Phenothiazine-based chalcones as potential dual-target inhibitors toward cholinesterases (AChE, BuChE) and monoamine oxidases (MAO-A, MAO-B)

Article abstract of DOI:10.1002/jhet.4156

Chalcones targeting neurodegenerative diseases have been known as attractive structures in drug design and discovery. In this study, phenothiazine-based chalcones as ChEs and MAOs inhibitors were designed and synthesized via base-catalyzed Claisen-Schmidt

Full text of DOI:10.1002/jhet.4156

Phenothiazine-based Chalcones as Potential Dual-Target
Inhibitors Towards Cholinesterases (AChE, BuChE) and
Monoamine Oxidases (MAO–A, MAO–B)
Cem Yamali*[1,2], Feyza Sena Engin[1], Sinan Bilginer[1], Mehtap Tugrak[1], Dilan Ozmen Ozgun[3],
Gulsen Ozli[4], Serkan Levent[5], Begum Nurpelin Saglik[5], Yusuf Ozkay[5], Halise Inci Gul*[1]
Abstract:
1. INTRODUCTION
Chalcones targeting neurodegenerative diseases have been known
as attractive structures in drug design and discovery. In this study,
phenothiazine-based chalcones as ChEs and MAOs inhibitors were
designed and synthesized via base-catalyzed Claisen-Schmidt
condensation, and chemical structures of the compounds were
elucidated by NMRs and HRMS. Compounds 3 and 9 showed
promising inhibition potency against AChE enzyme with IC50 values
of 0.221 µM and 0.053 µM while compound 9 displayed remarkable
inhibition potency towards MAO-B enzyme with IC50 value of 0.048
µM. Compound 9, as a dual-target inhibitor, selectively inhibited
AChE and MAO-B enzymes. This promising behavior is an
advantage for the compound since MAO-B and AChE inhibition
have a role in Alzheimer‟s disease (AD). Fused tricyclic ring
systems such as phenothiazine incorporated with chalcone moiety
being multitargeting ligands may help scientists for the rational
design of novel lead compounds targeting neurodegenerative
illnesses.
The investigation of therapeutic compounds to modify the
pathogenesis of neurodegenerative diseases has been a hot
topic in drug development. The improvement in learning
molecular mechanisms of Alzheimer‟s disease (AD) which is the
most prevalent disorder in older persons with mental
deterioration has been fascinating[1, 2]
.
The current pharmaceuticals used in the treatment of AD
are cholinesterase inhibitors (ChEIs) (Donepezil, Rivastigmine,
Galantamine) and N-methyl-D-aspartate (NMDA) receptor
antagonists (Memantine)[3]. ChEIs have been used to prevent
the degradation of the neurotransmitter acetylcholine (ACh) by
inhibiting acetylcholinesterase (AChE, EC 3.1.1.7) and
butyrylcholinesterase (BuChE, EC 3.1.1.8) enzymes that
influence breakdown of acetylcholine in synapses. Therefore, to
treat some of the symptoms of AD, inhibition of ChEs is one of
the popular approaches[4]. The enzyme levels in AD have
differences. So, AChE levels are low in AD, while BuChE activity
is enhanced[5]. Functionally, both enzymes hydrolyze
acetylcholine efficiently at different rates. However, AChE has
higher hydrolytic activity than BChE‟s[6].
[1]*
Assist. Prof. Dr. Cem Yamali, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum,
Turkey
E-mail: c.yamali@yahoo.com
[2]
[1]
[1]
[1]
[3]
[4]
[5]
[5]
[5]
[1]*
Assist. Prof. Dr. Cem Yamali, Department of Basic Pharmaceutical
Sciences, Faculty of Pharmacy, Cukurova University, Adana, Turkey
E-mail: c.yamali@yahoo.com
Feyza Sena Engin, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
E-mail: feyzasenaengin@gmail.com
Assit. Prof. Dr.Sinan Bilginer, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum,
Turkey. E-mail: sinanbilginer25@yahoo.com
Dr. Mehtap Tugrak, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University, Erzurum, Turkey.
E-mail: mehtaptugrak@hotmail.com
Dilan Ozmen Ozgun, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ibrahim Cecen University, Agri, Turkey.
E-mail: eczdilan@yahoo.com
Gulsen Ozli, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Erzincan University, Erzincan, Turkey.
E-mail: gulsengunesacar@gmail.com
Serkan Levent, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Anadolu University, Eskisehir, Turkey.
E-mail: serkanlevent@anadolu.edu.tr
Dr. Begum Nurpelin Saglik, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Anadolu University, Eskisehir,
Turkey. E-mail: bnsaglik@anadolu.edu.tr
Unfortunately, the current ChEIs remain as palliative
treatments of AD and are ineffective as a long-term treatment for
AD. Tacrine, the first ChEI, was withdrawn due to hepatotoxicity.
On the other hand, donepezil and rivastigmine show
dose_depended adverse effects[7]. Therefore, it is very desirable
to investigate safe and effective pharmaceuticals for AD.
Although the pathogenesis of AD is not fully clear, the
clinical hallmarks such as cholinergic dysfunction, amyloid
aggregation, tau hyperphosphorylation, and oxidative stress that
lead to neurodegeneration and loss of memory and cognitive
were reported[8, 9]. Current drug design strategies and targets
have been developed to obtain the most potent and selective
anti-AD drugs with less side effects due to the multi_factorial
structure of AD.
Prof. Dr. Yusuf Ozkay, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey.
E-mail: yozkay@anadolu.edu.tr
Prof. Dr. Halise Inci Gul, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University, Erzurum, Turkey.
E-mail: incigul1967@yahoo.com
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As a new target of AD, monoamine oxidases (MAOs) are
Methylene blue has been extended to phase 2 trials in
humans with AD.[19]. Phenothiazine-derived compounds also
show inhibitory potency against both cholinesterases, tau protein,
and neuroprotective effects[15,19,20]. Besides, tacrine, the first
ChEIs, is an example of a fused tricyclic drug against AD as a
lead compound to be inspired.
attracting attention. MAOs play a crucial role in the process of
deactivation of the monoamines in the body. Monoamines
convert to ammonia, hydrogen peroxide and corresponding
aldehyde[10]. Two types of MAOs in mammals are reported as
MAO-A and MAO-B enzymes, which bound covalently to their
co-factor flavin adenine dinucleotide (FAD). MAO-A inhibitors
are considered for the treatment of anxiety and depression,
while MAO-B inhibitors could be prefered as therapeutic agents
Chalcones, as well as their synthetic analogues, display
favorable biological activities against AD[21-26]. As they have
flexible structures, chalcones can effectively bind to many kinds
in Parkinson‟s and Alzheimer‟s diseases[11]
.
of enzymes or receptors targeting AD[27,28]
phenothiazine-based chalcones to modulate specific enzymes
may be powerful strategy for the therapeutic aim of
.
