Novel thiazolo[5,4-b]phenothiazine derivatives: Synthesis, structural characterization, and in vitro evaluation of antiproliferative activity against human leukaemia

Article abstract of DOI:10.3390/ijms18071365

The molecular frame of the reported series of new polyheterocyclic compounds was intended to combine the potent phenothiazine and benzothiazole pharmacophoric units. The synthetic strategy applied was based on oxidative cyclization of N-(phenothiazin-3-yl

Full text of DOI:10.3390/ijms18071365

International Journal of
Molecular Sciences
Article
Novel Thiazolo[5,4-b]phenothiazine Derivatives:
Synthesis, Structural Characterization, and In Vitro
Evaluation of Antiproliferative Activity against
Human Leukaemia
Balazs Brem 1, Emese Gal 1, Luiza Găină 1, Luminit¸a Silaghi-Dumitrescu 1, Eva Fischer-Fodor 2,3
,
Ciprian Ionu¸t Tomuleasa 2,4, Adriana Grozav 5, Valentin Zaharia 5, Lorena Filip 5,* and
Castelia Cristea 1,
*
1
Faculty of Chemistry and Chemical Engineering, Babes¸-Bolyai University, 400028 Cluj-Napoca, Romania;
bbrem@chem.ubbcluj.ro (B.B.); emese@chem.ubbcluj.ro (E.G.); gluiza@chem.ubbcluj.ro (L.G.);
lusi@chem.ubbcluj.ro (L.S.-D.)
2
3
4
5
Tumor Biology Department, Ion Chiricuta Oncology Institute, 400015 Cluj-Napoca, Romania;
efischerfodor@yahoo.com (E.F.-F.); ciprian.tomuleasa@umfcluj.ro (C.I.T.)
Medfuture Research Center, Iuliu Hatieganu University of Medicine and Pharmacy,
400012 Cluj-Napoca, Romania
Research Center for Functional Genomics and Translational Medicine,
Iuliu Hatieganu University of Medicine and Pharmacy, 400012 Cluj-Napoca, Romania
Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, 400012 Cluj-Napoca, Romania;
adriana.ignat@umfcluj.ro (A.G.); vzaharia@umfcluj.ro (V.Z.)
*
Correspondence: lfilip@umfcluj.ro (L.F.); castelia@chem.ubbcluj.ro (C.C.); Tel.: +40-264-583833 (C.C.)
Received: 17 May 2017; Accepted: 16 June 2017; Published: 26 June 2017
Abstract: The molecular frame of the reported series of new polyheterocyclic compounds
was intended to combine the potent phenothiazine and benzothiazole pharmacophoric units.
The synthetic strategy applied was based on oxidative cyclization of N-(phenothiazin-3-yl)-thioamides
and it was validated by the preparation of new 2-alkyl- and 2-aryl-thiazolo[5,4-b]phenothiazine
derivatives. Optical properties of the series were experimentally emphasized by UV-Vis
absorption/emission spectroscopy and structural features were theoretically modelled using density
functional theory (DFT). In vitro activity as antileukemic agents of thiazolo[5,4-b]phenothiazine and
N-(phenothiazine-3-yl)-thioamides were comparatively evaluated using cultivated HL-60 human
promyelocytic and THP-1 human monocytic leukaemia cell lines. Some representatives proved
selectivity against tumour cell lines, cytotoxicity, apoptosis induction, and cellular metabolism
impairment capacity. 2-Naphthyl-thiazolo[5,4-b]phenothiazine was identified as the most effective of
the series by displaying against THP-1 cell lines a cytotoxicity close to cytarabine antineoplastic agent.
Keywords: phenothiazine; thiazole; antiproliferative activity; structure-activity relationship
1. Introduction
Heterocyclic compounds containing nitrogen and sulphur were frequently evidenced in
many biological active compounds of both natural and synthetic origins. Two heteroaromatic
compounds commonly recognized for a broad spectrum of pharmacological activities are tricyclic
phenothiazine (PTZ) [1] and bicyclic benzothiazole (BTA) [2], each of them with individual synthetic
representatives sharing high therapeutic potency as antimicrobial, antimalarial, anthelmintic, analgesic,
or anti-inflammatory drugs. The antimicrobial activity of the phenothiazinium salts was exploited
in medical and environmental research [3], while the electronic properties of the neutral PTZ-based
Int. J. Mol. Sci. 2017, 18, 1365; doi:10.3390/ijms18071365
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 1365
2 of 14
chromophores finely tuned by variable substitution on the peripheral benzene rings [
versatile substrate that is recommended for materials scientific investigations.
4–6
] make this a
Recently, the potential anticancer activity of both PTZ and BTA seemed to be of high
research interest. PTZ derivatives, commonly highly related to their neuroleptic action, also
exhibited anti-proliferative effect, anti-calmodulin (CAM) action, and inhibition of protein kinases or
P-glycoprotein (Pgp) transport function [
correlated to the substituents attached to the carbon atoms of the ring, which increased lipophilicity,
and less correlated to the nature of the side chain connected to the heterocyclic nitrogen atom [ ].
7]. The anti-proliferative activity appeared more strongly
8
A direct interaction between PTZ derivatives capable of creating hydrogen bonds and Pgp was also
documented as crucial for inhibition of the transport function of the protein and increase of cellular
chemosensitivity [
anticancer effects on various cell lines [10
leukaemia cells [12 13]. BTA derivatives were also evidenced as anticancer agents based on their
9]. Synthetic azaphenothiazines and benzo[a]phenothiazines presented in vitro
,11]. Some antipsychotic PTZ drugs showed effects on
,
topoisomerase, microtubule polymerization, or Cytochrome P450 enzyme inhibition activity [14].
Merging the structure of BTA with other heterocycles appeared as a versatile approach in the design of
drug-like molecules with a new pharmacological profile, action, or toxicity and therefore, 2-substituted
BTA derivatives were encompassed in the design of several novel drug candidates [15–17].
As a synthetic target, 2-substituted BTA may be available by alternative routes involving the
condensation of 2-aminothiophenols with carbonyl/carboxylic acid derivatives (acyl chlorides, esters,
nitriles), or the cyclization of thiobenzanilides, with a wide range of methodologies developed in order
to improve the reaction selectivity, purity, and yield [18]. The intramolecular cyclization of anilides
appears as a convenient alternative prompted by copper- [19–24] or iron-based [25] catalysts.
The association of the potent PTZ and thiazole pharmacophores under the same molecular
frame was the target of our on-going concern for a rational design of new biologically active
compounds. In some of our previous reports, these heterocyclic units appeared separated by a spacer
(antitumoral phenothiazinyl-thiazolyl-hydrazine derivatives [26]) or directly joined (antimicrobial
benzothiazolyl-phenothiazine derivatives [27,28]). In this work, we described the synthesis and
characterization of new representatives of polyheterocyclic compounds containing benzo-fused
BTA/PTZ. In the structure of the new 2-substituted-Thiazolo[5,4-b]phenothiazine derivatives (TAPTZ),
heterocyclic PTZ and BTA units share a central benzene ring and thus offer a similar substitution
pattern that is significant for their biological activity. In support of our reasoning, Figure 1 shows the
chemical structures of selected PTZ and BTA derivatives with anti-proliferative activity, together with
their beneficial overlap in the new TAPTZ derivatives. The in vitro biological experiments described
herein were designated to comparatively evaluate the anti-proliferative efficacy of target TAPTZ versus
their PTZ precursors as antileukemic agents in human leukaemia cell growth.
Figure 1. Generic chemical structure and significant substitution pattern for antiproliferative agents
derived from PTZ and BTA and their structural complementarities with the target polyheterocyclic
TAPTZ structure.

