3,6-Diazaphenothiazines as potential lead molecules – synthesis, characterization and anticancer activity

Article abstract of DOI:10.3109/14756366.2016.1151014

3,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3′-nitro-2,4′-dipyridinyl sulfide and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed by alkylation and heteroarylation. The thiazine ring formation r

Full text of DOI:10.3109/14756366.2016.1151014

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
3,6-Diazaphenothiazines as potential lead
molecules – synthesis, characterization and
anticancer activity
Beata Morak-Młodawska, Krystian Pluta, Małgorzata Latocha, Kinga
Suwińska, Małgorzata Jeleń & Dariusz Kuśmierz
To cite this article: Beata Morak-Młodawska, Krystian Pluta, Małgorzata Latocha, Kinga
Suwińska, Małgorzata Jeleń & Dariusz Kuśmierz (2016): 3,6-Diazaphenothiazines as potential
lead molecules – synthesis, characterization and anticancer activity, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1151014
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1151014
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Date: 17 July 2016, At: 08:49

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1151014
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RESEARCH ARTICLE
3,6-Diazaphenothiazines as potential lead molecules – synthesis,
characterization and anticancer activityy
1
1
2
3
1
Beata Morak-Młodawska , Krystian Pluta , Małgorzata Latocha , Kinga Suwi n´ ska , Małgorzata Jele n´ , and Dariusz
2
Ku s´ mierz
1
Department of Organic Chemistry, School of Pharmacy with the Division of Laboratory Medicine, The Medical University of Silesia, Sosnowiec,
2
Poland, Department of Cell Biology, School of Pharmacy with the Division of Laboratory Medicine, The Medical University of Silesia, Sosnowiec,
3
Poland, and Faculty of Mathematics and Natural Sciences, Cardinal Stefan Wyszy n´ ski University, Warszawa, Poland
Abstract
,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3 -nitro-2,4 -dipyridinyl sulfide
and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed
by alkylation and heteroarylation. The thiazine ring formation ran via the Smiles rearrangement.
The structure elucidation was based on 2D NMR and X-ray analysis of N-methylated product.
Keywords
0
0
3
2
D NMR spectra, anticancer activity, gene
expressions, phenothiazines, the Smiles
rearrangement, X-ray analysis
3
,6-Diazaphenothiazines were investigated for antitumor activity using glioblastoma SNB-19,
History
melanoma C-32 and breast cancer MCF-7 cells. 10H-3,6-diazaphenothiazine was 10 times more
active (IC5050.72 mg/mL) than cisplatin. Two diazaphenothiazines with the 2-pyrimidinyl and
dimethylaminopropyl substituents were selectively active against MCF-7 and C-32 cells. The
expressions of H3 (proliferation marker), TP53, CDKN1A (cell cycle regulators), BAX and BCL-2
Received 24 November 2015
Revised 22 January 2016
Accepted 1 February 2016
Published online 26 February 2016
(proapoptopic and antiapoptopic genes) were detected by RT-QPCR method. The expression
analysis suggests the cell cycle arrest and the mitochondrial apoptosis pathway activation in
MCF-7 and SNB-19 cells.
Introduction
benzene rings with the homoaromatic and heteroaromatic rings to
form benzophenothiazines and azaphenothiazines. Such modifi-
cations are directed to alter a biological profile (its activity and
potency). Many reports of last decade showed that both classical
and modified phenothiazines exhibited very promising anticancer,
antibacterial, antifungal, anti-inflammatory activities and reversal
Cancer has been one of the main causes of death worldwide for
the last many years. The cancer therapy involves such a curative
treatment as surgery, radiation, chemotherapy and biotherapy
1
,2
.
Although chemotherapy has been still improved in cancer therapy
and the survival has been greatly increased, there is need to find
new potent antitumor agents with better selectivity and minor or
no side effects. Heterocyclic ring system plays an important role
in the development of novel scaffold with improved pharmaceut-
of multidrug resistance. This rich experimental material was
6–14
summarized in review articles and chapters in monographs
.
Phenothiazines are also reported to exert a potential benefit in the
3
,4
.
treatment of Alzheimer’s, Creutzfeldt-Jakob’s and AIDS-asso-
15–17
ical properties
ciated diseases
.
Tricyclic phenothiazines (as 10-dialkylaminoalkyldibenzo-1,4-
thiazines) are important class of fused heterocycles possessing
important biological actions and interesting chemical properties.
For many years, they have been recognized as drugs exhibiting
The phenothiazine modification with the azine ring is most
perspective as a new phenothiazine scaffold is formed (azaphe-
nothiazine), which can be altered by introduction of new
substituents at the thiazine nitrogen atom. The modification
with the pyridine ring led to form pyridobenzothiazines (mono-
azaphenothiazines) and dipyridothiazines (diazaphenothiazines).
One of pyridobenzothiazines is prothipendyl (1-azaphenothia-
zine,10-dimethylaminopropylpyrido[3,2-b][1,4]benzothiazine), a
5
neuroleptic, antihistaminic, antitussive and antiemetic activities .
Classical phenothiazines are low toxic, inexpensive, easy to
obtain, and some 10H-substituted compounds are even commer-
cially available. The chemical modifications of the phenothiazine
structures have been carried out mainly by introduction of new
substituents at the thiazine nitrogen atom (using
5
well-known drug possessing sedative and antiemetic properties .
Recently, prothipendyl was found to exhibit antiviral activity
against chikungunya virus (CHIKV), a mosquito-transmitting
1
0H-phenothiazines) and by the replacement of one or two
1
8,19
alphavirus causing CHIK fever
.
One of our strategies for phenothiazine modifications is based
on the replacement of two benzene rings with the pyridine rings.
We found new dipyridothiazines of the 1,6-, 1,8- and
2,7-diazaphenothiazine structures to exhibit promising anticancer
activity against lung cancers HOP-62 and HOP-92, colon cancers
yPart CXLVII in the series of Azinyl Sulfides.