Designing
In the last few years, MAO-B enzyme has been proved as
an essential target for research of AD since MAO-B catalyzes
formation of neurotoxic products[12]. This induces generation of
free radicals that lead to oxidative stress, neuronal cell death,
and β-amyloid plaques[13, 14]. As MAO-B inhibitors can regulate
neurotransmitters and inhibit oxidative damage, novel selective
MAO-B inhibitors can be developed as therapeutic agents
targeting AD.
a
neurodegenerative disorders.
Due to the complexity of AD, the multitarget-directed
ligands, which possess two or more complementary AD-related
targets, have been considered as an effective way for the
treatment of AD[29]. When compared with the clinical drugs which
show their effects on a single biological target, the hybride
structures that target multiple pathways can decrease toxicity
and adverse effects. A study also reported synthesis of novel
structures targeting AChE and MAO-B enzymes as dual
inhibitors for the treatment of AD[30]. The obtained results
encouraged many research groups to develop multitarget-
directed ligands against AD.
Phenothiazines (Figure 1), nitrogen- and sulfur-containing
tricyclic compounds, have been used as dyes, biological stains,
and pharmaceutical agents to treat various diseases[15]
.
Chlorpromazine (Figure 1) was the first drug successfully
employed as a neuroleptic in the treatment of psychotic
disorders such as schizophrenia as an antagonist of dopamine
receptors[16]
.
Phenothiazine bearing methylene blue, FDA-
By considering the points mentioned above, we designed
phenothiazine-based chalcones, which include a fused tricyclic
ring system similar to tacrine based on the hybrid approach to
design multitarget-directed ligands. We aim to synthesize dual-
targeting phenothiazine-based chalcones as ChEs and MAOs
inhibitors as possible lead compounds for Alzheimer‟s disease
as well as neurodegenerative diseases.
approved drug, is a therapeutic agent for malaria, fear and
claustrophobia, and psychiatric diseases (Figure 1). Since
methylene blue can easily cross the blood-brain barrier, its
analogues can be considered as a main structure to design new
compounds for diseases affecting the central nervous system[17]
Besides, phenothiazine-based compounds are described as
mostly well tolerated and have few side effects[18]
.
.
2. RESULTS AND DISCUSSION
2.1. Chemistry
In the present study, compounds were freshly synthesized by
the reaction of 2-acetylphenothiazine with substituted-
benzaldehydes
via
base-catalyzed
Claisen-Schmidt
condensation according to our previous studies[22,23,31] (Scheme
1). Chemical structures of compounds 1–16 were elucidated by
spectral techniques such as 1H NMR, 13C NMR, and HRMS.
Compounds 7, 12, and 15 were reported for the first time by this
study.
1
_
Figure 1. Chemical structures of phenothiazine and its
Analysis of H NMR spectra of compounds 1 16 showed
that all synthesized were in E isomers with coupling constants
derivatives
(J) between 15.4 – 20.5 Hz for α,β-protons, observed in the
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aromatic region of spectra. The proton of the NH group on the
and 9 [(E)-3-(4-nitrophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-
1-one] attracted great attention since their inhibition percentages
phenothiazine ring appeared in the range of 8.74–8.81 ppm as a
singlet. According to the 13C NMR spectra of the compounds,
the carbonyl carbon of the compounds resonated at around 188
ppm, as expected. HRMS results also confirmed the chemical
structures of the compounds. Spectrums of the compounds were
given as a supplementary file.
(% inhibition) were in the range of 87–93 % against AChE while
Donepezil‟s % inhibition was 99 %. These two compounds were
forwarded for further experiments to calculate their half-maximal
–
3
–
9
inhibitory concentrations (IC50) in the range of 10 –10 against
AChE (Table 2). The compounds showed promising inhibitory
potency when compared to donepezil. IC50 values of compounds
3, 9, and donepezil were as follows: 0.221, 0.053, and 0.02 µM,
respectively. These results indicate that the AChE inhibitory
potential of compound 9 is close to that of donepezil, and also,
this compound can be modified to enhance enzyme inhibitory
potency for having more potent lead compounds for future
studies.
The compounds having different substituted-phenyl ring or
bioisosteric ring were evaluated at different concentrations to
see their enzyme inhibitory effects. The majority of the
compounds were found less active as ChEs inhibitors, except for
3 and 9. Some modifications or substituents made significant
i : Methanol, aqueous solution of KOH (40 %), 70 °C.
Scheme 1. Synthesis of the phenothiazine-based chalcones
2.2. Cholinesterase (AChE, BuChE) Inhibitory Effects Of The
Compounds
differences
in
bioactivity.
Primary
structure-bioactivity
relationships were made according to the % inhibition of each
compound obtained at 10_3 M to determine which modification is
better and can be used for further designing of new enzyme
inhibitors.
When compounds 1 (H, 55 %), 2 (4_CH3, 60 %), and 3 (4_OCH3,
93 %) were compared, the electron-releasing 4_OCH3 group is
favorable than 4_CH3 since it increased activity. Among the
It is known that ChEs enzymes regulate cholinergic transmission.
Furthermore, the hydrolytic activity of the AChE enzyme is
higher than the BuChE‟s. Therefore, compounds targeting the
AChE enzyme are favorable in the treatment of AD[32]. The
compounds synthesized were evaluated against two ChEs to
determine enzyme selectivity of the compounds. Donepezil and
tacrine were used as reference drugs since the inhibitors differ in
selectivity for different forms of ChEs [33]. Biological evaluation of
halogenated compounds, fluorine substitution (73–77 %) led to
increased activity compared to the parent compound 1 (H, 55 %),
while the substitution pattern of the bromine or chlorine-bearing
compounds did not increase the activity excessively. On the
other hand, the substitution pattern of the electron-withdrawing
NO2 group has dramatically affected the activity. Compound 9
(p_NO2, 91 %) was considered as one of the most potent in this
study. p_NO2 substitution increased inhibitory enzyme potency
approximately 1.6 fold compared to compounds 10 (m_NO2,
58 %) and 1 (H, 55 %). Based on these results, it can be stated
that phenothiazine bearing chalcone-based compounds can be
used for future studies to obtain more potent ChE inhibitors
having increased pharmacological profile.
compounds 1 – 16 as potential AChE and BuChE inhibitors was
performed using in vitro modified-Ellman‟s assay [34]. The
bioassay results are shown in Tables 1 and 2.