Int. J. Mol. Sci. 2017, 18, 1365
3 of 14
2. Results
2.1. Synthesis of Thiazolo[5,4-b]phenothiazine Derivatives
The synthetic strategy applied for the preparation of the new TAPTZ heterocyclic structure was
founded on the selection of PTZ as a building block laid open for assembling the BTA unit, as illustrated
in Scheme 1.
Scheme 1. Synthetic path to Thiazolo[5,4-b]phenothiazine derivatives TAPTZ.
The starting N-(phenothiazin-3-yl)-amides 1a
(conveniently accessible according to our reported microwaves assisted amination procedure [29]) by
acylation with different acyl chlorides. The N-(phenothiazine-3-yl)-thioamides 2a were synthesized
in variable yields using Lawesson’s reagent for the conversion of amide functional group into its
thio-analogue. In the last step TAPTZ 3a were obtained by thiazole ring closure under oxidative
–e, were prepared from 3-amino-PTZ precursor
–
e
–
e
conditions [25]. The best cyclization yields were obtained in the synthesis of 3d by taking benefit of the
electron withdrawing effect of the nitro substituent which disabled the competitive conversion to 1d.
The structure of each new TAPTZ 3a
starting amide 1a
were unambiguously assigned by 1H-/13C-NMR, UV-vis, and IR spectroscopy
and confirmed by high resolution mass spectrometry.(1H-/13C-NMR and ESI-HRMS spectra of 3a
are presented in supplementary material). In 1H-NMR spectra of 3a
, the key signals arising from
–e, N-(phenothiazine-3-yl)-thioamide 2a–e intermediate, and
–
e
–e
–
e
the protons attached to the central benzene ring gave two distinct singlets situated in the regions
7.74–7.84 and 7.18–7.20 ppm, respectively. The more shielded singlet corresponded to the proton
situated in the spatial proximity of the N-methyl group, as confirmed by homonuclear two-dimensional
Nuclear Overhauser Effect Spectroscopy 2D NOESY experiments involving saturation of the transitions
corresponding to the three equivalent protons of the methyl group (2D-NOESY spectrum of 3a is
presented in supplementary material). The chemical shift value of the low field singlet appeared
slightly influenced by the nature of the substituent attached to the marginal thiazole ring. (NMR
spectra of 3a–e are presented in supplementary material).
2.2. Spectral Properties
Each TAPTZ 3a–e exhibited two UV absorption bands situated in the range 250–264 and
326–424 nm, respectively (Table 1). The allowed electronic transitions typical to the phenothiazine
core were responsible for the higher energy absorption band also observable in the spectra of
N-(phenothiazine-3-yl)-thioamides 2a–e and amides 1a–e, respectively, as exemplified in Figure 2a
by the overlaid UV absorption spectra of the phenyl substituted derivatives. The second absorption
band situated at longer wavelengths was characterized by lower intensity and a strong dependence
of its location on the electronic properties of the substituents attached in position 2 of the thiazole
unit (Figure 2b). The absorption of t-butyl-TAPTZ 3a occurred at higher energy, while the aromatic
substituents enabled a bathochromic shift of 44–98 nm.

Int. J. Mol. Sci. 2017, 18, 1365
4 of 14
Figure 2. UV Ultraviolet absorption spectra recorded in acetonitrile solution at concentration 10−5 M:
) phenyl-TAPTZ 3b, N-(phenothiazine-3-yl)-thiobenzamide 2b, and N-(phenothiazine-3-yl)-benzamide
(a
1b; (b) normalized longest wavelength absorption band for TAPTZ 3a–e (a.u. = arbitrary units).
Table 1. Experimental UVultraviolet-vis spectral properties and computational data for TAPTZ derivatives.
1
Stokes Shift 3
Egap,opt
(eV)
EHOMO
(eV)
ELUMO
(eV)
Dipole
2
4
5
5
λmax,abs (nm)
λ
max, em
(nm)
Cpd
3a
(cm
)
Moment 5 (D)
−1
(ε (M cm−1))
−1
263 (28,900)
326 (7400)
447
550
564
8300
4.49
4.28
4.18
−5.07
−5.07
−5.17
−0.78
−1.54
−1.77
2.68
2.67
3.92
261 (43,400)
370 (15,800)
3b
8800
8800
262 (32,800)
377 (12,200)
3c
264 (29,600)
300 (12,600)
424 (9300)
3d
-
-
−5.36
−2.72
7.63
250 (23,300)
294 (8900)
373 (8900)
3e
608
2
10,400
4.07
−5.07
−1.69
2.55
10−5 M in acetonitrile solution; after irradiation with the longest absorption wavelength; ∆ν = 1/λmax,abs
determined from the cross-section of absorption and emission spectra; computed using density
functional theory (DFT) level of theory B3LYP hybrid functional with 6-31G* basis set.
1
3
4
5
−
1/λmax,e
;
In acetonitrile solution, upon excitation with the corresponding longest wavelength absorption
maxima, TAPTZ derivatives generated broad, unstructured emission bands situated in the visible
range (Figure 3), excepting the most polar nitro-phenyl-TAPTZ 3d, with emission quenched by dipolar
interactions with the solvent. Large Stokes shift values (Table 1) typical to phenothiazine derivatives
were detected, suggesting a significant structural reorganization of the polarized excited state as
compared to the ground state [30].

Int. J. Mol. Sci. 2017, 18, 1365
5 of 14
Figure 3. Normalized emission bands of 2-substituted-Thiazolo[5,4-b]phenothiazine derivatives 3a−3e
(excitation with the corresponding longest wavelength absorption maxima).
2.3. Computational Data
The molecular structures of the new TAPTZ 3a–e have been studied by using density functional
theory (DFT) as implemented in the Spartan 06 software package (Version 06, Wavefunction, Inc.,
Irvine, CA, USA). The energies of frontier molecular orbitals (FMOs) (EHOMO, ELUMO) corresponding
to the optimized geometry of the lowest energy structure were computed at the B3LYP/6-31G* level of
theory and the results were summarized in Table 1. The FMO plots for each TAPTZ indicated that
highest occupied molecular orbital HOMO was located on the PTZ core, while lowest unoccupied
molecular orbital LUMO included the BTA and the aromatic substituent. Figure 4 shows the frontier
molecular orbital density plots for naphthyl-TAPTZ 3e (frontier molecular orbital density plots for
compounds 3a-d are presented in supplementary material).
HOMO
LUMO
Figure 4. Frontier molecular orbital density plots for TAPTZ 3e resulted from DFT calculations
performed at B3LYP/6-31G* level of theory (the iso-contour value was set to 0.02). The orbital wave
functions are positive in regions coloured in red and negative in regions coloured in blue. The ball and
stick molecular models display carbon atoms in grey, hydrogen atoms in white, sulfur atoms in yellow
and nitorogen atoms in blue.
The variation of electron density in TAPTZ 3a–e was modelled by generating the molecular
electrostatic potential surface (EPS), as illustrated in Figure 5, which displays the overall molecular
shape with electron rich regions coloured in red and electron poor regions in blue colour on the surface.
In each case, the heteroatoms of the phenothiazine core shared comparable electron densities, while
the thiazole ring appeared much more polarized, accumulating the negative charge on the nitrogen
atom, and, consequently, it may be designated as the most probable site for intermolecular electrostatic
interactions. The substituent attached in position 2 slightly modulates the electrostatic charge on
heteroatoms, as depicted in Figure 5.
The overall polarization of the polyheterocyclic structure expressed in terms of dipole moment is
presented in Table 1.

