 Hz, 2H, Ar-H), 7.245 (s, 1H, Triazole-H), 7.148-7.108 (m, 5H, Ar-H), 7.098-7.012 (m, 2H,
Ar-H), 6.915-6.876 (m, 2H, Ar-H), 6.764-6.745 (d, J = 7.6 Hz, 1H, Ar-H), 5.527 (s, 2H, CH2),
1
3
5
1
.289 (s, 2H, CH
2
); C NMR (100 MHz, CDCl
3
) δ (ppm): 161.8, 145.3, 139.3, 131.7, 130.2,
+
29.8, 128.6, 125.0, 122.9, 119.0, 117.7, 116.5, 51.8, 44.6; ESI-MS (m/z) = 389.10 (M+H) ;
calculated for C H FN S; C, 68.02; H, 4.41; N, 14.42; S, 8.25. Found: C, 67.93; H, 4.43; N,
2
2
17
4
1
4.51; S, 8.26.
4
.1.10.8.
4-((4-((2-(Trifluoromethyl)-10H-phenothiazin-10-yl)methyl)-1H-1,2,3-triazol-1-
1
yl)methyl)benzonitrile (11h). Grey solid. Yield: 80%; m.p: 142-143 °C; H NMR (CDCl , 400
3
MHz, δ in ppm): 7.580-7.559 (d, J = 8.4 Hz, 2H, Ar-H), 7.424-7.7.404 (d, J = 8.0 Hz, 2H, Ar-H),
7
.254 (s, 1H, Triazole-H), 7.091-7.214 (m, 4H, Ar-H), 6.973-6.871 (m, 2H, Ar-H), 6.771-6.752
2
1

ACCEPTED MANUSCRIPT
13
(d, J = 7.6 Hz, 1H, Ar-H), 5.514 (s, 2H, CH
2 2 3
), 5.238 (s, 2H, CH ); C NMR (100 MHz, CDCl )
δ (ppm): 147.5, 145.3, 142.8, 139.1, 133.2, 132.8, 131.9, 130.2, 129.1, 128.5, 127.6, 125.2,
1
24.6, 123.6, 122.8, 120.3, 118.7, 117.6, 113.4, 111.3, 51.5, 44.7; ESI-MS (m/z) = 464.10
+
(
M+H) ; calculated for C24
H
16
F
3
N
5
S; C, 62.19; H, 3.48; N, 15.11; S, 6.92. Found: C, 62.21; H,
3
.50; N, 15.09; S, 7.01.
4
1
.1.11. General procedure for synthesis of 4-((4-((3-formyl-10H-phenothiazin-10-yl) methyl)-
H-1,2,3-triazol-1-yl)methyl)benzonitrile derivatives (12 and 13). The above procedure
(synthesis of 2) was adopted to synthesize the compounds 12 and 13 by formylation of
compounds 11e and 11h respectively.
4
.1.11.1.
4-((4-((3-Formyl-10H-phenothiazin-10-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)
1
benzonitrile (12). Yellow solid. Yield: 74%; H NMR (CDCl , 400 MHz, δ in ppm): 9.816 (s,
3
1
H, Aldehyde-CH), 7.585-7.542 (m, 3H, Ar-H), 7.252 (s, 1H, Triazole-H), 7.187-7.134 (m, 4H,
Ar-H), 7.114-7.081 (m, 3H, Ar-H), 6.965-6.947 (m, 1H, Ar-H), 5.524 (s, 2H, CH ), 5.282 (s, 2H,
2
13
CH ); C NMR (100 MHz, CDCl ) δ (ppm): 191.4, 146.4, 142.9, 139.4, 137.9, 133.0, 132.1,
2
3
1
30.5, 130.2, 129.2, 128.6, 125.3, 122.8, 121.7, 120.4, 117.6, 113.9, 113.4, 51.5, 44.6; ESI-MS
+
(
m/z) = 424.4 (M+H) ; calculated for C H N OS; C, 68.07; H, 4.05; N, 16.54; S, 7.57. Found:
2
4
17
5
C, 68.05; H, 4.07; N, 16.52; S, 7.55.
