Synthesis and selected immunological properties of substituted quino[3,2-b]benzo[1,4]thiazines

Article abstract of DOI:10.1016/j.ejmech.2013.02.023

A new type of azaphenothiazines-tetracyclic quino[3,2-b]benzo[1,4] thiazines, possessing common substituents (H, CH₃, Cl, Br, F, CF _3, SCH₃) in positions 8-10 and pharmacophoric aminoalkyl substituents in position 6, were obtained from diquinodithiin and 2,2′-dichloro-3,3′-diquinolinyl disulfide in several-step syntheses. Sixty one compounds, grouped as the 6H, 6-dialkylaminoalkyl, 6-acylaminoalkyl and sulfonylaminoalkyl derivatives, were tested for cytotoxicity, their effects on phytohemagglutin A (PHA)-induced proliferative response of human peripheral blood mononuclear cells (PBMC) and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production by these cells. The compounds exhibited differential inhibitory activities in these tests and significantly varied in terms of cytotoxicity. The most promising compounds were tested for growth inhibition of leukemia L-1210 cells, colon cancer SV-948 cells and epidermal carcinoma A-341 cells. The most active compounds exhibited anticancer activity against these cell lines comparable to that of cisplatin. The structure-activity relationship of the compounds were discussed.

Full text of DOI:10.1016/j.ejmech.2013.02.023

European Journal of Medicinal Chemistry 63 (2013) 444e456
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and selected immunological properties of substituted
quino[3,2-b]benzo[1,4]thiazinesq
a
,
b
a
ꢀ a
*
Ma1gorzata Jelen , Krystian Pluta , Micha1 Zimecki , Beata Morak-M1odawska ,
Jolanta Artym b, Maja Kocie˛ba b
a
ꢀ
The Medical University of Silesia, Department of Organic Chemistry, Jagiellonska 4, 41-200 Sosnowiec, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Therapy, R. Weigla 12,
53-114 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A new type of azaphenothiazines e tetracyclic quino[3,2-b]benzo[1,4]thiazines, possessing common
substituents (H, CH3, Cl, Br, F, CF3, SCH3) in positions 8e10 and pharmacophoric aminoalkyl substituents
in position 6, were obtained from diquinodithiin and 2,20-dichloro-3,30-diquinolinyl disulfide in several-
step syntheses. Sixty one compounds, grouped as the 6H, 6-dialkylaminoalkyl, 6-acylaminoalkyl and
sulfonylaminoalkyl derivatives, were tested for cytotoxicity, their effects on phytohemagglutin A (PHA)-
induced proliferative response of human peripheral blood mononuclear cells (PBMC) and lipopolysac-
Received 10 October 2012
Received in revised form
18 February 2013
Accepted 21 February 2013
Available online 1 March 2013
charide (LPS)-induced tumor necrosis factor alpha (TNF-a) production by these cells. The compounds
Keywords:
exhibited differential inhibitory activities in these tests and significantly varied in terms of cytotoxicity.
The most promising compounds were tested for growth inhibition of leukemia L-1210 cells, colon cancer
SV-948 cells and epidermal carcinoma A-341 cells. The most active compounds exhibited anticancer
activity against these cell lines comparable to that of cisplatin. The structureeactivity relationship of the
compounds were discussed.
Phenothiazines
Azaphenothiazines
Antiproliferative activity
Anticancer activity
Thiazine ring formation
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
In continuation of our search for pharmaceutically active aza-
phenothiazines, we modified the phenothiazine structure with the
Tricyclic phenothiazines with aminoalkyl substituents at the
nitrogen atom are an important class of drugs exhibiting neuro-
leptic, antihistaminic, antitussive and antiemetic activities [1].
Recent reports describe very promising anticancer and antibacte-
rial activities, reversal of multidrug resistance and possibility of
treatment of Alzheimer’s, CreutzfeldteJakob’s and AIDS diseases,
not only for classical but also for newly synthesized phenothiazines
[2e6]. The modification strategies of the parent phenothiazine
structures include two approaches: a) introduction of a new sub-
stituent, mainly at the thiazine nitrogen atom at position 10, and
rarely at the carbon atom in the benzene ring, and b) substitution of
one or two benzene rings with homoaromatic and heteroaromatic
rings. The second way enables formation of new heterocyclic ring
systems: tri-, tetra- and pentacyclic azaphenothiazines containing
one, two, three and even four nitrogen atoms in the aromatic rings
[2,3,7e10]. Such phenothiazine structure modifications can change
potency and directions of activities of the basic structures.
pyridine and quinoline rings to form dipyridothiazines, quino-
benzothiazines and diquinothiazines [11e15]. Some diquinothia-
zines exhibited promising anticancer activities against human
tumor cell lines deriving from colon, breast, kidney, lung, ovary,
prostate, central nervous system, melanoma and leukemia [16]. In
addition, 10H-dipyridothiazine (10H-2,7-diazaphenothiazine) was
found to be a universal, low-toxic immunosuppressant, inhibiting
both humoral and cellular immune responses [17].
Angularly fused tetracyclic azaphenothiazines with the quino-
line moiety, such as substituted quino[3,4-b]benzothiazines and 5-
alkylquino[3,4-b]benzothiazinium salts, were obtained in an orig-
inal way from pentacyclic diquinodithiin (thioquinanthrene) and
its ammonium salt via the dithiin ring opening e the thiazine ring
closure reactions or via intramolecular cyclization of 1-methyl-4-
(arylamino)quinolinium-3-thiolates and (o-phenylthio)enamino-
quinolones. Some of these compounds exhibited anticancer and
antioxidant activities [11,18e20]. Recently, we elaborated a conve-
nient synthesis of a new type of azaphenothiazines with the
quinoline ring, linearly fused tetracyclic 8e10-substituted 6H-
quino[3,2-b]benzo[1,4]thiazines, in the reactions of isomeric pen-
tacyclic diquinodithiin 1 or 2,20-dichloro-3,30-diquinolinyl disulfide
q
Part CXXXV in the series of Azinyl Sulfides.
* Corresponding author. Tel.: þ48 32 364 1606; fax: þ48 32 364 1600.
E-mail address: pluta@sum.edu.pl (K. Pluta).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.023

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
445
(2) with substituted anilines via the dithiin ring opening in com-
pound 1 and the disulfide cleavage in compound 2 followed by the
thiazine ring closure processes [21].
Whereas the N,N-dialkylaminoalkyl substituents were intro-
duced at the nitrogen atom in one step N-alkylation, the intro-
duction of two other pharmacophoric substituents required three-
step synthesis. 6H-quinobenzothiazines 3a, 3c and 3f were alky-
lated with phthalimidopropyl and phthalimidobutyl bromides in
dry toluene in the presence of sodium hydride into the phthali-
midoalkyl derivatives 12aef. Next, these compounds underwent
reactions with hydrazine in aqueous ethanol to give six aminoalkyl
derivatives 13aef (alkyl ¼ propyl and butyl) in 70e85% yield
(Scheme 3).
Aminoalkylquinobenzothiazines 13aef were transformed into
the N-acyl derivatives. The reactions with acetic anhydride, ethyl
chloroformate and 2-chloroethyl isocyanate gave six 6-acetylami-
noalkylquinobenzothiazines 14aef, six 6-ethoxycarbonylamino-
alkylquinobenzothiazines 15aef and six 6-chloroethylureido-
alkylquinobenzothiazines 16aef (possessing a half-mustard unit)
in 63e91% yield. Aminoalkylquinobenzothiazines 13 were also
transformed into the N-sulfonyl derivatives. The reactions with
methanesulfonyl and p-toluenesulfonyl chlorides led to the sul-
fonamide derivatives: six 6-methanesulfonylaminoalkyl- and six 6-
p-toluenesulfonylaminoalkylquino-benzothiazines 17aef and
18aef in 68e81% yield (Scheme 4).
