Synthesis and in vitro antiproliferative activity of 5-alkyl-12(H)-quino[3, 4-b] [1,4]benzothiazinium salts

Article abstract of DOI:10.1016/j.ejmech.2010.07.035

A novel method of synthesizing 1,4-thiazine ring has led to the series of 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium salts. The derivatives containing a butyl or decyl substituents on the quinoline nitrogen atom were obtained by alkylation of 12(H)-quino[3,4-b][1,4]benzothiazine with alkyl bromides. Antiproliferative activity in vitro of the compounds (3) was assessed using two cancer cell lines (Hct116 and LLC) and doxorubicin as a reference. Most of the studied phenothiazine derivatives showed activity against both cell lines investigated (2.2-19.6 μg/mL concentration range). A structure-activity relationship was established. Only the compounds with substituents in the 11-position of the quinobenzothiazine ring did not exhibit activity against either cell line.

Full text of DOI:10.1016/j.ejmech.2010.07.035

European Journal of Medicinal Chemistry 45 (2010) 4733e4739
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antiproliferative activity of 5-alkyl-12(H)-quino[3,4-b] [1,4]
benzothiazinium salts
,
b
c,
Andrzej Zie˛ba a , Aleksander Sochanik , Agnieszka Szurko d, Marzena Rams d, Anna Mrozek d,
*
Piotr Cmoch e
a
ꢀ
Department of Organic Chemistry, Medical University of Silesia, ul. Jagiellonska 4, 41-200 Sosnowiec, Poland
Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Wybrzeze AK 15, 44-101 Gliwice, Poland
b
_
c
_
Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Wybrzeze AK 15, 44-101 Gliwice, Poland
d Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland
e Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel method of synthesizing 1,4-thiazine ring has led to the series of 5-alkyl-12(H)-quino[3,4-b][1,4]
benzothiazinium salts. The derivatives containing a butyl or decyl substituents on the quinoline nitrogen
atom were obtained by alkylation of 12(H)-quino[3,4-b][1,4]benzothiazine with alkyl bromides. Anti-
proliferative activity in vitro of the compounds (3) was assessed using two cancer cell lines (Hct116 and
LLC) and doxorubicin as a reference. Most of the studied phenothiazine derivatives showed activity
Received 9 April 2010
Received in revised form
28 June 2010
Accepted 18 July 2010
Available online 24 July 2010
against both cell lines investigated (2.2e19.6 mg/mL concentration range). A structureeactivity rela-
tionship was established. Only the compounds with substituents in the 11-position of the quino-
benzothiazine ring did not exhibit activity against either cell line.
Ó 2010 Published by Elsevier Masson SAS.
Keywords:
Aromatic nucleophilic substitution
Phenothiazine
Azaphenothiazine
Antiproliferative activity
Doxorubicin
1. Introduction
analogies. We thus investigated the reactions of thioquinan-
threnediinium salts (1) with nucleophilic amine reagents that
Phenothiazines are an important group of three-ring heterocy-
clic compounds with interesting chemical and biological properties
[1]. Numerous phenothiazine derivatives possess neuroleptic,
antimalarial, immunopotentiating, antibacterial, antiviral, anti-
fungal, antiproliferative or antitumor activities; they also stimulate
penetration of anticancer agents via blood-brain barrier [2e8].
Phenothiazine derivatives with aminoalkyl ligands attached to the
nitrogen atom of the thiazine ring represent a significant class of
neuroleptic drugs [9,10].
Modifications of basic structural fragments of drugs often lead
to a change in strength and direction of their interactions. The
search for novel pharmacologically active phenothiazine deriva-
tives continues, together with their new applications [11e13].
Antineoplastic properties of four-ring heterocyclic compounds, the
derivatives of benzo[c]acridine and benzo[a]phenothiazine were
reported in the literature [14]. These results certainly encourage the
synthesis and studies of novel compounds bearing structural
ultimately led to a novel method of synthesizing four-ring 1,4-
thiazine systems. Structural fragments that cannot be otherwise
incorporated into a phenothiazine system can be introduced via
this method [15,16]. We describe in here the synthesis of novel
phenothiazine derivatives having the structure of 12(H)-quino[3,4-
b][1,4]benzothiazinium salts 3. Finally, the results of investigating
their anticancer activity are presented.
2. Results and discussion
2.1. Chemistry
5,12-(Dimethyl)thioquinanthrenediinium bis-chloride (1), the
principal substrate for synthesis of compounds (3) was obtained
according to our earlier described method [17]. The reaction of bis-
chloride (1) with amines proceeds via a nucleophilic attack of
amine nitrogen atom on aza-activated positions 7a and 14a of salt
(1), which leads to a cleavage of the dithiine ring [15,16,18]. Our
studies show that the structure of reaction product salts (1) with
aromatic amines depends on the presence of oxygen in the reaction
* Corresponding author. Tel.: þ48 32 364 1603; fax: þ48 32 266 7860.
E-mail address: zieba@edu.sum.pl (A. Zie˛ba).