4
1
9
.1.11.2. 4-((4-((7-Formyl-2-(trifluoromethyl)-10H-phenothiazin-10-yl)methyl)-1H-1,2,3-triazol-
1
-yl)methyl)benzonitrile (13). Yellow solid. Yield: 76%; H NMR (CDCl , 400 MHz, δ in ppm):
3
.813 (s, 1H, Aldehyde-CH), 7.579-7.546 (m, 3H, Ar-H), 7.256 (s, 1H, Triazole-H), 7.182-7.139
(m, 4H, Ar-H), 7.107-7.092 (d, J = 6.0 Hz, 1H, Ar-H), 6.976-6.932 (m, 2H, Ar-H), 5.527 (s, 2H,
13
2 2 3
CH ), 5.289 (s, 2H, CH ); C NMR (100 MHz, CDCl ) δ (ppm): 191.6, 147.5, 144.2, 142.9,
1
5
1
4
37.6, 133.2, 131.0, 130.3, 130.1, 128.7, 125.3, 124.5, 123.6, 121.7, 120.0, 114.2, 113.5, 111.4,
+
1.6, 44.7; ESI-MS (m/z) = 492.1 (M+H) ; calculated for C H F N OS; C, 61.09; H, 3.28; N,
2
5
16
3
5
4.25; S, 6.52. Found: C, 60.9; H, 3.28; N, 14.19; S, 6.41.
.1.12 General procedure for synthesis of N'-((10-((1-(4-cyanobenzyl)-1H-1,2,3-triazol-4-
yl)methyl)-8-(trifluoromethyl)-10H-phenothiazin-3-yl)methylene)hydrazide derivatives (14a-n).
A mixture of compound 12/13 (1 mmol) and hydrazine derivative (1 mmol) was taken in ethanol
(
10 mL) in a dry 50 mL RB flask. A catalytic amount of conc. H SO was added and the mixture
2 4
was stirred for 10-15 minutes at RT. After the complete consumption of both starting materials
2
2

ACCEPTED MANUSCRIPT
(monitored by TLC), the reaction mixture was poured into ice cold water (10 mL). The solid
separated was filtered off under suction and washed with ethanol and water.
4
.1.12.1.
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)nicotinohydrazide (14a). Pale yellow solid. Yield: 89%; m.p: 250-251 °C. H NMR
(
DMSO-d , 400 MHz, δ in ppm): 11.945 (s, 1H, Amide-NH), 9.029 (s, 1H, Ar-H), 8.741-8.733
6
(d, J = 3.2 Hz, 1H, Ar-H), 8.282 (s, 1H, C=N-H), 8.230-8.211(m, 1H, Ar-H ), 8.107 (s, 1H, Ar-
H), 7.803-7.782 (d, J = 8.4 Hz, 2H, Ar-H), 7.564-7.442 (m, 3H, Ar-H), 7.303-7.282 (d, J = 8.4
Hz, 2H, Ar-H), 7.151-7.100 (m, 2H, Ar-H), 6.971-6.911 (m, 3H, Ar-H), 5.671 (s, 2H, -CH ),
.192 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.7, 150.4, 149.6, 148.7, 144.2,
2
1
3
5
1
1
2
6
41.8, 141.7, 138.2, 137.8, 134.1, 132.0, 131.8, 130.7, 129.1, 128.0, 127.5, 124.9, 124.5, 124.0,
+
22.8, 121.7, 119.1, 116.6, 116.3, 112.4, 50.2, 43.5; ESI-MS (m/z) = 543.10 (M+H) ; calculated
for C H N OS; C, 66.40; H, 4.09; N, 20.65; S, 5.91. Found: C, 66.28; H, 4.14; N, 20.58; S,
3
0
22
8
5
.94.
.1.12.2.