In this study, we describe and discuss the synthesis and the
structure of new 6-substituted 9-X-quino[3,2-b]benzo[1,4]thiazines
(X ¼ H, Cl and SCH3) with three main types of pharmacophoric
groups: dialkiloaminoalkyl, acylaminoalkyl and sulfonylaminoalkyl.
First we evaluated cytotoxicity of these compounds against human
peripheral blood mononuclear cells (PBMC). Next we investigated
inhibition of phytohemagglutinin A (PHA)-induced proliferation of
PBMC and regulation of lipopolysaccharide (LPS)-induced tumor
necrosis factor alpha (TNF-a) production in human whole blood cell
cultures. Selected compounds were also used for evaluation of
anticancer activities against several tumor cell lines in vitro. The
same biological activities were also studied for 6-unsubstituted 6H-
quinobenzothiazines, possessing additional substituents in the
benzene ring in positions 8e10. Altogether sixty one variously
substituted quinobenzothiazines were tested.
2. Results and discussion
2.1. Chemistry
The analysis of the quinobenzothiazine structures was a crucial
problem, as the synthesis may run with the Smiles rearrangement
or by other unknown processes and may form also 5H-tautomers
and 5-substituted products. In the reaction with p-anilines, other
possible isomeric product structures 19e23 were depicted in
Scheme 5.
New azaphenothiazines, 6H-quino[3,2-b]benzo[1,4]thiazines,
were obtained in a few-step syntheses. The substrates for these
syntheses, pentacyclic diquinodithiin 1 and 2,20-dichloro-3,30-
diquinolinyl disulfide (2), were obtained from 3,4-dihydro-2(1H)-
quinolone with thionyl chloride in DMF [22,23]. The reactions of
compounds 1 and 2 with substituted anilines and their hydrochlo-
rides without a solvent or in monomethyl ether of diethylene glycol
led to thirteen 8-, 9- and 10-substituted 6H-quinobenzothiazines
(3e5) with the hydrogen, chlorine, bromine and fluorine atoms and
the methyl, methylthio and trifluoromethyl groups (Scheme 1) [21].
Next we introduced the pharmacophoric substituents in the
quinobenzothiazine system at the thiazine nitrogen atom in posi-
tion 6. We selected diverse N,N-dialkylaminoalkyl, N-acylami-
noalkyl and N-sulfonylaminoalkyl groups, found in pharmacoactive
compounds and anticancer phenothiazines [2,3,16]. As the pattern
quinobenzothiazine substrates, we chose 6H-quinobenzothiazines
3a, 3c and 3f with the hydrogen and chlorine atoms, and the
methylthio group in position 9. The N,N-dialkylaminoalkyl sub-
stituents were introduced in the N-alkylation reactions with hy-
drochlorides of acyclic and cyclic dialkylaminoalkyl chlorides in
boiling dioxane in the presence of sodium hydroxide. In this way,
eighteen different 6-dialkylaminoalkyl derivatives 6e11 were ob-
tained in 73e83% yield (Scheme 2).
The kind of fusion of the quinoline ring with the thiazine ring,
[3,2-b] or [2,3-b], was initially solved using the homonuclear NOE
experiment for the N-methyl derivative. Irradiation of the methyl
protons gave an enhancement of the only one proton signal (the H7
proton) what pointed at the [3,2-b] ring fusion. A discrimination of
the isomeric products of the reactions with m-anilines as 8- and 10-
substituted quinobenzothiazines were also initially assigned
basing on the coupling constants Jortho and Jmeta [21]. However, the
1H NMR analysis did not exclude 7- and 9-substituted quino-
benzothiazines as the result of the reverse Smiles rearrangement.
Our supposition of the 8- and 10-substituted structures was
recently confirmed by X-ray analysis of compound 4c and the
benzyl derivative of 5c [24]. The structural problems of the prod-
ucts of the reactions with p-anilines were also solved using X-ray
analysis of the product 14e (the reaction with p-chloroaniline),
possessing strong biological activities (vide infra). X-ray study
confirmed the fused quinoline and thiazine system as [3,2-b], the
chlorine atom in position 9 and the acetylaminobutyl substituent
Scheme 1. Synthesis if 6H-quinobenzothiazines 3e5 from diquinodithiin 1 and 2,20-dichloro-3,30-diquinolinyl disulfide (2).

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
Scheme 2. Synthesis of 6-dialkylaminoalkylquinobenzothiazines 6e11.
at the thiazine nitrogen atom in position 6 [25]. X-ray analysis
excluded definitely structures 19e23 and confirmed that the N-
alkylation reactions of 6H-quinobenzothiazines (3a, 3c and 3f)
with dialkylaminoalkyl and phthalimidoalkyl chlorides occurred at
the thiazine nitrogen atom.
25% inhibition at 10 mg/mL. The nature of the substituents and their
location in the benzene ring (8e10) did not show strong direct
influence on the activity. Compounds 6e11 with the dia-
lkylaminoalkyl groups exhibited extremely strong activities at the
concentration of 10 mg/mL, with exception of compound 11a. There
were no differences in the activities between the propylene and
butylene linkages and acyclic and cyclic amino groups in com-
pounds 6e11. N-Acylaminoalkyl- and N-sulfonylaminoalkyl de-
rivatives 14e17 exhibited also strong activities but generally lesser
than dialkylaminoalkyl derivatives 6e11. The exceptions were
chloroethylureidopropyl and chloroethylureidobutyl derivatives
16b, 16c, 16e and 16f which were as strongly active as dia-
lkyaminoalkyl derivatives 6e11. It is of interest that compounds 16a
and 16d with the same chloroethylureidoalkyl groups were much
less active what is probably attributed to the weak influence of the
hydrogen atom in position 9 in comparison with the chlorine atom
or the methylthio group. The kind of the alkyl group (propyl or
butyl) had no significant influence on the antiproliferative activity.
Only compounds 18aef with the p-toluenesulfonylaminoalkyl
2.2. Biological activities
For the biological tests, sixty one quinobenzothiazines were
selected being 8e10-substituted 6H-quinobenzothiazines 3e5
and 9-substituted quinobenzothiazines 6e18 with the N,N-dia-
lkylaminoalkyl, N-acetylaminoalkyl, N-etoxycarbonylalkyl, chlor-
oethylureidoalkyl,
N-methanesulfonylaminoalkyl
and
N-p-
toluenesulfonylaminoalkyl groups in position 6. The tests included
the proliferative response of human peripheral blood mononuclear
cells induced by phytohemagglutinin A, the cytotoxic effect on
human peripheral blood mononuclear cells and lipopolysaccharide-
induced production of tumor necrosis factor
results of the tests are presented in Table 1, which lists only com-
pounds exhibiting low cytotoxicity (less than 20% of toxicity) at
a. The combined
groups were less active or inactive even at 50 mg/mL.
As the inhibitory effects of the compounds could be caused by
their high cytotoxicity, all the compounds were tested for their
effects on peripheral blood mononuclear cells. Generally, inactive
and less active compounds were non-toxic. The very strongly
antiproliferative compound 3e turned out to be moderately cyto-
10 mg/mL. Three most promising compounds, selected on the basis
of their strong antiproliferative effects, were tested for anticancer
activities against leukemia L-1210 cells, epidermal carcinoma A-431
cells and colon carcinoma SV-948 cells (the results shown in Fig. 1).