0223-5234/$ e see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.07.035

4734
A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
mixture (Scheme 1). If the reaction of bis-chloride (1) with
aromatic amines is carried out in the presence of atmospheric
oxygen introduced with vigorous stirring, its products are the
respective 12(H)-quino [3,4-b][1,4]benzothiazinium salts (3). When
access of atmospheric oxygen to the reaction mixture is prevented,
the reaction leads to 1-alkyl-4-(arylamine)quinolinium-3-thiolates
(2). In the presence of aniline hydrochloride (a hydrochloride
donor) and atmospheric oxygen the 3-thiolates (2) undergo cycli-
zation to respective derivatives of quinobenzothiazinium (3). Such
results indicate that the 3-thiolates (2) act as intermediates in the
reactions of bis-salts (1) with aromatic amines in the presence of
atmospheric oxygen and that these reactions lead to quinobenzo-
thiazinium salts (3). By using such approach we synthesized
assignmentwasespecially theproton signal multiplicities (forH-8, H-
9 and H-11) and the magnitudes of their coupling. The values of these
constants ((3h) dH9 ¼ 6.49e6.59 ppm (doublet of doublet),
3J ¼ 8.7 Hz, 4J ¼ 2.4 Hz, dH8 ¼ 6.82e6.90 ppm (doublet), 3J ¼ 8.7 Hz),
dH11 ¼6.97e7.06 ppm
(doublet),
4J ¼ 2.4 Hz;
(3k)
dH9 ¼ 6.43e6.47 ppm (doublet of doublet), 3J ¼ 8.3 Hz, 4J ¼ 2.1 Hz,
dH11 ¼6.57e6.60 ppm (doublet), 4J ¼ 2.1 Hz), dH8 ¼ 6.66e6.70 ppm
(doublet), 4J ¼ 8.3 Hz are typical of coupling constants for 1,2,4-
trisubstituted benzene ring protons. The compounds (3h) and (3k)
feature the structural fragment shown above, whereas the
compounds (3n) and (3o) contain the 1,2,3-trisubstituted benzene
fragment (Scheme 2) [19].
The synthetic route described cannot be taken advantage of in
order to obtain compounds (3p) or (3q) which both contain
a halogen atom in the 11-position of the quinobenzothiazine ring.
In the case of bis-chloride 1 reactions with 2-fluoroaniline and
2-chloroaniline, their course was not dependent on the presence of
oxygen in the reaction mixture. The 3-thiolates 2n and 2o, forming
at the first stage of the reaction undergo cyclization to 1,4-thiazine
not via hydrogen atom substitution but via substitution of halogen
atom with sulfur (Scheme 3). In nucleophilic substitution reactions
the fluoride and chloride anions are easy-leaving groups and they
do not require the presence of an oxidant. Such reactions lead to the
product having the structure of 3a. In the post-reaction mixture no
presence of the compounds (3p) or (3q) was found; these
compounds result from cyclization reaction occurring via substi-
tution of the hydrogen atom in the 6-position with the thiolate
sulfur atom (Scheme 3).
a
series of 5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
chlorides (3ael) containing various substituents at different posi-
tions of the benzene ring, as well as 5-methyl-12(H)-pyrido[2,3-e]
quino[3,4-b][1,4]thiazinium chloride (3m) which contains a pyri-
dine ring instead of the benzene ring.
Cyclization of 3-thiolates (2) occurs via substitution of the
phenyl group hydrogen atom with a sulfur atom in the
a position
with respect to the amine group. The cyclization of 3-thiolates (2h)
and (2k), formed in the reaction of respective bis-salts (1) with 3-
substituted aniline derivatives (1,3-phenylenediamine and 3-ami-
nophenol), can occur at 2- or 6-position of the phenyl group
(Scheme 2) and this can lead to two different products. Only the
compounds (3h) and (3k), forming as a result of hydrogen atom
substitution at the 6-position of the phenyl group were isolated
from the post-reaction mixture. Such a reaction course can result
from steric hindrance exerted by substituents present in the 1- and
3-positions of the benzene ring.
The 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium bromides
(3r) and (3s) containing, respectively, a butyl or dodecyl substituent
at the quinoline nitrogen atom, were obtained with good yields by
alkylating 12(H)-quino[3,4-b][1,4]benzothiazine (4) [20] with
The structure of the obtained compounds (3h) and (3k) was
corroborated by 1H NMR. Of diagnostic value for the structure
Scheme 1. Synthesis of compounds (2aem) and (3aem).

A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
4735
Scheme 2. Cyclization reaction of 1-methyl-4-(arylamino)quinolinium-3-thiolates (2h) and (2k).
appropriate alkyl bromides. In the experimental part we provide 1H
and 13C NMR data for all newly obtained compounds (2).
Compounds (3), in particular amine and hydroxyl derivatives have
very poor solubility. We provide herein 1H NMR data for all
compounds (3). For two chosen derivatives (3d and 3r), obtained by
different synthetic routes, we provide full assignments of 13C NMR
signals (based on two-dimensional spectra analysis), which fully
corroborate their structures (Scheme 4).
which the thiazine ring is bent along the axis determined by sulfur
and nitrogen atoms. X-Ray analysis of the compounds (3a) [15] and
(3m) [16] has shown that their structures are completely planar
and that the greatest atomic deflection from the plane, determined
by the ring-forming atoms, is 0.068 Å. The planar structure of
compounds (3) is probably a result of large contribution of struc-
ture B to their resonance hybrid (Scheme 5). We assumed that
introducing either electron-acceptor or electron-donor substitu-
ents into various positions of the benzene ring would significantly
affect both the molecular shape and electron density distribution in
the investigated compounds, resulting in their altered activity.
The examined quinobenzothiazinium salts (3) had various
substituents (CH3, F, Cl, Br, OH and NH2) introduced into various
positions of the benzene ring. Additionally, they also contain
a nitrogen atom in the 8-position of the quinobenzothiazine ring.