4
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)pyrazine-2-carbohydrazide (14b). Yellow solid. Yield: 97%; m.p: 112-113 °C. H
NMR (DMSO-d , 400 MHz, δ in ppm): 12.220 (s, 1H, Amide-NH), 9.239 (s, 1H, Ar-H), 8.906-
.899 (d, J = 2.8 Hz, 1H, Ar-H), 8.771-8.761 (m, 1H, Ar-H), 8.489 (s, 1H, C=N-H), 8.112 (s,
H, Ar-H), 7.807-7.786 (d, J = 8.4 Hz, 2H, Ar-H), 7.464-7.412 (m, 2H, Ar-H), 7.309-7.289 (d, J
8.0 Hz, 2H, Ar-H), 7.155-7.104 (m, 2H, Ar-H), 6.990-6.915 (m, 3H, Ar-H), 5.675 (s, 2H,
6
8
1
=
13
CH ), 5.197 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 157.7, 148.7, 146.2, 145.7,
2
2
6
1
1
41.8, 141.7, 138.2, 134.1, 132.0, 131.8, 130.7, 129.1, 128.0, 127.5, 124.9, 124.5, 124.0, 121.7,
+
19.1, 118.2, 116.6, 112.4, 50.2, 43.5; ESI-MS (m/z) = 544.10 (M+H) ; calculated for
C
29
H
21
N
9
OS; C, 64.07; H, 3.89; N, 23.19; S, 5.90. Found: C, 64.05; H, 3.93; N, 23.14; S, 5.92.
4
.1.12.3.
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)isonicotinohydrazide (14c). Brown solid. Yield: 98%; m.p: 166-167 °C. H NMR
(
DMSO-d , 400 MHz, δ in ppm): 12.194 (s, 1H, Amide-NH), 8.975-8.961 (d, J = 5.6 Hz, 2H,
6
Ar-H), 8.475 (s, 1H, C=N-H), 8.105 (s, 1H, Ar-H), 7.815-7.793 (d, J = 8.8 Hz, 2H, Ar-H), 7.516-
7
2
1
.466 (m, 2H, Ar-H), 7.318-7.274 (m, 4H, Ar-H), 7.091-7.029 (m, 3H, Ar-H), 6.991-6.935 (s,
13
H, Ar-H), 5.682 (s, 2H, -CH ), 5.192 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm)
2
2
6
64.7, 151.4, 148.7, 144.2, 141.8, 141.7, 139.2, 138.2, 134.1, 132.0, 130.7, 129.1, 128.0, 127.5,
2
3

ACCEPTED MANUSCRIPT
1
5
6
24.9, 124.5, 124.0, 123.5, 121.7, 119.1, 118.2, 116.6, 116.3, 112.4, 50.2, 43.5; ESI-MS (m/z) =
+
43.10 (M+H) ; calculated for C H N OS; C, 66.40; H, 4.09; N, 20.65; S, 5.91. Found: C,
3
0
22
8
6.30; H, 4.12; N, 20.68; S, 5.86.
4
1
2
.1.12.4. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoromethyl)-
0H-phenothiazin-3-yl)methylene)nicotinohydrazide (14d). Brown solid. Yield: 92%; m.p: 208-
1
09 °C; H NMR (DMSO-d
6
, 400 MHz, δ in ppm): 11.949 (s, 1H, Amide-NH), 9.036 (s, 1H, Ar-
H), 8.745-8.737 (d, J = 3.2 Hz, 1H, Ar-H), 8.286 (s, 1H, C=N-H), 8.235-8.217 (m, 1H, Ar-H ),
8
7
.116 (s, 1H, Ar-H), 7.807-7.785 (d, J = 8.8 Hz, 2H, Ar-H), 7.576-7.485 (m, 3H, Ar-H), 7.342-
.301 (m, 4H, Ar-H), 7.119 (s, 1H, Ar-H), 7.071-7.052 (m, 1H, Ar-H), 5.664 (s, 2H, CH ), 5.185
2
1
3
(
s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.7, 150.4, 149.6, 148.7, 146.3, 141.8,
2
6
1
1
41.7, 138.2, 137.8, 134.1, 132.0, 131.8, 131.6, 130.7, 129.3, 127.5, 124.9, 124.5, 124.1, 123.4,
+
22.8, 122.5, 119.1, 116.3, 112.4, 110.9, 50.2, 43.5; ESI-MS (m/z) = 611.40 (M+H) ; calculated
21 3 8
for C31H F N OS; C, 60.98; H, 3.47; N, 18.35; O, 2.62; S, 5.25. Found: C, 60.96; H, 3.47; N,
1
8.37; S, 5.23.