The proliferation test was performed at the concentrations of 10
toxic (50% inhibition at 10
proliferative dialkylaminoalkyl compounds 6e11 with over 93%
inhibition at 10 g/mL showed also strong cytotoxicity (over 82%
inhibition, respectively). Only two compounds 10b and 10c
exhibited low cytotoxicity at 10 g/mL. Similarly, very strongly
mg/mL). The most strongly anti-
and 50
mg/mL for all compounds (initially at 1 mg/mL only for
selected compounds). Only few compounds were inactive and
about twenty compounds were weakly inhibitory. Thirty four
compounds exhibited distinct inhibitory activities up to 50% in
comparison with the control test, most of them at the concentra-
m
m
antiproliferative chloroethylureidoalkyl compounds (16b, 16c, 16e
and 16f) and significantly antiproliferative N-acetylaminoalkyl 14,
N-etoxycarbonylaminoalkyl 15 and N-methanesulfonylaminoalkyl
17 compounds did not exhibit toxic effects at the concentration of
tion of 10 mg/mL. Out of compounds 3e5, possessing the hydrogen
atom at the thiazine nitrogen atom and selected groups in the
benzene ring, compound 3e, with the fluorine atom in position 9,
was found extremely active (100% inhibition at 10 mg/mL). How-
ever, this compound was not included in Table 1 because of its
cytotoxicity (see below). Other compounds with the hydrogen,
chlorine, bromine and fluorine atoms, and the methyl, methylthio
and trifluoromethyl groups exhibited lower activities with at most
10
14c, 14e, 14f, 15a, 15d, 17b, 17c, 17e and 17f) were non-toxic even at
the concentration of 50 g/mL. Some of the compounds, designated
as NT, even enhanced survival of the tested PBMC.
mg/mL. It is worth noting that some of these compounds (14b,
m
Scheme 3. Synthesis of 6-aminoalkylquinobenzothiazines 13.

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
447
Scheme 4. Transformations of 6-aminoalkylquinobenzothiazines 13 into N-acyl 14e16 and N-sulfonyl 17e18 derivatives.
The inhibitory effects of the compounds on LPS-induced TNF-
production were tested at the concentration of 5 and 25 g/mL.
Twenty quinobenzothiazines of all types exhibited significant in-
hibition of at least 50% at 5 g/mL. The strong suppressive effect (at
a
TNF-
low dose and strong TNF-
or little inhibition of proliferation associated with strong inhibition
of inducible TNF- production. It seems, therefore, that the mech-
a
production, ii/ 16f with exceptional antiproliferative action at
m
a
inhibition and iii/ 3b, 4c, 5c showing no
m
a
least 70% inhibition) was observed for nine compounds.
anisms of action among the studied derivatives are differential.
Motohashi and co-workers found that new anticancer phenothia-
zines with the chloroethylureidoalkyl and acylaminoalkyl sub-
stituents and tetracyclic benzophenothiazines effectively inhibited
the Con-A-induced T cell proliferation, slightly but selectively
inhibited the antibody-dependent cellular cytotoxicity (ADCC) ac-
tivity and enhanced the NK cell activity in non-transformed cells.
The effects of these compounds on cancer cell growth and differ-
entiation varied depending on the cell growth and differentiation
stages. Only a few benzophenothiazines induced nucleosome-size
DNA fragmentation. Other benzophenothiazines, classical neuro-
leptic phenothiazines and chloroethylureidoalkylphenothiazines
did not act in this way. The last compounds were supposed to act on
the cancer cells via alkylurea induced alkylation of proteins or on
DNA by a particular intercalation [2,28e30]. As our quinobenzo-
thiazines have structures close to the Motohashi’s phenothiazines
their cytotoxic activities probably are induced by the same mecha-
nisms. The X-ray study of compound 14e revealed unexpectedly
planar tetracyclic ring system [25], what may suggest its mechanism
of the antiproliferative effect by DNA intercalation for this type of
phenothiazines.
The most promising compounds 14c, 16b and 16f (being
strongly antiproliferative and relatively low cytotoxic) were
selected for evaluation of anticancer activities against three cancer
cell lines. The anticancer activity was tested at the concentrations of
0.1e50
compounds exhibited the anticancer activities similar to the
cisplatin activity at the concentration of 10e50 g/mL, and in some
cases quinobenzothiazines were found even more active (91.8e
97.0% inhibition at 10 g/mL). Compounds 16b and 16f were also
more active (96% inhibition) against colon carcinoma SV-948 cells
at the concentration of 5 g/mL. When evaluating the anticancer
mg/mL using cisplatin as the reference drug (Fig. 1). All these
m
m
m
effects of the compounds in vitro one has to consider high toxicity
[26] and side-effects [27] of cisplatin, the reference drug, used in
this study. Cisplatin showed, for example, LD50 (killing of 50% of
granulocyte/macrophage progenitor cells) already at 0.9
mg/mL af-
ter 1 h of culture and that value was as low as 0.12
continuous cell culture [26].
mg/mL for a
In general, several activity patterns among the studied com-
pounds can be described: i/ 14e, 16b which are strongly anti-
proliferative already at low dose but little inhibitory with regard to
Scheme 5. Other possible structures of quinobenzothiazines.

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
448
Table 1
Activities of the compounds in selected immunological assays. The table shows: the degree of cytotoxicity against PBMC of the selected compounds e exhibiting low cyto-
toxicity (less than 20% of toxicity) at 10 g/mL or being non toxic (NT), effects on phytohemagglutin A (PHA)-induced proliferative response of human peripheral blood
m
mononuclear cells and lipopolysaccharide-induced TNF-alpha production by these cells. The results are given in percentage inhibition as compared with appropriate DMSO
controls. Positive values denote inhibition, negative stimulation.
No.
Cytotoxicity
Antiproliferative
activity
TNF-
a
No.
Cytotoxicity
Antiproliferative
activity
TNF-a
10
m
g/mL
50
m
g/mL
10
mg/mL
50
m
g/mL
5
m
g/mL
25
mg/mL
10
m
g/mL
50
m
g/mL
10
mg/mL
50
m
g/mL
5
m
g/mL
25 mg/mL
3a
3b
3c
3d
3f
3g
4a
4b
4c
5a
5b
5c
10b
10c
11a
14a
14b
14c
14d
14e
14f
15a
15b
NT
2.4
NT
NT
NT
NT
NT
NT
NT
NT
NT
5.0
NT
15.4
NT
NT
NT
NT
NT
NT
7.1
14.4
ꢁ6.2
5.7
19.0
2.9
61.0
27.1
20.6
0.3
10.8
16.4
13.6
98.2
92
39.8
92.4
58.3
68.8
94.3
92.6
42.0
88.4
60.4
25.6
54.1
2.0
16.1
66.6
9.6
10.1
19.2
ꢁ5.6
19.2
ꢁ25.0
48.8
4.6
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
NT
NT
NT
NT
8.5
NT
NT
NT
NT
NT
89.5
92.7
NT
86.3
95.1
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
45.4
73.9
ꢁ0.7
44.3
11.2
98.6
93.0
16.6
98.4
94.8
36.6
73.4
57.3
42.3
59.4
53.3
6.6
38.8
83.7
ꢁ12.0
27.8
77.9
100.0
93.7
88.7
94.6
92.7
74.6
74.4
65.4
94.3
43.9
62.7
ꢁ0.8
22.7
ꢁ46.4
2.4
74.9
0.2
13.2
50.9
14.6
9.2
50.7
ꢁ1.8
54.8
73.3
8.1
70.1
12.3
8.41
33.1
5.0
72.1
1.0
ꢁ17.9
ꢁ0.3
ꢁ9.7
0.0
24.9
16.3
4.3
21.7
10.7
0.1
16.2
22.5
51.1
ꢁ1.2
11.2
72.3
17.0
13.6
74.9
ꢁ70.5
49.2
49.1
ꢁ0.4
11.4
71.7
0.0
15.9
NT
7.9
19.2
4.2
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
4.3
21.5
13.0
NT
56.0
47.5
60.6
30.5
47.5
42.3
24.5
13.2
15.6
20.9
20.8
12.5
ꢁ12.7
37.5
8.4
NT
2.3
ꢁ2.6
56.7
34.6
62.4
32.2
60.6
64.5
20.2
53.4
60.4
14.1
31.7
1.2
19.2
18
83.8
91.7
4.5
91.9
NT
NT
NT
NT
7.9
NT
96.3
93.8
ꢁ23.5
68.7
50.8
72.5
72.7
71.2
54.7
69.7
45.0
73.7
14.0
ꢁ7.5
72.8
3.8
NT
NT
NT
NT
NT
NT
NT
NT
NT
12.9
ꢁ5.3
3.3
13.2
ꢁ5.0
5.9
72.1
15.8
20.7
70.4
13.2
46.8
7.7
19.8
ꢁ34.7
NT
NT
10.3
3. Conclusion
10-Substituted 6H-quinobenzothiazines 3aeg, 4aec and 5aec
were obtained as in the described synthesis from dithiin 1 and
disulfide 2 with p- and m-substituted anilines [21].