One of the mechanisms involved in antiproliferative effects of
chemotherapeutics is DNA intercalation. This mode of action is
typical for antiproliferative anthracycline antibiotics (e.g. doxoru-
bicin) that feature planar tetracyclic (aromatic or heteroaromatic)
fused rings. Compounds (3) investigated by us do have this type of
structure. It might be supposed then that these compounds would
share a similar mode of antiproliferative action. This mode of action
affecting cancer cells’ DNA has been indeed suggested in reports
2.2. Antiproliferative activity
The activity of the obtained 5-alkyl-12(H)-quino[3,4-b][1,4]
benzothiazinium salts (3) was investigated in vitro using cultured
Hct116 and LLC cell lines. Biological activity of
a chemical
compound depends, among other things, on its electron, steric and
lipophilic characteristics [21]. Introducing novel substituents and
functional groups at various positions of aromatic or hetero-
aromatic fragments of a potential drug may lead to changes of its
molecular shape allowing optimum binding to the receptor as well
as to alteration of physical chemical properties that affect drug
distribution and metabolism [21]. The spatial arrangements in
compounds (3) differ from those of the so far reported neuroleptic
phenothiazines, azaphenothiazines or benzo[a]phenothiazines, in
Scheme 3. Reaction of bis-salt (1) with 2-fluoroaniline and 2-chloroaniline.

4736
A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
Table 1
Antiproliferative activity in vitro of 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium
salts (3) and doxorubicin (reference) against the cancer cell lines studied
Compound
Antiproliferative activity IC50
HCT116
[m
g/mL]
LLC
3a
3b
3c
7.8 ꢀ 1.2
4.4 ꢀ 0.7
Neg
2.3 ꢀ 0.6
8.8 ꢀ 0.8
Neg
3d
3e
3f
3g
3h
3i
6.7 ꢀ 0.5
7.4 ꢀ 0.5
6.9 ꢀ 0.9
6.7 ꢀ 0.5
7.6 ꢀ 1.4
Neg
6.4 ꢀ 1.3
5.4 ꢀ 3.1
6.5 ꢀ 1.8
7.2 ꢀ 2.8
5.7 ꢀ 0.9
Scheme 4. Synthesis of 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium bromide (3r)
and (3s).
Neg
concerning antiproliferative properties of phenothiazine and benzo
[a]phenothiazine derivatives [6,22,23]. Structurally, compounds (3)
studied herein are their analogs.
3j
3k
3l
5.1 ꢀ 1.1
2.2 ꢀ 0.3
Neg
8.9 ꢀ 2.2
4.7 ꢀ 0.7
Neg
Most of the compounds (3) were active against both cell lines
3m
3r
3s
17.2 ꢀ 2.7
14.4 ꢀ 0.8
19.6 ꢀ 1.5
3.3 ꢀ 0.2
13.2 ꢀ 2.3
14.0 ꢀ 1.5
17.1 ꢀ 1.6
3.7 ꢀ 0.3
studied (IC50 ¼ 2.2e19.6
mg/mL). Their activity appears to depend,
to a significant extent, on the type of substituent present and its
ring location. Compound (3k), which demonstrated the highest
Doxorubicin
Neg - negative at the concentration used.
activity overall towards either cell line studied (IC50 ¼ 2.2e4.7
mg/
mL), contains additionally an amine group at the 10-position of the
quinobenzothiazine ring. Assuming such mechanism of anti-
proliferative action the outstanding activity of the compound (3k)
may be the result of additional interactions of amine group proton,
via hydrogen bonds, with purine or pyrimidine bases in the DNA
molecule. On the other hand, the amine group (as in the case of
doxorubicin or amine derivatives of acridine) may form, following
ionization, ionic bonds with negatively charged phosphate groups
in the DNA backbone. This would stabilize complexes between 3k
and cellular DNA. Lesser activity has been shown by compound (3j),
containing an amine group in the 9-position of the quinobenzo-
thiazine ring (located further away from the NH group in the
thiazine ring). In what concerns compound (3l), the presence of an
amine group in the 11-position of the quinobenzothiazine ring
results in lack of activity towards either cell line studied. Similar
effects have been observed for compounds (3c) and (3i) containing
methyl or hydroxyl group, respectively, in the 11-position of the
ring. Similar effects (no activity or abrogated activity) occurring in
case of compounds featuring electron donor-type substituents
(NH2, OH) capable of forming hydrogen bonds and for compounds
unable of forming such bonds (CH3) suggest that the cause of
inactivity of compounds (3c), (3i), (3l) lies in the steric hindrance of
their substituents at 11- position with respect to thiazine ring NH
group. All the compounds having substituents at the 10-position
show activity towards both cell lines used herein. Compounds (3g)
and (3h) featuring hydroxyl group at the 9- and 10-position of the
thiazine ring have similar activity towards both cell lines. One of the
quinobenzothiazinium salts (3), namely compound (3m), with an
additional nitrogen atom in the 8-position of the quinobenzothia-
zine ring, showed a decreased antiproliferative activity in both
studied cell lines, as compared to compound (3a) which does not
contain this additional nitrogen atom.
Lipophilicity of a compound determines, to a great extent, its
ability to penetrate the cell membrane and it can significantly affect
drug interactions. In a previous study we reported lipophilicity
parameters determined by reversed-phase thin-layer chromatog-
raphy (log PTLC), for most of the compounds investigated herein
(falling in the 2.12e3.15 range) [24]. Increased length of alkyl chain
at the quinoline nitrogen atom in the compound (3r) (butyl,
log PTLC ¼ 2.65) and (3s) (decyl, log PTLC ¼ 3.15) augments their lip-
ophilicity parameters, as compared to the compound (3a) (methyl,
log PTLC ¼ 2.36) and significantly decreasing at the same time their
activity against the cell lines studied (Table 1). On the other hand,
the compound (3m) which has the lowest lipophilicity parameter
among the investigated compounds (log PTLC ¼ 2.12), also showed
significantly lower activity as compared to the compound (3a). This
suggests that lipophilicity parameters are not a decisive factor in
antiproliferative activity of quinobenzothiazinium salts (3) against
Hct116 and LLC cell lines in vitro. Physical chemical properties,
including lipophilicity can be, however, of great importance for in
vivo testing of these compounds.