Int. J. Mol. Sci. 2017, 18, 1365
6 of 14
Figure 5. Molecular electrostatic potential surface (EPS) plots of TAPTZ 3a–e hierarchized in order of
computed energy of the most stable conformer; blue colour on the surface represents highly positive
potential regions, red colour represents highly negative potentials, while orange, yellow and green
colours depict intermediate values of potential. Electrostatic charge is depicted on heteroatoms.
2.4. Biological Properties
The antiproliferative activity of the obtained series of N-(phenothiazine-3-yl)-thioamides 2a
–e and
polyheterocyclic TAPTZ 3a was investigated in vitro using cultured HL-60 human promyelocytic
–e
and THP-1 human monocytic leukaemia cell lines, versus cytarabine, an antineoplastic agent used
in the treatment of leukaemia. The cytotoxicity of the studied compounds was assessed using the
MTT colorimetric assay and quantified using the parameter median inhibitory concentration (IC50);
their inhibitory activity against tumour and normal cells in vitro covered a wide range of values, as
summarized in Table 2.
Table 2. Half inhibitory concentration (IC50) calculated using sigmoidal dose-response relationship for
N-(phenothiazine-3-yl)-thioamides (2a, 2c, 2e), TAPTZ 3a, 3c, 3e, and cytarabine, respectively, against
THP-1 and HL-60 tumour cells and normal peripheral blood mononuclear cells (PBMCs) in vitro.
Antiproliferative Activity IC50 (µM)
Cpd
THP-1
HL-60
PBMC
101.70 ± 0.11 b
175.8 ± 0.23 b
2a
2b
2c
2e
3a
3b
3c
3e
>2000
1121
1481 a
>2000
88.9 ± 0.03 b
>2000
69.1 ± 0.19 b
>2000
>2000
227 a
463.4 ± 0.16 b
194.3 ± 0.17 b
338.9 ± 0.14 b
1066 a
>2000
1906 a
>1000
>2000
1241 a
>1000
21.6 ± 0.06 b
67.2 ± 0.09 b
9.0 ± 0.05 b
13.5 ± 0.05 b
Cytarabine
Estimated outside the 95% confidence interval; b Standard deviation of three time independent tests.
a
Nitro-derivatives 2d, 3d did not demonstrate significant growth inhibition against either tumour
cells or normal cells. A close inspection of the IC50 values listed in Table 2 shows that naphthyl
substituted TAPTZ 3e displayed the most noticeable cytotoxic effect, with an estimated IC50 value
closest to cytarabine standard against THP-1 and a less pronounced anti-proliferative activity against
HL-60 cells, while the corresponding N-(phenothiazin-3-yl)-thioamide 2e seemed completely inactive.
None of the compounds with anti-proliferative activity against tumour cells (3e, 2a, 2c) influenced the

Int. J. Mol. Sci. 2017, 18, 1365
7 of 14
survival of the normal peripheral blood mononuclear cells (PBMCs) during the same time period and
concentration range, thus proving a marked selectivity.
The most efficient compounds (3e, 2a, 2c) were tested for their capacity to trigger the programmed
cell death in the leukaemia cell lines (Figure 6). The early apoptotic process was revealed within 4 h after
the exposure of the cells to the tested compounds, and the tendency rose when cells were treated for 8 h.
A remarkable apoptotic effect was observed for TAPTZ 3e on both THP-1 and HL-60 cell lines (one-way
analysis of variance, Dunnet multiple comparison test, p < 0.0001). N-(Phenothiazin-3-yl)-pivalamide
2a was also effective against both cell lines. The parallel measurement of necrotic cells stained with
propidium iodide (PI) also displayed the largest number of necrotic cells in populations treated with
TAPTZ 3e, while 2a caused necrosis mainly in THP-1 cells. The apoptosis induction capacity of 2c
,
although inferior to 3e in THP-1 cells (one-way analysis of variance ANOVA, Bonferroni post-test,
p < 0.001) and at the 8-h time point even in HL-60 cells, is well balanced by the low proportion of
necrotic cells, showing a prevalence to programmed cell death induction. A similar behaviour of
PTZ derivatives was previously mentioned with respect to compound selectivity towards normal
lymphocytes [31,32] and therefore we presume that the mechanism of apoptosis induction in treated
THP-1 and HL-60 cells may be associated with the inhibition of mitochondrial DNA polymerase,
decreased ATP production, and caspases fragmentation.
Figure 6. Apoptosis induction capacity of N-(phenothiazin-3-yl)-thioamides 2a, 2c, and TAPTZ
3e monitored by fluorescence emission measured at 530–575 nm (488 nm excitation) upon human
monocytic leukaemia cell THP-1 and human promyelocytic leukaemia cell HL-60 populations stained
with: Annexin V (above), propidium iodide (below).
The alteration of cell reducing capacity after the treatment with N-(phenothiazin-3-yl)-thioamides
in series
as an indicator of cellular metabolism impairment [33] (supplementary material Figure S1). The THP-1
cell reducing capacity diminished significantly after treatment with 2a 2c 3a 3c 3e. Compounds 2c
3a 3b 3e showed similar activity against HL-60 populations, and according to the correlation with the
2 and TAPTZ in series 3 was evaluated based on Alamar Blue stain reduction in viable cells,
,
,
,
,
,
,
,
minor modifications of PBMC viability, none of the tested compounds had the capacity to influence the
metabolism of the normal leukocytes. It is known that PTZ influence the drug transportation through
Pgp pumps [34] and therefore the metabolic alteration can be related to this capacity. Compounds with

Int. J. Mol. Sci. 2017, 18, 1365
8 of 14
significant cytotoxicity 2c
,
3e against tested cell lines typically affected the cellular metabolic activity
as well, while 2a (which presented a lower toxicity upon HL-60 populations) did not influence the
reducing environment of the cells, even though its capability to induce apoptosis was evidenced.
3. Discussion
The described synthetic protocol offers a reliable route to the heterocyclic system containing
benzo-fused thiazole and phenothiazine units with linear arrangement. Apart from the preparation
of various 2-substituted TAPTZ derivatives, the scope of this methodology can be broadened to the
preparation of novel constitutional isomers of TAPTZ when starting with different position isomers of
the amino-phenothiazine precursors.
The optical absorption and emission properties of TAPTZ derivatives appear slightly modulated
by the nature of the substituent attached to the thiazole unit. UV-Vis absorption and emission
spectroscopy can be conveniently employed in the detection of the TAPTZ derivatives in various
environments. The large Stokes shift values recommend TAPTZ derivatives approaching near IR
emission maxima (e.g., 3e) for imagistic applications.
The tested series of TAPTZ derivatives and N-(phenothiazine-3-yl)-thioamides displayed
generally modest biological activity. Based on structural similarity with previously reported PTZ
derivatives with anti-proliferative activity, TAPTZs emphasized a favourable substitution pattern of
the PTZ core induced by the fusion of the thiazole ring, possible hydrogen bond associations involving
mainly the nitrogen atom of the thiazole unit, and biological activity increased by the presence of
hydrophobic substituents.
4. Materials and Methods
All chemicals used were of reagent grade. The reaction progress was monitored by thin layer
chromatography on Merck DC Alufolien, silica gel 60 F254 and components were visualized by UV
lamp VL-4LC. Purification by flash chromatography was performed on silica gel 60 (particle size
0.032–0.063 mm) and recrystallization.
EI-MS spectra were recorded on a GC-MS QP 2010 Shimadzu mass spectrometer and HRMS
spectra on Thermo LTQ Orbitrap XL. NMR spectra were recorded at room temperature in solution on
400 or 600 MHz Bruker Avance instruments. Chemical shifts are expressed in terms of
to standard tetramethylsilane (TMS).
δ (ppm) relative
4.1. General Procedure for Obtaining N-Acyl-3-Aminophenothiazines (1a–e)
To a mixture of 3-amino-10-methyl-10H-phenothiazine [22] (6.5 mmol) and TEA (7 mmol) in
◦
CH2Cl2 (20 mL) at 0 C and under inert atmosphere was added the corresponding acyl-chloride
(6.5 mmol). After stirring at room temperature for 12 h, the reaction mixture was concentrated and
further diluted with brine (50 mL). The two phases were separated and the water phase was extracted
with dichloromethane (3
evaporated. The crude product was subjected to column chromatography (silica gel, toluene, or
×
100 mL). The combined organic phases were dried and the solvent was
toluene/acetone = 10/1) which provided 1a–e as white or cream powder.
N-(10-Methyl-10H phenothiazin-3-yl)pivalamide (1a). Purification by flash chromatography gave 1a (1.6 g,
◦
80%) as white solid, m.p. 188–190 C. 1H-NMR (400 MHz; DMSO-d6; Me4Si): δH 1.19 (9H, s, CH3), 3.26
(3H, s, NCH3), 6.87 (1H, d, 3J = 8.7 Hz, 1-H), 6.91–6.95 (2H, m, 9-H, 7-H), 7.14 (1H, d, 3J = 7.3 Hz, 6-H),
7.19 (1H, t, 3J = 8.0 Hz, 8-H), 7.45 (1H, dd, 3J = 8.7 Hz, 4J = 2.0 Hz, 2-H), 7.5 (1H, d, 4J = 2.0 Hz, 4-H),
9.12 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6): δC 27.2 (3C), 35.0, 39.5, 114.2, 114.3, 118.7, 119.5,
121.7, 121.8, 122.2, 126.7, 127.7, 134.4, 140.7, 145.4, 176.2. HRMS (ESI) m/z: Calcd. for C18H21N2OS
[M + H]+ 313.1369; Found 313.1358.