We report here a few step synthesis of sixty novel tetracyclic 9-
substituted 6-aminoalkylquinobenzothiazines. The kind of the ami-
noalkyl substituent at the thiazine nitrogen atom such as dia-
lkylaminoalkyl, acylaminoalkyl, sulfonylaminoalkyl, played a crucial
role in the antiproliferative and anticancer activities. The additional
substituentinthebenzenering(forexamplethe chlorineandfluorine
atoms and the methylthio group) enhanced the activities in com-
parison with the hydrogen atom. The 9-fluoroquinobenzothiazine
ring system was a significant pharmacophoric unit, as compound
3e without any aminoalkyl groups at the thiazine nitrogen atom,
exhibited strong antiproliferative activity. The most active com-
pounds 14e, 16b and 16f possessed the acetylaminobutyl, chloror-
oethylureidopropyl and chlororoethylureidobutyl groups and
exhibited activities comparable to that of cisplatin. A combination of
the quinobenzothiazine system and two kinds of substituents (the
aminoalkyl at the thiazine nitrogen atom and some simple groups in
the benzene ring) is a useful starting point for further study to found
more potent anticancer agents and their mechanism of action.
4.2.2. Synthesis of 9-substituted 6-dialkylaminoalkylquinobenzothia-
zines 6e11
A mixture of 6H-quinobenzothiazine 3a (0.13 g, 0.5 mmol) [or 9-
chloro-6H-quinobenzothiazine 3c (0.14 g, 0.5 mmol) or 9-
methylthio-6H-quinobenzothiazine 3f (0.15 g, 0.5 mmol)], sodium
hydroxide (0.30 g, 7.5 mmol) and hydrochloride of dialkyla-
minoalkyl chloride (1.5 mmol, 2-diethylaminoethyl e 0.26 g, 3-
dimethylaminopropyl e 0.24 g, 3-dimethylamino-2-methylpropyl e
0.26 g, 2-(1-pyrrolidyl)ethyl e 0.26 g, 2-(1-piperidyl)ethyl e 0.28 g, 2-
(1-methyl-2-piperydinyl)ethyl e 0.30 g) in dry dioxane (5 mL) was
refluxed for 3 h. After cooling the reaction mixture was poured into
water (25 mL) and extracted with chloroform (3 ꢀ 10 mL). The
combined extracts were washed with water to pH ¼ 7 and dried over
Na2SO4. Chloroform was evaporated in vacuo and the residue was
purified by column chromatography (Al2O3, CHCl3) to give com-
pounds 6e11.
4. Experimental
4.2.2.1. 6-(2-Diethylaminoethyl)quinobenzothiazine(6a). (0.13 g, 74%),
an oil. 1H NMR (CDCl3)
d: 1.17 (t, 6H, 2CH3), 2.73 (m, 4H, 2CH2), 2.95 (t,
4.1. General techniques
2H, CH2), 4.23 (m, 2H, NCH2), 6.89 (t, 1H, H-9), 7.03 (2d, 2H, H-7, H-
10), 7.14 (t, 1H, H-8), 7.23 (t, 1H, H-2), 7.46 (m, 2H, H-1, H-3), 7.54 (s,
Melting points were determined in open capillary tubes on a
Boetius melting point apparatus and were uncorrected. The NMR
spectra were recorded on Bruker DRX and Bruker Fourier 300
spectrometers (1H at 500 MHz, 13C at 125 and 75 MHz) in CDCl3 or
DMSO-d6. Fast Atom Bombardment (FAB MS) mass spectra were
run on a Finnigan MAT 95 spectrometer at 70 eV.
1H, H-12), 7.67 (d, 1H, H-4). 13C NMR (75 MHz, CDCl3)
d: 12.14, 44.60,
47.82, 48.48, 112.09, 115.56, 118.32, 119.86, 122.49, 124.07, 126.09,
126.67, 127.25, 127.68, 128.98, 131.29, 141.44, 145.78, 151.77. FAB MS
m/z: 350 (M þ 1, 100), 277 [M þ1 ꢁ NH(C2H5)2, 58], 250
[M þ 1 ꢁ C2H4N(C2H2)2, 50]. Anal. calcd. for C21H23N3S: C 72.17, H
6.63, N 12.02, S 9.17. Found: C 72.01, H 6.75, N 11.87, S 9.01.
4.2.2.2. 6-(2-Diethylaminoethyl)-9-chloroquinobenzothiazine (6b).
4.2. Chemistry
(0.15 g, 78%), m.p. 74e75 ꢂC. 1H NMR (CDCl3)
d: 1.17 (t, 6H, 2CH3),
2.74 (m, 4H, 2CH2), 2.94 (t, 2H, CH2), 4.31 (m, 2H, NCH2), 6.97 (d, 1H,
H-7), 7.03 (d, 1H, H-10), 7.10 (dd, 1H, H-8), 7.25 (t, 1H, H-2), 7.48 (t,
1H, H-3), 7.49 (d, 1H, H-1), 7.57 (s, 1H, H-12), 7.67 (d, 1H, H-4). FAB
4.2.1. Synthesis of substrates 1, 2 and 6H-quinobenzothiazines 3e5
Diquino-1,4-dithiin 1 and 2,20-dichloro-3,30-diquinolinyl disul-
fide (2) were obtained according to the described procedures. 8e

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
449
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A-431
*
*
5
*
*
*
*
*
*
*
*
*
*
*
K
K
K
0.1
0.1
0.1
1
1
1
5
10 25 50 0.1
1
10 25 50 0.1
1
5
10 25 50 0.1
14e
1
1
1
5
10 25 50 0.1
16b
1
1
1
5
10 25 50
16f
DMSO
Cisplatin
0.6
0.5
0.4
0.3
0.2
0.1
0.0
L-1210
#
*
*
*
*
*
*
*
*
*
*
*
*
5
10 25 50 0.1
1
5
10 25 50 0.1
1
5
10 25 50 0.1
14e
5
10 25 50 0.1
16b
5
10 25 50
16f
DMSO
Cisplatin
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
SV-948
*
*
*
5
*
*
*
5
*
5
*
*
*
*
*
*
*
*
*
*
5
10 25 50 0.1
1
10 25 50 0.1
1
5
10 25 50 0.1
14e
10 25 50 0.1
16b
10 25 50
16f
DMSO
Cisplatin
Fig. 1. The anticancer activities of selected compounds at concentrations of 0.1e50
the mean optical density ꢃSE (*p < 0.001, #p < 0.05).
mg/mL. A-431, L-1210 and SW-948 cell lines were used in the study. The results are presented as

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
450
MS m/z: 384 (M þ 1, 82), 311 [M þ 1 ꢁ NH(C2H5)2, 100], 284
[(M þ 1 ꢁ C2H4N(C2H2)2, 30]. Anal. calcd. for C21H22ClN3S: C 65.70,
H 5.78, N 10.94. Found: C 65.48, H 5.71, N 10.78.