Address for correspondence: Prof. Krystian Pluta, Department of Organic
Chemistry, The Medical University of Silesia, Jagiello n´ ska 4, 41-200
Sosnowiec, Poland. Tel: 48 32 364 1606. Fax: 48 32 364 1600. E-mail:
pluta@sum.edu.pl

2
B. Morak-Młodawska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
1
COLO 205, HCT-116 and SW-948, renal cancers RXF393 and
2
H NMR (CDCl ) d: 4.38 (broad s, 2H, NH ), 6.72(d,
3
2
0,21
A498, and leukemia HL-60(TB) and L-1210
diazaphenothiazine also shows immunosuppressant, inhibiting (dd, J ¼ 7.7 Hz, J ¼ 4.8 Hz, 1H), 8.18 (dd, J ¼ 7.8 Hz, J ¼ 1.3 Hz,
both humoral and cellular immune responses, and antioxidant 1H), 8.41 (d, J ¼ 5.4 Hz, 1H), 9.39 (s, 1H). EI MS m/z: 248 (M, 27),
. 10H-2,7- J ¼ 5.7 Hz, 1H), 7.18 (dd, J ¼7.7 Hz, J ¼ 1.3 Hz, 1H), 7.29
2
2–24
properties
.
In the literature, one can find the synthesis of two 7-substituted 3.25, N 22.57. Found: C 48.49, H 3.32, N 22.47.
202 (M + 1-NO 100). Anal. Calcd for: C H N O S, C 48.38, H
2 10 8 4 2
(Cl, OCH ) 10H-1-nitro-3,6-diazaphenothiazines in the reaction
3
of 6-substituted 3-amino-2(1H)-pyridinethione with 3,5-dinitro-4-
chloropyridine in methanolic potassium hydroxide. As the 1,4-
thiazine ring formation can proceed directly as the Ullmann
cyclization or indirectly through the Smiles rearrangement of the
S–N type, the differentiation between possible structures, i.e. 7-
substituted 10H-1-nitro-3,6-diazaphenothiazine and 10H-4-nitro-
Synthesis of 10H-3,6-diazaphenothiazine (6)
From sodium 3-amino-2-pyridinethiolate (1) and 4-chloro-3-
nitropyridine (2). To a solution of sodium 3-amino-2-pyridi-
nethiolate (1) (148 mg, 1 mmol) in dry DMF (10 mL), 4-chloro-3-
nitropyridine (2) (158 mg, 1 mmol) was added. The reaction
mixture was stirred at room temperature for 1 h and next was
refluxed for 3 h. After cooling, the reaction mixture was
2
bonding between the NH and NO groups .
,6-diazaphenothiazine, was based on the strong hydrogen
2
5
2
evaporated in vacuo. The dry residue was dissolved in CHCl3
The aim of this paper is the synthesis of unknown 10H-3,6-
diazaphenothiazine, transformation of this parent compound into
and purified by column chromatography (aluminum oxide,
CHCl ) to give: 10H-3,6-diazaphenothiazine (6) (140 mg, 69%);
1
5 varied 10-substituted derivatives and determination of their
3
ꢀ
m.p. 149–150 C.
1
anticancer activity against selected tumor cell lines. Since the
synthesis can lead to one of the two 10H-diazaphenothiazines (2,6
or 3,6) and transformation intovarious N-substituted derivatives via
alkylation and arylation of the thiazine nitrogen atom can be
disturbed by the reaction of the pyridine nitrogen atom, the
unquestionable elucidation of the product structure seems to be a
crucial challenge.
H NMR (DMSO ) d: 6.48 (d, J ¼ 5.4 Hz, 1H, H ), 6.83 (dd,
d6
1
J ¼ 7.8 Hz, J ¼ 1.2 Hz, 1H, H ), 6.95 (dd, J ¼ 7.8 Hz, J ¼ 4.8 Hz,
9
1
H, H ), 7.79 (dd, J ¼ 7.8 Hz, J ¼ 1.2 Hz, 1H, H ), 7.86 (s, 1H,
4 2
8
7
1
3
H ), 7.95 (d, J ¼ 5.4 Hz, 1H, H ), 9.14 (broad s, 1H, NH).
C
NMR (DMSO ) d: 109.22 (C ), 114.05 (C ), 121.08 (C ),
d6
1
4a
9
1
1
23.33 (C ), 136.30 (C ), 140.52 (C ), 143.28 (C ), 146.28 (C ),
8 9a 7 5a 4
46.46 (C ), 149.52 (C10a). EI MS m/z: 201 (M, 100). Anal Calcd
2
for: C H N S, C 59.68, H 3.51, N 20.88. Found: C 59.61, H 3.59,
10 7 3
N 20.78.
Methods
Chemistry
0
0
In cyclization of 3-amino-3 -nitro-2,4 -dipyridinyl sulfide
0
Melting points were determined in open capillary tubes on a
Boetius melting point apparatus (Stuart Equipment, Stone, UK) (3). The brown solution of 3-amino-3 -nitro-2,4 -dipyridinyl
0
1
and are uncorrected. The H NMR, COSY, ROESY, HSQC, sulfide (3) (124 mg, 0.5 mmol) in dry DMF (5 mL) was refluxed
for 3 h. After cooling, the reaction mixture was evaporated in
600 spectrometer at 300 and 600 MHz (Bruker, vacuo. The dry residue was dissolved in CHCl and purified by
Rheinstetten, Germany) in deuteriochloroform and dimethylsulf- column chromatography (aluminum oxide, CHCl ) to give 10H-
,6-diazaphenothiazine (6) (90 mg, 90%).
HMBC spectra were recorded on a Bruker Fourier 300 i
TM
Ascend
3
3
3
oxide-d with tetramethylsilane as the internal standard. The
C NMR spectrum was recorded at 75 MHz. Electron impact
6
1
3
mass spectra (EI MS) and chemical ionization mass spectra (CI
MS) were run on a Finnigan MAT 95 spectrometer (Thermo
Finnigan MAT, Bremen, Germany) at 70 eV. The thin layer
chromatography was performed on silica gel 60 F254 (Merck
Synthesis of 10-substituted 3,6-diazaphenothiazines (7–10,12,13)
To a solution of 10H-3,6-diazaphenothiazine (6) (100 mg,
0
.5 mmol) in dry DMF (5 mL), NaH (24 mg, 1 mmol, 60% NaH
in mineral oil was washed out with hexane) was added. The
reaction mixture was stirred at room temperature for 1 h and then
alkyl or heteroaryl halide (methyl iodide, allyl bromide, benzyl
chloride, 5-chloro-1-methyl-4-nitroimidazole, 2-chloropyrimi-
dine, 1.5 mmol) was added and the stirring was continued for
1
aluminum oxide 60 F254 neutral (type E) (Merck 1.05581)
with CHCl -EtOH (10:1 v/v) as eluents.