3. Conclusions
The reaction of thioquinantrenediinium bis-salts (1) with
aromatic amines used herein occured in two stages and led with
good yield to 12(H)-quino[3,4-b][1,4]benzothiazinium salts (3). The
3-thiolates (2) forming in the first stage of the reaction undergo
cyclization via hydrogen atom substitution in the benzene ring with
thiolate sulfur atom, and this requires presence of atmospheric
oxygen in the reaction mixture. Cyclization of 3-thiolates (2n) and
(2o) containing halogen atoms in the phenyl group in position
a
respective to the amine group occurs via halogen atom substi-
Such results may suggest that the structural fragment exerting
the most decisive impact on antiproliferative activity of compounds
(3) is the 1,4-thiazine ring. Activity has been demonstrated for
compound (3a) which does not contain additional substituents.
tution with thiolate sulfur atom and leads to salts having the
structure of compound (3a). These reactions allow obtaining
several quinobenzothiazinium salt derivatives (3aek) containing
substituents (CH3, F, Cl, Br, OH, NH2) in various positions of the
benzene ring and compound (3m) containing an additional
nitrogen atom in the 8-position of the quinobenzothiazine ring. The
derivatives (3res) containing butyl and decyl groups at the quin-
oline nitrogen atom were obtained with good yield by alkylating
12(H)-quino[3,4-b][1,4]benzothiazine (4) with appropriate alkyl
bromides. All of the obtained compounds (3) were investigated for
their antiproliferative activity in vitro using Hct116 and LLC cell
lines. The results obtained demonstrated a structureeactivity
Scheme 5. Mesomeric structures of compounds (3).

A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
4737
relationship. The greatest activity was shown by the compound
with substituents in 9- and 10-position of the quinobenzothiazine
ring and by the compound (3a) which does not feature any addi-
tional substituents. The compounds (3) containing substituents in
the 11-position of the quinobenzothiazine ring did not exhibit
activity against either cell line examined.
Substitution of butyl and decyl moieties at the quinoline
nitrogen atom of compounds (3r) and (3s) increased their lip-
ophilicity parameters lowering at the same time antiproliferative
activity when compared to the compound (3a) which features
a methyl substituent. The presence of additional nitrogen atom in
the 8-position of the quinobenzothiazine ring in the compound
(3m) also decreases its antiproliferative activity as compared to the
compound (3a) which lacks an additional nitrogen atom. The
obtained results justify further studies on the synthesis of quino-
benzothiazine derivatives with the aim of attempting to optimize
their pharmacological (antineoplastic) usefulness.
4.2.1.3. 1-Methyl-4-(4-hydroxyphenylamino)quinolinium-3-þthiolate
(2g). Yield: 69%; m.p. 190 ꢁC. MS, EI (70 eV) (m/z): 282 (M , 100%);
1H NMR (DMSOd-6, 600 MHz) : 4.17 (s, 3H, CH3), 6.80 (m, 2H, H-30,
d
H-50), 7.00 (m, 2H, H-20, H-60), 7.24 (m, 1H, H-6), 7.50 (m, 1H, H-5),
7.59 (m, 1H, H-7), 7.89 (m, 1H, H-8), 8.67 (s, 1H, H-2), 9.65 (s, 1H,
OH), 10.05 (s, 1H, NH); 13C NMR (DMSOd-6, 151 MHz)
d: 41.7 (CH3),
113.93 (C-4a), 116.10 (2C, C-30, C-50), 118.27 (C-8), 124.11 (C-6),
124.19 (C-5), 124.91 (2C, C-20, C-60), 129.03 (C-7), 132.52 (C-10),
135.90 (C-8a),136.80 (C-3), 144.04 (C-2), 153.20 (C-4), 155.50 (C-40);
Anal. Calcd for C16H14N2OS: C 68.06, H 5.00, N 9.92, S 11.35. Found:
C 68.11, H 4.92, N 9.84, S 11.30.
4.2.1.4. 1-Methyl-4-(3-hydroxyphenylamino)quinolinium-3-thiolate
(2h). Yield: 67%; m.p. 201e202 ꢁC. MS, EI (70 eV) (m/z): 282 (Mþ,
100%): 1H NMR (DMSOd-6, 600 MHz)
d: 4.21 (s, 3H, CH3), 49 (m, 1H,
H-20), 6.54 (m, 1H, H-60), 6.62 (m, 1H, H-40), 7.15 (m, 1H, H-50), 7.33
(m, 1H, H-6), 7.55 (m, 1H, H-5), 7.62 (m, 1H, H-7), 7.96 (m, 1H, H-8),
8.78 (s,1H, H-2), 9.62 (s,1H, OH), 9.88 (s,1H, NH); 13C NMR (DMSOd-
4. Experimental
6, 151 MHz) d
: 41.95(CH3), 108.93(C-20), 112.22(C-40), 112.84(C-60),
114.92(C-4a), 118.45(C-8), 124.42(C-5), 124.68(C-6), 128.96(C-7),
130.18(C-50), 134.90(C-8a), 138.90(C-3), 142.57(C-10), 145.30(C-2),
152.25(C-4), 158.27(C-30); Anal. Calcd for C16H14N2OS: C 68.06, H
5.00, N 9.92, S 11.35. Found: C 67.92, H 5.12, N9.86, S 11.27.
4.1. General techniques
Melting points are uncorrected. NMR spectra were recorded
using a Varian VXR 300, Bruker DRX 500 and Varian-NMR-Varian
VNMRS 600 spectrometer. To assign the structures, the following
2D experiments were employed: 1He13C gradient selected HSQC
and HMBC sequences. Standard experimental conditions and
standard Bruker or Varian programs were used. The 1H and 13C
NMR spectral data are given relative to the TMS signal at 0.0 ppm. EI
MS spectra were recorded using an LKB GC MS 20091 spectrometer
at 75 eV.
