Synthesis, spectroscopic characterization, and anticancer activity of new 10-substituted 1,6-diazaphenothiazines

Article abstract of DOI:10.1007/s00044-016-1646-3

New phenothiazine derivatives as 10-substituted dipyridothiazines of the 1,6-diazaphenothiazine structure were obtained in the cyclization reaction of 3-amino-3′-nitro-2,2′-dipyridinyl sulfide and 3,3′-dinitro-2,2′-dipyridinyl disulfide, and in the reaction of 2-chloro-3-ntropyridine with sodium 3-amino-2-pyridinethiolate followed by various alkylation and arylation reactions. The reaction of the thiazine ring formation ran via the Smiles rearrangement of the S-N type. As the alkylation reactions could proceed at the thiazine, azine or both nitrogen atoms, the product structure elucidation was based on the 2D NMR (Rotating-frame Overhauser Effect Spectroscopy, Correlated Spectroscopy, Heteronuclear Single Quantum Coherence, and Heteronuclear Multiple Bond Correlation) spectra of the N-methylated product. Some 10-substituted 1,6-diazaphenothizines (5, 10, 12, 13) were at least anticancer active against melanoma C-32 and breast cancer MCF-7 cell lines as a reference drug – cisplatin. The monoazaphenothiazine drug, prothipendyl, turned out to be less active than least 6 derivatives of the 1,6-diazaphenothiazine structure.

Full text of DOI:10.1007/s00044-016-1646-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1646-3
ORIGINAL RESEARCH
Synthesis, spectroscopic characterization, and anticancer activity of new
10-substituted 1,6-diazaphenothiazines
Beata Morak-Młodawska1 Krystian Pluta1 Małgorzata Latocha2
Małgorzata Jeleń1
●
●
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Received: 6 July 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
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Abstract New phenothiazine derivatives as 10-substituted
dipyridothiazines of the 1,6-diazaphenothiazine structure
were obtained in the cyclization reaction of 3-amino-3′-
nitro-2,2′-dipyridinyl sulfide and 3,3′-dinitro-2,2′-dipyr-
idinyl disulfide, and in the reaction of 2-chloro-3-ntropyr-
idine with sodium 3-amino-2-pyridinethiolate followed by
various alkylation and arylation reactions. The reaction of
the thiazine ring formation ran via the Smiles rearrange-
ment of the S-N type. As the alkylation reactions could
proceed at the thiazine, azine or both nitrogen atoms, the
product structure elucidation was based on the 2D NMR
(Rotating-frame Overhauser Effect Spectroscopy, Corre-
lated Spectroscopy, Heteronuclear Single Quantum
Coherence, and Heteronuclear Multiple Bond Correlation)
spectra of the N-methylated product. Some 10-substituted
1,6-diazaphenothizines (5, 10, 12, 13) were at least antic-
ancer active against melanoma C-32 and breast cancer
MCF-7 cell lines as a reference drug – cisplatin. The
monoazaphenothiazine drug, prothipendyl, turned out to be
less active than least 6 derivatives of the 1,6-diazaphe-
nothiazine structure.
Keywords Phenothiazines Dipyridothiazines NMR
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structure elucidation 2D NMR spectra The Smiles
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rearrangement Anticancer activity
Introduction
Tricyclic phenothiazines (dibenzo-1,4-thiazines) are
important class of heterocycles possessing significant bio-
logical activities and interesting chemical features. Classical
10-substituted phenothiazines with the aminoalkyl groups at
the nitrogen atom have been for many years valuable drugs
exhibiting neuroleptic, antihistaminic, antitussive, and
antiemetic activities (Gupta and Kumar, 1988). They are
relatively easy-obtainable, inexpensive, and low toxic, and
they can be valuable source for searching new drugs of
other biological activities. The chemical structure mod-
ifications of these compounds were carried out mainly by
introduction of new substituents at the thiazine nitrogen
atom and substitution of one or two benzene rings with
homoaromatic and heteroaromatic rings. Such modifications
are expected to change not only potency but also types of
activities. Both classical and modified phenothiazines are
found to exhibit very promising anticancer, antibacterial,
antifungal, anti-inflammatory, and multidrug resistance
reversal activities, summarized recently in the review arti-
cles and chapters in monographs (Motohashi et al., 2000,
2006; Mitchell, 2006; Dasgupta et al., 2008; Aaron et al.,
2009; Sudeshna and Parimal, 2010; Pluta et al., 2011;
Wesołowska, 2011; Jaszczyszyn et al., 2012). They show
also a potential benefit in treatment of Alzheimer’s,
Creutzfeldt-Jakob’s, and AIDS-associated diseases (Mos-
naim et al., 2006; González-Muñoz et al., 2010).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1646-3) contains supplementary material,
which is available to authorized users.
* Krystian Pluta
pluta@sum.edu.pl
1
School of Pharmacy with the Division of Laboratory Medicine,
Department of Organic Chemistry, The Medical University of
Silesia, Jagiellońska 4, Sosnowiec 41-200, Poland
2
School of Pharmacy with the Division of Laboratory Medicine,
The last type of the modification with azine rings lead to
formation of azaphenothiazines. Our strategy for modification
Department of Cell Biology, The Medical University of Silesia,
Jedności 8, Sosnowiec 41-200, Poland

Med Chem Res
of the phenothiazine structure is based on the introduction of
two pyridine rings instead of the benzene ones to form various
dipyrido[1,4]thiazines. We found new dipyrido[1,4]thiazines
of the 1,8- and 2,7-diazaphenothiazine structures to exhibit
promising anticancer activity against lung cancers HOP-62
and HOP-92, colon cancers COLO 205, HCT-116 and SW-
948, renal cancers RXF393 and A498, and leukemia HL-60
(TB) and L-1210 (Pluta et al., 2010; Morak-Młodawska et al.,
2015). 10H-2,7-diazaphenothiazine shows also immunosup-
pressant, inhibiting both humoral and cellular immune
responses, and antioxidant properties (Zimecki et al., 2009;
Morak-Młodawska et al., 2010).