Int. J. Mol. Sci. 2017, 18, 1365
9 of 14
N-(10-Methyl-10H-phenothiazin-3-yl)benzamide (1b). Purification by flash chromatography gave 1b
1
(3.78 g, 81%) as grey solid, m.p. 220–222 ◦C (lit [35]). H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.29
(3H, s, NCH3), 6.93–6.96 (3H, m, 9-H, 1-H, 7-H), 7.17 (1H, dd, 3J = 8.0 Hz, 4J = 1.4 Hz, 6-H), 7.21 (1H, td,
3J = 8.5 Hz, 4J = 1.2 Hz, 8-H), 7.50–7.58 (3H, m, Ph), 7.6 (1H, dd, 3J = 8.8 Hz, 4J = 2.2 Hz, 2-H), 7.67 (1H,
d, 4J = 2.2 Hz, H), 7.94 (2H, d, 3J = 7.0 Hz, Ph,), 10.19 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6):
δC 35.5, 114.8 (2C), 119.1, 120.1, 122.1, 122.5, 122.7, 127.2, 128.0 (2C), 128.2, 128.8 (2C), 131.9, 134.7, 135.2,
141.6, 145.8, 165.6. HRMS (ESI) m/z: Calcd. for C20H17N2OS [M + H]+ 333.1055; Found 333.1065.
N-(10-Methyl-10H-phenothiazin-3-yl)-4-bromobenzamide (1c). Purification by flash chromatography gave
1c (0.9 g, 56%) as grey solid, m.p. 202–204◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.29 (3H, s,
NCH3), 6.93–6.95 (3H, m, 9-H, 1-H, 7-H), 7.16 (1H, d, 3J = 7.0 Hz, 6-H), 7.21 (1H, t, 3J = 7.3 Hz, 8-H),
7.45 (2H, d, 3J = 8.1 Hz, Ph), 7.60 (1H, d, 3J = 8.0 Hz, 2-H), 7.67 (1H, s, 4-H), 7.81 (2H, d, 3J = 8.12 Hz,
Ph) 10.46 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6): δC 35.5, 113.6, 114.4, 118.8, 119.9, 121.6, 122.0,
122.3, 124.2, 126.8, 127.8 (2C), 129.7 (2C), 133.8, 140.8, 141.3, 144.8, 145.3, 164.2. HRMS (ESI) m/z: Calcd.
for C20H16BrN2OS [M + H]+ 411.0161/ 413.0140; Found 411.0168/ 413.0145.
N-(10-Methyl-10H-phenothiazin-3-yl)-4-nitrobenzamide (1d). Purification by flash chromatography gave
1d (2.21 g, 89%) as grey solid, m.p. 245–247 ◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.30 (3H, s,
NCH3), 6.93–6.97 (3H, m, 1-H, 9-H, 7-H), 7.16 (1H, d, 3J = 7.0 Hz, 6-H), 7.21 (1H, t, 3J = 7.3 Hz, 8-H),
7.60 (1H, d, 3J = 8.0 Hz, 2-H), 7.67 (1H, s, 4-H), 8.16 (2H, d, 3J = 8.1 Hz, Ph), 8.35 (2H, d, 3J = 8.1 Hz,
Ph) 10.50 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6): δC 35.5, 114.4 (2C), 118.8, 119.8, 121.8, 122.1,
122.3, 123.5 (2C), 126.8, 127.8, 129.1 (2C), 133.7, 140.4, 141.6, 145.2, 149.1, 163.4. HRMS (ESI) m/z: Calcd.
for C20H16N3O3S [M + H]+ 378.0907; Found 378.0899.
N-(10-Methyl-10H-phenothiazin-3-yl)-1-naphthamide (1e). Purification by flash chromatography gave 1e
(1.37 g, 60%) as grey solid, m.p. 230–232 ◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.31 (3H, s,
NCH3), 6.94–6.98 (3H, m, 9-H, 1-H, 7-H), 7.18 (1H, d, 3J = 7.0 Hz, 6-H), 7.22 (1H, t, 3J = 7.5 Hz, 8-H),
7.58–7.63 (3H, m, 4-H, Napth), 7.64 (1H, dd, 3J = 8.7 Hz, 4J = 2 Hz, 2-H), 7.74–7.75 (2H, m, Naph), 8.01
(1H, m, Naph), 8.05 (1H, d, 3J = 8.2 Hz, Naph), 8.21 (1H, m, Naph), 10.53 (1H, brs, NH). 13C-NMR
(100 MHz; DMSO-d6): δC 35.5, 114.4, 114.5, 118.2, 119.1, 121.6, 122.2, 122.3, 125.0, 125.1, 125.4, 126.3,
127.0, 127.8, 128.3, 129.6, 130.1, 130.6, 133.1, 134.4, 134.6, 141.2, 145.4, 166.9. HRMS (ESI) m/z: Calcd.
for C24H19N2OS [M + H]+ 383.1213; Found 383.1232.
4.2. General Procedure for Obtaining N-(Phenothiazin-3-yl)tioamides (2a–e)
The amide 1a–e (1mmol) and the Lawesson’s reagent [2,4-bis(4-methoxyphenyl)-1,3,2,4-
dithiadiphosphetane]-2,4-dithione (0.5 mmol)] were heated to reflux in toluene (70 mL). A clear
solution was quickly formed; after a 4 h reflux period the reaction mixture was concentrated. The crude
product was purified by flash chromatography (silica gel, toluene, or toluene/ethyl acetate = 20/1) to
afford compounds 2a–e as yellowish powders.
N-(10-Methyl-10H-phenothiazin-3-yl)pivalthioamide (2a). Purification by flash chromatography gave 2a
(0.7 g, 65%) as yellow solid, m.p. 138–140 ◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 1.44 (9H,
s, t-Bu), 3.34 (3H, s, NCH3), 6.76 (1H, d, 3J = 8.6 Hz, 1-H), 6.79 (1H, dd, 3J = 8 Hz, 4J = 0.6 Hz, 9-H),
6.92 (1H, td, 3J = 7.5 Hz, 4J = 0.6 Hz, 7-H), 7.11 (1H, dd, 3J = 7.5 Hz, 4J = 1.4 Hz, 6-H), 7.17 (1H, td,
3J = 8 Hz, 4J = 1.4 Hz, 8-H), 7.25 (1H, d, 4J = 2.4 Hz, 4-H), 7.33 (1H, dd, 3J = 8.6 Hz, 4J = 2.4 Hz, 2-H),
8.63 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6): δC 30.3 (3C), 35.4, 45.3, 113.8, 114.2, 119.7, 122.7,
123.7, 123.9, 124.4, 127.2, 127.7, 133.7, 144.6, 145.4, 213.6. HRMS (ESI) m/z: Calcd. for C18H21N2S2
[M + H]+ 329.1141; Found 329.1137.
N-(10-Methyl-10H-phenothiazin-3-yl)benzothioamide (2b). Purification by flash chromatography gave 2b
(0.9 g, 70%) as yellow solid, m.p. 172–174 ◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.38 (3H, s,
NCH3), 6.82 (1H, d, 3J = 7.8 Hz, 9-H), 6.83 (1H, d, 3J = 8.5 Hz, 1-H), 6.94 (1H, t, 3J = 7.3 Hz, 7-H), 7.14
(1H, d, 3J = 7.3 Hz, 6-H), 7.18 (1H, t, 3J = 7.8 Hz, 8-H), 7.4–7.44 (2H, m, Ph), 7.48–7.51 (2H, m, Ph, 4-H),