3
.05735) with CHCl -EtOH (5:1 and 10:1 v/v) and on
3
Synthesis of sodium 3-amino-2-pyridinothiolate (1)
2
4 h. The mixture was poured into water (15 mL), extracted with
(3 ꢁ 10 mL) and dried using anhydrous Na SO . The
obtained product was purified by column chromatography (alu-
0 mmols) were added carefully and the mixture was refluxed for minum oxide, CHCl ) to give:
h. After cooling, the solvent was evaporated in vacuo. The dry (1) 10-Methyl-3,6-diazaphenothiazine (7) (87 mg, 79%); m.p.
0
0
To a solution of 3,3 -dinitro-2,2 -dipyridinyl disulfide (310 mg, CHCl
3
2
4
1
mmol) in dry ethanol (30 mL), 2 tablets of NaBH (378 mg,
4
1
2
3
ꢀ
1
1
58–159 C: H NMR (CDCl ) d: 3.30 (s, 3H, CH ), 6.60 (d,
residue was recrystallized from ethanol yielding 230 mg (71%) of
brown crystals of sodium 3-amino-2-pyridinethiolate (1) (230 mg,
3
3
J ¼ 5.4 Hz, 1H, H
), 6.98 (dd, J ¼7.8 Hz, J ¼ 1.2 Hz, 1H, H
),
9
1
ꢀ
7
salt 1 with 10% solution of HCl, 3-aminopyridine-2(1H)-thione
1%), m.p.4260 C. After acidification of the aqueous solution of
7.07 (dd, J ¼ 7.8 Hz, J ¼ 4.8 Hz, 1H, H
8
), 8.06 (dd,
J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.15 (s, 1H, H ), 8.27 (d,
7
4
1
3
). C NMR (CDCl
o
was obtained, m.p. 131–132 C ( ; m.p. 131–132 C).
26
o
J ¼ 5.4 Hz, 1H, H
) d: 34.36 (NCH ),
3
2
3
1
08.52 (C ), 118.61 (C ), 120.37 (C ), 122.14 (C ), 139.05
1 4a 9 8
(
1
C ), 143.52 (C ), 145.79 (C ), 146.76 (C ), 149.15 (C ),
9a 7 5a 4 2
0
0
Synthesis of 3-amino-3 -nitro-2,4 -dipyridinyl sulfide (3)
50.36 (C10a). EI MS m/z: 215 (M, 100), 200 (M-CH , 58).
3
To a solution of sodium 3-amino-2-pyridinethiolate (1) (148 mg,
Anal Calcd for: C11
C 61.26, H 4.27, N 19.41.
158 mg, 1 mmol) was added. The mixture was stirred at room (2) 10-Allyl-3,6-diazaphenothiazine (8) (92 mg, 78%); an oil: H
H N S C 61.37, H 4.21, N 19.52. Found:
9 3
1
mmol) in dry ethanol (10 mL), 2-chloro-3-nitropyridine (2)
1
(
temperature for 3 h and next the resulting brown crystals were
0
NMR (CDCl
5.39 (m, 1H, ¼CH), 5.97 (m, 1H, CH), 6.59 (d, J ¼ 5.4 Hz,
1H, H ), 6.97 (m, 2H, H , H
), 8.02 (dd, 1H, J ¼ 4.8 Hz,
J ¼ 1.2 Hz, H ), 8.06 (s, 1H, H ), 8.17 (d, J ¼ 5.4 Hz, 1H, H ).
3
) d: 4.37 (m, 2H, N-CH
2
), 5.21 (m, 1H, ¼CH),
filtered off, washed with ethanol and air dried to give 3-amino-3 -
0
nitro-2,4 -dipyridinyl sulfide (3). The dry product was recrystal-
ꢀ
1
9
8
lized from ethanol yielding 218 mg (88%), m.p. 149–150 C.
7
4
2

DOI: 10.3109/14756366.2016.1151014
3,6-Diazaphenothiazines as potential lead molecules
3
EI MS m/z: 241 (M, 45), 39 (C H , 100). Anal Calcd for: CI MS m/z: 389 (M + H, 100), 201 (M + 1-(CH ) N(CO) C H ,
3
3
2 3
2 6 4
C H N S C 64.70, H 4.59, N 17.41. Found: 64.72, H 4.54, 10). Anal Calcd for: C H N O S: C 64.93, H 4.15, N 14.42.
1
3
11
3
21 16 4 2
N 17.29.
3) 10-Benzyl-3,6-diazaphenothiazine (10) (85 mg, 58%); an oil:
Found: C 64.82, H 4.22, N 14.21.
(
(
(
1
H NMR (CDCl ) d: 5.05 (s, 2H, CH ), 6.49 (d, J ¼ 5.4 Hz, Synthesis of 10-substituted 3,6-diazaphenothiazines (14–20)
3
2
1
H, H ), 6.83 (dd, J ¼7.8 Hz, J ¼ 1.2 Hz, 1H, H ), 7.07 (dd,
8 6 5
1
9
To a solution of 10H-3,6-diazaphenothiazine (6) (100 mg,
0
J ¼ 7.8 Hz, J ¼ 4.8 Hz, 1H, H ), 7.39 (m, 5H, C H ), 8.08 (m,
.5 mmol) in dry dioxane (10 mL), NaOH (200 mg, 5 mmol) was
3
6
7
H, H , H H ). EI MS m/z: 291 (M, 20), 200 (M-CH C H ,
7 4, 2 2 6 5
added. The mixture was refluxed for 2 h and hydrochloride of
dialkylaminoalkyl chloride (2-diethylaminoethyl, 3-dimethylami-
nopropyl, 3-dimethylamino-2-methylpropyl) or hydrochloride
of cycloaminoethyl chloride [1–(2-chloroethyl)pyrrolidine, 2–(2-
0), 91 (C H CH , 100). Anal Calcd for: C H N S C
2
6
5
17 13 3
0.08, H 4.50, N 14.42. Found: C 70.10, H 4.59, N 14.31.