It is well known that the synthesis of phenothiazine and
azaphenothiazine ring system may proceed via 1,4-thiazine
ring formation with the use of diphenyl sulfides, phenyl
azinyl sulfides or diazinyl sulfides directly in the Ullmann
cyclization or indirectly through the Smiles rearrangement
of the S-N type to diphenylamines, phenylazinylamines,
and diazinylamines followed by cyclization. During the
rearrangement, the phenyl or azinyl part migrates from the
sulfur atom to the nitrogen atom (Pluta et al., 2009; Silberg
et al., 2006). There only two reports of double Smiles
rearrangement during those syntheses (Morak et al., 2002;
Morak-Młodawska et al., 2012).
imidoalkyl groups. In this work, we discuss the synthesis
and the structure elucidation of the NH- and N-alkyl-1,6-
diazaphenothiazines, and their anticancer activity.
Results and discussion
Chemistry
The possibility of the Smiles rearrangement depends on the
sulfide structure and reaction conditions. The most often the
rearrangement proceeds under basic conditions (sodium
hydroxide in ethanol), rarely under neutral or acidic media. It
is sometimes difficult to state if the rearrangement took place
as the rearranged and non-rearranged products can have the
same or similar structure. In the last case, the structure dif-
ference is in the location of a substituent or a nitrogen atom
(in the azaphenothiazine structure) (Pluta et al., 2009).
In our case, 3-amino-3′-nitro-2,2′-dipyridinyl sulfide 4
(obtained from 2-chloro-3-nitropyridine 1 and sodium 3-
amino-2-pyridinethiolate 3 in ethanol) heated in refluxing
N,N-dimethylformamide (DMF) solution did not undergo
cyclization to symmetrical 10H-4,6-diazaphenothiazine 5
1
(giving only three aromatic proton signals in the H NMR
The synthesis of 10-substituted derivatives from 10H-
diazaphenothiazines by the alkylation of the thiazine
nitrogen atom can be disturbed by alkylation of the azine
nitrogen atom.
For those reasons, the unquestioned elucidation of the
structure of the direct product, NH-azaphenothiazine, and
its N-substituted derivatives is crucial.
In continuation of those studies we have worked out an
efficient synthesis of another type of dipyridothiazines,
10H-1,6-diazaphenothiazine, and the transformation of this
parent compound into 10-substituted derivatives, possessing
alkyl, arylalkyl, heteroaryl and dialkylaminoalkyl, and
spectrum) but to 10H-1,6-diazaphenothiazine 7 in 90 %
yield. It means that the synthesis proceeded through the
Smiles rearrangement to dipyridinylamine 6 (which was not
isolated, Scheme 1). We found 3,3′-dinitro-2,2′-dipyridinyl
disulfide 2 (obtained from compound 1 to give compound 3)
to be quite a good substrate to form 1,6-diazaphenothiazine 7
(in 72 % yield) in boiling DMF in the presence of sodium
hydroxide. This is very useful synthesis because disulfide can
be obtained from commercially available 2-chloro-3-nitro-
pyridine 1 in 89 % yield. The same phenothiazine product
was obtained directly (in 64 % yield) from pyridines 1 and 3
in boiling DMF. It is not the first synthesis of 10H-1,6-
Scheme 1 Synthesis of 10H-
1,6-diazaphenothiazine 7

Med Chem Res
diazaphenothiazine 7 as Rodig and coworkers obtained
compound 7 in low yield (43 %) in cyclization of 3-acet-
ylamino-3′-nitro-2,2′-dipyridinyl sulfide in ethanolic solution
of potassium hydroxide and in very good yield (92 %) in
cyclization of dipyridinylamine 6 in DMSO-ethanol solution
of potassium hydroxide. In both cases the reactions substrates
were obtained in two steps using 2-chloro-3-nitropyridine 1
(Rodig et al., 1966).
(γ-pyridinyl proton) and pointed at the structure 8. Only
spatial 1H–1H connectivity with a proton at about 8 ppm (α-
pyridinyl proton) could point at the structure 8A. The full
proton signal assignment was achieved by study of other
proton spatial proximity (ROESY) and 1H—1H con-
nectivities (COSY). The signal at 6.98 ppm was assigned as
H-9 proton. The confirmation of the proton assignment
came from the 13C NMR spectrum which was solved by the
use of HSQC and HMBC spectra indicating the 13C–1H
relationship. The HSQC spectrum showed which proton
was bonded to the carbon atom (the C–H relationship
The next step was transformation of compound 7 into
N-substituted derivatives mainly by alkylation. Although
the alkylation of phenothiazines proceeds mainly at the
thiazine nitrogen atom, there are a few reports on the
alkylation of azaphenothiazines at the azine nitrogen atom
giving azaphenothiazinium salts and neutral N-alkylaza-
phenothiazines (Clarke et al., 1961; Werle et al., 1962;
Pappalardo et al., 1973; Carter and Cheeseman, 1977; Saari
et al., 1983). We studied the reaction of compound 7 with
methyl iodide in dry DMF in the presence of sodium
hydride. The methylated product possessed only one methyl
group (observed in the 1H NMR spectrum) without the
ammonium function what could point at the structure 8. To
exclude alternative neutral N-methylazaphenothiazine
structure i.e. 1-methyl-1,6-diazaphenothiazine 8A, we
recorded 2D NMR (Rotating-frame Overhauser Effect
Spectroscopy (ROESY), Correlated Spectroscopy (COSY),
Heteronuclear Single Quantum Coherence (HSQC), and
Heteronuclear Multiple Bond Correlation (HMBC)) spectra
of the N-methyl product. The ROESY experiment with
irradiation of the methyl protons at 3.40 ppm showed the
proximity of the methyl group to the proton at 6.98 ppm
1
through one bond, JC,H connectivity) and the HMBC
spectrum indicated the C–H relationship through three
(predominantly), two and four (exceptionally) bonds (3JC,H
,
2JC,H and JC,H connectivities). Selected spatial proton-
proton proximity, proton-proton and proton-carbon con-
nectivities for compound 8 were shown in Scheme 2. The
all 1H–1H and 1H–13C connectivities were included in
Table 1. The resulted product was identified as 10-
methyldipyrido[2,3-b;2′,3′-e][1,4]thiazine 8. The 1H and
13C NMR spectra of the rest compounds were solved using
the HSQC and HMBC experiments.