This study is the first report for the synthesized compounds
in terms of AChE (except 1, 2, 3, 5, and 10) and the BuChE
enzyme inhibitory profile. Compounds 1, 2, 3, 5, and 10 were
reported with AChE inhibition study in 2012. According to this
study [35], the compounds showed an inhibitory potency with
IC50 values between 1.0 to 6.4 µg/ml when compared to the
neostigmine with IC50 value of 8.3 µg/ml.
Initially, the compounds‟ inhibitory potency was evaluated
at 10– and 10– M (Table 1). It shows that the compounds were
more potent against the AChE enzyme when compared with
BuChE. Among the compounds, particularly, compounds 3 [(E)-
3-(4-methoxyphenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one]
3
4
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2.3. Monoamine Oxidase (MAO-A, MAO-B) inhibitory effects
of synthesized compounds
MAOs enzyme inhibitory profiles of phenothiazine-based
chalcones 1–16 were reported here firstly. The compounds were
tested against both ChEs and MAOs to see whether they have
dual inhibitory effects since MAO-B was reported as one of the
hallmarks of AD[14]
.
If the compounds show their effects on both enzymes, they
can be considered as potent dual inhibitors. From this point of
view, the compounds were evaluated against MAO-A and MAO-
B enzymes, and their inhibition results (% inhibition) was
reported in Tables 3 and 4. Chlorgiline, a selective MAO-A
inhibitor, and selegiline, a selective MAO-B inhibitor, were used
in the bioassay as standard MAOs inhibitors. Firstly, compounds
Figure 2. Lead compounds of the study: 3 (AChE) and 9 (AChE
and MAO-B)
3. CONCLUSIONS
were tested at 10– and 10– M to see whether they have
inhibitory potency against MAOs (Table 3). Then, the MAO-B
enzyme was treated with different concentrations of the most
3
4
The chalcone-based compounds targeting neurodegenerative
diseases have been reported as promising structures in the field
of drug design and discovery. In this present study,
phenothiazine-based chalcones as ChEs and MAOs inhibitors
were freshly synthesized via base-catalyzed Claisen-Schmidt
condensation. The lead compounds, preferably inhibited AD-
related AChE and MAO-B enzymes. The compounds 3 and 9
showed remarkable inhibition potency against AChE enzyme
with IC50 values of 0.221 µM and 0.053 µM while the compound
–
3
–
9
potent compound in the range of 10 –10 for calculation IC50
values (Table 4).
Interestingly, compound 9, which had significant AChE
inhibitory potency, displayed inhibition potency towards MAO-B
enzyme in the range of 88–92 % while reference drug
selegiline‟s potency was in the range of 94–98 %. IC50 values of
9 and selegiline were 0.048 and 0.037 µM, respectively. Results
showed that compound 9 could be considered as the most
potent inhibitor of MAO-B enzyme, among others. Moreover,
compound 9 was found more selective towards MAO-B when
compared with MAO-A. This situation is an advantage for
compound 9 since MAO-B inhibition has role in AD. It may show
anti-AD effects by inhibiting AChE and MAO-B enzymes which
need to be approved by further assays.
9
displayed significant inhibition potency towards MAO-B
enzyme with IC50 values of 0.048 µM. The most potent and
selective compound 9 can be declared as a promising dual-
targeting lead inhibitor towards AChE and MAO-B enzymes.
Hopefully, these results can help researchers for the rational
design of novel phenothiazine-based lead compounds targeting
neurodegenerative diseases.
As a result of this study, the dual-target inhibitors showing
enzyme inhibition properties selectively against both AChE and
MAO-B enzymes were reported. For future concepts, further
molecular modifications can be made to direct the lead
compounds‟ physicochemical properties, enzyme selectivity, and
pharmacological profile. (Figure 2).
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Table 1. Cholinesterases (AChE, BuChE) inhibitory effects (% inhibition) of compounds 1–16
% AChE
% BChE
Compound
–
–
–
–
10 3 M
10 4 M
10 3 M
10 4 M
1
55.128 ± 0.956
60.965 ± 0.851
93.125 ± 1.185
77.255 ± 1.024
55.129 ± 0.962
76.255 ± 0.956
73.955 ± 0.942
69.529 ± 0.741
91.528 ± 1.118
58.955 ± 0.852
41.488 ± 0.753
68.264 ± 0.951
59.621 ± 0.842
60.250 ± 0.943
47.206 ± 0.654
54.859 ± 0.679
99.156 ± 1.302
-
36.755 ± 0.529
25.472 ± 0.637
87.256 ± 1.056
39.623 ± 0.419
30.640 ± 0.511
31.477 ± 0.492
37.258 ± 0.365
29.217 ± 0.660
87.642 ± 1.247
21.367 ± 0.407
36.850 ± 0.510
30.140 ± 0.628
29.475 ± 0.453
34.161 ± 0.467
27.488 ± 0.617
26.147 ± 0.477
97.395 ± 1.255
-
38.165 ± 0.820
36.521 ± 0.784
41.066 ± 0.693
39.527 ± 0.467
27.394 ± 0.835
34.288 ± 0.726
33.018 ± 0.798
28.750 ± 0.697
25.623 ± 0.547
27.985 ± 0.447
29.748 ± 0.451
31.488 ± 0.572
34.170 ± 0.740