4.2.1.5. 1-Methyl-4-(2-hydroxyphenylamino)quinolinium-3-thiolate
(2i). Yield: 63%; m.p. 212e214 ꢁC, MS, EI (70 eV) (m/z): 282 (Mþ,
100%); 1H NMR (DMSOd-6, 600 MHz)
d: 4.17 (s, 3H, CH3), 6.76 (m,
1H, H-50), 6.91 (m, 1H, H-6), 7.00 (m, 1H, H-30), 7.07 (m, 1H, H-40),
7.26 (m, 1H, H-6), 7.57 (m, 2H, H-5), 7.60 (m, 1H, H-7), 7.91 (m, 1H,
H-8), 8.69 (s, 1H, H-2), 9.96 (s, 1H, OH), 9.91 (s, 1H, NH); 13C NMR
(DMSOd-6, 151 MHz) d
: 41.69 (CH3), 114.72 (C-4a), 116.19 (C-30),
118.16 (C-8), 119.12 (C-50), 122.94 (C-60), 123.78 (C-5), 124.21 (C-6),
126.14 (C-40), 128.54 (C-10), 128.97 (C-7), 134.91 (C-8a), 137.69 (C-3),
144.34 (C-2), 150.31 (C-20), 152.68 (C-4); Anal. Calcd for
C16H14N2OS: C 68.06, H 5.00, N 9.92, S 11.35. Found: C 67.92, H 4.86,
N 9.84, S 11.28.
4.2. Chemistry
4.2.1. Synthesis of 1-methyl-4-(arylamino)quinolinium-3-thiolates (2)
Argon was passed through the suspension of bis-chloride (1)
(0.419 g, 1 mmol) in dry pyridine (15 mL) at RT over 15 min. Amine
(2.5 mmol) was added and argon passed for further 15 min. The
mixture was then stirred at RT for 7 days. The solid product was
filtered off and washed with dry ether. The raw product was puri-
fied through recrystallization from ethanol.
4.2.1.6. 1-Methyl-4-(4-aminophenylamino)quinolinium-3-thiolate
(2j). Yield: 84%; m.p. 190e192 ꢁC. MS, EI (70 eV) (m/z): 281 (Mþ,
100%); 1H NMR (DMSOd-6, 600 MHz)
d: 4.12 (s, 3H, CH3), 6.40 (broad
signal, 2H, NH2), 6.60 (m, 2H, H-30, H-50), 6,87 (m, 2H, H-20, H-60),
7.22 (m, 1H, H-6), 7.58 (m, H, H-7), 7.59 (m, 1H, H-5), 7.87 (m, 1H, H-
8), 8,60 (s, 1H, H-2), 10.04 (s, 1H, NH); 13C NMR (DMSOd-6, 151 MHz)
4.2.1.1. 1-Methyl-4-(4-fluorophenylamino)quinolinium-3-thiolate
(2d). Yield: 70%; m.p. 215e217 ꢁC. MS, EI (70 eV) (m/z): 284 (Mþ,
d
: 41.53 (CH3), 119.73 (C-4a), 114.46 (2C, C-30, C-50), 118.10 (C-8),
100%); 1H NMR (DMSOd-6, 600 MHz)
d
: 4.21 (s, 3H, CH3), 7.18 (m,
123.77 (C-6), 124.27(C-5), 124.88 (2C, C-20, C-60), 129.08 (C-7),
129.29 (C-10), 135.46 (C-8a), 136.11 (C-3), 143.41 (C-2), 147.22(C-40),
153.41 (C-4); Anal. Calcd for C16H15N3S: C 68.30, H 5.37, N 14.93, S
11.39. Found: C 68.24, H 5.30, N 14.87, S 11.25.
2H, H-20, H-60), 7.21 (m, 2H, H-30, H50), 7.32 (m, 1H, H-6), 7.32 (m,
1H, H-5), 7.62 (m, 1H, H-7), 7.96 (m, 1H, H-8), 8.80 (s, 1H, H-2), 9.94
(s, 1H, NH); 13C NMR (DMSOd-6, 151 MHz)
d: 41.97 (CH3), 114.77 (C-
4a), 116.16(d, 2JC-F ¼ 22.8 Hz, 2C, C-30, C-50), 118.63 (C-8), 124.09 (C-
5),124.25 (d, 3JC-F ¼ 8.0 Hz, 2C, C-20, C-60),124.85 (C-6),128.83 (C-7),
134.96 (C-8a), 138.03 (C-10), 138.98 (C-3), 145.41 (C-2), 152.40 (C-4),
4.2.1.7. 1-Methyl-4-(3-aminophenylamino)quinolinium-3-thiolate
(2k). Yield: 67%; m.p. 204e206 ꢁC. MS, EI (70 eV) (m/z): 281(Mþ,
1
159.24 (d, JC-F ¼ 243.8 Hz, C-40); Anal. Calcd for C16H13FN2S: C
100%); 1H NMR (DMSOd-6, 600 MHz)
d: 4.18 (s, 3H, CH3), 5.42 (broad
67.58, H 4.61, N 9.85, S 11.27. Found: C 67.51, H 4.57, N 9.79, S 11.24.
signal, 2H, NH2), 6.26 (m, 1H, H-60), 6.33 (m, 2H, H-20), 6.44 (m, 1H,
H-40), 7.01 (m, 1H, H-50), 7.30 (m, 1H, H-6), 7.50 (m, 1H, H-7), 7.61
(m,1H, H-5), 7.92 (m,1H, H-8), 8,72 (s,1H, H-2), 9.90 (s,1H, NH); 13C
4.2.1.2. 1-Methyl-4-(4-bromophenylamino)quinolinium-3-thiolate
(2f). Yield: 64%; m.p. 213e214 ꢁC. MS, EI (70 eV) (m/z): 345 (Mþ,
NMR (DMSOd-6,151 MHz) d
: 41.80 (CH3),107.63 (C-20),109.89 (C-60),
100%); 1H NMR (DMSOd-6, 600 MHz)
d: 4.23 (s, 3H, CH3), 7.05 (m,
111.24 (C-40), 114.56 (C-4a), 118.23 (C-8), 124.37 (C-6), 124.59 (C-5),
129.02 (C-7), 129.79 (C-50), 135.01 (C-8a), 138.00 (C-3), 142.01 (C-
30), 144.63 (C-2), 149.84 (C-10), 152.64 (C-4); Anal. Calcd for
C16H15N3S: C 68.30, H 5.37, N 14.93, S 11.39. Found: C 68.19, H 5.31,
N 14.72, S 11.32.