4
The parent product 7 was transformed into new deriva-
tives possessing the allyl (9), propargyl (10), benzyl (11),
nitropyridinyl (12), phthalimidopropyl (17) and the dia-
lkylaminoethyl (13–16, with cyclic and non-cyclic amine
group) substituents in the reactions of appropriate dialky-
laminoethyl halides in the presence of base (NaH, NaOH,
t-BuOK) in neutral solvents. The propargyl derivative 10
was converted into the dialkylaminobutynyl derivatives 18
Scheme 2 The 2D NMR
experiments for compound 8:
ROESY, COSY, HSQC, and
HMBC (selected connectivities)

Med Chem Res
Table 1 The H and 13C NMR
1H NMR δ
ROESY
COSY
13C NMR δ
HSQC
HMBC
1
assignment, and full proton–
proton and proton–carbon
connectivities (ROESY, COZY,
HSQC, and HMBC) for 10-
methyl-1,6-diazaphenothiazine 8
(ppm)
(ppm)
CH3 3.40
H3 6.81
3.40–6.98
—
CH3 32.84
C4a 116.62
3.40–32.84 3.40–120.31/139.73/
153.49
6.98–3.40/7.05 6.98–7.05
6.81–118.19 6.81–116.62/134.66/
145.29
H9 6.98
H8 7.05
H4 7.31
H7 8.02
6.81–7.31
7.05–6.98
7.31–6.81
—
6.81–7.31/8.04 C3 118.19
7.05–7.31/6.98 C9 120.31
6.98–122.11 6.98–142.62/144.76
7.05–122.11 7.05–139.73/142.62
7.31–134.66 7.31–145.29/153.49
7.31–6.81
8.02–7.05
C8 122.11
C4 134.66
8.02–142.62 8.02–122.11/144.76/
120.31
H2 8.04
—
8.04–6.81
C9a 139.73
8.04–145.29 8.04–118.19/134.66/
153.49
C7 142.62
C5a 144.76
C2 145.29
C10a 153.49
Scheme 3 Synthesis of 10-substituted 1,6-diazaphenothiazines
and 19 (with the triple bond) via the Mannich reaction with
formaldehyde and secondary amine (non-cyclic and cyclic)
in the presence of copper(I) chloride in dioxane (Scheme 3).
compounds were more active than cisplatin. Compound 19
(with the methylpiperazinylbutynyl group) was as active as
cisplatin and compounds 13, and 17 (with the diethylami-
noethyl and phthalimidopropyl groups) were slightly less
active.
Anticancer activity
The parent compound 7 and derivative 13 (with the
diethylaminoethyl group) were as active as cisplatin against
melanoma C-32 cell line. The SNB-19 cell line was the
most resistant for the tested compounds. The most active
derivative 9 (with the allyl group) exhibited IC50 close to
20 μg/mL.
Compounds 11, 15 and 18 (with the benzyl, piper-
idinylethyl and diethylaminobutynyl groups) were com-
pletely inactive against all cell lines with IC50 4 50 μg/mL.
Prothipendyl, containing only one pyridine ring, was
moderately active only against the MCF-7 cell line but
about 5-6 times less active than the most active diazaphe-
nothiazines. It worth noting that the most active compounds
7, 12 and 13 (together with six other compounds) were non-
The anticancer activity of 1,6-diazaphenothiazines 7–19 was
investigated in vitro using cultured glioblastoma SNB-19,
melanoma C-32 and breast cancer MCF-7 cell lines. Normal
human fibroblast (HFF-1) cell line was used as a control and
cisplatin as a reference drug. To compare the influence of
the nitrogen atoms in the azaphenothiazine system on the
anticancer activity, the classical monoazaphenothiazine
drug, prothipendyl (10-dimethylaminopropyl-1-azaphe-
nothiazine), was also tested. The tested compounds exhib-
ited different activities against the cell lines. The MCF-7
cell line was found as very sensitive for most compounds.
Eight derivatives exhibited good anticancer activity with
IC50 o 10 μg/mL (Table 2). The most active (IC50 o 5 μg/
mL) were compounds 7, 10 and 12 with the hydrogen atom,
and the propargyl and nitropyridinyl groups. Those
toxic against normal fibroblasts (HFF-1) with the IC50
50 μg/mL whereas cisplatin turned out to be toxic.
4

Med Chem Res
were recorded on a Bruker AscendTM 600 spectrometer at
600 MHz in deuteriochloroform with tetramethylsilane as
the internal standard. The 13C NMR spectra were recorded
at 150 MHz. Electron impact mass spectra (EI MS) and fast
atom bombardment mass spectra (FAB MS, in glycerol)
were run on a Finnigan MAT 95 spectrometer at 70 eV. The
thin layer chromatography was performed on silica gel 60
Table 2 The anticancer activity of derivatives of 1,6-
diazaphenothiazines
No.
Anticancer activity IC50 (μg/mL)
SNB-19
C-32
MCF-7
HFF-1
7
32.8
33.3
18.9
>50
>50
28.2
24.2
>50
>50
31.3
>50
>50
49.1
48.7
7.7
7.5
4.8
>50
>50
31.6
6.1
8
>50
44.1
27.1
>50
32.0
6.6
9.1
9
42.3
3.9
F254 (Merck 1.05735) with CHCl3–EtOH (5:1 and 10:1 v/v)
10
and on aluminum oxide 60 F254 neutral (type E) (Merck
1.05581) with CHCl3–EtOH (10:1 v/v) as eluents.
11
>50
4.6
>50
>50
>50
>50
>50
10.0
>50
>50
46.6
>50
8.2
12
13
8.2
Synthesis of 3,3′-dinitro-2,2′-dipyridinyl disulfide (2)
14
>50
>50
16.3
>50
>50
35.3
>50
7.8
10.7
>50
9.4
A solution of 2-chloro-3-nitropyridine (1) (158 mg, 1 mmol)
and thiourea (152 mg, 2 mmol) in ethanol (10 mL) was
refluxed for 3 h. After cooling the resulting crystals were
filtered off, washed with water and air dried to give 3,3′-
dinitro-2,2′-dipyridinyl disulfide (2) as orange needles (140
mg, 89 %) m.p. 249–250 °C. 1H NMR (CDCl3) δ: 7.32 (dd,
J = 7.8 Hz, J = 4.2 Hz, 2H, H5, H5′), 8.57 (dd, J = 7.8 Hz, J
= 1.2 Hz, 2H, H4, H4′), 8.61 (dd, J = 4.2 Hz, J = 1.2 Hz, 2H,
H6, H6′). 13C NMR (CDCl3) δ: 120.82 (2CH, C5, C5′),
133.80 (2CH, C4, C4′), 146.71 (2CH, C3, C3′), 153.54
(2CH, C6, C6′), 161.02 (2C, C2, C2′, CS). EI MS m/z: 310
(M, 5), 156 (M+1-NO2C5H3N, 25) 92 (100). Anal. calcd.
for: C10H6N4O4S2, C 38.71; H 1.95, N 18.06. Found: C
38.51, H 1.63, N 17.99.