[M þ 1 ꢁ C4H8N(CH3)2, 22]. Anal. calcd. for C21H22ClN3S: C 65.70, H
5.78, N 10.94. Found: C 65.41, H 5.70, N 10.71.
4.2.2.9. 6-(3-Dimethylamino-2-methylpropyl)-9-methylthioquino-
4.2.2.3. 6-(2-Diethylaminoethyl)-9-methylthioquinobenzothiazine
benzothiazine (8c). (0.15 g, 76%), an oil. 1H NMR (CDCl3)
d: 1.02 (d,
(6c). (0.16 g, 81%), m.p. 48e49 ꢂC. 1H NMR (CDCl3)
d: 1.18 (t, 6H,
3H, CH3), 2.30 (s, 6H, 2CH3), 2.46 (s, 3H, CH3), 4.31 (m, 5H, 2CH2,
CH), 7.04 (d, 1H, H-7), 7.07 (d, 1H, H-10), 7.10 (d, 1H, H-8), 7.26 (t, 1H,
H-2), 7.52 (t, 1H, H-3), 7.53 (d, 1H, H-1), 7.68 (s, 1H, H-12), 7.70 (d,
1H, H-4). FAB MS m/z: 396 (M þ 1, 95), 351 (M ꢁ N(CH3)2, 100), 309
[M ꢁ C3H7N(CH3)2, 60], 296 [M þ 1 ꢁ C4H8N(CH3)2, 30]. Anal. calcd.
for C22H25N3S2: C 66.80, H 6.37, N 10.62. Found: C 66.65, H 6.32, N
10.35.
2CH3), 2.44 (s, 3H, CH3), 2.75 (m, 4H, 2CH2), 2.95 (m, 2H, CH2), 4.32
(m, 2H, NCH2), 6.98 (m, 2H, H-7, H-10), 7.07 (d, 1H, H-8), 7.23 (t, 1H,
H-2), 7.46 (m, 2H, H-1, H-3), 7.55 (s, 1H, H-12), 7.66 (d, 1H, H-4). 13
C
NMR (75 MHz, CDCl3) d: 12.13, 17.10, 44.64, 47.81, 48.55, 115.95,
117.86, 120.81, 124.11, 125.76, 125.97, 126.10, 127.22, 127.24, 129.09,
131.45, 131.55, 139.38, 145.78, 151.55. FAB MS m/z: 396 (M þ 1, 100),
380 (M
þ
1
ꢁ
CH3), 323 (M
þ
1
ꢁ
N(C2H5)2, 90), 310
[M þ 1 ꢁ CH2N(C2H5)2, 15], 296 [M þ 1 ꢁ C2H4N(C2H5)2, 30]. Anal.
calcd. for C22H25N3S2: C 66.80, H 6.37, N 10.62. Found: C 66.62, H
6.39, N 10.38.
4.2.2.10. 6-(1-Pyrrolidylethyl)quinobenzothiazine (9a). (0.14 g, 82%),
m.p. 68e69 ꢂC. 1H NMR (CDCl3)
d: 1.88 (m, 4H, 2CH2), 2.80 (m, 4H,
2CH2), 3.04 (m, 2H, CH2), 4.46 (m, 2H, NCH2), 6.90 (t, 1H, H-9), 7.06
(2d, 2H, H-7, H-10), 7.15 (t, 1H, H-8), 7.24 (t, 1H, H-2), 7.47 (m, 2H, H-
1, H-3), 7.57 (s, 1H, H-12), 7.68 (m, 1H, H-4). 13C NMR (75 MHz,
4.2.2.4. 6-(3-Dimethylaminopropyl)quinobenzothiazine
(7a).
(0.13 g, 78%), m.p. 65e66 ꢂC. 1H NMR (CDCl3)
d: 2.08 (m, 2H, CH2),
CDCl3) d: 23.55, 44.62, 52.12, 54.50, 115.71, 118.43, 120.00, 122.58,
2.30 (s, 6H, 2CH3), 2.49 (m, 2H, CH2), 4.26 (t, 2H, NCH2), 6.96 (t, 1H,
H-9), 6.89 (d,1H, H-7), 7.07 (d,1H, H-10), 7.15 (t,1H, H-8), 7.24 (t,1H,
H-2), 7.48 (t,1H, H-3), 7.49 (d,1H, H-1), 7.59 (s,1H, H-12) 7.69 (d,1H,
124.14, 126.08, 126.73, 127.31, 127.73, 129.00, 131.47, 141.35, 145.71,
151.93. FAB MS m/z: 348 (M þ 1, 52), 277 (M þ 1 ꢁ C4H9N, 100), 250
(M þ 1 ꢁ C2H4NC4H8, 28). Anal. calcd. for C21H21N3S: C 72.59, H
6.09, N 12.09. Found: C 72.41, H 6.14, N 11.89.
H-4). 13C NMR (125 MHz, CDCl3)
d: 24.38, 43.93, 45.51, 57.43, 115.77,
118.70, 120.36, 122.56, 124.09, 126.03, 126.14, 126.81, 127.33, 127.69,
129.01, 131.58, 141.50, 145.79, 152.38. FAB MS m/z: 336 (M þ 1, 100),
291 [M þ 1 ꢁ NH(CH3)2, 52], 250 [M þ 1 ꢁ C3H6NH(CH3)2, 28]. Anal.
calcd. for C20H21N3S: C 71.61, H 6.61, N 12.53. Found: C 71.40, H 6.69,
N 12.28.
4.2.2.11. 6-(1-Pyrrolidylethyl)-9-chloroquinobenzothiazine
(9b).
(0.15 g, 79%), m.p. 68e69 ꢂC. 1H NMR (CDCl3)
d
: 1.96 (m, 4H, 2CH2),
2.95 (m, 4H, 2CH2), 3.15 (m, 2H, CH2), 4.51 (m, 2H, NCH2), 6.04
(m, 2H, H-7, H-10), 7.13 (d, 1H, H-8), 7.28 (t, 1H, H-2), 7.50 (t, 1H,
H-3), 7.51 (d, 1H, H-1), 7.61 (s, 1H, H-12), 7.67 (d, 1H, H-4). FAB
MS m/z: 382 (M þ 1, 76), 311 (M þ 1 ꢁ HNC4H8, 100), 284
(M þ 1 ꢁ C2H4NC5H10, 15). Anal. calcd. for C21H20ClN3S: C 66.04, H
5.28, N 11.00. Found: C 65.88, H 5.31, N 10.81.
4.2.2.5. 6-(3-Dimethylaminopropyl)-9-chloroquinobenzothiazine (7b).
(0.15 g, 81%), an oil. 1H NMR (CDCl3)
d: 2.09 (m, 2H, CH2), 2.36 (s, 6H,
2CH3), 2.57 (m, 2H, CH2), 4.25 (t, 2H, CH2), 6.82 (d, 1H, H-7), 7.06 (d,
1H, H-10), 7.11 (dd,1H, H-8), 7.27 (t, 1H, H-2), 7.50 (t, 1H, H-3), 7.51 (s,
1H, H-1), 7.61 (s,1H, H-12), 7.69 (d,1H, H-4). FAB MS m/z: 370 (M þ 1,
100), 325 [M þ 1 ꢁ NH(CH3)2, 51], 298 [M þ 1 ꢁ C2H4NH(CH3)2, 20],
284 [M þ 1 ꢁ C3H6NH(CH3)2, 18]. Anal. calcd. for C20H20ClN3S: C
64.94, H 5.45, N 11.36. Found: C 64.81, H 5.40, N 11.08.
4.2.2.12. 6-(1-Pyrrolidylethyl)-methylthioquinobenzothiazine (9c).