0
0
4) 10-(1’-Methyl-4 -nitro-5 -imidazolyl)-3,6-diazaphenothia-
1
ꢀ
zine (12) (100 mg, 69%); m.p. 82–83 C: H NMR (DMSO-
d ) d: 3.65 (s, 3H, CH ), 6.03 (d, J ¼ 5.4 Hz, 1H, H ), 6.85
chloroethyl)-1-methylpiperidine, 1–(2-chloroethyl)piperidine,
–(2-chloroethyl)mor-pholine 1.5 mmol] was added. The reaction
6
3
1
1
mixture was refluxed for 24 h. After cooling, dioxane was
evaporated in vacuo and residue was dissolved in CHCl3
(
m, 2H, H , H ), 7.49 (s, 1H), 7.86 (dd, 1H, J ¼ 4.8 Hz,
7 2 4
9
8
J ¼ 1.2 Hz, 1H, H ), 8.13 (m, 2H, H , H ). EI MS m/z: 326
(
M, 45), 42 (C H N, 100). Anal Calcd for: C H N SO C
2 4 14 10 6 2
(10 mL). The solution was washed with water (10 mL), dried
with anhydrous Na SO and evaporated in vacuo. The obtained
5
5) 10-(2 -Pyrimidinyl)-3,6-diazaphenothiazine (13) (119 mg,
1.53, H 3.09, N 25.75. Found: C 51.29, H 3.13, N 25.61.
0
2
4
1
product was purified by column chromatography (aluminum
oxide, CH Cl ) to give:
ꢀ
8
5%); m.p. 199–200 C: H NMR (DMSO ) d: 6.09 (d,
d6
2
2
J ¼ 5.4 Hz, 1H, H ), 6.97 (m, 2H, H , H ), 7.43 (t, J ¼4.5 Hz,
1
9
8
0
(
1) 10-(2 -Diethylaminoethyl)-3,6-diazaphenothiazine
1
(14)
1
1
H, H
H, H ) 8.34 (d, J ¼ 5.4 Hz, 1H, H ) 8.84 (d, J ¼ 4.5 Hz, 2H,
0
), 7.79 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.08 (s,
5
7
(
102 mg, 71%); an oil: H NMR: d 1.10 (t, J ¼ 7.2 Hz, 6H,
4
,
2
2
2
1
CH ), 2.65 (q, J ¼ 7.2 Hz, 4H, 2CH ), 2.82 (t, J ¼ 7.2 Hz,
2 2
3
2
H ). EI MS m/z: 279 (M, 100), 200 (M-C H N , 25).
Anal Calcd for: C H N S C 60.20, H 3.25, N 25.07. Found:
0
,H
0
4
6
4 3 2
H, CH ), 3.94 (t, J ¼ 7.2 Hz, 2H, CH ), 6.73 (d, J ¼ 5.4 Hz,
1
4 9 5
H, H ), 7.03 (m, 2H, H , H ), 8.02 (dd, 1H, J ¼ 4.8 Hz,
1
9
8
C 60.31, H 3.29, N 24.95.
J ¼ 1.2 Hz, H ), 8.10 (s, 1H, H ), 8.24 (d, J ¼ 5.4 Hz, 1H, H ).
7
4
2
CI MS m/z: 301 (M + 1, 100), Anal Calcd for: C H N S C
6 20 4
1
6
2) 10-(3 -Dimethylaminopropyl)-3,6-diazaphenothiazine (15)
3.97; H 6.71; N 18.65. Found: C 63.92; H 6.78; N 18.50.
0
Synthesis of 10-propargyl-3,6-diazaphenothiazines (9)
(
To a suspension of 10H-3,6-diazaphenothiazine (6) (100 mg,
.5 mmol) in dry DMF (10 mL), potassium tert-butoxide (80 mg,
1
(100 mg, 70%); an oil: H NMR: d 1.83 (m, 2H, CH2),
0
2
.21 (s, 6H, 2CH ), 2.37 (t, J ¼ 7.5 Hz, 2H, NCH ), 3.79 (t,
3
2
0
.72 mmol) was added. The mixture was stirred at room
J ¼ 7.5 Hz, 2H, NCH ), 6.64 (d, J ¼ 5.4 Hz, 1H, H ), 7.01 (m,
2
1
temperature for 1 h. Then the 80% solution of propargyl bromide
80 mg, 0.64 mmol) in dry toluene was added dropwise. The
2
H, H , H ), 7.97 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.06
4 2
9
8
7
(
(
(
s, 1H, H ), 8.17 (d, J ¼ 5.4 Hz, 1H, H ).CI MS m/z: 287
3 6 2 6
solution stirred at room temperature for 24 h and poured into
water (20 mL), extracted with methylene chloride (20 mL), dried
with anhydrous Na SO , evaporated to the brown oil. The residue
M + 1, 100), 202 (M + 1-C H NC H , 15). Anal Calcd for
C H N S C 62.91; H 6.33; N 19.56. Found: C 62.99; H
15 18 4
2
4
6.27; N 19.45.
0
was purified by column chromatography (silica gel, CHCl ) to
3
0
(3) 10-(3 -Dimethylamino-2 -methylpropyl)-3,6-diazaphenothia-
1
yield 70 mg (61%) of 10-propargyl-3,6-diazaphenothiazine (9),
ꢀ
zine (16) (120 mg, 82%); an oil: H NMR: d 1.02 (d,
m.p. 139–141 C.
1
J ¼ 6.5 Hz, 3H, CH ), 2.29 (m, 9H, 2CH CH , CH), 4.05
3
3,
2
H NMR (CDCl ): d 2.31 (t, J ¼ 2.4 Hz, 1H), 4.43 (d,
3
(m, 2H, CH ), 6.79 (d, J ¼ 5.4 Hz, 1H, H ), 7.05 (m, 2H, H ,
8 7
2
1
9
J ¼ 2.4 Hz, 2H), 6.95 (d, J ¼ 5.7 Hz, 1H, H ), 7.09 (dd,
1
H ), 8.08 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.10 (s, 1H,
4 2
J ¼ 7.8 Hz, J ¼ 4.8 Hz, 1H, H ), 7.35 (dd, J ¼7.8 Hz, J ¼ 1.2 Hz,
8
H ), 8.26 (d, J ¼ 5.4 Hz, 1H, H ). CI MS m/z: 301 (M + 1,
16 20 4
1
H, H ), 8.11 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.20 (s, 1H,
4 2
9
7
100). Anal Calcd for: C H N S C 63.97; H 6.71; N 18.65.
Found: C 63.87; H 6.79; N 18.52.
H ), 8.32 (d, J ¼ 5.7 Hz, 1H, H ). CI MS: 240 (M + 1, 100). Anal
Calcd for: C H N S C 65.25, H 3.79, N 17.56. Found: C 65.22,
3
0
1
3
9
(4) 10-(2 -Pyrrolidinylethyl)-3,6-diazaphenothiazine
1
(17)
H 3.71, N 17.46.