2H, H-30, H-50), 7.39 (m,1H, H-6), 7.45 (m,1H, H-5), 7.51(m, 2H, H-30,
H-50), 7.64 (m, 1H, H-7), 8.00 (m, 1H, H-8), 8.85 (s, 1H, H-2), 9.82
(broad signal, 1H, NH); 13C NMR (DMSOd-6, 151 MHz)
d: 42,14 (CH3),
115.48 (C-4a), 116.17 (C-40), 118.79 (C-8), 123.46 (2C, C-20, C-60),
124.11 (C-5), 125.28 (C-6), 128.88 (C-7), 132.08 (2C, C-30, C-50),
134.75 (C-8a), 140.04 (C-3), 141.18 (C-10), 146.24 (C-2), 151.57 (C-4);
Anal. Calcd for C16H13BrN2S: C 55.66, H 3.80, N 8.11, S 9.29. Found: C
55.61, H 3.73, N 8.15, S 9.20.
4.2.1.8. 1-Methyl-4-(2-aminophenylamino)quinolinium-3-thiolate
(2l). Yield: 49%; m.p. 229e230 ꢁC. MS, EI (70 eV) (m/z): 281(Mþ,
100%); 1H NMR (DMSOd-6, 600 MHz)
d: 4.16 (s, 1H, CH3), 5.17 (broad

4738
A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
signal, 2H, NH2), 6.52 (m, 1H, H-50), 6.71 (m, 1H, H-60), 6.87 (m, 1H,
H-30), 7.03 (m, 1H, H-40), 7.22 (m, 1H, H-6), 7.53 (m, 2H, H-5), 7.58
(m, 1H, H-7), 7.88 (m, 1H, H-8), 8.70 (s, 1H, H-2), 9.75 (s, 1H, NH);
4.2.2.5. 11-Hydroxy-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazi-
nium chloride (3i). Yield: Procedure A: 60%, Procedure B: 68%; m.p.
276e277 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.14 (s, 3H, CH3),
NMR (DMSOd-6, 151 MHz)
d
: 41.70 (CH3), 114.37 (C-4a), 115.53 (C-
6.53e6.62 (m, 1H, Harom), 6.79e6.88 (m, 1H, Harom), 6.91e6.70 (m,
1H, H-9), 7.76e7.90 (m, 1H, H-2), 7.99e814 (m, 2H, H-3, H-4),
8.54e8.62 (m, 1H, H-5), 8.70 (s, 1H, H-6), 9.95(s, 1H, OH), 10.97 (s,
1H, NH); Anal. Calcd for C16H13ClN2OS: C 60.66, H 4.14, N 8.84, S
10.12. Found: C 60.54, H 4.06, N 8.70, S 10.07.
30), 116.44 (C-50), 118.07 (C-8), 123.89 (C-5), 124.3 (C-6), 124.67 (C-
60), 1226.45 (C-10), 127.17 (C-40), 129.00 (C-7), 135.06 (C-8a), 137.36
(C-3), 143.25 (C-20), 144.11 (C-2), 153.86 (C-4); Anal. Calcd for
C16H15N3S: C 68.30, H 5.37, N 14.93, S 11.39. Found: C 68.26, H 5.32,
N 14.88, S 11.38.
4.2.2.6. 9-Amino-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
4.2.2. Synthesis of 12(H)-quino[3,4-b][1,4]benzothiazinium salts (3)
Procedure A: Aniline hydrochloride (0.155 g, 1.2 mmol) was
added to the mixture of 3-thiolate (2) (1 mmol) in 10 mL of dry
pyridine and the whole was mixed at 70 ꢁC for 12 h. After cooling it
down to RT the formed precipitate was filtered off and washed with
ether. The raw product was recrystallized from ethanol.
Procedure B: Amine (2.5 mmol) was added to the mixture of bis-
chloride (1) (0.419 g,1 mmol) in 10 mL of dry pyridine and the whole
was mixed at 70 ꢁC for 12 h. The mixture was cooled down to RT, the
formed precipitate was filtered off and washed with ether. The raw
product was purified through recrystallization from ethanol.
chloride (3j). Yield: Procedure A: 67%, Procedure B: 70%; m.p.
288e290 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.01 (s, 3H, CH3), 5.41
(s, 2H, NH2), 6.18e6,24 (d, 4J ¼ 2.1 Hz, 1H, H-8), 6.28e6.35 (dd,
3J ¼ 8.7 Hz, 4J ¼ 2.1 Hz, 1H, H-10), 7.04e7.12 (d, 3J ¼ 8.7 Hz, 1H, H-
11), 7.71e7.79 (m. 1H, H-2), 7.90e8.01 (m, 2H, H-3, H-4), 8.37 (s, 1H,
H-6), 8.62e8.70 (m, 1H, H-1), 10.69 (s, 1H, NH); Anal. Calcd for
C16H14ClN3S: C 60.85, H 4.47, N 13.31, S 10.15. Found: C 60.73, H
4.36, N 13.23, S 10.08.