15
16
17
8.2
18
>50
7.5
19
Prothipendyl
Cisplatin
23.2
7.4
Conclusion
We report here synthesis of new 10-substituted 1,6-diaza-
phenothiazines. Parent compound, 10H-1,6-diazaphe-
nothiazine 7, was obtained in three ways from appropriate
dipyridinyl sulfide and disulfide, and a pair of 2,3-dis-
ubstituted pyridines. The thiazine ring formation ran via the
Smiles rearrangement of the S-N type. The parent com-
pound was transformed into 10-subtituted derivatives with
the alkyl, heteroaryl, dialkylaminoalkyl, dialkylaminoalk-
ynyl and imidoalkyl groups in the alkylation and hetero-
arylation reactions. As the alkylation reactions could
proceed at the thiazine, azine or both nitrogen atoms, the
product structure elucidation was based on the 2D NMR
(ROESY, COSY, HSQC, and HMBC) spectra of the N-
methylated product. Some 1,6-diazaphenothiazines (7, 10,
12, 13) were at least anticancer active against melanoma C-32
and breast cancer MCF-7 cell lines as a reference drug –
cisplatin. Monoazaphenothiazine drug, prothipendyl, turned
out to be less active than at least six derivatives of 1,6-
diazaphenothiazines against all three cancer cell lines.
Synthesis of sodium 3-amino-2-pyridinethiolate (3)
To a solution of 3,3′-dinitro-2,2′-dipyridinyl disulfide (2)
(310 mg, 1 mmol) in absolute ethanol (30 mL) two tablets of
NaBH4 (10 mmol) were added carefully and the solution
was refluxed for 2 h. After cooling the solvent was removed
and evaporated in vacuo. The dry residue was recrystallized
from ethanol, yielding brown crystals of sodium 3-amino-2-
pyridinethiolate (3) (230 mg, 71 %), m.p. 4 260 °C. After
acidification with 10 % solution of HCl, 3-aminopyridine-2
(1H)-thione was obtained, m.p. 131–132 °C (lit. (Rodig et
al., 1964) 131–132 °C).
Synthesis of 3-amino-3′-nitro-2,2′-dipyridinyl sulfide (4)
To a solution of sodium 3-amino-2-pyridinethiolate (3)
(148 mg, 1 mmol) in dry ethanol (10 mL) 2-chloro-3-nitro-
pyridine (1) (158 mg, 1 mmol) was added. The mixture was
stirred at room temperature for 3 h and next the resulting
brown crystals were filtered off, washed with ethanol, air
dried and recrystallized from ethanol, yielding 3-amino-3′-
nitro-2,2′-dipyridinyl sulfide (4) as yellow needles (220 mg,
89 %), m.p. 166 °C (lit. [27] 167–168 °C)).
Experimental
Chemistry
Melting points were determined in open capillary tubes on a
Boetius melting point apparatus and are uncorrected. The
1H, 13C NMR, COSY, NOESY, HSQC, HMBC spectra
1H NMR (CDCl3) δ: 7.12 (m, 1H), 7.24 (m, 1H), 7.49 (m,
1H), 8.13 (broad s, 2H, NH2), 8.23 (m, 1H), 8.53 (m, 1H),

Med Chem Res
8.64 (m, 1H). 1H NMR (CDCl3) δ: 4.27 (broad s, 2H, NH2),
7.12 (m, 1H, H4), 7.24 (m, 2H, H5, H5′), 8.14 (m, 1H, H4′),
8.55 (m, 2H, H6, H6′). 13C NMR (CDCl3) δ: 119.87 (CH,
C5), 122.56 (CH, C4), 125.80 (CH, C5′), 133.80 (CH, C6),
135.66 (C, C3), 140.79 (CH, C4′), 142.29 (C, C2), 146.81 (C,
C3′), 153.62 (CH, C6′), 156.52 (C, C2′). EI MS m/z: 248 (M,
25), 202 (M+1-NO2 100). Anal. calcd. for: C10H8N4O2S, C
48.38, H 3.25, N 22.57. Found: C 48.42, H 3.39, N 22.51.
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 halides (methyl iodide,
allyl bromide, benzyl chloride, 4-chloro-3-pyridine, 1.5
mmol) was added and the stirring was continued for 24 h.
The mixture was poured into water (15 mL), extracted with
CHCl3 (3 × 10 mL) and dried using anhydrous Na2SO4. The
obtained product was purified by column chromatography
(aluminum oxide, CHCl3) to give:
Synthesis of 10H-1,6-diazaphenothiazine (7)
10-Methyl-1,6-diazaphenothiazine (8) (98 mg, 91 %); light
brown needles (EtOH), m.p. 105–106 °C 1H NMR
(CDCl3) δ: 3.40 (s, 3H, CH3), 6.81 (dd, J = 7.2 Hz, J = 4.8
Hz, 1H, H3), 6.98 (d, J = 7.8 Hz, 1H, H9), 7.05 (dd, J = 7.8
Hz, J = 4.8 Hz, 1H, H8), 7.31 (dd, 1H, J = 7.2 Hz, J = 1.2
Hz, H4), 8.02 (dd, J = 4.2 Hz, J = 1.2 Hz, 1H, H7), 8.04 (dd,
J = 4.2 Hz, J = 1.2 Hz, 1H, H2). 13C NMR (CDCl3) δ: 32.84
(NCH3), 116.62 (C4a), 118.19 (C3), 120.31 (C9), 122.11
(C8), 134.66 (C4), 139.73 (C9a), 142.62 (C7), 144.76 (C5a),
145.29 (C2), 153.49 (C10a). EI MS m/z: 215 (M, 100), 200
(M-CH3, 45). Anal. calcd. for: C11H9N3S C 61.37, H 4.21,
N 19.52. Found: C 61.24, H 4.21, N 19.49.