(
110 mg, 74%); an oil: H NMR (CDCl ) d: 1.85 (m, 4H,
3
2
3
7
H
CH ), 2.65 (m, 4H, 2CH ), 2.86 (t, J ¼ 7.5 Hz, 2H, CH ),
2
2
2
.93 (t, J ¼ 7.5 Hz, 2H, NCH ), 6.71 (d, J ¼ 5.4 Hz, 1H, H ),
2
1
Synthesis of 10-phthalimidopropyl-3,6-diazaphenothiazines (11)
To a stirred solution of 10H-3,6-diazaphenothiazine (6) (100 mg,
.04 (m, 2H, H , H ), 8.02 (dd, 1H, J ¼ 4.8 Hz, J ¼ 1.2 Hz,
9
8
), 8.10 (s, 1H, H
), 8.23 (d, J ¼ 5.4 Hz, 1H, H
). CI MS
2
S C 64.40;
7
4
0.5 mmol) in dry toluene (20 mL), NaH (120 mg, 5 mmol, washed
out with hexane) was added. The mixture was stirred at room
m/z: 299 (M + 1, 100). Anal Calcd for: C16 N
H 6.08; N 18.78. Found: C 64.25; H 6.09; N 18.65.
H
18
4
0
temperature for 30 min, then refluxed for 1 h and a solution of N- (5) 10-(2 -Piperydinylethyl)-3,6-diazaphenothiazine
(18)
) d: 1.48 (m, 2H,
) 2.55 (m, 4H, 2CH ), 2.68 (t,
), 3.87 (t, J ¼ 6.8 Hz, 2H, NCH ), 6.72
(d, J ¼ 5.4 Hz, 1H, H ), 7.03 (m, 2H, H , H ), 8.01 (dd,
J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.08 (s, 1H, H ), 8.20 (d,
J ¼ 5.4 Hz, 1H, H ). CI MS m/z: 313 (M + 1, 100). Anal
Calcd for: C17 S: C 65.35; H 6.45; N 17.93. Found: C
1
(
(
3-bromopropyl)phthalimide (405 mg, 1.5 mmol) in toluene
10 mL) was added. The mixture was refluxed for 48 h. After
(120 mg, 72%); an oil: H NMR (CDCl
CH ), 2.48 (m, 4H, 2CH
J ¼ 6.8 Hz, 2H, CH
3
2
2
2
cooling, the resulted solid was filtered off, toluene was evaporated
in vacuo and the residue was purified by column chromatography
2
2
1
9
8
0
(aluminum oxide, CHCl ) to give 10-(3 -phthalimidopropyl)-3,6-
diazaphenothiazine (11) (117 mg, 74%), m.p. 45–47 C.
7
4
3
ꢀ
2
1
H NMR (DMSO ) d: 2.11 (m, 2H, CH ), 3.58 (t, J ¼ 6.1 Hz,
H N
20 4
d6
2
2
H, NCH ), 3.77 (t, J ¼ 6.0 Hz, 2H, NCH ), 5.92 (d, J ¼ 7.2 Hz,
65.22; H 6.49; N 17.80.
0
2
2
1
H, H ), 6.42 (d, J ¼ 1.5 Hz, 1H, H ), 6.68 (dd, J ¼ 7.8 Hz, (6) 10-(1’-Methyl-2 -piperydinylethyl)-3,6-diazaphenothiazine
1
4
1
J ¼ 4.7 Hz, 1H, H ), 6.81 (dd, J ¼ 7.8 Hz, J ¼ 1.5 Hz, 1H, H ),
(19) (0.119 g, 74%); an oil: H NMR (CDCl
(m, 12H), 2.26 (s, 3H, NCH ), 2.87 (m, 1H, CH), 3.85 (m,
2H, NCH ), 7.01 (m, 2H, H
), 6.63 (d, J ¼ 5.4 Hz, 1H, H
) d: 1.25–2.20
3
8
9
6
.85 (dd, J ¼ 7.2 Hz, J ¼ 1.5 Hz, 1H, H ), 7.69 (dd, J ¼ 4,7 Hz,
3
2
J ¼ 1.5 Hz, 1H, H ), 7.71 (m, 2H
), 7.86 (m, 2H ).
phthalimide
2
1
,
9
7
phthalimide

4
B. Morak-Młodawska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
H ), 8.02 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H, H ), 8.11 (s, 1H, absorbance of the samples against a background control was read
8
7
H ), 8.22 (d, J ¼ 5.4 Hz, 1H, H ). CI MS m/z 327 (M + H, at 450 nm using with a reference wavelength ꢄ ¼ 600 nm a
4
2
1
Found: C 66.15; H 6.79; N 17.11.
00). Anal Calcd for: C H N S C 66.22; H 6.79; N 17.16. microplate reader. Results are expressed as means of at least two
18 22 4
independent experiments performed in triplicate.
0
(
7) 10-(2 -Morpholinylethyl)-3,6-diazaphenothiazine
1
(20)
(0.112 g, 69%); an oil: H NMR (CDCl ) d: 1.67 (m, 4H,
The RT-QPCR method
3
2
4
7
CH ), 2.59 (m, 4H, 2CH ), 2.82 (t, J ¼ 6.6 Hz, 2H, CH ),
2
2
2
Genes transcriptional activity (H3, TP53, CDKN1A, BCL-2, BAX) was
evaluated by real time RT-QPCR method with OPTICON TM
.22 (t, J ¼ 6.6 Hz, 2H, NCH ) 6.73 (d, J ¼ 5.4 Hz, 1H, H ),
2
1
.01 (m, 2H, H , H ), 8.04 (dd, J ¼ 4.8 Hz, J ¼ 1.2 Hz, 1H,
9
8
Õ
DNA Engine (MJ Research, Watertown, NY) and QuantTect
Õ
H ), 8.10 (s, 1H, H ), 8.25 (d, J ¼ 5.4 Hz, 1H, H ). CI MS
7
4
2
SYBR Green RT-PCR Kit (Quiagen, Valencia, CA). Cells were
exposed to compounds 6 and 13 at concentration of 0.5 mg/mL for
m/z: 315 (M + 1, 40), 202 (M + 1-C H NOC H , 15), 114
2
4
4 8
(C H NC H , 100). Anal Calcd for: C H N OS: C 61.12;
H 5.77; N 17.82. Found: C 61.19; H 5.57; N 17.71.