(0.15 g, 76%), m.p. 90e91 ꢂC. 1H NMR (CDCl3)
d: 1.93 (m, 4H, 2CH2),
2.45 (s, 3H, SCH3), 1.74 (m, 4H, 2CH2), 2.88 (m, 4H, 2CH2), 3.09 (m,
2H, CH2), 4.49 (m, 2H, NCH2), 7.00 (d, 1H, H-7), 7.05 (d, 1H, H-10),
7.09 (d, 1H, H-8), 7.26 (t, 1H, H-2), 7.49 (t, 1H, H-3), 7.50 (d, 1H, H-
1), 7.59 (s, 1H, H-12), 7.68 (d, 1H, H-4). FAB MS m/z: 394 (M þ 1,
100), 323 (M ꢁ NC4H8, 90), 309 [M ꢁ CH2N(CH2)4, 10], 296
[M þ 1 ꢁ C2H4N(CH2)4, 30]. Anal. calcd. for C22H23N3S2: C 67.14, H
5.89, N 10.68. Found: C 66.95, H 5.91, N 10.42.
4.2.2.6. 6-(3-Dimethylaminopropyl)-9-methylthioquinobenzothiazine
(7c). (0.15 g, 79%), m.p. 55e56 ꢂC. 1H NMR (CDCl3)
d: 2.18 (m, 2H,
CH2), 2.45 (s, 9H, 3CH3), 2.72 (m, 2H, CH2), 4.27 (m, 2H, NCH2), 6.90
(d,1H, H-7), 7.02 (d,1H, H-10), 7.08 (d,1H, H-8), 7.25 (t,1H, H-2), 7.49
(t, 1H, H-3), 7.50 (s, 1H, H-1), 7.61 (s, 1H, H-12), 7.67 (d, 1H, H-4).
FAB MS m/z: 382 (M þ 1, 100), 337 [M ꢁ N(CH3)2, 95], 309
[M ꢁ C2H4N(CH3)2, 35], 296 [M þ 1 ꢁ C3H6N(CH3)2, 35]. Anal. calcd.
for C21H23N3S2: C 66.11, H 6.08, N 11.01. Found: C 66.00, H 6.011,
N 10.87.
4.2.2.13. 6-(1-Piperidylethyl)quinobenzothiazine (10a). (0.15 g, 83%),
m.p. 71e72 ꢂC. 1H NMR (CDCl3)
d: 1.58 (m, 2H, CH2), 1.87 (m, 4H,
2CH2), 2.90 (m, 4H, 2CH2), 3.13 (m, 2H, CH2), 4.60 (m, 2H, NCH2),
6.92 (t, 1H, H-9), 7.07 (d, 1H, H-7), 7.21 (d þ t, 2H, H-8, H-10), 7.26 (t,
1H, H-2), 7.46 (m, 2H, H-1, H-3), 7.59 (s, 1H, H-12), 7.67 (d, 1H, H-4).
FAB MS m/z: 362 (M þ 1, 100), 277 (M þ 1 ꢁ C5H11N, 55), 250
(M þ 1 ꢁ C2H4NC5H10, 21). Anal. calcd. for C22H23N3S: C 73.10, H
6.41, N 11.62. Found: C 73.02, H 6.29, N 11.37.
4.2.2.7. 6-(3-Dimethylamino-2-methylpropyl)quinobenzothiazine (8a).
(0.13 g, 74%), an oil. 1H NMR (CDCl3)
d: 0.99 (t, 3H, CH3), 2.20 (m, 1H,
CH2), 2.26 (s, 6H, 2CH3), 2.27 (m, 1H, CH), 2.35 (m, 1H, CH2), 4.35 (m,
2H, NCH2), 6.95 (t,1H, H-9), 7.07 (d,1H, H-7), 7.14 (d,1H, H-10), 7.19 (t,
1H, H-8), 7.27 (t, 1H, H-2), 7.50 (t, 1H, H-3), 7.53 (d, 1H, H-1), 7.68 (s,
1H, H-12), 7.73 (d, 1H, H-4). FAB MS m/z: 350 (M þ 1, 100), 305
[M þ 1 ꢁ NH(CH3)2, 62], 363 [M þ 1 ꢁ C3H7N(CH3)2, 36], 250
[M þ 1 ꢁ C4H8N(CH3)2, 24]. Anal. calcd. for C20H21N3S: C 71.61, H 6.61,
N 12.53. Found: C 71.35, H 6.71, N 12.31.
4.2.2.14. 6-(1-Piperidylethyl)-9-chloroquinobenzothiazine (10b).
(0.16 g, 81%), m.p. 93e94 ꢂC. 1H NMR (CDCl3)
d: 1.53 (m, 2H, CH2),
1.74 (m, 4H, 2CH2), 2.67 (m, 4H, 2CH2), 2.92 (m, 2H, CH2), 4.45
(m, 2H, NCH2), 7.04 (d, 1H, H-7), 7.12 (m, 2H, H-8, H-10), 7.27 (t,
1H, H-2), 7.49 (t, 1H, H-3), 7.50 (d, 1H, H-1), 7.59 (s, 1H, H-12),
7.67 (d, 1H, H-4). FAB MS m/z: 396 (M
þ
1, 32), 311
4.2.2.8. 6-(3-Dimethylamino-2-methylpropyl)-9-chloroquinobenzo-
(M þ 1 ꢁ HNC5H10, 100), 284 (M þ 1 ꢁ C2H4NC5H10, 21). Anal.
calcd. for C22H22ClN3S: C 66.74, H 5.60, N 10.61. Found: C 66.49, H
5.64, N 10.36.
thiazine (8b). (0.15 g, 78%), an oil. 1H NMR (CDCl3)
d: 0.98 (d, 3H,
CH3), 2.20 (m,1H, CH2), 2.26 (s, 6H, 2CH3), 2.28 (m, 1H, CH), 2.33 (m,
1H, CH2), 4.35 (m, 2H, NCH2), 6.98 (d, 1H, H-7), 7.13 (d, 1H, H-10),
7.14 (dd, 1H, H-8), 7.26 (t, 1H, H-2), 7.52 (t, 1H, H-3), 7.54 (d, 1H, H-
1), 7.68 (s,1H, H-12), 7.72 (d,1H, H-4). FAB MS m/z: 384 (M þ 1,100),
339 [M þ 1 ꢁ NH(CH3)2, 78], 297 [M þ 1 ꢁ C3H7N(CH3)2, 35], 284
4.2.2.15. 6-(1-Piperidylethyl)-9-methylthioquinobenzothiazine (10c).
(0.16 g, 79%), m.p. 59e60 ꢂC. 1H NMR (CDCl3)
d
: 1.66 (m, 2H, CH2),
1.70 (m, 6H, 3CH2), 2.44 (s, 3H, CH3), 2.59 (m, 2H, CH2), 2.88 (m, 2H,

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451
CH2), 4.41 (m, 2H, NCH2), 6.99 (d, 1H, H-7), 7.03 (d, 1H, H-10), 7.08
(dd, 1H, H-8), 7.24 (t, 1H, H-2), 7.48 (t, 1H, H-3), 7.49 (d, 1H, H-1),
7.57 (s, 1H, H-12), 7.67 (d, 1H, H-4). FAB MS m/z: 408 (M þ 1, 100),
323 (M þ 1 ꢁ HNC5H10, 10), 284 (M þ 1 ꢁ C2H4NC5H10, 20). Anal.
calcd. for C23H25N3S2: C 67.78, H 6.18, N 10.31. Found: C 67.40, H
6.21, N 10.09.