4
1
8
9
1
.1.12.5. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoromethyl)-
0H-phenothiazin-3-yl)methylene)pyrazine-2-carbohydrazide (14e). Pale yellow solid. Yield:
1
5%; m.p: 232-233 °C. H NMR (DMSO-d , 400 MHz, δ in ppm): 12.252 (s, 1H, Amide-NH),
6
.241 (s, 1H, Ar-H), 8.909-8.904 (d, J = 2.0 Hz, 1H, Ar-H), 8.774-8.764 (m, 1H, Ar-H), 8.503 (s,
H, C=N-H), 8.161 (s, 1H, Ar-H), 7.781-7.760 (d, J = 8.4 Hz, 2H, Ar-H), 7.489-7.464 (m, 2H,
Ar-H), 7.365-7.250 (m, 4H, Ar-H), 7.115 (s, 1H, Ar-H), 7.057-7.036 (m, 1H, Ar-H), 5.688 (s,
1
3
2
1
1
H, -CH ), 5.266 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 157.7, 148.7, 146.3,
2 2 6
45.7, 141.8, 141.7, 138.2, 134.1, 132.0, 131.8, 131.6, 130.7, 129.3, 127.5, 124.9, 124.5, 124.1,
+
23.4, 122.5, 119.1, 118.2, 112.4, 110.1, 50.2, 43.5; ESI-MS (m/z) = 612.1 (M+H) ; calculated
for C H F N OS; C, 58.91; H, 3.30; N, 20.61; O, 2.62; S, 5.24. Found: C, 58.88; H, 3.25; N,
3
0
20
3
9
2
0.59; S, 5.25.
4
1
.1.12.6.
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoromethyl)-
0H-phenothiazin-3-yl)methylene)isonicotinohydrazide (14f). Reddish brown solid. Yield: 96%;
1
m.p: 218-219 °C. H NMR (DMSO-d , 400 MHz, δ in ppm): 11.967 (s, 1H, Amide-NH), 9.058
6
(s, 1H, Ar-H), 8.754-8.745 (d, J = 3.6 Hz, 1H, Ar-H), 8.301 (s, 1H, C=N-H), 8.254-8.237 (m,
1
H, Ar-H ), 8.128 (s, 1H, Ar-H), 7.842-7.821 (d, J = 8.4 Hz, 2H, Ar-H), 7.597-7.458 (m, 3H, Ar-
2
4

ACCEPTED MANUSCRIPT
H), 7.386-7.796 (m, 4H, Ar-H), 7.128 (s, 1H, Ar-H), 7.094-7.081 (m, 1H, Ar-H), 5.668 (s, 2H,
13
CH ), 5.187 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.7, 151.4, 148.7, 146.3,
2
2
6
1
1
41.8, 141.7, 139.2, 138.2, 134.1, 132.0, 131.6, 130.7, 129.3, 127.5, 124.9, 124.5, 124.1, 123.5,
+
23.4, 122.5, 119.1, 118.2, 116.3, 112.4, 110.1, 50.2, 43.5; ESI-MS (m/z) = 611.1 (M+H) ;
calculated for C H F N OS; C, 60.98; H, 3.47; N, 18.35; S, 5.25. Found: C, 60.97; H, 3.45; N,
3
1
21
3
8
1
8.34; S, 5.24.