2 4 5 10 16 18 4
2
4 h. The RNA extraction was made by using Quick-RNAÔ Kit
MiniPrep (ZYMO RESEARCH, Irvine, CA). Total RNA integrity
was analyzed in 1.2% agarose electrophoresis with added
ethidium bromide compound. The quantity and purity of extracted
total RNA were determined by using spectrophotometric analysis
with HP845 (Hewlett Packard, Waldbronn, Germany) spectro-
photometer. The statistical analysis was performed using the
Statistica 8.0 software (StatSoft, Tulsa, OK). All values were
expressed as means ± SE.
Crystal data
C H N S, M ¼ 215.27, orange needle, 0.48 ꢁ 0.22 ꢁ 0.15 mm ,
3
1
1 9 3
3
˚
orthorhombic, space group P2 2 2 , V ¼ 941.6(1) A , Z ¼ 4,
1
1 1
3
D ¼ 1.519 g/cm , F ¼448, KappaApexII, Mo-Kꢀ radiation,
c
000
˚
ꢀ
l ¼ 0.71073 A, T ¼ 100(2) K, 2ꢁ ¼ 55.0 , 4688 reflections
max
collected, 2123 unique (R ¼ 0.030). The structure was solved
int
2
and refined using the programs SHELXS-97 and SHELXL-
7
Results and discussion
Chemistry
2
8
2
013 , respectively. Final GooF ¼ 1.11, R ¼ 0.056, wR ¼ 0.113, R
2
indices based on 1907 reflections with I42ꢂ(I) (refinement on F ),
37 parameters, 0 restraints. Lp and absorption corrections applied,
Synthesis and 2D NMR analysis
1
ꢂ 1
29
ꢃ ¼ 0.307 mm . Absolute structure parameter ¼ 0.2(1) .
The synthetic routes to phenothiazines proceed mainly through
the Smiles rearrangement depending on the substrate structures
and reaction conditions. Very often the rearrangement was
observed under basic conditions (sodium or potassium hydroxide
in methanol or ethanol), rarely under neutral or acidic media. In
some cases, the rearrangement is hard to monitor for the
rearranged and non-rearranged products that can have the same
structure or can differ very subtly (the place of a substituent or a
Cytotoxic and antiproliferative effects in vitro
Cell culture
Compounds were evaluated for their anticancer activity using
three cultured cell lines: SNB-19 (human glioblastoma, DSMZ –
German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany), C32 (human amelanotic melanoma,
ATCC – American Type Culture Collection, Manassas, VA) and
3
0–33
nitrogen atom in the case of the azaphenothiazines
We started the azaphenothiazine synthesis with the cyclization
.
0
0
of 3-amino-3 -nitro-2,4 -dipyridinyl sulfide (3) (obtained from
sodium 3-amino-2-pyridinethiolate (1) and 4-chloro-3-nitropyr-
idine (2) in ethanol) in refluxing DMF solution to give 90% yield
of the product. The same product was obtained (in 69%) directly
from sodium 3-amino-2-pyridinethiolate (1) and 4-chloro-3-
nitropyridine (2) in refluxing DMF (Scheme 1). Those reactions
were monitored by TLC analysis as the product chromatogram,
unlike to the substrate chromatograms, showed characteristic for
MCF-7 (human breast cancer, ATCC, Manassas, VA). The
ꢀ
cultured cells were kept at 37 C and 5% CO . The cells were
2
4
seeded (1 ꢁ 10 cells/well/100 mL DMEM supplemented with
1
(
0% FCS and streptomycin and penicillin) using 96-well plates
Corning).
Cell proliferation and viability
In recent years, tetrazolium salts have been described to be azaphenothiazines color changing during irradiation with UV
used for the measurement of cell proliferation and viability. light from pale yellow (6–11) and yellow-celadon (14–20) to
The tetrazolium salts are cleaved to formazan by cellular beige, and yellow (12,13) to orange-red. All azaphenothiazine
enzymes. An expansion in the number of viable cells results in chromatograms gave yellow color after sprayed with the mixture
an increase in the overall activity of mitochondrial dehydro- of sulfuric acid-water-ethanol (1:1:8). To distinguish the struc-
genases in the sample. This augmentation in enzyme activity ture of the azaphenothiazine (4 or 6), the obtained compound was
leads to an increase in the amount of formazan dye formed, methylated with methyl iodide. The alkylation of azaphenothia-
which directly correlates to the number of metabolically active zines was reported as the process occurring mainly at the thiazine
cells in the culture. The formazan dye produced by metabol- nitrogen atom but one can find a few reports on alkylation at the
ically active cells is quantified by a scanning ELISA reader by azine nitrogen atom giving azaphenothiazinium salts and neutral
3
4–38
measuring the absorbance of the dye solution at appropriate N-alkylazaphenothiazines
. The reaction of the product with
wavelengths (ꢄ ¼ 420–480 nm with a reference wavelength methyl iodide in dry DMF in the presence of sodium hydride led
ꢄ ¼ 600 nm).
to the methylated product, which possessed only one methyl
1
group (observed in the H NMR spectrum). The lack of the
ammonium function could point at 10-methyl-3,6-diazaphe-
nothiazine (7) (the Smiles product) or 10-methyl-2,6-diazaphe-
WST-1 assay
The WST-1 assay (Roche Diagnostics, Mannheim, Germany) was nothiazine (4A) (the Ullmann product) or 3-methyl-3,6-
used to evaluate the effect of compounds on the number of cells in
cultures, which has the cytotoxic effect of the tested compounds
diazaphenothiazine (7A) (an alternative alkylation product). To
elucidate the product structure, we recorded 2D NMR (ROESY,
and their influence on the proliferation of cells. After exposure to COSY, HSQC and HMBC) spectra of the N-methyl product. The
tested compounds (at concentrations between 0 and 100 mg/mL) ROESY experiment with irradiation of the methyl protons at
for 72 h, cells were incubated with WST-1 (10 mL) for 1 h, and the
3
.30 ppm showed the proximity of the methyl group to the protons

DOI: 10.3109/14756366.2016.1151014
3,6-Diazaphenothiazines as potential lead molecules
5
at 6.60 ppm (a doublet) and 6.98 ppm (a doublet of doublet), was carried out. The X-ray analysis fully confirmed the product
1
which excludes structures 4A and 7A (a singlet proton signal structure concluded from the H NMR spectra as the 3,6-
would be involved) and pointed at structure 7. The full proton diazaphenothiazine system 7 and showed spatial arrangement in
signal assignment was achieved by study of other proton spatial the molecule in a solid state (Figure 1).