From 2-chloro-3-nitropyridine (1) and sodium 3-amino-2-
pyridinethiolate (3) To a solution of sodium 3-amino-4-
pyridinethiolate (3) (148 mg, 1 mmol) in dry DMF (10 mL) 2-
chloro-3-nitropyridine (1) (158 mg, 1 mmol) was added. The
mixture was stirred at room temperature for 1 h and next was
refluxed for 4 h. After cooling the reaction mixture was eva-
porated in vacuo. The dry residue was dissolved in CHCl3
and purified by column chromatography (aluminum oxide,
CHCl3) to give 10H-1,6-diazaphenothiazine (7) as beige
needles (EtOH) (130 mg, 64 %), m.p. 191–192 °C (lit. (Rodig
et al., 1966) 223–225 °C). 1H NMR (CDCl3) δ: 6.68 (dd, J
= 7.8 Hz, J = 1.2 Hz, 1H, H9), 6.72 (1H, NH), 6.73 (dd, J =
7.2 Hz, J = 4.8 Hz, 1H, H3), 6.88 (dd, J = 7.2 Hz, J = 4.8 Hz,
1H, H8), 7.18 (dd, 1H, J = 7.8 Hz, J = 1.2 Hz, H4), 7.81 (dd,
J = 4.8 Hz, J = 1.2 Hz, 1H, H7), 7.92 (dd, J = 4.8 Hz, J = 1.2
Hz, 1H, H2). 13C NMR (CDCl3) δ: 114.49 (C4a), 118.54 (C3),
120.21 (C9), 122.20 (C8), 134.31 (C4), 136.31 (C9a), 141.37
(C5a), 143.28 (C7), 145.45 (C2), 151.22 (C10a). EI MS m/z:
201 (M, 100). Anal. calcd. for: C10H7N3S, C 59.68, H 3.51, N
20.88. Found: C 59.51, H 3.53, N 20.82.
10-Allyl-1,6-diazaphenothiazine (9) (103 mg, 86 %); a dark
yellow oil 1H NMR (CDCl3) δ: 4.63 (m, 2H, NCH2), 5.27
(m, 2H, =CH2), 5.99 (m, 1H, CH), 6.76 (dd, J = 7.5 Hz, J
= 4.8 Hz, 1H, H3), 6.97 (m, 2H, H8, H9), 7.23 (dd, J = 7.5
Hz, J = 1.5 Hz, 1H, H4), 7.96 (m, 2H, H2, H7). 13C NMR
(CDCl3) δ: 47.82 (CH, CH2), 117.20 (CH, CH2=), 132.58
(CH, CH=), 115.77 (C, C4a), 118.48 (CH, C3), 121.77 (CH,
C9), 122.09 (CH, C8), 134.62 (CH, C4), 139.02 (C, C9a),
141.40 (CH, C7), 143.98 (C, C5a), 145.29 (CH, C2), 152.04
(C, C10a). EI MS m/z: 241 (M, 55), 200 (M-CH2CHCH2,
100). Anal. calcd. for: C13H11N3S C 64.70, H 4.59, N
17.41. Found: 64.67, H 4.56, N 17.32.
In cyclization of 3-amino-3′-nitro-2,2′-dipyridinyl sulfide (4)
The brown solution of 3-amino-3′-nitro-2,2′-dipyridinyl sul-
fide (4) (124 mg, 0.5 mmol) in dry DMF (5 mL) was refluxed
for 4 h. After cooling the reaction mixture was evaporated in
vacuo. The dry residue was dissolved in CHCl3 and purified
by column chromatography (aluminum oxide, CHCl3) to give
10H-1,6-diazaphenothiazine (7) (90 mg, 90 %).
10-Benzyl-1,6-diazaphenothiazine (11) (95 mg, 65 %); a
dark yellow oil 1H NMR (CDCl3) δ: 5.29 (s, 2H, CH2),
6.68 (m,1H, H3), 6.88 (m, 2H, H9, H8), 7.27 (dd, J = 7.2 Hz,
J = 1.4 Hz, 1H, H4), 7.38 (m, 5H, C6H5), 7.92 (m, 2H, H2,
H7). 13C NMR (CDCl3) δ: 48.78 (CH, CH2), 116.22 (C,
C4a), 118.43 (CH, C3), 121.45 (CH, C9), 121.83 (CH, C8),
126.45 (2CH, o-CH), 126.98 (CH, p-CH), 128.82 (2CH, m-
CH), 134.46 (CH, C4), 136.60 (C, CCH2), 138.44 (C, C9a),
142.66 (CH, C7), 144.38 (C, C5a), 145.34 (CH, C2), 152.52
(C, C10a). EI MS m/z: 291 (M, 30), 200 (M-CH2C6H5, 100).
Anal. calcd. for: C17H13N3S C 70.08, H 4.50, N 14.42.
Found: C 70.04, H 4.54, N 14.30.
In cyclization of 3,3′-dinitro-2,2′-dipyridinyl disulfide (2)
To a solution of 3,3′-dinitro-4,4′-dipyridinyl disulfide (2)
(310 mg, 1 mmol) in dry DMF (10 mL) NaOH (120 mg,
3 mmol) was added and refluxed for 48 h. After cooling the
reaction mixture was evaporated in vacuo. The dry residue
was dissolved in CHCl3 and purified by column chroma-
tography (aluminum oxide, CHCl3) to give 10H-1,6-diaza-
phenothiazine (7) (144 mg, 72 %).