.1.12.7. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
4
yl)methylene)-4-nitrobenzohydrazide (14g). Reddish brown solid. Yield: 98%; m.p: 132-133 °C;
1
6
H-NMR (DMSO-d , 400 MHz, δ in ppm): 12.071 (s, 1H, Amide-NH), 8.836-8.823 (d, J = 5.2
Hz, 2H, Ar-H), 8.316 (s, 1H, Ar-H), 8.116 (S, 1H, C=N-H), 7.916-7.868 (m, 2H, Ar-H), 7.807-
7
2
.763 (m, 2H, Ar-H), 7.490-7.454 (m, 2H, Ar-H), 7.309-7.223 (m, 2H, Ar-H), 7.155-7.106 (m,
13
H, Ar-H), 6.989-6.898 (m, 3H, Ar-H), 5.677 (s, 2H, -CH ), 5.199 (s, 2H, CH ); C NMR (100
2
2
MHz, DMSO-d ) δ (ppm): 164.3, 149.2, 148.7, 144.1, 141.8, 141.7, 138.2, 138.1, 134.1, 132.0,
6
1
1
3
30.7, 129.1, 129.0, 128.0, 127.5, 124.9, 124.4, 124.1, 124.0, 121.7, 119.1, 118.2, 116.5, 116.3,
+
12.4, 50.2, 43.5; ESI-MS (m/z) = 587.10 (M+H) ; calculated for C H N O S; C, 63.47; H,
3
1
22
8
3
.78; N, 19.10; S, 5.47. Found: C, 63.55; H, 3.76; N, 19.07; S, 5.47.
4
.1.13.8.
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)benzohydrazide (14h). Pale green solid. Yield: 97; m.p: 152-153 °C; H-NMR
(
DMSO-d , 400 MHz, δ in ppm): 12.052 (s, 1H, Amide-NH), 8.312 (s, 1H, C=N-H), 8.106 (S,
6
1
H, Ar-H), 8.074- 8.063 (d, J = 4.4 Hz, 2H, Ar-H), 7.812-7.716 (m, 4H, Ar-H), 7.691-7.625 (m,
3
H, Ar-H), 7.523-7.458 (m, 2H, Ar-H), 7.314-7.272 (m, 1H, Ar-H), 7.102-7.029 (m, 2H, Ar-H),
13
6
.957-6.868 (m, 2H, Ar-H), 5.681 (s, 2H, -CH ), 5.210 (s, 2H, CH ); C NMR (100 MHz,
2 2
6
DMSO-d ) δ (ppm): 164.3, 148.7, 144.1, 141.8, 141.7, 138.2, 134.1, 132.7, 132.0, 131.9, 130.7,
1
4
5
29.1, 128.5, 128.3, 128.0, 127.5, 124.9, 124.4, 121.7, 119.1, 118.2, 116.5, 116.3, 112.4, 50.2,
+
3.5; ESI-MS (m/z) = 542.1 (M+H) ; calculated for C H N OS; C, 68.74; H, 4.28; N, 18.10; S,
3
1
23
7
.92. Found: C, 68.72; H, 4.31; N, 18.15; S, 5.93.
4
.1.12.9.
(E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)-4-fluorobenzohydrazide (14i). Pale yellow solid. Yield: 89%; m.p: 107-108 °C; H
NMR (DMSO-d , 400 MHz, δ in ppm): 12.065 (s, 1H, Amide-NH), 8.311 (s, 1H, C=N-H), 8.112
6
(S, 1H, Ar-H), 8.085- 8.073 (d, J = 4.8 Hz, 2H, Ar-H), 7.899-7.852 (m, 2H, Ar-H), 7.791-7.746
2
5

ACCEPTED MANUSCRIPT
(m, 2H, Ar-H), 7.412-7.369 (m, 2H, Ar-H), 7.294-7.209 (m, 2H, Ar-H), 7.098-7.051 (m, 2H, Ar-
13
H), 6.943-6.854 (m, 3H, Ar-H), 5.686 (s, 2H, -CH ), 5.216 (s, 2H, CH ); C NMR (100 MHz,
2
2
DMSO-d ) δ (ppm): 167.0, 164.3, 148.7, 144.1, 141.8, 141.7, 138.2, 134.1, 132.0, 130.7, 130.5,
6
1
5
1
4
29.1, 129.0, 128.0, 127.5, 124.9, 124.4, 124.0, 121.7, 119.1, 118.2, 116.5, 116.3, 115.9, 112.4,
+
0.2, 43.5; ESI-MS (m/z) = 560.1 (M+H) ; calculated for C H FN OS; C, 66.53; H, 3.96; N,
3
1
22
7
7.52; S, 5.73. Found: C, 66.57; H, 3.95; N, 17.45; S, 5.75.