(d, 1H, H-10), 7.12 (dd, 1H, H-8), 7.28 (t, 1H, H-2), 7.47 (t, 1H, H-3),
7.50 (d, 1H, H-1), 7.62 (s, 1H, H-12), 7.67 (m, 3H, H-4, 2Hphthal.), 7.75
(m, 2H, 2Hphthal.). 13C NMR (75 MHz, CDCl3)
d: 25.34, 35.97, 42.81,
116.28, 117.79, 122.49, 123.13, 124.34, 125.90, 126.12, 126.33,
127.38, 127.68, 129.22, 131.92, 132.04, 133.87, 139.98, 145.54,
151.73, 168.33. FAB MS m/z: 472 (M þ 1, 100), 311 (M ꢁ C9H6O2N,
20), 297 (M ꢁ C10H8O2N, 20), 284 (M þ 1 ꢁ C11H10O2N, 30). Anal.
calcd. for C26H18ClN3O2S: C 66.17, H 3.84, N 8.90. Found: C 66.01, H
3.88, N 8.69.
4.2.2.16. 6-(1-Methyl-2-piperidylethyl)quinobenzothiazine (11a).
(0.15 g, 80%), m.p. 109e110 ꢂC. 1H NMR (CDCl3)
d: 1.43 (m, 4H,
2CH2), 1.83 (m, 2H, CH2), 2.21 (m, 4H, 2CH2), 2.44 (s, 3H, CH3),
2.88 (m, 1H, CH), 4.18 (m, 1H, NCH2), 4.30 (m, 1H, NCH2), 6.89 (t,
1H, H-9), 6.93 (d, 1H, H-7), 7.05 (d, 1H, H-10), 7.14 (t, 1H, H-8),
7.23 (t, 1H, H-2), 7.47 (m, 2H, H-1, H-3), 7.57 (s, 1H, H-12), 7.66
4.2.3.3. 9-Methylthio-6-phthalimidopropylquinobenzothiazine (12c).
(0.42 g, 87%), m.p. 141e142 ꢂC. 1H NMR (CDCl3)
d: 2.27 (m, 2H, CH2),
2.46 (s, 3H, CH3), 3.85 (t, 2H, NCH2), 4.56 (m, 2H, NCH2), 6.97 (d, 1H,
H-7), 7.02 (d, 1H, H-10), 7.12 (dd, 1H, H-8), 7.38 (t, 1H, H-2), 7.57 (t,
1H, H-3), 7.58 (d, 1H, H-1), 7.63 (m, 2H, 2Hphthal), 7.69 (m, 3H, H-12,
2Hphthal.), 7.80 (d, 1H, H-4). FAB MS m/z: 484 (M þ 1, 100), 296
(M ꢁ C3H6C8H4O2 N, 10). Anal. calcd. for C27H21N3O2S2: C 67.06, H
4.38, N 8.69. Found: C 66.91, H 4.36, N 8.45.
(m, 1H, H-4). FAB MS m/z: 376 (M
þ
1, 100), 277
(M þ 1 ꢁ C5H10NCH3, 19), 263 (M þ 1 ꢁ C6H12NCH3, 48), 250
(M þ 1 ꢁ C2H4NC5H9CH3, 37). Anal. calcd. for C23H25N3S: C 73.56,
H 6.71, N 11.19. Found: C 73.44, H 6.70, N 11.02.
4.2.2.17. 6-(1-Methyl-2-piperidylethyl)-9-chloroquinobenzothiazine
(11b). (0.15 g, 73%), m.p. 139e140 ꢂC. 1H NMR (CDCl3)
2H, CH2), 1.92 (m, 8H, 4CH2), 2.66 (s, 3H, CH3), 3.16 (m, 1H, CH), 4.31
(m, 2H, NCH2), 6.93 (d, 1H, H-7), 7.12 (d, 1H, H-10), 7.18 (d, 1H, H-8),
7.33 (t, 1H, H-2), 7.55 (t, 1H, H-3), 7.57 (d, 1H, H-1), 7.67(s, 1H, H-12),
d: 1.51 (m,
4.2.3.4. 6-Phthalimidobutylquinobenzothiazine (12d). (0.40 g, 89%),
m.p. 111e112 ꢂC. 1H NMR (CDCl3)
d: 1.92 (m, 4H, 2CH2), 3.78 (t, 2H,
CH2), 4.28 (t, 2H, NCH2), 6.88 (t, 1H, H-9), 6.90 (d, 1H, H-7), 7.05 (d,
1H, H-10), 7.13 (t, 1H, H-8), 7.22 (t, 1H, H-2), 7.44 (t, 1H, H-3), 7.47 (d,
1H, H-1), 7.58 (s, 1H, H-12), 7.63 (d, 1H, H-4), 7.69 (m, 2H, 2Hphthal.),
7.81 (m, 2H, 2Hphthal.). FAB MS m/z: 452 (M þ 1, 100), 263
(M þ 1 ꢁ C3H6NO2C8H4, 20), 250 (M ꢁ C12H12O2N, 50). Anal. calcd.
for C27H21N3O2S: C 71.82, H 4.69, N 9.31. Found: C 71.60, H 4.67, N
9.08.
7.69 (d, 1H, H-4). FAB MS m/z: 410 (M
þ
1, 100), 311
(M þ 1 ꢁ C5H10NCH3, 26), 297 (M þ 1 ꢁ C6H12NCH3, 60), 284
(M þ 1 ꢁ C2H4C5H9NCH3, 49). Anal. calcd. for C23H24ClN3S: C 67.38,
H 5.90, N 10.25. Found: C 67.19, H 5.88, N 10.02.
4.2.2.18. 6-(1-Methyl-2-piperidylethyl)-9-methylthioquinobenzo-
thiazine (11c). (0.17 g, 81%), m.p. 129e130 ꢂC. 1H NMR (CDCl3)
d:
4.2.3.5. 9-Chloro-6-phthalimidobutylquinobenzothiazine
(12e).
1.40 (m, 2H, CH2), 1.72 (m, 4H, 2CH2), 1.84 (m, 2H, CH2), 2.17 (m, 2H,
CH2), 2.41 (s, 3H, SCH3), 2.53 (s, 3H, NCH3), 3.02 (m, 1H, CH), 4.24
(m, 2H, NCH2), 6.87 (d, 1H, H-7), 7.00 (d, 1H, H-10), 7.09 (d, 1H, H-
8), 7.25 (t, 1H, H-2), 7.48 (t, 1H, H-3), 7.49 (d, 1H, H-1), 7.59 (s,
1H, H-12), 7.64 (d, 1H, H-4). FAB MS m/z: 422 (M þ 1, 100), 309
[M þ 1 ꢁ CHN(CH3) C5H9, 15], 296 [M þ 1 ꢁ (CH2)2NCH3C5H9, 15].
Anal. calcd. for C24H27N3S2: C 68.37, H 6.45, N 9.97. Found: C 68.14,
H 6.47, N 9.73.
(0.42 g, 88%), m.p. 139e140 ꢂC. 1H NMR (CDCl3)
d
: 1.90 (m, 4H,
2CH2), 3.77 (m, 2H, NCH2), 4.28 (m, 2H, NCH2), 6.80 (d, 1H, H-7),
7.01 (d, 1H, H-10), 7.08 (dd, 1H, H-8), 7.25 (m, 1H, H-2), 7.46 (d, 1H,
H-1), 7.48 (t, 1H, H-3), 7.61 (s, 1H, H-12), 7.70 (m, 3H, H-4,
2Hphthal.), 7.81 (m, 2H, 2Hphthal.). FAB MS m/z: 486 (M þ 1, 100),
284 (M ꢁ C12H12O2N, 30). Anal. calcd. for C27H20ClN3O2S: C 66.73,
H 4.15, N 8.65. Found: C 66.59, H 4.17, N 8.51.
4.2.3.6. 9-Methylthio-6-phthalimidobutylquinobenzothiazine (12f).