10-(3′-Nitro-4′-pyridinyl)-1,6-diazaphenothiazine
(12)
(125 mg, 75 %); as red needles (EtOH), m.p. 167–169 °C
1H NMR (CDCl3) δ: 6.32 (d, 1H), 6.81 (m, 1H), 6.89 (m,
1H), 7.33 (d,1H, H4), 7.53 (d, 1H), 7.72 (d, 1H), 8.06 (d,
1H), 9.04 (d, 1H), 9.42 (s, 1H). 13C NMR (CDCl3) δ:
116.07 (C, C4a), 119.74 (CH, C3), 121.95 (CH, C8), 123.21
Synthesis of 10-substituted 1,6-diazaphenothiazines 8–12
To a solution of 10H-1,6-diazaphenothiazine (7) (100 mg,
0.5 mmol) in dry DMF (5 mL) NaH (24 mg, 1 mmol, 60 %

Med Chem Res
(CH, C9), 126.36 (CH, C5′), 135.08 (CH, C4), 138.19 (C,
C9a), 141.06 (C, C4′), 144.01 (C, C5a) 144.06 (CH, C7),
144.68 (CH, C2), 146.09 (C, C3′), 146.85 (C, C2′), 149.50
(C, C10a) 156.24 (CH, C6′), 145.34 (CH, C2), 152.52 (C,
6H, 2CH3), 2.65 (q, J = 7.2 Hz, 4H, 2CH2), 2.78 (t, J = 7.2
Hz, 2H, CH2), 4.08 (t, J = 7.2 Hz, 2H, CH2), 6.72 (dd, J =
7.8 Hz, J = 4.8 Hz, 1H, H3), 6.97 (dd, J = 7.8 Hz, J = 4.8
Hz, 1H, H8), 7.13 (dd, J = 7.2 Hz, J = 1.2 Hz, 1H, H4), 7.19
(dd, J = 7.2 Hz, J = 1.2 Hz, 1H, H9), 7.94 (m, 2H, H2, H7).
13C NMR (CDCl3) δ: 11.96 (2CH, 2CH3), 44.00 (CH,
NCH2), 47.69 (2C, 2CH2CH3), 48.98 (CH, NCH2), 116.30
(C, C4a), 118.07 (CH, C3), 120.66 (CH, C9), 122.00 (CH,
C8), 134.25 (CH, C4), 138.77 (C, C9a), 142.39 (CH, C7),
144.39 (C, C5a), 145.13 (CH, C2), 152.42 (C, C10a). FAB
MS m/z: 301 (M+1, 20), 228 (M+1-NC4H10, 100), 200 (M
+1-C2H4NC4H10, 25). Anal. calcd. for: C16H20N4S C 63.97;
H 6.71; N 18.65. Found: C 63.88; H 6.74; N 18.43.
C10a). EI MS m/z: 323 (M, 60), 277 (M+1-NO2, 100), 200
(M-NO2C6H4, 10). Anal. calcd. for: C15H9N5O2S C 55.72,
H 2.81, N 21.66 Found: C 56.04, H 2.96, N 22.01.
Synthesis of 10-propargyl-1,6-diazaphenothiazines (10)
To a suspension of 10H-1,6-diazaphenothiazine (7) (100
mg, 0.5 mmol) in DMF (10 mL) potassium tert-butoxide
(80 mg, 0.72 mmol) was added. The mixture was stirred at
room temperature for 1 h. Then to the solution 80 % solu-
tion of propargyl bromide (80 mg, 0.64 mmol) in dry
toluene (0.12 mL) was added dropwise. The solution stirred
at room temperature for 24 h and poured into water (20 mL),
extracted with methylene chloride (20 mL), dried with
anhydrous Na2SO4, evaporated to the brown oil. The resi-
due was purified by column chromatography (silica gel,
CHCl3) to yield 10-propargyl-1,6-diazaphenothiazine (10)
(83 mg, 70 %); dark yellow needles (EtOH), m.p. 96–97 °C.
1H NMR: δ 2.31 (t, J = 2.4Hz, 1H), 4.69 (d, J = 2.4 Hz,
2H), 6.84 (dd, J = 7.5 Hz, J = 5.1 Hz, 1H, H3), 7.06 (dd, J
= 7.5 Hz, J = 5.1 Hz, 1H, H8), 7.31 (m, 2H, H4, H9), 8.10
(d, J = 5.5 Hz, 1H, H7), 8.3 (dd, J = 5.1 Hz, J = 1.2 Hz, 1H,
H7), 8.15 (dd, J = 5.1 Hz, J = 1.3 Hz, 1H, H2). 13C NMR
(CDCl3) δ: 35.03 (CH, NCH2), 72.50 (CH, CH2CCH),
79.10 (C, CH2CCH), 116.40 (C, C4a), 118.69 (CH, C3),
121.04 (CH, C9), 122.02 (CH, C8), 134.65 (CH, C4), 137.84
(C, C9a), 143.13 (CH, C7), 144.54 (C, C5a), 145.34 (CH,
C2), 151.87 (C, C10a). EI MS: 239 (M, 90), 200 (M-
CH2CCH, 100). Anal. calcd. for: C13H9N3S C 65.25, H
3.79, N 17.56. Found: C 65.21, H 3.74, N 17.38.
10-(2′-Pyrrolidinylethyl)-1,6-diazaphenothiazine (14) (110 mg,
75 %); a beige oil 1H NMR (CDCl3) δ: 1.48 (m, 2H, CH2),
1.60 (m, 4H, 2CH2), 2.52 (m, 4H, 2CH2), 2.68 (t, J = 7.5 Hz,
2H, CH2), 4.15 (t, J = 7.5 Hz, 2H, NCH2), 6.73 (dd, J = 7.8
Hz, J = 4.8 Hz, 1H, H3), 6.96 (dd, J = 7.8 Hz, J = 4.8 Hz, 1H,
H8),7.12 (m, 2H, H4, H9), 7.94 (m, 2H, H2, H7). 13C NMR
(CDCl3) δ: 23.35 (2CH, 2CH2) 40.90 (CH, NCH2), 49.42 (CH,
NCH2), 53.57 (2CH, 2CH2), 115.99 (C, C4a), 118.66 (CH, C3),
120.11 (CH, C9), 122.01 (CH, C8), 135.78 (CH, C4), 137.94
(C, C9a), 143.15 (CH, C7), 144.66 (C, C5a), 145.36 (CH, C2),
152.29 (C, C10a). FAB MS m/z: 299 (M+1, 100), 202 (M+1-
C2H4NC4H8, 29). Anal. calcd. for: C16H18N4S C 64.40; H
6.08; N 18.78. Found: C 64.25; H 6.05; N 18.55.