.1.12.10. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazin-3-
1
yl)methylene)-4-methylbenzohydrazide (14j). Brown solid. Yield: 89%; m.p: 175-176 °C; H-
NMR (DMSO-d
6
, 400 MHz, δ in ppm): 12.054 (s, 1H, Amide-NH), 8.304 (s, 1H, C=N-H), 8.104
S, 1H, Ar-H), 8.067- 8.055 (d, J = 4.8 Hz, 2H, Ar-H), 7.862-7.814 (m, 2H, Ar-H), 7.756-7.712
m, 2H, Ar-H), 7.401-7.357 (m, 2H, Ar-H), 7.276-7.192 (m, 2H, Ar-H), 7.044-6.996 (m, 2H, Ar-
(
(
1
3
H), 6.912-6.824 (m, 3H, Ar-H), 5.681 (s, 2H, -CH ), 5.212 (s, 2H, CH ), 2.335 (s, 3H, CH ); C
2
2
3
NMR (100 MHz, DMSO-d ) δ (ppm): 164.3, 148.7, 144.1, 142.8, 141.8, 141.7, 138.2, 134.1,
6
1
32.0, 131.2, 130.7, 129.1, 128.8, 128.3, 128.0, 127.5, 124.9, 124.4, 124.0, 121.7, 119.1, 118.2,
+
1
16.5, 116.3, 112.4, 50.2, 43.5, 21.1; ESI-MS (m/z) = 556.1 (M+H) ; calculated for C H N OS;
3
2
25
7
C, 69.17; H, 4.53; N, 17.65; S, 5.77. Found: C, 69.21; H, 4.54; N, 17.63; S, 5.71.
4
1
1
.1.12.11. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoro methyl)-
0H-phenothiazin-3-yl)methylene)-4-nitrobenzohydrazide (14k). Yellow solid. Yield: 84%; m.p:
1
90-191 °C; H-NMR (DMSO-d , 400 MHz, δ in ppm): 11.257 (s, 1H, Amide-NH), 8.149-8.093
6
(m, 3H, Ar-H), 7.913 (S, 1H, Ar-H), 7.777-7.536 (m, 2H, Ar-H), 7.496-7.469 (m, 2H, Ar-H),
7
5
1
1
.360-7.244 (m, 4H, Ar-H), 7.153-7.112 (m, 3H, Ar-H), 7.012-6.991 (d, J = 8.4 Hz, 1H, Ar-H),
13
.689 (s, 2H, -CH ), 5.256 (s, 2H, CH ); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.3, 149.2,
2
2
6
48.7, 146.3, 141.9, 141.7, 138.2, 138.1, 134.1, 132.0, 130.7, 129.3, 129.0, 127.5, 124.9, 124.4,
24.1, 124.0, 123.4, 122.5, 119.1, 118.2, 116.3, 110.1, 112.4, 50.2, 43.5; ESI-MS (m/z) = 655.1
+
(
M+H) ; calculated for C H F N O S; C, 58.71; H, 3.23; N, 17.12; S, 4.90. Found: C, 58.62;
32
21
3
8
3
H, 3.25; N, 17.21; S, 4.89.