Int. J. Mol. Sci. 2017, 18, 1365
10 of 14
7.58 (1H, dd, 3J = 8.5 Hz, 4J = 1.8 Hz, 2-H), 7.83 (2H, d, 3J = 7.3 Hz, Ph), 8.87 (1H, brs, NH). 13C-NMR
(100 MHz; DMSO-d6): δC 35.5, 113.8, 114.1, 122.6, 122.70, 122.76, 123.2, 124.1, 126.6 (2C), 127.2, 127.6,
128.6 (2C), 131.2, 133.8, 142.9, 144.7, 145.4, 195.1. HRMS (ESI) m/z: Calcd. for C20H17N2S2 [M + H]+
349.0828; Found 349.0831.
N-(10-Methyl-10H-phenothiazin-3-yl)-4-bromobenzothio-amide (2c). Purification by flash chromatography
◦
1
gave 2c (0.5 g, 44%) as yellow solid, m.p. 148–150 C. H-NMR (400 MHz, DMSO-d6, Me4Si): δH
3.33 (3H, s, NCH3), 6.95–7.02 (3H, m, 9-H, 1-H, 7-H), 7.17 (1H, dd, 3J = 7.8 Hz, 4J = 1.3 Hz, 6-H), 7.23
(1H, td, 3J = 8.0 Hz, 4J = 1.3 Hz, 8-H), 7.37–7.39 (3H, m, Ph, 2-H, 4-H), 7.75 (2H, d, 3J = 8.1 Hz, Ph),
11.74 (1H, brs, NH). 13C-NMR (100 MHz; DMSO-d6): δC 35.7, 114.4, 114.9, 116.7, 121.4, 121.9, 122.2,
123.1, 124.2, 124.8, 127.3, 129.7 (2C), 130.8 (2C), 135.5, 141.8, 143.8, 145.5, 195.8. HRMS (ESI): Calcd. for
C20H16BrN2S2 [M + H]+ 426.9932/428.9912; Found 426.9947/ 428.9923.
N-(10-Methyl-10H-phenothiazin-3-yl)-4-nitrobenzothioamide (2d). Purification by flash chromatography
gave 2d (1.6 g, 90%) as brown solid, m.p. 165–167◦C. 1H-NMR (400 MHz, DMSO-d6, Me4Si): δH 3.34
(3H, s, NCH3), 6.96–7.03 (3H, m, 1-H, 9-H, 7-H), 7.18 (1H, dd, 3J = 8.0 Hz, 4J = 1.5 Hz, 6-H), 7.24 (1H, td,
3J = 7.9 Hz, 4J = 1.5 Hz, 8-H), 7.67 (1H, dd, 3J = 8.7 Hz, 4J = 2.4 Hz, 2-H), 7.74 (1H, d, 4J = 2.4 Hz, 4-H),
7.98 (2H, d, 3J = 8.8 Hz, Ph), 8.30 (2H, d, 3J = 8.8 Hz, Ph), 12.02 (1H, brs, NH). 13C-NMR (100 MHz;
DMSO-d6): δC 35.7, 114.7, 115.2, 121.8, 122.3, 122.4, 123.1, 123.7 (2C), 123.8, 127.3, 128.4, 129.1 (2C),
134.9, 144.0, 145.4, 147.8, 148.2, 194.6. HRMS (ESI) m/z: Calcd. for C20H16N3O2S2 [M + H]+ 394.0678;
Found 394.0659.
N-(10-Methyl-10H-phenothiazin-3-yl)naphthalene-1-carbothioamide (2e). Purification by flash chromatography
gave 2e (1.12 g, 82%) as yellow solid, m.p. 130–133◦C. 1H-NMR (600 MHz, CDCl3, Me4Si): δH 3.40 (3H,
3
3
s, NCH3), 6.84 (2H, d, J = 8.5 Hz, 1-H, 9-H), 6.98 (1H, t, J = 7.5 Hz, 7-H7), 7.16 (1H, dd, 3J = 7.5 Hz
,
4J = 1.2 Hz, 6-H), 7.21 (1H, td, J = 8.5 Hz, J = 1.2 Hz, 8-H), 7.50–7.56 (3H, m, Naph), 7.62 (1H, d,
4J = 2.3 Hz, 4-H), 7.65 (1H, d, 3J = 7.0 Hz, Naph), 7.69 (1H, dd, 3J = 8.5 Hz, 4J = 2.3 Hz, 2-H), 7.89 (2H, d,
3J = 8.2 Hz, Naph), 8.2 (1H, d, 3J = 8.2 Hz, Naph), 8.96 (1H, brs, NH). 13C-NMR (125 MHz, CDCl3): δC
35.7, 114.7, 115.1, 121.8, 122.0, 122.3, 123.0, 123.2, 124.3, 125.4, 125.7, 126.6, 127.1, 127.3, 128.4, 128.5, 128.7,
129.2, 133.5, 136.1, 143.3, 143.5, 145.6, 196.4. HRMS (ESI) m/z: Calcd. for C24H19N2S2 [M + H]+ 399.0984;
Found 399.0997.
3
4
4.3. General Procedure for Obtaining Thiazolo[5,4-b]phenothiazine (3a–e)
The previously reported procedure for benzothiazole synthesis by iron catalysed oxidative C-S
bond formation [18] was adapted. Thus, a mixture of thioamide, potassium persulfate (K2S2O8),
2 equivalents of pyridine, and 10 mol % FeCl3 in dimethylsulfoxide DMSO (20 mL) was stirred at
80 ◦C for 4 h. The reaction mixture was cooled down to room temperature, and diluted with water.
After extraction with ethyl acetate (3 × 25 mL), the combined organic layers were washed with brine
(20 mL), and dried over anh. MgSO4. The solvent was evaporated and the residue was purified
by column chromatography (silica gel, toluene, or toluene/ethylacetate = 20:1) which gave 3a–e as
yellowish solids which were recrystallized from ethanol.
2-(t-Butyl)-10-methy◦l-10H-thiazolo[5,4-b]phenothiazine (3a). Thioamide 2a gave 3a (0.338 g, 26%) as grey
solid, m.p. 183–185 C. 1H-NMR(400 MHz, CDCl3, Me4Si): δH 1.48 (9H, s, tBu), 3.41 (3H, s, NCH3), 6.83
(1H, dd, 3J = 8.2Hz, 4J = 0.6 Hz, 9-H), 6.95 (1H, td, 3J = 7.6 Hz, 4J = 1.0 Hz, 7-H), 7.16–7.2 (3H, m, 6-H,
8-H, 11-H), 7.74 (1H, s, 4-H). 13C-NMR (100 MHz, CDCl3): δC 30.8 (3C), 36.0, 38.3, 105.7, 114.5, 120.4,
122.6, 123.4, 123.7, 127.2, 127.6, 134.9, 143.6, 145.6, 149.2, 180.0. IR (KBr, ν
, cm−1) 3030 (w), 2961(m),
1574 (m), 1506 (m), 1470 (m), 1321 (m), 1278 (m), 870 (s), 846 (s), 753 (s). HRMS (ESI) m/z: Calcd. for
C18H19N2S2 [M + H]+ 327.0984; Found 327.0995.