10-(2′-Piperydinylethyl)-1,6-diazaphenothiazine (15) (112
mg, 72 %); a beige oil 1H NMR (CDCl3) δ: 1.48 (m, 2H,
CH2), 1.61 (m, 4H, 2CH2) 2.52 (m, 4H, 2CH2), 2.68 (t, J =
6.8 Hz, 2H, CH2), 4.13 (t, J = 6.8 Hz, 2H, NCH2), 6.73 (dd,
J = 7.8 Hz, J = 4.8 Hz, 1H, H3), 6.96 (dd, J = 7.8 Hz, J =
4.8 Hz, 1H, H8), 7.12 (m, 2H, H4, H9), 7.94 (m, 2H, H2,
H7). 13C NMR (CDCl3) δ: 23.86 (CH, CH2), 25.32 (2CH,
2CH2), 42.47 (CH, NCH2), 54.41 (2CH, 2CH2), 54.86 (CH,
NCH2), 116.43 (C, C4a), 118.21 (CH, C3), 120.91 (CH, C9),
122.22 (CH, C8), 134.34 (CH, C4), 138.50 (C, C9a), 142.59
(CH, C7), 144.22 (C, C5a), 145.10 (CH, C2), 152.26 (C,
Synthesis of 10-substituted 1,6-diazaphenothiazines 13–16
To a solution of 10H-1,6-diazaphenothiazine (7) (100 mg,
0.5 mmol) in dry dioxane (10 mL) NaOH (200 mg, 5 mmol)
was added. The mixture was refluxed for 1,5 h and hydro-
chloride of dialkylaminoalkyl chloride (2-diethylami-
noethyl) and hydrochloride of cycloaminoethyl chloride 1-
(2-chloroethyl)pyrrolidine, 1-(2-chloroethyl)piperidine, 2-
(2-chloroethyl)-1-methylpiperidine 1.5 mmol) was added.
The reaction mixture was refluxed for 24 h. After cooling
dioxane was evaporated in vacuo and residue was dissolved
in CHCl3 (10 mL). The extracts were washed with water,
dried with anhydrous Na2SO4 and evaporated in vacuo. The
obtained product was purified by column chromatography
(aluminum oxide, CH2Cl2) to give:
C10a). FAB MS m/z: 313 (M+1, 100), 202 (M+1-
C2H4NC5H10, 20). Anal. calcd. for: C17H20N4S: C 65.35; H
6.45; N 17.93. Found: C 65.22; H 6.47; N 17.80.
10-(1′-Methyl-2′-piperydinylethyl)-1,6-diazaphenothiazine
(16) (119 mg, 74 %); a beige oil 1H NMR (CDCl3) δː
1.30–2.15 (m, 7H), 2.38 (s, 3H, NCH3), 2.94 (m, 1H, CH),
4.02 (m, 2H, NCH2), 6.73 (dd, J = 7.6 Hz, J = 5.1 Hz, 1H,
H3), 6.96 (m, 2H, H8, H4), 7.20 (m, 1H, H9), 7.94 (m, 2H,
H2, H7). 13C NMR (CDCl3) δ: 23.93 (CH, CH2), 25.11 (CH,
CH2), 28.58 (CH, CH2), 30.38 (CH, CH2), 41.00 (CH,
NCH3), 42.50 (CH, CH2), 56.79 (CH, CH), 62.34 (CH,
NCH2), 116.38 (C, C4a), 118.10 (CH, C3), 120.28 (CH, C9),
122.02 (CH, C8), 134.37 (CH, C4), 138.57 (C, C9a), 142.41
(CH, C7), 144.61 (C, C5a), 145.14 (CH, C2), 152.47 (C,
10-(2′-Diethylaminoethyl)-1,6-diazaphenothiazine
(13)
(110 mg, 72 %); a beige oil 1H NMR: δ 1.06 (t, J = 7.2 Hz,

Med Chem Res
C10a). FAB MS. 327 (M+H, 80), 313 (M+1-CH3 100).
Anal. calcd. for: C18H22N4S C 66.22; H 6.79; N 17.16.
Found: C 66.17; H 6.75; N 17.05.
(2CH, 2CH2), 47.45 (CH, NCH2), 79.01 (C, CH2C), 80.01
(C, CH2C), 116.47 (C, C4a), 118.68 (CH, C3), 121.07 (CH,
C9), 121.90 (CH, C8), 134.68 (CH, C4), 137.78 (C, C9a),
143.13 (CH, C7), 144.66 (C, C5a), 145.16 (CH, C2), 151.92
(C, C10a). FAB MS m/z: 325 (M+1, 15), 252 (M+1-C4H10N,
100), 201 (M+1-C8H14N, 20). Anal. calcd. for: C18H20N4S
C 66.63, H 6.21, N 17.27. Found: C 66.41, H 6.27, N 17.12.
Synthesis of 10-phthalimidopropyl-1,6-diazaphenothiazines
(17)
To a stirred solution of 10H-1,6-diazaphenothiazine (7)
(100 mg, 0.5 mmol) in dry toluene (20 mL) NaH (0.12 g,
5 mmol, washed out with hexane) was added. The mixture
was stirred for 30 min at room temperature, then refluxed
for 1 h and a solution of N-(3-bromopropyl)phthalimide
(405 mg, 1.5 mmol) in toluene (10 mL) was added. The
mixture was refluxed for 48 h. After cooling the resulted
solid was filtered off, toluene was evaporated in vacuo and
the residue was purified by column chromatography (alu-
minum oxide, CHCl3) to give 10-(3′-phthalimidopropyl)-
1,6-diazaphenothiazine (17) (115 mg, 73 %), reddish nee-
10-[4-(4-Methylpiperazin-1-yl)but-2-ynyl]-1,6-diazaphe-
nothiazine (19) (120 mg, 69 %); a yellowish oil 1H NMR
(CDCl3) δ: 1.25 (s, 3H, NCH3), 2.69 (m, 8H, 4CH2), 3.32
(s, 2H, CH2), 4.57 (s, 2H, CH2), 6.80 (dd, J = 7.8 Hz, J =
4.8 Hz, 1H, H3), 7.09 (dd, J = 7.8 Hz, J = 4.8 Hz, 1H, H8),
7.33 (m, 2H, H4, H9), 8.03 (m, 2H, H2, H7). 13C NMR
(CDCl3) δ: 23.83 (2CH, 2CH2), 25.75 (2CH, 2CH2), 35.17
(CH, CH2), 47.98 (CH, NCH3), 53.27 (CH, CH2), 79.80 (C,
CH2C), 80.10 (C, CH2C), 116.26 (C, C4a), 118.51 (CH, C3),
121.33 (CH, C9), 121.86 (CH, C8), 134.63 (CH, C4), 137.90
(C, C9a), 142.95 (CH, C7), 144.50 (C, C5a), 145.22 (C2),
151.98 (C10a). FAB MS m/z: 351 (M+1, 100), 201 (M+1-
C9H15N2, 40). Anal. calcd. for: C19H21N5S C 64.93, H
6.02, N 19.93. Found: C 64.77, H 5.94 N 19.79.