1
1
proximity (ROESY) and H– H connectivities in COSY spec-
The tricyclic ring system is not planar but folded along the S–
ꢀ
trum. The signals at 6.60 and 6.98 ppm were assigned as H and N axis with the butterfly angle of 134.7(2) between two pyridine
1
H protons, respectively. The confirmation of the proton assign- ring planes. The central thiazine ring is in boat conformation with
9
1
3
ment came from the C NMR spectrum, which was solved by the the angle between two halves (SCCS) of 139.3(2) . The methyl
ꢀ
1
3
1
use of HSQC and HMBC spectra indicating the C– H relation- group is located in equatorial position with the S5ꢃꢃꢃN10–C11
ꢀ
ship. The HSQC spectra showed which proton was bonded to the angle of 170.5(2) . The important bond angles of the central ring
1
carbon atom (the C–H relationship through one bond, JC,H C9a–N10–C10a and C4a–S5–C5a are 120.11(18) and 98.1(2) ,
ꢀ
ꢀ
ꢀ
connectivity) and the HMBC spectra indicated the C–H relation- respectively. The sum of three C–N10–C angles is 353.1
ship through three (predominantly), two and four (exceptionally) indicating the pyramidal configuration of the bonds around the
4
bonds ( JC,H, JC,H and JC,H connectivities). Selected spatial central nitrogen atom. Whereas the N–C bond lengths in the
3
2
˚
proton–proton proximity, proton–proton and proton–carbon con- pyridine rings are in the range of 1.328–1.354 A, the N–C bond
1
nectivities for compound 7 are shown in Scheme 2. The all H– H lengths in the thiazine ring are significantly longer 1.409–1.410 A.
1
˚
1
13
and H– C connectivities are included in the ‘‘Supplementary The N–C bond to the methyl group was the longest one (1.460 A)
˚
3
Material’’ (Table 3, Scheme 4). The methylated product was being a result of the sp hybridization of the carbon atom. The
identified as 10-methyl-3,6-diazaphenothiazine (10-methyldipyr- only known X-ray structures of dipyridothiazines, deposited in the
39
0
0
ido[2,3-b;4 ,3 -e][1,4]thiazine) (7).
Cambridge Structural Database , concern 10-substituted 1,8- and
,7-diazaphenothiazines, and their methyl iodides. Whereas 1,8-
2
and 2,7-diazaphenothiazines are folded, the methyl iodide salts
are folded or planar depending on the character of the nitrogen
The X-ray structure analysis
3
2,33,40,41
As the indirect method of the structure elucidation based on the atom and number of alkylated atoms
.
In solid state, the molecules are arranged into layer-type stacks
1
H NMR analysis is subtle and can lead to false conclusions, a
single crystal X-ray diffraction study of the methyl derivative along the c-axis. The arrangement within a layer involves four
Scheme 1. Synthesis of 10H-3,6-diazaphe-
nothiazine 6.
Scheme 2. The ROESY NMR experiments
for compound 7.

6
B. Morak-Młodawska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
.
weak C–H N hydrogen bonding to neighboring molecules Similar strong and selective action was found for 10-pyrimidinyl
. .
(
Supplementary Material).
derivative (13) against breast cancer MCF-7 cell line. 10-
Dimethylaminopropyl derivative (15) exhibited as good activity
against melanoma C-32 cell line as cisplatin and moderate activity
against MCF-7 cell line. Other 3,6-diazaphenothiazines exhibited
weaker activity with IC 428 mg/mL. Prothipendyl turned out to
The transformation into 10-substituted derivatives
The parent product 6 was transformed into other derivatives
possessing the allyl (8), propargyl (9), benzyl (10), phthalimido-
propyl (11), imidazolyl (12), pyrimidinyl (13) and dialkylami-
noalkyl (with cyclic and non-cyclic amine groups) (14–20)
substituents in the reactions with appropriate halides in neutral
solvents (DMF, toluene, dioxane) in the presence of base (NaH,
NaOH, t-BuOK) (Scheme 3).
5
0
be less active than 3, 6, and 8 derivatives of 3,6-diazaphenothia-
zine against investigated cell lines, respectively. All 3,6-diazaphe-
nothiazines were found to be non-toxic (IC 450 mg/mL) or
5
0
almost non-toxic (IC50 38.7–43.9 mg/mL) against normal human
fibroblast (HFF-1) cell line in comparison with toxic cisplatin
(IC50 ¼ 8.2 mg/mL).
Anticancer activity
Apoptosis assay
The anticancer activity of 3,6-diazaphenothiazines (6–20) was
investigated in vitro using cultured glioblastoma SNB-19, It has been known that the growth, division and eventual death of
melanoma C-32 and breast cancer MCF-7 cell lines and cisplatin the cells in the body are processes that are controlled by hundreds
4
2
as a reference drug. Normal human fibroblast (HFF-1) cell line of genes working together . The most active compounds 6 and 13
was used as a control. To compare the influence of the nitrogen (with the hydrogen and pyrimidinyl substituents) were selected for
atoms in the azaphenothiazine system on the anticancer activity, efforts to understand the mechanism of anticancer action. To
the classical monoazaphenothiazine drug, prothipendyl, was also determine one of the antiproliferative mechanisms, the gene
tested. The tested 3,6-diazaphenothiazines exhibited different transcriptional activities of proliferation marker (H3) cell cycle
activities against the cell lines. Two derivatives exhibited very regulator (TP53 and CDKNIA) and intracellular apoptosis path-
strong activity with IC 51 mg/mL (Table 1).
way (BACL-2 and BAX) were analyzed with the use of RT-QPCR
5
0
The parent compound (10H-phenothiazine) (6) exerted very method. The results of analysis of H3, TP53, CDKN1A, BCL-2,
strong action against all tumor lines with IC ¼0.46–0.72 mg/mL), BAX genes in MCF-7, SNB-19 and C-32 cells after 24 h of
5
0
being over 10 times more active than a reference drug – cisplatin. treatment are collected in Table 2.
Figure 1. ORTEP drawing of 10-methyl-3,6-diazaphenothiazine, showing the atom labeling.