31.629 ± 0.559
29.118 ± 0.735
26.578 ± 0.603
-
24.562 ± 0.651
20.970 ± 0.425
35.276 ± 0.451
24.281 ± 0.748
20.128 ± 0.881
21.641 ± 0.547
25.475 ± 0.756
21.623 ± 0.841
18.240 ± 0.462
22.331 ± 0.632
24.205 ± 0.787
24.617 ± 0.510
26.557 ± 0.529
24.875 ± 0.421
20.362 ± 0.460
17.621 ± 0.435
-
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Donepezil
Tacrine
99.827 ± 1.378
98.651 ± 1.402
Table 2. % Inhibition and IC50 (µM) values of the most potent compounds 3 and 9 against AChE
–
–
–
–
–
–
–
10 3 M
10 4 M
10 5 M
10 6 M
10 7 M
10 8 M
10 9 M
22.148
± 0.459
19.623
± 0.575
18.410
± 0.411
Compound
3
IC50 (µM)
93.125
± 1.185
91.528
± 1.118
99.156
± 1.302
87.256
± 1.056
87.642
± 1.247
97.395
± 1.255
70.148
± 1.248
78.116
± 1.063
93.583
± 1.167
58.117
± 0.985
71.185
± 0.974
91.277
± 1.074
40.205
± 0.859
62.478
± 0.851
76.982
± 0.951
35.622
± 0.751
42.027
± 0.632
35.459
± 0.453
0.221
± 0.008
0.053
9
± 0.001
0.020
Donepezil
± 0.001
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Table 3. Monoamine oxidases (MAO_A, MAO_B) inhibitory effects (% inhibition) of compounds 1–16
% MAO_A
% MAO_B
Compound
–
–
–
–
10 3 M
10 4 M
10 3 M
10 4 M
1
33.015 ± 0.658
28.114 ± 0.529
39.250 ± 0.754
40.158 ± 0.602
27.529 ± 0.498
40.744 ± 0.511
34.708 ± 0.475
29.265 ± 0.409
37.529 ± 0.614
34.525 ± 0.674
28.955 ± 0.491
36.754 ± 0.510
39.852 ± 0.637
32.369 ± 0.499
27.753 ± 0.487
29.753 ± 0.417
99.411 ± 2.955
-
24.157 ± 0.514
20.605 ± 0.411
33.451 ± 0.588
34.758 ± 0.684
23.479 ± 0.477
35.621 ± 0.618
30.525 ± 0.455
23.525 ± 0.575
32.207 ± 0.416
27.928 ± 0.507
20.411 ± 0.531
30.521 ± 0.619
28.456 ± 0.488
24.159 ± 0.579
22.951 ± 0.412
21.477 ± 0.497
98.257 ± 2.824
-
45.258 ± 0.988
41.597 ± 0.850
46.753 ± 0.751
37.264 ± 0.688
39.314 ± 0.749
50.116 ± 0.963
49.276 ± 0.811
51.741 ± 0.729
92.365 ± 1.158
44.358 ± 0.617
36.719 ± 0.527
38.955 ± 0.432
48.260 ± 0.517
42.637 ± 0.631
40.957 ± 0.718
49.855 ± 0.871
-
37.255 ± 0.716
30.356 ± 0.621
40.159 ± 0.549
30.349 ± 0.533
31.753 ± 0.451
35.627 ± 0.481
41.310 ± 0.507
45.779 ± 0.562
88.126 ± 1.248
40.749 ± 0.477
31.528 ± 0.469
29.746 ± 0.488
42.749 ± 0.591
35.411 ± 0.317
30.177 ± 0.467
42.608 ± 0.475
-
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Chlorgiline
Selegiline
98.589 ± 2.055
94.850 ± 2.114
Table 4. % Inhibition and IC50 (µM) value of the most potent compound 9 against MAO-B
–
–
–
–
–
–
–
10 3 M
10 4 M
10 5 M
10 6 M
10 7 M
10 8 M
10 9 M
Compound
9
IC50 (µM)
92.365
± 1.158
98.589
± 2.055
88.126
± 1.248
94.850
± 2.114
76.425
± 1.051
87.412
± 2.028
70.628
± 0.956
79.558
± 1.057
66.328
± 0.854
66.248
± 1.112
41.259
± 0.557
43.015
± 1.014
20.128
± 0.632
15.107
± 0.340
0.048
± 0.001
0.037
Selegiline
± 0.001
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122.6, 122.3, 115.7, 115.1, 113.4. HRMS (ESI–MS) m/z
C21H15NOS calculated [M + H]+ 329.0869; measured 329.0872.
1-(10H-Phenothiazine-2-yl)-3-(4-methylphenyl)-prop-2-en-1-
one (2)
4. MATERIALS AND METHODS
4.1. Chemistry
NMR spectra of compounds 1–8 and 10 were recorded with a
Bruker 300 MHz digital FT-NMR spectrometer (Bruker
Bioscience, Billerica, MA, USA) while spectras of compounds 9
1
Yield 48 %. Mp: 229–230 ºC. H NMR (DMSO–d6) δ (ppm) 8.78
and 11–16
were recorded using a Varian Mercury Plus
(s, 1H, NH), 7.76-7.69 (m, 4H, olefinic H, arom.H), 7.60 (dd, 1H,
arom. H, J1 = 8.0 Hz, J2 = 1.8 Hz), 7.30 (d, 1H, arom. H, J = 1.7
Hz), 7.26 (d, 2H, arom. H, J = 8.0 Hz), 7.07 (d, 1H, arom. H, J =
8.0 Hz), 7.00 (td, 1H, arom. H, J1 = 7.7 Hz, J2 = 1.5 Hz), 6.91 (dd,
1H, arom. H, J1 = 7.7 Hz, J2 = 1.5 Hz), 6.75 (td, 1H, arom. H, J1 =
7.4 Hz, J2 = 1.2 Hz), 6.65 (dd, 1H, arom. H, J1 = 7.9 Hz, J2 = 1.1
Hz), 2.34 (s, 3H, -CH3). 13C NMR (DMSO_d6) δ (ppm) 188.4,
144.3, 142.6, 141.6, 141.2, 137.4, 132.4, 130.0, 129.3, 128.5,
126.7, 126.6, 123.9, 122.9, 122.6, 121.2, 115.7, 115.1, 113.4,
21.56. HRMS (ESI–MS) m/z C22H17NOS calculated [M + H]+
343.1025; measured 343.1028.
spectrometer 400 MHz (Varian Inc., Palo Alto, CA) in DMSO-d6.
High Resolution Mass Spectra (HRMS) was taken using a liquid
chromatography ion trap-time of flight tandem mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with an
electrospray ionization (ESI) source, operating in both positive
and negative ionization mode. Shimadzu's LCMS Solution
software was used for data analysis. HRMS of compound 9 was
recorded on an Agilent 6530 Accurate-Mass Q-TOF LCMS.
Melting points were determined using an Electrothermal 9100
instrument (IA9100, Bibby Scientific Limited, Staffordshire, UK)
and are uncorrected. Reactions were monitored by Thin Layer
Chromatography (TLC) using silicagel HF254 (Merck Art 5715)
plate under UV lamb (254 and 365 nm, Spectroline, Model ENF-
240C/FE, Spectronics Corporation Westbury, New York U.S.A).