Int. J. Mol. Sci. 2017, 18, 1365
11 of 14
2-Phenyl-10-methyl-10H-thiazolo[5,4-b]phenothiazine (3b). Thioamide 2b gave 3b (0.386 g, 44%) as
◦
1
pale-yellow solid, m.p. 163–165 C. H-NMR (400 MHz, CDCl3, Me3Si): δH 3.42 (3H, s, NCH3),
6.84 (1H, d, 3J = 7.8 Hz, 9-H), 6.97 (1H, td, 3J = 8.2 Hz, 4J = 0.76 Hz, 7-H), 7.17–7.21 (3H, m, 6-H, 8-H,
11-H), 7.45–7.47 (3H, m, Ph), 7.8 (1H, s, 4-H), 8.01 (2H, m, Ph). 13C-NMR (100 MHz, CDCl3): δC 36.0,
105.7, 114.6, 120.8, 122.8, 123.4, 124.2, 127.30, 127.32 (2C), 127.7, 129 (2C), 130.7, 133.7, 135.1, 144.1, 145.4,
150.2, 165.5. IR (KBr,
ν
cm−1) 3020 (w), 2961 (m), 1571 (m), 1506 (m), 1478 (m), 1326 (m), 1284 (m), 761
,
(s), 744 (s). HRMS (ESI) m/z: Calcd. for C20H15N2S2 [M + H]+ 347.0671; Found 347.0678.
2-(4-Bromophenyl)-10-methyl-10H-thiazolo[5,4-b]-phenothiazine (3c). Thioamide 2c gave 3c (0.13 g, 26%)
◦
as pale-yellow solid, m.p. 176–178 C. 1H-NMR (400 MHz, CDCl3, Me3Si): δH 3.44 (3H, s, NCH3), 6.85
(1H, dd, 3J = 7.7 Hz, 4J = 0.9 Hz, 9-H), 6.97 (1H, td, 3J = 7.6 Hz, 4J = 0.9 Hz, 7-H), 7.18–7.22 (3H, m, 6-H,
8-H, 11-H), 7.58 (2H, d, 3J = 8.5 Hz, Ph), 7.78 (1H, s, 4-H), 7.87–7.89 (2H, d, 3J = 8.5 Hz, Ph). 13C-NMR
(100 MHz, CDCl3): δC 36.1, 105.7, 114.7, 120.9, 122.9, 123.4, 124.5, 125, 127.3, 127.7, 128.7 (2C), 132.29
(2C), 132.5, 135.2, 144.4, 145.3, 150.1, 164.6. IR (KBr, ν
, cm−1) 3059 (w), 2962 (m), 1558 (m), 1505 (m),
1471 (m), 1327 (m), 1265 (m), 942 (s), 868 (s), 825 (s), 753 (s). HRMS (ESI) m/z: Calcd. for C20H14BrN2S2
[M + H]+ 424.9776/426.9756, Found 424.9791/426.9768.
2-(4-Nitrophenyl)-10-methyl-10H-thiazolo[5,4-b]-phenothiazine (3d). Thioamide 2d gave 3d (0.6 g, 51%) as
pale-yellow solid, m.p. 206–208 ◦C. 1H-NMR (400 MHz, CDCl3, Me3Si): δH 3.47 (3H,s, NCH3), 6.88
(1H, d, 3J = 8.0 Hz, 9-H), 6.99 (1H, t, 3J = 7.1 Hz, 7-H), 7.16–7.26 (3H, m, 6-H, 8-H, 11-H), 7.84 (1H, s,
4-H4), 8.16 (2H, d, 3J = 8.8 Hz, Ph), 8.30 (2H, d, 3J = 8.8 Hz, Ph). 13C-NMR (100 MHz, CDCl3): δC 36.2,
105.5, 114.8, 121.3, 123.1, 123.2, 124.4 (2C), 125.3, 127.3, 127.9 (2C), 128.3, 129.1, 135.9, 139.3, 145.1, 148.7,
150.2, 162.5. IR (KBr, ν
, cm−1) 3059 (w), 2920 (m), 1595 (m), 1502 (s), 1466 (m), 1324 (s), 1291 (m), 871 (s),
851 (s), 829 (s), 749 (s). HRMS (ESI) m/z: Calcd. for C20H14N3S3 [M + H]+ 392.0522; Found 392.0529.
2-(Naphthalen-1-yl)-10-methyl-10H-thiazolo[5,4-b]-phenothiazine (3e). Thioamide 2e gave 3e (0.24 g, 32%)
◦
as pale-yellow solid, m.p. 204–206 C. 1H-NMR (600 MHz, CDCl3, Me3Si): δH 3.46 (3H, s, NCH3), 6.87
(1H, d, 3J = 7.8 Hz, 9-H), 6.98 (1H, t, 3J = 7.4 Hz, 7-H), 7.20–7.23 (2H, m, 6-H, 8-H), 7.28 (1H, s, 11-H),
7.52–7.57 (2H, m, Naph), 7.61 (1H, td, 3J = 8.4 Hz, 4J = 1.0 Hz, Naph), 7.88 (1H, d, 3J = 7.0 Hz, Naph),
7.91 (1H, d, 3J = 8.4 Hz, Naph), 7.93 (1H, s, 4-H), 7.96 (1H, d, 3J = 8.3 Hz, Naph), 8.96 (1H, d, 3J = 8.4
Hz, Naph). 13C-NMR (125 MHz, CDCl3): δC 36.1, 105.4, 114.6, 121.1, 122.9, 123.5, 124.3, 125.1, 126.0,
126.6, 127.3, 127.73, 127.75, 128.5, 129.3, 130.7, 130.8, 131.0, 134.1, 135.5, 144.3, 145.5, 150.3, 165.6. IR
(KBr, ν
, cm−1) 3059 (w), 2962 (m), 1574 (m), 1472 (m), 1440 (m), 1265 (m), 1141 (m), 925 (s), 876 (s), 829
(s), 750 (s); HRMS (ESI) m/z: Calcd. for C24H17N2S2 [M + H]+ 397.0828; Found 397.0834.
4.4. Biologic Assay
Instrumentation for in vitro testing: Lamil Plus class II Laminary Hoods (from Karstulan Metalli
Oy, Karstula, Finland), Uniequip incubator with CO2 (Martinsried, Germany), Synergy II microplate
reader from BioTek Instruments (Winooski, VT, USA), Universal 32R centrifuge with swing-out rotor
(from Hettich, Tuttlingen, Germany), Titramax 1000 incubator with shaker (from Heidolph Instruments,
Schwabach, Germany), BX40 optical microscope and CKX41 inverted phase fluorescence microscope
(from Olympus Corporation, Center Valley, PA, USA).
4.4.1. Cell Cultures
HL-60 human promyelocytic- and THP-1 human monocytic leukaemia cell lines were acquired
from European Cell Culture Collection. Cells were cultivated in RPMI-1640 media supplemented with
10% fetal bovine serum, 2 mM glutamine, and 1% 2-[4-(2-hydroxyethyl)piperazin-1-yl]etansulfonic
acid HEPES buffer (all reagents from Sigma Aldrich, St. Louis, MO, USA). Human peripheral blood
mononuclear cells (PBMCs) were isolated from whole blood, obtained by venepuncture from a
43-year-old female donor, following her informed written consent. PBMCs were isolated in density
gradient, using Histopaque 1077 separation media and Hanks Modified Eagle’s Media (both from
Sigma Aldrich) for cell wash, using our previously described method [36].