4.2.2.7. 10-Amino-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
chloride (3k). Yield: Procedure A: 70%, Procedure B: 75%; m.p.
261e262 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.13 (s, 1H,CH3),
5.20e5.80 (broad signal, 2H, NH2), 6.30e6.37 (dd, 3J ¼ 8.4 Hz,
4J ¼ 2.1 Hz, 1H, H-9), 6.65e6.74 (m, 2H, H-8, H-11), 7.78e7.87 (m,
1H, H-2), 7.99e8.11 (m, 2H, 3-H, 4-H), 8.62 (s, 1H, H-6), 8.80 (m, 1H,
4.2.2.1. 9-Fluoro-12(H)-quino[3,4-b][1,4]benzothiazinium
(3d). Yield: Procedure A: 63%, Procedure B: 70%; m.p. 265e267 ꢁC.
1H NMR (DMSOd-6, 300 MHz)
: 4.11 (s, 3H, CH3), 6.98e7.04 (m, 1H,
chloride
d
H-1), 10.71 (s, 1H, NH). 1H NMR (CD3OD, 500 MHz)
d: 4.17 (s, 1H,
Harom), 7.08e7.13 (m, 1H, Harom), 7.45e7.52 (m, 1H, Harom),
7,78e7.85 (m, 1H, Harom), 8.01e8.08 (m, 2H, Harom), 8.64(s, 1H, H-6),
8,84e8.92 (m, 1H, H-1), 11.02 (s, 1H, NH); 13C NMR (CD3OD,
CH3), 6.43e6.47 (dd, 3J ¼ 8.3 Hz, 4J ¼ 2.1 Hz, 1H, H-9), 6.57e6.60 (d,
4J ¼ 2.1 Hz, 1H, H-11), 6.66e6.70 (d, 3J ¼ 8.3 Hz, 1H, H-8), 7.79e7.84
(m, 1H, H-2), 7.98e8.19 (m, 2H, H-3, H-4), 8.36 (s, 1H, H-6),
8.45e8.51 (m, 1H, H-1); Anal. Calcd for C16H14ClN3S: C 60.85, H 4.47,
N 13.31, S 10.15. Found: C 60.77, H 4.31, N 13.25, S 10.10.
151 MHz)
d
: 43.45 (CH3), 107.55 (C-6a), 115.03 (d, 2JF-C ¼ 26.7 Hz, C-
2
3
8), 115.99 (d, JF-C ¼ 23.2 Hz, C-10), 117.09 (C-12a), 117.66 (d, JF-
¼ 7.8 Hz, C-7a), 119.55 (C-1), 120.68 (d, 3JF-C ¼ 8.8 Hz, C-11), 120.96
C
(C-11a),124.06 (C-4), 129.46 (C-2), 136.98 (C-3), 140.62 (C-4a),143.8
(C-6), 153.52 (C-12a), 162.81 (d, 1JF-C ¼ 247.6, Hz, C-9); Anal. Calcd
for C16H12ClFN2S: C 60.28, H 3.79, N 8.79, S 10.06. Found: C 60.21, H
3.82, N 8.64, S 9.97.
4.2.2.8. 11-Amino-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
chloride (3l). Yield: Procedure A: 67%, Procedure B: 74%; m.p.
267e269 ꢁC. 1H NMR (CD3OD, 300 MHz)
d: 4.18 (s, 3H, CH3),
6.34e6.39 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.3 Hz, 1H, Harom), 6.66e6.69 (dd,
3J ¼ 8.2 Hz, 4J ¼ 1.3 Hz, 1H, Harom), 6.85e6.89 (dd, 3J ¼ 8.2 Hz,
3J ¼ 7.6 Hz, 1H, H-9), 7.78e7.82 (m, 1H, H-2), 7.98e8.03 (m, 2H, H-3,
H-4), 8.32 (s, 1H, H-6), 8.43e8.46 (m, 1H, H-1); Anal. Calcd for
C16H14ClN3S: C 60.85, H 4.47, N 13.31, S 10.15. Found: C 60.74, H
4.36, N 13.21, S 10.08.
4.2.2.2. 9-Bromo-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
chloride (3f). Yield: Procedure A: 68%, Procedure B: 75%; m.p.
252e254 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.12 (s, 3H, CH3),
7.32e7.39 (m, 3H, Harom), 7.81e7.87 (m, 1H, Harom), 8.03e8.10 (m,
2H, Harom), 8.67 (s, 1H, H-6), 8.80e8.86 (m, 1H, H-1), 10.94 (s, 1H,
NH); Anal. Calcd for C16H12BrClN2S: C 50.61, H 3.19, N 7.38, S 8.44.
Found: C 50.46, H 3.11, N 7.26, S 8.36.
4.2.3. Synthesis of 5-Alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium
bromide (3r) and (3s)
A mixture of 12(H)-quino[3,4-b][1,4]benzothiazine (4) (1 mmol)
and alkyl bromide (5 mmol) was refluxed for 24 h. It was then
allowed to cool to rt and was triturated with diethyl ether (10 mL).
The solid produced was collected by filtration, washed with diethyl
ether, and dried in vacuo to give crude (3r) or (3s), which was
purified by recrystallization from ethanol.
4.2.2.3. 9-Hydroxy-5methyl-12(H)-quino[3,4-b][1,4]benzothiazinium
chloride (3g). Yield: Procedure A: 61%, Procedure B: 68%; m.p.
304e306 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.06 (s, 3H, CH3),
6.44e6.50 (d, 4J ¼ 2.7 Hz, 1H, H-8), 6.52e6.60 (dd, 3J ¼ 8.7,
4J ¼ 2.7 Hz,1H, H-10), 7.20e7.27 (d, 3J ¼ 8.7, 1H, H-11), 7.74e7.84 (m,
1H, H-2), 7.96e8.04 (m, 2H, H-3, H-4), 8.50 (s, 1H, H-6), 8.70e8.78
(m, 1H, H-1), 9.89 (s, 1H, OH), 10.80 (broad signal, 1H, NH); Anal.