1
dles (Et2O), m.p. 42–44 °C. H NMR (CDCl3) δː 2.28 (m,
2H, CH2), 3.43 (t, J = 6.1 Hz, 2H, NCH2), 3.87 (t, J = 6.0
Hz, 2H, NCH2), 6.72 (m, 2H, H3, H9), 6,88 (m, 1H, H8),
7.18 (m, 1H, H4), 7.74 (m, 2Hphthalimide), 7.79 (m, 1H, H7),
7.87 (m, 2Hphthalimide) 7.92 (m, 2H, H2). 13C NMR (CDCl3)
δ: 29.82 (CH, CH2), 31.65 (CH, NCH2), 36.74 (CH,
NCH2), 114.95 (C, C4a), 118.40 (CH, C9), 120.42 (CH, C3),
122.26 (CH, C8), 123.36 (2CH, 2CHphthalimide), 132.00 (2C,
2Cphthalimide), 134.09 (2CH, 2CHphthalimide), 135.66 (CH,
C4), 136.11 (C, C10a), 141.23 (C, C9a), 143.39 (CH, C2),
144.68 (CH, C7), 151.16 (C, C5a), 168.29 (2C, 2CO). FAB
MS m/z: 389 (M+1, 100), 201 (M+1-(CH2)3N(CO)2C6H4,
30). Anal. calcd. for C21H16N4O2S: C 64.93, H 4.15, N
14.42. Found: C 64.84, H 4.19, N 14.31.
Cytotoxic and antiproliferative effects in vitro
Cell culture
Compounds were evaluated for their anticancer activity
using three cultured cell lines: SNB-19 (human glio-
blastoma, DSMZ - German Collection of Microorganisms
and Cell Cultures, Braunschweig, Germany), C-32 (human
amelanotic melanoma, ATCC - American Type Culture
Collection, Manassas, VA, USA), MCF-7 (human breast
cancer, ATCC, Manassas, VA, USA) and HFF-1 (human
fibroblast cell line, ATCC, Manassas, VA, USA). The cul-
tured cells were kept at 37 °C and 5 % CO2. The cells were
seeded (1 × 104 cells/well/100 μL Dulbecco's modified
Eagle's medium supplemented with 10 % FCS and strepto-
mycin and penicillin) using 96-well plates (Corning).
General procedure for synthesis of 10-dialkylaminobutynyl-
1,6-diazaphenothiazines (18, 19)
A mixture of 10-propargyl-1,6-diazaphenothiazine (10)
(100 mg, 0.5 mmol), paraformaldehyde (0.5 mmol), amine
(0.7 mmol) and copper(I) chloride (catalytic amount) in
peroxide-free, dry dioxane (10 mL) was heated with con-
tinuous stirring at 70–80 °C for 3 h. After cooling water (20
mL) was add and mixture was extracted with chloroform,
dried with anhydrous Na2SO4, and evaporated in vacuo.
The dry residue was dissolved in CH2Cl2 and purified by
column chromatography (aluminum oxide, CH2Cl2) to give:
Cell proliferation and viability
In recent years tetrazolium salts have been described to be
used for the measurement of cell proliferation and viability.
The tetrazolium salts are cleaved to formazan by cellular
enzymes. An expansion in the number of viable cells results
in an increase in the overall activity of mitochondrial
dehydrogenases in the sample. This augmentation in enzyme
activity leads to an increase in the amount of formazan dye
formed, which directly correlates to the number of metabo-
lically active cells in the culture. The formazan dye produced
by metabolically active cells is quantified by a scanning
enzyme-linked immunosorbent assay reader by measuring
10-(4-diethylaminobut-2-ynyl)-1,6-diazaphenothiazine (18)
(130 mg, 80 %); a yellowish oil 1H NMR (CDCl3) δ: 1.03
(t, J = 7.2 Hz, 6H, 2CH3), 2.50 (q, J = 7.2 Hz, 4H, 2 NCH2),
3.42 (s, 2H, CH2), 4.70 (s, 2H, CH2), 6.79 (dd, J = 7.8 Hz,
J = 4.8 Hz, 1H, H3), 7.03 (dd, J = 7.8 Hz, J = 4.8 Hz, 1H,
H8), 7.33 (m, 2H, H4, H9), 8.03 (m, 2H, H2, H7). 13C NMR
(CDCl3) δ: 11.68 (2CH, 2CH3), 35.18 (CH, CH2), 40.65

Med Chem Res
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the absorbance of the dye solution at appropriate wave-
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Morak-Młodawska B, Suwińska K, Pluta K, Jeleń M (2012) 10-(3′-
Nitro-4′-pyridyl)-1,8-diazaphenothiazine as the double Smiles
rearrangement. J Mol Struct 1015:94–98
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WST-1 assay
The WST-1 assay (Roche Diagnostics, Mannheim, Ger-
many) was used to evaluate the effect of compounds on the
number of cells in cultures, which as the cytotoxic effect of
the tested compounds and their influence on the prolifera-
tion of cells. After exposure to tested compounds (at con-
centrations between 0 and 100 μg/mL) for 72 h, cells were
incubated with WST-1 (10 μL) for 1 h, and the absorbance
of the samples against a background control was read at
450 nm with a reference wavelength λ = 600 nm using a
microplate reader UVM340 (Biogenet). Results are
expressed as means of at least two independent experiments
performed in triplicate.
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potential and possible targets of phenothiazine-related com-
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potential of phenothiazines. Curr Drug Target 7:1055–1066
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Pluta K, Morak-Młodawska B, Jeleń M (2011) Recent progress in
biological activities of synthesized phenothiazines. Eur J Med
Chem 46:3179–3189
Acknowledgments The work was supported by the Medical Uni-
versity of Silesia (grant KNW-1–004/K/4/0).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Pluta K, Jeleń M, Morak-Młodawska B, Zimecki M, Artym J, Kocięba
M (2010) Anticancer activity of newly synthesized azaphe-
nothiazines in NCI’s anticancer screening. Pharmacol Rep
62:319–332
Pluta K, Morak-Młodawska B, Jeleń M (2009) Synthesis and prop-
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