4.1.1. General synthesis method of 1-(10H-Phenothiazin-2-
yl)-3-aryl-2-propen-1-ones, 1–16
1-(10H-Phenothiazine-2-yl)-3-(4-methoxyphenyl)-prop-2-en-
1-one (3)
1
Yield 65 %. Mp: 226–227 ºC. H NMR (DMSO_d6) δ (ppm) 8.79
(s, 1H, NH), 7.70 (d, 1H, olefinic H, J = 15.7 Hz), 7.63 (d, 1H,
olefinic H, J = 15.7 Hz), 7.59 (dd, 1H, arom. H, J1 = 8.1 Hz, J2 =
1.7 Hz), 7.30 (d, 1H, arom. H, J = 1.7 Hz), 7.06 (d, 1H, arom. H,
J = 8.0 Hz), 7.03_6.97 (m, 3H, arom. H), 6.91 (dd, 1H, arom. H,
J1 = 7.7 Hz, J2 = 1.4 Hz), 6.76 (td, 1H, arom. H, J1 = 7.5 Hz, J2 =
1.2 Hz), 6.66 (dd, 1H, arom. H, J1 = 7.9 Hz, J2 = 1.1 Hz), 3.82 (s,
3H, -OCH3).13C NMR (DMSO_d6) δ (ppm) 188.3, 161.8, 144.3,
142.6, 141.7, 137.6, 131.2, 128.4, 127.8, 126.7, 126.6, 123.7,
122.9, 122.5, 119.8, 115.7, 115.1, 114.9, 113.4, 55.9. HRMS
(ESI–MS) m/z C22H17NO2S calculated [M + H]+ 359.0975;
measured 359.0982.
Compounds 1–16 were synthesized according to the our
pevious study[36]. To the mixture of 2-acetylphenothiazine (4.14
mmol) and a suitable arylaldehyde in methanol (20 ml) in 1:1
mol ratios, an aqueous solution of KOH (40%, 4 mL) was added
(Scheme 1). The mixture was refluxed at 70 ºC for 3 h.
Reactions were followed by TLC. The mixture was cooled to
room temperature and poured onto crushed ice containing few
drops of concentrated HCl (37 %) solution and left overnight in a
cooler. The precipitated solid product was collected by filtration
and washed with cold methanol. The crude compounds were
purified by crystallization from suitable amount of DMF:water
mixtures.
1-(10H-Phenothiazine-2-yl)-3-(4-fluorophenyl)-prop-2-en-1-
one (4)
1
Yield 60 %. Mp: 224–225 ºC. H NMR (DMSO_d6) δ (ppm) 8.78
(s, 1H, NH), 7.97_7.92 (m, 2H, arom. H), 7.82 (d, 1H, arom. H, J
= 8.8 Hz), 7.77 (d, 1H, olefinic H, J = 15.8 Hz), 7.70 (d, 1H,
olefinic H, J = 15.8 Hz), 7.66_7.61 (m, 1H, arom. H), 7.33_7.27
(m, 3H, arom. H), 7.09_7.05 (m, 1H, arom. H), 7.00 (td, 1H, arom.
H, J1 = 7.3 Hz, J2 = 1.5 Hz), 6.91 (dd, 1H, arom. H, J1 = 7.7 Hz,
J2 = 1.4 Hz), 6.76 (td, 1H, arom. H, J1 = 7.5 Hz, J2 = 1.2 Hz), 6.66
(dd, 1H, arom. H, J1 = 7.9 Hz, J2 = 1.0 Hz).13C NMR (DMSO_d6)
δ (ppm) 188.3, 165.5, 162.0, 144.3, 142.8, 141.6, 137.5, 131.7,
131.2, 128.5, 126.6, 124.1, 123.0, 122.4, 119.8, 116.4, 115.7,
115.0, 113.4. HRMS (ESI–MS) m/z C21H14FNOS calculated [M +
H]+ 347.0775; measured 347.0775.
1-(10H-Phenothiazine-2-yl)-3-phenyl-prop-2-en-1-one (1)
1
Yield 57 %. Mp: 225–226 ºC. H NMR (DMSO–d6) δ (ppm) 8.79
(s, 1H, NH), 7.87_7.84 (m, 2H, arom. H), 7.79 (d, 1H, olefinic H,
J = 15.7 Hz), 7.71 (d, 1H, olefinic H, J = 15.7 Hz), 7.62 (dd, 1H,
arom. H, J1 = 8.0 Hz, J2 = 1.7 Hz), 7.47_7.45 (m, 3H, arom. H),
7.31 (d, 1H, arom. H, J =1.7 Hz), 7.08 (d, 1H, arom. H, J = 8.0
Hz), 7.00 (td, 1H, arom. H, J1 = 7.6 Hz, J2 = 1.5 Hz), 6.92 (dd, 1H,
arom. H, J1 = 7.7 Hz, J2 = 1.3 Hz), 6.77 (td, 1H, arom. H, J1 = 7.5
Hz, J2 = 1.1 Hz), 6.66 (dd, 1H, arom. H, J1 = 7.9 Hz, J2 = 1.1 Hz).
13C NMR (DMSO_d6) δ (ppm) 188.5, 144.2, 142.6, 141.6, 137.3,
135.1, 131.1, 129.4, 129.3, 128.5, 126.7, 126.6, 124.1, 123.0,
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1-(10H-Phenothiazine-2-yl)-3-(4-chlorophenyl)-prop-2-en-1-
one (5)
1-(10H-Phenothiazine-2-yl)-3-(3-bromophenyl)-prop-2-en-1-
one (8)
1
1
Yield 64 %. Mp: 259–260 ºC. H NMR (DMSO_d6) δ (ppm) 8.79
Yield 69 %. Mp: 234–236 ºC. H NMR (DMSO–d6) δ (ppm) 8.79
(s, 1H, NH), 7.90 (d, 2H, arom. H, J= 8.5 Hz), 7.82 (d, 1H,
olefinic H, J = 15.7 Hz), 7.69 (d, 1H, olefinic H, J = 15.7 Hz),
7.63 (dd, 1H, arom. H, J1 = 8.1 Hz, J2 = 1.7 Hz), 7.52 (d, 2H,
arom. H, J = 8.5 Hz), 7.29 (d, 1H, arom. H, J = 1.7 Hz), 7.08 (d,
1H, arom. H, J = 8.0 Hz), 7.03_6.98 (m, 1H, arom. H), 6.92 (dd,
1H, arom. H, J1 = 7.6 Hz, J2 = 1.1 Hz), 6.79_6.74 (m, 1H, arom.
H), 6.66 (dd, 1H, arom. H, J1 = 7.4 Hz, J2 = 1.0 Hz).13C NMR
(DMSO_d6) δ (ppm) 188.4, 142.8, 142.6, 141.6, 137.2, 135.5,
134.1, 131.0, 129.4, 128.5, 126.7, 126.6, 124.3, 123.13, 123.07,
122.6, 115.6, 115.1, 113.3. HRMS (ESI–MS) m/z C21H14ClNOS
calculated [M + H]+ 363.0479; measured 363.0477.