Int. J. Mol. Sci. 2017, 18, 1365
12 of 14
For colorimetric testing, cells were seeded on 96-well micro plates, at a concentration of
3 × 104 cells/100 µL media, and incubated for 24 h (Nunclon Delta surface plates from Thermo Fischer
Scientific, Waltham, MA, USA). For fluorescence measurements, black, clear-bottom 96-well plates
were used (from Corning, Tewksbury, MA, USA).
4.4.2. Cell Viability Tests
Cell viability was tested with Trypan Blue staining and optical microscope examination.
Stock solutions of 40 mM in dimethyl sulfoxide (DMSO, from Titolchimica, Pontevecchio Polesine,
RO, Italy) are prepared. Compounds were diluted in a mixture of DMSO:PEG 400 in a proportion of
1:1. Twenty micromolar (20 mM) solutions of tested compounds were prepared in a sterile glucose
5% solution (Braun Melsungen AG, Melsungen, Germany), slightly acidified to pH 6.8–6.9 to obtain a
better solubility; the solutions were sonicated. Cells were treated with the tested compounds at dilution
series of 1000 to 1.25 µM final concentrations in cell suspension. As positive reference, 0.01–250 µM
solutions of cytarabine (Cytosar, from Actavis, Dublin, Ireland)—an antineoplastic agent used in
leukaemia, which inhibits the synthesis of deoxyribonucleic acid—were used.
The compounds potential for auto-fluorescence and colour background was tested.
The cytotoxicity of the compounds was assessed using the MTT colorimetric method [37]; the IC50
parameter was calculated from the dose-response curve (GraphPad Prism 5 software, version 5,
GraphPad Company, San Diego, CA, USA).
The metabolic activity of the treated cells was evaluated using the resazurin-based quantitative
method (Alamar Blue reagent purchased from Life Technologies, Carlsbad, CA, USA), as an alternative
to cell viability testing. The cytoplasm of viable cells is able to reduce resazurin to resorufin, a red-colour
and highly fluorescent dye, whose intensity is related to the number of living cells within the
population. The cells were plated on 96-well microplates and treated in the same way and with
identical concentrations as for the MTT test, the wells were stained with Alamar Blue in a proportion
of 20:100 µL, and after 2 h of incubation the fluorescence emission was measured at 620 nm.
Apoptosis was measured using Alexa Fluor 480-labelled AnexinV marker, and with the propidium
iodide stain which binds the DNA by random intercalation between nuclear bases in death cells
(reagents from Life Technologies). The cells were seeded in 24-well plates at a concentration of
106 cells/mL media and treated for 4 and 8 h, respectively, with a final concentration of 250
µM
tested compound. The harvested cells were washed in cold PBS and suspended in staining buffer.
Samples were divided in two aliquots, stained with Alexa Fluor Annexin V, or with PI, respectively.
After 15 min incubation, the cells were washed in cold PBS, suspended in 100
fluorescence was measured in triplicate at 530–575 nm using 488 nm excitation.
µL buffer, and the
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/7/1365/s1.
Acknowledgments: The partial financial support from the Romania–European Economical Space (Norway)
International Grant No. 1/2014 (Ciprian Ionu¸t Tomuleasa) and European Social Found, Human Resources
Development Operational Programme 2007–2013, project No. POSDRU/159/1.5/S/136893 (Adriana Grozav)
as well as from Babes-Bolyai University (GTC-31791/2016) by the author Emese Gal are greatly acknowledged.
Funds for covering the costs to publish in open access were assumed by the Faculty of Pharmacy, I. Ha¸tieganu
University of Medicine and Pharmacy, Cluj-Napoca (Lorena Filip).
Author Contributions: Balazs Brem performed the synthetic chemistry experiments; Emese Gal and
Lumini¸ta Silaghi-Dumitrescu contributed analysis tools; Luiza Găină contributed computational tools;
Eva Fischer-Fodor and Ciprian Ionu¸t Tomuleasa performed the in vitro biological experiments; Adriana Grozav
and Lorena Filip contributed reagents/materials; Valentin Zaharia analysed the data; Castelia Cristea wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Pluta, K.; Morak-Młodawska, B.; Jelen´, M. Recent progress in biological activities of synthesized
phenothiazines. Eur. J. Med. Chem. 2011, 46, 3179–3189. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2017, 18, 1365
13 of 14
2.
3.
Keri, R.S.; Patil, M.R.; Patil, S.A.; Budagupi, S. A comprehensive review in current developments of
benzothiazole-based molecules in medicinal chemistry. Eur. J. Med. Chem. 2015, 89, 207–251. [CrossRef]
[PubMed]
Wainwright, M. Phenothiazinium salts as antimicrobial photosensitizing agents. In Photodynamic Inactivation
of Microbial Pathogens: Medical and Environmental Applications; Hamblin, M.R., Jori, G., Eds.; RSC Publishing:
Cambbridge, UK, 2011; Volume 11, pp. 19–43.
4.
5.
6.
7.
Pereteanu, I.S.; Müller, T.J.J. Synthesis and electronic properties of 3,7-dianilino substituted N-hexyl
phenothiazines. Org. Biomol. Chem. 2013, 11, 5127–5135. [CrossRef] [PubMed]
Sailer, M.; Franz, A.W.; Müller, T.J.J. Synthesis and electronic properties of monodisperse oligophenothiazines.
Chem. Eur. J. 2008, 14, 2602–2614. [CrossRef] [PubMed]
Meyer, T.; Ogermann, D.; Pankrath, A.; Müller, T.J.J. Phenothiazinyl rhodanylidene merocyanines for
dye-sensitized solar cells. J. Org. Chem. 2012, 77, 3704–3715. [CrossRef] [PubMed]
´
Jaszczyszyn, A.; Ga˛siorowski, K.; Swia˛tek, P.; Malinka, W.; Cies´lik-Boczula, K.; Petrus, J.;
Czarnik-Matusewicz, B. Chemical structure of phenothiazines and their biological activity. Pharmacol. Rep.
2012, 64, 16–23. [CrossRef]
8.
9.
Hendrich, A.B.; Wesolowska, O.; Motohashi, N.; Molnar, J.; Michalak, K. New phenothiazine-type
multidrug resistance modifiers: Anti-MDR activity versus membrane perturbing potency. Biochem. Biophys.
Res. Commun. 2003, 304, 260–265. [CrossRef]
Seelig, A. A general pattern for substrate recognition by P-glycoprotein. Eur. J. Biochem. 1998, 251, 252–261.
[CrossRef] [PubMed]
10. Pluta, K.; Jeleñ, M.; Morak-Modawska, B.; Zimecki, M.; Artym, J.; Kociêba, M. Anticancer activity of newly
synthesized azaphenothiazines from NCI’s anticancer screening bank. Pharmacol. Rep. 2010, 62, 319–332.
[CrossRef]
11. Motohashi, N.; Kawase, M.; Satoh, K.; Sakagami, H. Cytotoxic potential of phenothiazines. Curr. Drug Targets
2006, 7, 1055–1066. [CrossRef]
12. Shin, S.Y.; Choi, B.H.; Kim, J.R.; Kim, J.H.; Lee, Y.H. Suppression of P-glycoprotein expression by
antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells. Eur. J. Pharm. Sci.
2006, 28, 300–306. [CrossRef] [PubMed]
13. Syed, S.K.; Christopherson, R.I.; Roufogalis, B.D. Reversal of vinblastine transport by chlorpromazine in
membrane vesicles from multidrug-resistant human CCRF-CEM leukaemia cells. Br. J. Cancer 1998, 78,
321–327. [CrossRef] [PubMed]
14. Jena, J. Significance of benzothiazole moiety in the field of cancer. Int. J. Pharm. Pharm. Sci. 2014, 6, 16–22.
15. Li, H.; Wang, X.-M. Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole
to discover novel anticancer agents. Bioorg. Med. Chem. 2014, 22, 3739–3748. [CrossRef] [PubMed]
16. Lindgren, E.B.; de Brito, M.A.; Vasconcelos, T.R.A.; de Moraes, M.O.; Montenegro, R.C.; Yoneda, J.D.;
Leal, K.Z. Synthesis and anticancer activity of (E)-2-benzothiazole hydrazones. Eur. J. Med. Chem. 2014, 86,
12–16. [CrossRef] [PubMed]
17. Weekes, A.A.; Westwell, A.D. 2-Arylbenzothiazole as a privileged scaffold in drug discovery. Curr. Med. Chem.
2009, 16, 2430–2440. [CrossRef] [PubMed]
18. Prajapati, N.P.; Vekariya, R.H.; Borad, M.A.; Patel, H.D. Recent advances in the synthesis of 2-substituted
benzothiazoles: A review. RSC Adv. 2014, 4, 60176–60208. [CrossRef]
19. Saha, P.; Ramana, T.; Purkait, N.; Ashif, A.M.; Paul, R.; Punniyamurthy, T. Ligand-free copper-catalyzed
synthesis of substituted benzimidazoles, 2-aminobenzimidazoles, 2-aminobenzothiazoles, and benzoxazoles.
J. Org. Chem. 2009, 74, 8719–8725. [CrossRef] [PubMed]
20. Evindar, G.; Batey, R.A. Parallel synthesis of a library of benzoxazoles and benzothiazoles using
ligand-accelerated copper-catalyzed cyclizations of ortho-halobenzanilides. J. Org. Chem. 2006, 71, 1802–1808.
[CrossRef] [PubMed]
21. Yang, D.; Fu, H.; Hu, L.; Jiang, Y.; Zhao, Y. Copper-catalyzed synthesis of benzimidazoles via cascade
reactions of o-haloacetanilide derivatives with amidine hydrochlorides. J. Org. Chem. 2008, 73, 7841–7844.
[CrossRef] [PubMed]
22. Peng, J.; Ye, M.; Zong, C.; Hu, F.; Feng, L.; Wang, X.; Wang, Y.; Chen, C. Copper-catalyzed intramolecular
C-N bond formation: A straightforward synthesis of benzimidazole derivatives in water. J. Org. Chem. 2011
76, 716–719. [CrossRef] [PubMed]
,