Calcd for C16H13ClN2OS: C 60.66, H 4.14, N 8.84, S 10.12. Found: C
60.58, H 4.09, N 8.78, S 10.08.
4.2.3.1. 5-Buthyl-12(H)-quino[3,4-b][1,4]benzothiazinium bromide
(3r). Yield: 89%; m.p. 241e242 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d:
0.89e0.94 (t, J ¼ 7.2 Hz, 3H, CH3), 1.33e1.41 (m, 2H, CH2), 1.76e1.83
(m, 2H, CH2), 4.51e4.62 (t, J ¼ 7.8 Hz, 2H, NCH2), 7.03e7.12 (m, 2H,
Harom), 7.16e7.20 (m, 1H, Harom), 7.29e7.34 (m, 1H, Harom),
7.82e7.86 (m, 1H, H-2), 8.02e8.07 (m, 1H, H-3), 8.18e8.20 (m, 1H,
H-4), 8.66e8.73 (m, 2H, H-1, H-6), 10.31 (s, 1H, NH); 13C NMR
4.2.2.4. 10-Hydroxy-5-methyl-12(H)-quino[3,4-b][1,4]benzothiazi-
nium chloride (3h). Yield: Procedure A: 63%, Procedure B: 65%; m.p.
284e285 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d: 4.14 (s, 3H, CH3);
6.49e6.59 (dd, 3J ¼ 8.7 Hz, 4J ¼ 2.4 Hz, 1H, H-9), 6.82e6.90 (d,
3J ¼ 8.7 Hz, 1H, H-8), 6.97e7.06 (d, 4J ¼ 2,4, 1H, H-11), 7.75e7.88 (m,
1H, H-2), 7.98e8.12 (m, 2H, H-3, H-4), 8,65 (s, 1H, H-6), 8.85e8.94
(m, 1H, H-1), 9.97 (s, 1H, OH), 10.87 (s, 1H, NH); Anal. Calcd for
C16H13ClN2OS: C 60.66, H 4.14, N 8.84, S 10.12. Found: C 60.49, H
4.11, N 8.80, S 10.10.
(DMSOd-6, 151 MHz) d: 13.90 (CH3), 20.75 (CH2), 32.46 (CH2), 56.54
(NCH2), 109.19 (C-6a), 117.35 (C-12b), 118.51 (C-11a), 119.44 (C-11),
119.57 (C-4), 124.31 (C-1), 127.94 (C-8), 128.63 (C-9), 129.35 (C-2),
129.73 (C-10), 135.89 (C-3), 137.31 (C-7a), 139.64 (4-Ca), 142.98 (C-
6), 153.54 (C-12a); Anal. Calcd for C19H19BrN2S: C 58.92, H 4.94, N
7.23, S 8.28. Found: C 58.75, H 4.81, N 7.11, S 8.21.

A. Zie˛ba et al. / European Journal of Medicinal Chemistry 45 (2010) 4733e4739
4739
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4.2.3.2. 5-Decyl-12(H)-quino[3,4-b][1,4]benzothiazinium
bromide
(3s). Yield: 85%; m.p. 143e145 ꢁC. 1H NMR (DMSOd-6, 300 MHz)
d:
0.82e0.87 (t, J ¼ 6.6 Hz, 3H, CH3),1.18e1.37 (m,14H, CH2), 1.77e1.84
(m, 2H, CH2), 4.53e4.58 (t, J ¼ 7.5 Hz, 2H, NCH2), 7.06e7.11 (m, 2H,
Harom), 7.16e7.21 (m, 1H, Harom), 7.31e7.35 (m, 1H, Harom), 7.82e7.87
(m, 1H, H-2), 8.02e8.07 (m, 1H, H-3), 8.18e8.20 (m, 1H, H-4),
8.68e8.74 (m, 2H, H-1, H-6), 10.61 (s, 1H, NH); Anal. Calcd for
C25H31BrN2S: C 63.69, H 6.63, N 5.94, S 6.80. Found: C 63.53, H 6.51,
N 5.84, S 6.73.
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4.3.1. Cell culture
The synthesized compounds were evaluated for their anticancer
activity using two cultured cell lines: HCT 116 (human colon
carcinoma, American Type Culture Collection, Rockville, MD, USA)
and LLC (Lewis lung carcinoma, Institute of Immunology and
Experimental Therapy, Wroc1aw, Poland). The cultured cells were
kept at 37 ꢁC and 5% CO2. The cells were seeded (1 ꢂ104 cells/well/
100 mL D-MEM supplemented with 12% FCS and streptomycin and
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penicillin) using 96-well plates (Corning). After 24 h, when the cells
were approximately 80% confluent, aqueous stock solutions of the
studied compounds (with DMSO added, if necessary) or doxoru-
bicin were medium-diluted and added at various concentrations to
the cultures which were then incubated for further 12 or 24 h. At
the conclusion of incubation an MTS assay was performed.
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4.3.2. MTS assay
Viability was monitored by the MTS-tetrazolium reduction
assay (Promega). This colorimetric method measures mitochondrial
dehydrogenase activity and relies on the ability of metabolically
active cells to reduce the MTS reagent, a colorless tetrazolium salt
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazol), to a colored soluble compound (formazan),
proportionally to the number of viable cells. This cytotoxicity assay
was performed after 24-h exposure to varying concentrations (from
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m
bance was measured at 490 nm using a spectrophotometric plate
reader (Elx800, Biotek Instruments, Inc.). Background absorbance
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1
Part CXXI in the series of azinyl sulfides.