(s, 1H, NH), 8.16 (d, 1H, arom. H, J = 1.4 Hz), 7.87 (d, 1H,
olefinic H, J = 15.7 Hz), 7.84-7.79 (m, 1H, arom. H), 7.69_7.61
(m, 3H, arom. H), 7.40 (t, 1H, arom. H, J = 7.9 Hz), 7.30 (d, 1H,
arom. H, J = 1.7 Hz), 7.07 (d, 1H, arom. H, J = 8.0 Hz), 7.00 (td,
1H, arom. H, J1 = 7.5 Hz, J2 = 1.4 Hz), 6.91 (dd, 1H, arom. H, J1
= 7.7 Hz, J2 = 1.4 Hz), 6.76 (td, 1H, arom. H, J1 = 7.5 Hz, J2 = 1.2
Hz), 6.66 (dd, 1H, arom. H, J1 = 7.9 Hz, J2 = 1.2 Hz).13C NMR
(DMSO_d6) δ (ppm) 188.3, 142.6, 142.4, 141.6, 137.7, 137.1,
133.5, 131.4, 131.2, 128.6, 128.5, 126.7, 126.6, 124.4, 123.8,
123.3, 122.9, 122.6, 115.6, 115.1, 113.4. HRMS (ESI_MS) m/z
C21H14BrNOS calculated [M + H]+ 408.0052; measured 408.0044.
1-(10H-Phenothiazine-2-yl)-3-(4-nitrophenyl)-prop-2-en-1-
one (9)
1-(10H-Phenothiazine-2-yl)-3-(3-fluorophenyl)-prop-2-en-1-
one (6)
1
1
Yield 95 %. Mp: 230–232 ºC. H NMR (DMSO_d6) δ (ppm) 8.79
Yield 81 %. Mp: 285–287 ºC. H NMR (DMSO–d6) δ (ppm) 8.79
(s, 1H, NH), 7.86 (d, 1H, olefinic H, J = 15.6 Hz), 7.82_7.79 (m,
1H, arom. H), 7.69 (d, 1H, olefinic H, J = 15.6 Hz), 7.68_7.63 (m,
2H, arom. H), 7.53_7.45 (m, 1H, arom. H), 7.30 (d, 1H, arom. H,
J = 1.8 Hz), 7.28_7.24 (m, 1H, arom. H), 7.08 (d, 1H, arom. H, J
= 8.0 Hz), 7.00 (td, 1H, arom. H, J1 = 7.6 Hz, J2 = 1.5 Hz), 6.91
(dd, 1H, arom. H, J1 = 7.7 Hz, J2 = 1.4 Hz), 6.76 (td, 1H, arom. H,
J1 = 7.5 Hz, J2 = 1.2 Hz), 6.66 (dd, 1H, arom. H, J1 = 7.9 Hz, J2 =
(s, 1H, NH), 8.25 (d, 2H, arom. H, J = 8.05 Hz), 8.11 (d, 2H,
arom. H, J = 8.05 Hz), 7.96 (d, 1H, olefinic H, J = 15.7 Hz), 7.76
(d, 1H, olefinic H, J = 15.7 Hz), 7.64 (d, 1H, arom. H, J = 7.69
Hz), 7.27 (s, 1H, arom. H), 7.07 (d, 1H, arom. H, J = 7.7 Hz),
6.98 (t, 1H, arom. H, J = 7.3 Hz), 6.88 (d, 1H, arom. H, J = 7.3
Hz), 6.72 (t, 1H, arom. H, J = 7.3 Hz), 6.63 (d, 1H, arom. H, J =
7.7 Hz).13C NMR (DMSO_d6) δ (ppm) 188.5, 148.7, 142.9, 141.9,
1.2 Hz). 13C NMR (DMSO_d6) δ (ppm) 188.4, 164.6, 161.3, 142.7, 141.7, 141.5, 137.1, 130.5, 130.4, 128.7, 127.0, 126.8, 126.6,
141.6, 137.7, 137.1, 131.3, 128.5, 126.7, 126.6, 125.9, 124.4,
123.8, 123.2, 122.6, 117.8, 117.6, 115.6, 115.0, 113.3. HRMS
(ESI–MS) m/z C21H14FNOS calculated [M + H]+ 347.0775;
measured 347.0772.
124.62, 124.61, 123.6, 122.8, 115.3, 113.5. HRMS (ESI_MS)
m/z C21H14N2O3S calculated [M]+ 374.07251; measured
374.07134.
1-(10H-Phenothiazine-2-yl)-3-(3-nitrophenyl)-prop-2-en-1-
one (10)
1-(10H-Phenothiazine-2-yl)-3-(2-fluorophenyl)-prop-2-en-1-
1
one (7)
Yield 38 %. Mp: 250–252 ºC. H NMR (DMSO_d6) δ (ppm) 8.80
1
Yield 38 %. Mp: 219–220 ºC. H NMR (DMSO_d6) δ (ppm) 8.81
(s, 1H, NH), 8.74 (s, 1H, arom. H), 8.32-8.24 (m, 2H, arom. H),
8.01 (d, 1H, olefinic H, J = 15.7 Hz), 7.81 (d, 1H, olefinic H, J =
15.7 Hz), 7.75 (d, 1H, arom. H, J = 8.0 Hz), 7.71-7.68 (m, 1H,
arom. H), 7.30 (d, 1H, arom. H, J = 1.7 Hz), 7.08 (d, 1H, arom. H,
J = 8.0 Hz), 7.00 (td, 1H, arom. H, J1 = 7.6 Hz, J2 = 1.3 Hz), 6.90
(dd, 1H, arom. H, J1 = 6.3 Hz, J2 = 1.3 Hz), 6.76 (td, 1H, arom. H,
J1 = 7.6 Hz, J2 = 1.3 Hz), 6.65 (dd, 1H, arom. H, J1 = 8 Hz, J2 =
1.1 Hz).13C NMR (DMSO_d6) δ (ppm) 188.3, 148.9, 142.6, 141.7,
141.5, 137.1, 137.0, 135.5, 130.8, 128.5, 126.7, 126.6, 125.1,
125.0, 124.6, 123.4, 122.6, 115.6, 115.1, 113.3. HRMS (ESI–
MS) m/z C21H14N2O3S calculated [M + H]+ 375.0798; measured
375.0795.
(s, 1H, NH), 8.06 (td, 1H, arom. H, J1 = 7.8 Hz, J2 = 1.5 Hz), 7.85
(d, 1H, olefinic H, J = 15.8 Hz), 7.78 (d, 1H, olefinic H, J = 15.8
Hz), 7.59 (dd, 1H, arom. H, J1 = 8.0 Hz, J2 = 1.8 Hz), 7.55_7.48
(m, 1H, arom. H), 7.35_7.28 (m, 3H, arom. H), 7.08 (d, 1H, arom.