4
1
1
3
7
.1.12.12. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoro methyl)-
0H-phenothiazin-3-yl)methylene)benzohydrazide (14l). Colourless solid. Yield: 98%; m.p: 167-
1
68 °C; H-NMR (DMSO-d , 400 MHz, δ in ppm): 11.246 (s, 1H, Amide-NH), 8.105-8.049 (m,
6
H, Ar-H), 7.914 (S, 1H, Ar-H), 7.735-7.548 (m, 4H, Ar-H), 7.489-7.465 (m, 2H, Ar-H), 7.326-
.245 (m, 3H, Ar-H), 7.107-7.061 (m, 3H, Ar-H), 6.985-6.966 (d, J = 7.6 Hz, 1H, Ar-H), 5.685
2
6

ACCEPTED MANUSCRIPT
1
3
(
s, 2H, CH
2
), 5.245 (s, 2H, CH
2 6
); C NMR (100 MHz, DMSO-d ) δ (ppm): 164.3, 148.7, 146.3,
1
1
41.8, 141.7, 138.2, 134.1, 132.7, 132.0, 131.9, 131.6, 130.7, 129.3, 128.5, 128.3, 127.5, 124.9,
+
23.4, 122.5, 119.1, 118.2, 116.3, 112.4, 110.1, 50.2, 43.5; ESI-MS (m/z) = 610.1 (M+H) ;
OS; C, 63.05; H, 3.64; N, 16.08; S, 5.26. Found: C, 63.04; H, 3.65; N,
22 3 7
calculated for C32H F N
1
6.06; S, 5.27.
4
1
.1.12.13. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoro methyl)-
0H-phenothiazin-3-yl)methylene)-4-fluorobenzohydrazide (14m). Yellow solid. Yield: 90%;
1
m.p: 141-142 °C; H-NMR (DMSO-d
6
, 400 MHz, δ in ppm): 11.251 (s, 1H, Amide-NH), 8.094-
8
.039 (m, 3H, Ar-H), 7.907 (S, 1H, Ar-H), 7.741-7.514 (m, 2H, Ar-H), 7.472-7.446 (m, 2H, Ar-
H), 7.331-7.216 (m, 4H, Ar-H), 7.104-7.064 (m, 3H, Ar-H), 6.997-6.976 (d, J = 8.4 Hz, 1H, Ar-
1
3
H), 5.701 (s, 2H, CH
2
), 5.267 (s, 2H, CH
2 6
); C NMR (100 MHz, DMSO-d ) δ (ppm): 167.0,
1
1
6
6
64.3, 148.7, 146.3, 141.8, 141.7, 138.2, 131.6, 134.1, 132.0, 130.7, 130.5, 129.3, 129.0, 127.5,
24.9, 124.4, 123.4, 122.5, 119.1, 118.2, 116.3, 115.9, 112.4, 110.1, 50.2, 43.5; ESI-MS (m/z) =
+
28.1 (M+H) ; calculated for C H F N OS; C, 61.24; H, 3.37; N, 15.62; S, 5.11. Found: C,
3
2
21
4
7
1.26; H, 3.35; N, 15.61; S, 5.10.
4
1
.1.12.14. (E)-N'-((10-((1-(4-Cyanobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-8-(trifluoro methyl)-
0H-phenothiazin-3-yl)methylene)-4-methylbenzohydrazide (14n). Brown solid. Yield: 94%;
1
m.p: 168-169 °C. H-NMR (DMSO-d
6
, 400 MHz, δ in ppm): 11.245 (s, 1H, Amide-NH), 8.073-
8
.017 (m, 3H, Ar-H), 7.902 (S, 1H, Ar-H), 7.724-7.498 (m, 2H, Ar-H), 7.456-7.432 (m, 2H, Ar-
H), 7.298-7.184 (m, 4H, Ar-H), 7.095-7.056 (m, 3H, Ar-H), 6.974-6.954 (d, J = 8.0 Hz, 1H, Ar-
13
H), 5.694 (s, 2H, CH ), 5.256 (s, 2H, CH ), 2.344 (s, 3H, CH ); C NMR (100 MHz, DMSO-d )
2
2
3
6
δ (ppm): 164.3, 148.7, 146.3, 142.8, 141.8, 141.7, 138.2, 134.1, 132.0, 131.6, 131.2, 130.7,
1
4
1
29.3, 128.8, 128.3, 127.5, 124.9, 124.4, 123.4, 122.5, 119.1, 118.2, 116.3, 112.4, 110.1, 50.2,
+
3.5, 21.1; ESI-MS (m/z) = 624.1 (M+H) ; calculated for C H F N OS; C, 63.55; H, 3.88; N,
3
3
24
3
7
5.72; S, 5.14. Found: C, 63.57; H, 3.86; N, 15.77; S, 5.11.
5
. References
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