Int. J. Mol. Sci. 2017, 18, 1365
14 of 14
23. Jaseer, E.A.; Prasad, D.J.C.; Dandapat, A.; Sekar, G. An efficient copper(II)-catalyzed synthesis
of benzothiazoles through intramolecular coupling-cyclization of N-(2-chlorophenyl)benzothioamides.
Tetrahedron Lett. 2010, 51, 5009–5012. [CrossRef]
24. Ma, D.; Xie, S.; Xue, P.; Zhang, X.; Dong, J.; Jiang, Y. Efficient and economical access to substituted
benzothiazoles: Copper-catalyzed coupling of 2-haloanilides with metal sulfides and subsequent
condensation. Angew. Chem. Int. Ed. 2009, 48, 4222–4225. [CrossRef] [PubMed]
25. Wang, H.; Wang, L.; Shang, J.; Li, X.; Wang, H.; Gui, J.; Lei, A. Fe-catalysed oxidative C-H
functionalization/C-S bond formation. Chem. Commun. 2012, 48, 76–78. [CrossRef] [PubMed]
26. Ignat, A.; Lovasz, T.; Vasilescu, M.; Fischer-Fodor, E.; Tatomir, C.B.; Cristea, C.; Silaghi-Dumitrescu, L.; Zaharia, V.
Heterocycles 27. Microwave assisted synthesis and antitumour activity of novel phenothiazinyl-thiazolyl-
hydrazine derivatives. Arch. Pharm. Chem. Life Sci. 2012, 345, 574–583. [CrossRef] [PubMed]
27. Brem, B.; Gal, E.; Cristea, C.; Gaina, L.; Grozav, A.; Zaharia, V.; Silaghi-Dumitrescu, L. Synthesis of new
benzothiazolyl-phenothiazine derivatives. Studi. Univ. Babes Bolyai Chem. 2015, 60, 371–378.
28. Grozav, A.; Zaharia, V.; Cristea, C.; Fit, N.I. Antimicrobial activity screening of benzothiazolyl-phenothiazine
derivatives. Studia UBB Chem. 2015, 60, 283–289.
29. Găină, L.I.; Mătărângă-Popa, L.N.; Gal, E.; Boar, P.; Lonnecke, P.; Hey-Hawkins, E.; Silaghi-Dumitrescu, L.;
Cristea, C.; Bischin, C.; Lupan, I. Microwave-assisted catalytic amination of phenothiazine; reliable access to
phenothiazine analogues of Tröger’s base. Eur. J. Org. Chem. 2013, 24, 5500–5508. [CrossRef]
30. Yang, L.; Feng, J.K.; Ren, A.M. Theoretical study on electronic structure and optical properties of
phenothiazine-containing conjugated oligomers and polymers. J. Org. Chem. 2005, 70, 5987–5996. [CrossRef]
[PubMed]
31. Zhelev, A.M.; Ohba, A.M.; Bakalova, R.; Hadjimitova, V.; Ishikawa, M.; Shinohara, Y.; Baba, Y. Phenothiazines
suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability
of normal lymphocytes. Cancer Chemother. Pharmacol. 2004, 53, 267–275. [CrossRef] [PubMed]
32. Gil-Ad, I.; Shtaif, B.; Levkovitz, Y.; Dayag, M.; Zeldich, E.; Weizman, A. Characterization of
phenothiazine-induced apoptosis in neuroblastoma and glioma cell lines: Clinical relevance and possible
application for brain-derived tumors. J. Mol. Neurosci. 2004, 22, 189–198. [CrossRef]
33. Rampersad, S.N. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular
health in cell viability bioassays. Sensors 2012, 12, 12347–12360. [CrossRef] [PubMed]
34. Spengler, G.; Takács, D.; Horváth, A.; Riedl, Z.; Hajós, G.; Amaral, L.; Molnár, J. Multidrug resistance
reversing activity of newly developed phenothiazines on P-glycoprotein (ABCB1)-related resistance of
mouse T-lymphoma cells. Anticancer Res. 2014, 34, 1737–1741. [PubMed]
35. Cauquil, G.; Casadevall, E.; Casadevall, A. Recherches dans la serie de la phenothiazine(5e memoire).
Transposition de Bechmann et reaction de Schmidt appliquées à la synthèse d’amino-methyl 10-phenothiazines.
Bull. Soc. Chem. Fr. 1962, 602–616.
36. Fischer-Fodor, E.; Mot, A.; Deac, F.; Arkosi, M.; Silaghi-Dumitrescu, R. Towards hemerythrin-based blood
substitutes: Comparative performance to hemoglobin on human leukocytes and umbilical vein endothelial
cells. J. Biosci. 2011, 36, 215–221. [CrossRef] [PubMed]
37. Miklášová, N.; Fischer-Fodor, E.; Mikláš, R.; Kucková, L.; Kožíšek, J.; Liptaj, T.; Soritau, O.; Valentova, J.;
Devinsky, F. Synthesis and characterization of new biologically active palladium(II) complexes with
(1E,6E)-1,7-bis(3,4-diethoxyphenyl)-1,6-heptadiene-3,5-dione. Inorg. Chem. Commun. 2014, 46, 229–233.
[CrossRef]
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).