H, J = 8.0 Hz), 7.00 (td, 1H, arom. H, J1 = 7.6 Hz, J2 = 1.5 Hz),
6.91 (dd, 1H, arom. H, J1 = 7.7 Hz, J2 = 1.4 Hz), 6.76 (td, 1H,
arom. H, J1 = 7.5 Hz, J2 = 1.2 Hz), 6.66 (dd, 1H, arom. H, J1 = 8.0
Hz, J2 = 1.2 Hz). 13C NMR (DMSO_d6) δ (ppm) 188.2, 142.7,
141.5, 137.0, 135.5, 133.2, 133.0, 129.8, 128.5, 126.7, 125.5,
124.4, 123.1, 122.8, 122.7, 122.6, 116.7, 116.4, 115.6, 115.1,
113.3. HRMS (ESI–MS) m/z C21H14FNOS calculated [M + H]+
347.0775; measured 347.0766.
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1-(10H-Phenothiazine-2-yl)-3-(thiophene-2-yl)-prop-2-en-1-
one (11)
1-(10H-Phenothiazine-2-yl)-3-(2,4-dichlorophenyl)-prop-2-en-
1-one (14)
1
Yield % 72. Mp: 244–245 ºC. 1H NMR (DMSO–d6) δ (ppm) 8.79
(s, 1H, NH), 7.87 (d, 1H, olefinic H, J = 15.4 Hz), 7.77 (d, 1H,
arom. H, J = 5.1 Hz), 7.65 (d, 1H, arom. H, J = 3.7 Hz), 7.49 (dd,
1H, arom. H, J1 = 8.1 Hz, J2= 1.5 Hz), 7.39 (d, 1H, olefinic H, J =
15.4 Hz), 7.26_7.17 (m, 1H, arom. H), 7.05 (d, 1H, arom. H, J =
7.7 Hz), 7.01_6.97 (m, 2H, arom. H), 6.90 (dd, 1H, arom. H, J1 =
7.7 Hz, J2 = 1.1 Hz), 6.75 (td, 1H, arom. H, J1 = 7.3 Hz, J2 = 1.1
Hz), 6.64 (dd, 1H, arom. H, J1 = 7.5 Hz, J2 = 1.1 Hz).13C NMR
(DMSO_d6) δ (ppm) 188.1, 142.8, 141.8, 140.4, 137.4, 137.3,
133.8, 131.1, 129.5, 128.7, 127.0, 126.9, 124.2, 123.0, 122.8,
120.6, 115.9, 115.3, 113.6. HRMS (ESI–MS) m/z C19H13NOS2
calculated [M + H]+ 336.0511; measured 336.0496.
Yield 56 %. Mp: 228–230 ºC. H NMR (DMSO–d6) δ (ppm) 8.78
(s, 1H, NH), 8.13 (d, 1H, arom. H, J = 8.5 Hz), 7.89 (d, 1H,
olefinic H, J = 15.6 Hz), 7.80 (d, 1H, olefinic H, J = 15.6 Hz),
7.70 (d, 1H, arom. H, J = 2.0 Hz), 7.58 (dd, 1H, arom. H, J1 = 8.1
Hz, J2 = 1.8 Hz), 7.49 (dd, 1H, arom. H, J1 = 8.5 Hz, J2 = 2.0 Hz),
7.24 (s, 1H, arom. H), 7.05 (d, 1H, arom. H, J = 8.1 Hz), 7.0_6.95
(m, 1H, arom. H), 6.88 (d, 1H, arom. H, J = 7.7 Hz), 6.77_6.72
(m, 1H, arom. H), 6.62 (d, 1H, arom. H, J = 8.1 Hz). 13C NMR
(DMSO_d6) δ (ppm) 184.4, 142.9, 141.6, 137.82, 137.81, 137.1,
136.33, 136.32, 135.8, 132.0, 130.3, 130.2, 128.8, 128.7, 127.0,
125.7, 125.0, 123.5, 123.0, 115.3, 113.4. HRMS (ESI–MS) m/z
C21H13Cl2NOS calculated [M
398.0164.
+
H]+ 398.0168; measured
1-(10H-Phenothiazine-2-yl)-3-(4-trifluoromethylphenyl)-prop-
2-en-1-one (12)
1-(10H-Phenothiazine-2-yl)-3-(3,4-dichlorophenyl)-prop-2-en-
1-one (15)
1
Yield 85 %. Mp: 266–268 ºC. H NMR (DMSO–d6) δ (ppm) 8.80
1
(s, 1H, NH), 8.07 (d, 2H, arom. H, J = 8.1 Hz), 7.92 (d, 1H,
olefinic H, J = 15.7 Hz), 7.79 (d, 1H, arom. H, J = 8.4 Hz), 7.74
(d, 1H, olefinic H, J = 15.7 Hz), 7.64 (dd, 1H, arom. H, J1 = 8.1
Hz, J2 = 1.8 Hz), 7.28 (d, 1H, arom. H, J = 1.5 Hz), 7.08 (d, 1H,
arom. H, J = 8.05 Hz), 7.00_6.92 (m, 1H, arom. H), 6.76_6.74 (m,
1H, arom. H), 6.90 (d, 1H, arom. H, J = 7.6 Hz), 6.64 (d, 2H,
Yield 48 %. Mp: 236–238 ºC. H NMR (DMSO–d6) δ (ppm) 8.77
(s, 1H, NH), 8.2 (d, 1H, arom. H, J = 1.8 Hz), 7.83 (d, 1H, olefinic
H, J = 15.7 Hz), 7.79 (dd, 1H, arom. H, J1 = 8.4 Hz, J2 = 1.8 Hz),
7.67 (d, 1H, arom. H, J = 8.4 Hz), 7.66 (d, 1H, olefinic H, J =
15.7 Hz), 7.60 (dd, 1H, arom. H, J1 = 7.7 Hz, J2 = 1.8 Hz), 7.24 (d,
1H, arom. H, J = 1.8 Hz), 7.05 (d, 1H, arom. H, J = 8.1 Hz),
arom. H, J = 8.05 Hz).13C NMR (DMSO_d6) δ (ppm) 188.6, 142.9, 6.98_6.94 (m, 1H, arom. H), 6.89 (d, 1H, arom. H, J = 7.7 Hz),
142.4, 141.7, 139.4, 137.2, 130.1, 128.7, 127.4, 127.0, 126.9,
126.4, 126.3, 125.2, 124.8, 123.5, 122.8, 115.8, 115.3, 113.5.
6.76_6.72 (m, 1H, arom. H), 6.64 (d, 1H, arom. H, J = 7.3 Hz).
13C NMR (DMSO_d6) δ (ppm) 184.6, 142.8, 141.67, 141.66,
137.2, 136.2, 133.4, 132.5, 131.7, 130.8, 129.7, 127.0, 126.9,
124.8, 124.5, 123.5, 122.9, 115.8, 115.3, 113.5. HRMS (ESI–
HRMS (ESI–MS) m/z C22H14F3NOS calculated [M
+
H]+
398.0821; measured 398.0818.