Scheme 3. Synthesis of 10-substituted 3,6-diazaphenothiazines.

DOI: 10.3109/14756366.2016.1151014
3,6-Diazaphenothiazines as potential lead molecules
7
Table 1. The anticancer activity of 3,6-diazaphenothiazines.
Table 2. The influence of compounds 6 and 13 on the expression of genes
encoding: H3, TP53, CDKN1A, BCL-2, BAX in: breast cancer cell line
MCF-7, glioblastoma SNB-19 and melanoma C-32.
Anticancer activity IC50 (mg/mL)
No.
SNB-19
C-32
MCF-7
HFF-1
Number of mRNA copies/mg total RNA
6
7
8
9
0.58
450
45.7
45.1
34.7
450
45.6
450
450
31.1
450
46.8
450
38.9
450
48.7
7.7
0.72
450
29.9
32.9
43.4
450
450
450
42.1
6.3
450
450
450
450
450
450
7.8
0.46
450
32.9
37.9
30.2
450
43.9
0.73
450
11.3
450
450
450
28.4
450
23.2
7.4
38.6
450
450
450
450
450
450
450
450
450
450
450
450
450
450
450
8.2
Gene
MCF-7
SNB-19
C-32
H3
Control
6
13
TP53
Control
6
13
CDKN1A
Control
6
80840+/-11781
16163+/-2601
31503+/-2705
1759+/-69
367+/-43
221+/-13
12+/-2
6+/-3
5+/-1
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
106742+/-21545
95722+/-2235
138476+/-23716
39570+/-2490
41270+/-7086
26630+/-1578
678+/-63
722+/-46
855+/-150
70649+/-6536
138319+/-26200
127548+/-10964
25432+/-1347
35087+/-270
38987+/-3397
449+/-63
343+/-79
524+/-70
13
BCL-2
Control
6
Prothipendyl
Cisplatin
2555+/-520
1607+/-12
3283+/-400
3011+/-302
1724+/-300
1284+/-96
24397+/-297
11352+/-2483
12082+/-966
1
3
BAX
Control
100749+/-4311
94674+/-5319
90944+/-13237
122417+/-14571
110350+/-19616
99895+/-14766
3162+/-434
1690+/-627
1726+/-111
The gene encoding the histone H3 is considered as an indicator
proliferation, which plays an important role in regulation of the
6
1
3
4
3
expression of the genetic information encoded in DNA . For both BAX/BCL-2
compounds, the number of transcripts significantly decreased,
what could suggest the alteration in chromatin conformation.
The importance of P53 protein in cancer biology is undisputed.
This protein is recognized as the guardian of the genome and is
able to induce apoptosis. The P53 transcription factor is activated
by potentially oncogenic stimuli, such as ribosomal stress, DNA
damage, telomere erosion, nutrient deprivation and oncogene
hyperactivation. This protein influences cell cycle arrest by
changing the expression of CDKN1A gene encoding the P21
Control
6
39.4
58.9
27.7
40.7
64.0
77.8
0.13
0.15
0.14
1
3
The parent compound 10H-3,6-diazaphenothizine was over 10
times more active (IC 50.72 mg/mL) than a reference drug –
cisplatin against glioblastoma SNB-19, melanoma C-32 and
5
0
breast cancer MCF-7 cell lines. Two diazaphenothiazines with the
2
4
2,44
protein
. Whereas compound 13 generated significant changes
-pyrimidinyl and dimethylaminopropyl substituents were very
in the expression of TP53 gene (the increase in MCF-7 and C-32
cells but the decrease in SNB-19 cells); compound 6 did not show
any alterations. Both compounds show significant increase of
CDKN1A copies in MCF-7 and SNB-19 cells suggesting possi-
bility of participation in cell cycle arrest and apoptosis.
selectively antitumor active against breast cancer MCF-7 (10
times active) and melanoma C-32 (similarly active) cell lines,
respectively, in relation to cisplatin. 3, 6 and 8 derivatives of 3,6-
diazaphenothiazines turned out to be more active than mono-
azaphenothiazine drug, prothipendyl, against investigated cell
lines. All 3,6-diazaphenothiazines were found to be non-toxic or
almost non-toxic against normal human fibroblast (HFF-1) cell
line in comparison with toxic cisplatin. The analysis of gene
expressions (H3, TP53, CDKN1A, BCL-2, BAX) confirmed the
antiproliferative activity of both compounds and indicated the
activation of the p53 pathway in cancer cells, leading to the cell
cycle arrest. The gene expression ratio BAX/BCL-2 suggested the
mitochondrial apoptosis pathway activation in MCF-7 and SNB-
The P53 protein can also stimulate the cell to changes in
gene expression of proapoptopic BAX and antiapoptopic BCL-2
4
5–48
involved in mitochondrial pathway apoptosis
. Both com-
pounds reduced the expression of BCL-2 and BAX genes (with
one exception for compound 13). Analysis of the gene
expression ratio BAX/BCL-2 in the MCF-7 and SNB-19 cells
showed activation of the mitochondrial apoptosis for compound
6
(in both cells) and 13 (only in SNB-19). Transcriptional
activity of these genes in the C-32 cells suggests a different
way of cell death.
1
9 cells.
Declaration of interest
Conclusion
The work was supported by the Medical University of Silesia
(grant KNW-1–004/K/4/0).
We report here synthesis of 15 new 10-substituted 3,6-
diazaphenothiazines. The parent compound, 10H-3,6-diazaphe-
nothiazine, was obtained in cyclization reaction of 3-amino-3 -
0
0
nitro-2,4 -dipyridinyl sulfide and in the reaction of sodium 3- References
amino-2-pyridinethiolate with 4-chloro-3-nitropyridine. This
1
.
Cancer Facts and Figures, American Cancer Society, 2013.
Available from: http://www.cancer. org/acs/groups/content/@epide-
compound was transformed into 10-substituted derivatives with
the alkyl, imidoalkyl, heteroaryl and dialkylaminoalkyl groups in
the alkylation and heteroarylation reactions. The analysis of 2D
NMR (ROESY, COSY, HSQC and HMBC) spectra of the
N-methylated product showed that the thiazine ring formation
proceeded through the Smiles rearrangement of the S–N type and
the alkylation proceeded at the thiazine, not the azine nitrogen
atom. This supposition was fully confirmed by X-ray analysis.
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Supplementary material available online