1-(10H-Phenothiazine-2-yl)-3-(4-benzyloxyphenyl)-prop-2-en-
1-one (13)
MS) m/z C21H13Cl2NOS calculated [M
measured 398.0159.
+ H]+ 398.0168;
1
Yield 33 %. Mp: 217–219 ºC. H NMR (DMSO–d6) δ (ppm) 8.76
1-(10H-Phenothiazine-2-yl)-3-(3,4methylenedioxyphenyl)-
(s, 1H, NH), 7.76 (d, 2H, arom. H, J = 8.8 Hz), 7.63 (d, 1H,
olefinic H, J = 20.5 Hz), 7.57 (d, 1H, olefinic H, J = 20.5 Hz),
7.53 (d, 1H, arom. H, J = 8.1 Hz), 7.42 (d, 1H, arom. H, J = 7.3
Hz), 7.38_7.34 (m, 1H, arom. H), 7.32_7.30 (m, 1H, arom. H),
7.25 (d, 1H, arom. H, J = 0.7 Hz), 7.06_6.97 (m, 4H, arom. H),
6.88 (d, 2H, arom. H, J = 7.7 Hz), 6.75 (d, 1H, arom. H, J = 7.3
Hz), 6.64 (d, 2H, arom. H, J = 7.7 Hz), 5.13 (s, 2H, CH2).13C
NMR (DMSO_d6) δ (ppm) 188.7, 161.1, 144.5, 142.8, 141.8,
137.8, 137.3, 131.4, 129.2, 128.7, 128.44, 128.43, 128.1, 127.0,
126.9, 124.0, 123.1, 122.9, 120.0, 116.0, 115.9, 115.3, 113.5,
70.1. HRMS (ESI–MS) m/z C28H21NO2S calculated [M + H]+
436.1366; measured 436.1356.
prop-2-en-1-one (16)
Yield 87 %. Mp: 220–222 ºC. H NMR (DMSO_d6) δ (ppm) 8.74
1
(s, 1H, NH), 7.59 (s, 1H, arom. H), 7.55 (dd, 1H, arom. H, J1 =
8.05 Hz, J2 = 1.8 Hz), 7.51 (d, 1H, arom. H, J = 1.1 Hz), 7.26 (d,
1H, arom. H, J = 1.8 Hz), 7.24 (d, 1H, arom. H, J = 1.8 Hz), 7.23
(s, 1H, arom. H), 7.0 (d, 1H, arom. H, J = 8.1 Hz), 6.98 (d, 1H,
olefinic H, J = 16.1 Hz), 6.94 (d, 1H, olefinic H, J = 16.1 Hz),
6.74 (d, 1H, arom. H, J = 7.7 Hz), 6.87 (dd, 1H, arom. H, J1 = 7.8
Hz, J2 = 1.1 Hz), 6.63 (dd, 1H, arom. H, J1 = 7.8 Hz, J2 = 1.1 Hz),
6.05 (s, 2H, -CH2-). 13C NMR (DMSO–d6) δ (ppm) 188.6, 150.3,
148.8, 144.6, 142.8, 141.9, 137.7, 129.8, 128.7, 127.0, 126.8,
126.5, 124.1, 123.2, 122.8, 120.4, 115.9, 115.3, 113.5, 109.3,
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107.5, 102.3. HRMS (ESI–MS) m/z C22H15NO3S calculated [M +
H]+ 374.0845; measured 374.0847.
Declaration of Interest
“The authors declared no conflict of interest” in the manuscript.
Captions
4.2. Biological assays
4.2.1. Anticholinesterase and Butyrylcholinesterase
Inhibition Assay
Figure 1. Chemical structures of phenothiazine and its
derivatives
Acetylcholinesterase (AChE, E.C.3.1.1.7, from electric eel),
butyrylcholinesterase (BChE from equine serum), 5,5'-dithiobis-
(2-nitrobenzoic acide) (DTNB), donepezil hydrochloride and
tacrine were purchased from Sigma-Aldrich (Steinheim,
Germany). Acetylthiocholine iodide (ATC) and butyrylthiocholine
iodide (BTC) were obtained from Fluka (Germany). All pipetting
processes were performed using Biotek Precision XS robotic
system (USA). Measurements of the percentage inhibition were
carried out at 412 nm by using BioTek Synergy H1 microplate
reader (USA). The inhibitory activity of compounds against
AChE and BChE was determined in 96-well plates by modified
Ellman‟s method[32,34,37] using donepezil and tacrine as reference
drugs.
Figur 2. Lead compounds of the study: 3 (AChE) and 9 (AChE
and MAO-B)
Scheme 1. Synthesis of the phenothiazine-based chalcones
Table 1. Cholinesterases (AChE, BuChE) inhibitory effects (%
inhibition) of the compounds 1–16
Table 2. % Inhibition and IC50 (µM) values of the most potent
compounds 3 and 9 against AChE
Table 3. Monoamine oxidases (MAO-A, MAO-B) inhibitory
effects (% inhibition) of the compounds 1–16
Table 4. % Inhibition and IC50 (µM) value of the most potent
compound 9 against MAO-B
Key words: Cholinesterase, monoamine oxidase, chalcone,
phenothiazine, Alzheimer‟s disease, enzyme inhibitors,
Claisen-Schmidt
4.2.2. MAO-A and MAO-B Inhibition Assay
Ampliflu™
Red
(10-Acetyl-3,7-dihydroxyphenoxazine),
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peroxidase from horseradish, hMAO-A, hMAO-B, H2O2, tyramine
hydrochloride, selegiline and clorgiline were purchased from
Sigma-Aldrich (Steinheim, Germany) and retained under the
suggested conditions by supplier. All pipetting processes were
performed using a Biotek Precision XS robotic system (USA).
Measurements were carried out by a BioTek-Synergy H1
microplate reader (USA) based on the fluorescence generated
(excitation, 535 nm, emission, 587 nm) over a 30-min period, in
which the fluorescence increased linearly[38-40]. Blank, control
and all concentrations of inhibitors were analyzed in
quadruplicate and inhibition percent was calculated by using the
following equation:
[2]
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FCt2: Fluorescence of a control well measured at t2 time, FCt1:
Fluorescence of a control well measured at t1 time, FIt2:
Fluorescence of an inhibitor well measured at t2 time, FIt1:
Fluorescence of an inhibitor well measured at t1 time.
The IC50 values were calculated from a dose-response
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log concentration with the use of GraphPad „PRISM‟ software
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