4.2.3. Synthesis of 9-substituted 6-phthalimidoalkylquinobenzo-
thiazines 12
(0.43 g, 87%), m.p. 118e119 ꢂC. 1H NMR (CDCl3)
d: 2.27 (m, 2H, CH2),
2.46 (s, 3H, CH3), 3.85 (t, 2H, NCH2), 4.56 (m, 2H, NCH2), 6.97 (d, 1H,
H-7), 7.02 (d, 1H, H-10), 7.12 (dd, 1H, H-8), 7.38 (t, 1H, H-2), 7.57 (t,
1H, H-3), 7.58 (d, 1H, H-1), 7.63 (m, 2H, 2Hphthal.), 7.69 (m, 3H, H-12,
2Hphthal.), 7.80 (d, 1H, H-4). FAB MS m/z: 484 (M þ 1, 100), 296
(M ꢁ C3H6C8H4O2 N, 10). Anal. calcd. for C27H21N3O2S: C 67.58, H
4.66, N 8.44. Found: C 67.51, H 4.63, N 8.29.
To a stirred solution of 6H-quinobenzothiazine 3a (0.25 g,
1 mmol) [or 9-chloro-6H-quinobenzothiazine 3c (0.28 g,1 mmol) or
9-methylthio-6H-quinobenzothiazine 3f (0.30 g, 1 mmol)] in dry
toluene (10 mL) NaH (0.24 g,10 mmol, washed out with hexane) was
added. The mixture was refluxed for 30 min and a solution of N-
(bromoalkyl)phthalimide [2.2 mmol, N-(3-bromopropyl)phthali-
mide e 0.60 g, N-(4-bromobutyl)phthalimide e 0.62 g] in dry
toluene (5 mL) was added. The mixture was refluxed for 24 h. Next
toluene was evaporated in vacuo and the residue was extracted with
CHCl3 (2 ꢀ 5 mL). The extract was concentrated and purified by
column chromatography (silica gel, CHCl3) to give compounds 12.
4.2.4. Synthesis of 9-substituted 6-aminoalkylquinobenzothiazines 13
To a boiling solution of 6-phthalimidoalkylquinobenzothiazines
12aef (1 mmol) in EtOH (25 mL) 80% aqueous solution of hydrazine
(0.2 mL, 5 mmol) was added. The mixture was refluxed for 2 h. After
cooling the reaction mixture was acidified to pH ¼ 2 with conc.
hydrochloric acid and evaporated. Water (10 mL) was added to the
residue, the resulting solid was filtered off and washed with 10%
hydrochloric acid. Combined filtrates were alkalized to pH ¼ 10 and
the resulted solid was filtered off, washed with water, dried and
purified by column chromatography (SiO2, CHCl3eEtOH 10:1) to
give compounds 13.
4.2.3.1. 6-Phthalimidopropylquinobenzothiazine (12a). (0.39 g, 89%),
m.p. 104e105 ꢂC. 1H NMR (CDCl3)
d: 2.34 (m, 2H, CH2), 3.94 (t, 2H,
NCH2), 4.32 (t, 2H, NCH2), 6.90 (m, 1H, H-7), 6.92 (m, 1H, H-9), 7.05
(d, 1H, H-10), 7.14 (t, 1H, H-8), 7.22 (t, 1H, H-2), 7.42 (t, 1H, H-3), 7.46
(d, 1H, H-1), 7.55 (m, 2H, H-4, H-12), 7.66 (m, 2H, 2Hphthal.), 7.77 (m,
2H, 2Hphthal.). FAB MS m/z: 438 (M þ 1, 100), 437 (Mþ, 65), 250
(M ꢁ C11H9O2N, 30). Anal. calcd. for C26H19N3O2S: C 71.38, H 4.38, N
9.60. Found: C 71.23, H 4.35, N 9.43.
4.2.4.1. 6-Aminopropylquinobenzothiazine (13a). (0.25 g, 81%), m.p.
142e143 ꢂC. 1H NMR (CDCl3)
d: 2.16 (m, 2H, CH2), 3.03 (t, 2H, NCH2),
4.28 (t, 2H, NCH2), 6.88 (m, 1H, H-7), 6.90 (m, 1H, H-9), 7.03 (d, 1H,
H-10), 7.12 (t, 1H, H-8), 7.18 (t, 1H, H-2), 7.43 (m, 2H, H-1, H-3), 7.52
(s, 1H, H-12), 7.67 (d, 1H, H-4). FAB MS m/z: 308 (M þ 1, 100), 291
4.2.3.2. 9-Chloro-6-phthalimidopropylquinobenzothiazine
(0.40 g, 85%), m.p. 161e162 ꢂC. 1H NMR (CDCl3)
: 2.30 (m, 2H,
CH2), 3.91 (t, 2H, NCH2), 4.35 (t, 2H, NCH2), 6.84 (m, 1H, H-7), 7.05
(12b).
d
(M
þ
1
ꢁ
NH3, 50), 263 (M
þ
1
ꢁ
C2H5NH2, 40), 250

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M. Jelen et al. / European Journal of Medicinal Chemistry 63 (2013) 444e456
452
[(M þ 1 ꢁ C3H6NH2), 50]. Anal. calcd. for C18H17N3S: C 70.33, H 5.57,
131.96, 141.00, 145.50, 152.58, 170.12. FAB MS m/z: 350 (M þ 1, 100),
263 (M þ 1 ꢁ C2H5NHCOCH3, 10), 250 (M þ 1 ꢁ C3H6NHCOCH3, 25).
Anal. calcd. for C20H19N3OS: C 68.74, H 5.48, N 12.02. Found: C 68.51,
H 5.50, N 11.79.
N 13.67. Found: C 70.21, H 5.59, N 13.48.
4.2.4.2. 6-Aminopropyl-9-chloroquinobenzothiazine (13b). (0.26 g,
76%), m.p. 84e85 ꢂC. 1H NMR (DMSO-d6)
d: 2.08 (m, 2H, CH2), 2.97 (t,
2H, NCH2), 4.24 (m, 2H, NCH2), 7.14 (d, 1H, H-7), 7.25 (dd,1H, H-8), 7.34
(m, 2H, H-2, H-10), 7.56 (t, 1H, H-3), 7.68 (m, 2H, H-1, H-4), 8.01 (s, 1H,
H-12). FAB MS m/z: 342 (M þ 1, 100), 325 (M þ 1 ꢁ NH3, 40), 295
(M þ 1 ꢁ C4H6N, 20), 284 (M þ 1 ꢁ C3H8N, 35). Anal. calcd. for
4.2.5.2. 6-Acetylaminopropyl-9-chloroquinobenzothiazine (14b).
(0.32 g, 84%), m.p. 172e173 ꢂC. 1H NMR (CDCl3)
d: 2.00 (s, 3H,
CH3), 2.08 (m, 2H, CH2), 3.43 (m, 2H, NCH2), 4.50 (m, 2H, NCH2),
6.92 (d, 1H, H-7), 7.12 (d, 1H, H-10), 7.16 (d, 1H, H-8), 7.36 (t, 1H,
H-2), 7.58 (m, 2H, H-1, H-3), 7.74 (s, 1H, H-12), 8.05 (m, 1H, H-4).
FAB MS m/z: 384 (M þ 1, 100), 311 (M þ 1 ꢁ CH3NHCOCH3, 40),
297 (M þ 1 ꢁ C2H5NHCOCH3, 45), 284 (M þ 1 ꢁ C3H6NHCOCH3,
65). Anal. calcd. for C20H18ClN3OS: C 62.57, H 4.73, N 10.95.
Found: C 62.29, H 4.75, N 10.77.
C18H16ClN3S: C 63.24, H 4.72, N 12.29. Found: C 63.10, H 4.75, N 12.03.
4.2.4.3. 6-Aminopropyl-9-methylthioquinobenzothiazine
(13c).
(0.30 g, 85%), m.p. 94e95 ꢂC. 1H NMR (DMSO-d6)
d
: 2.40 (m, 2H,
CH2), 2.49 (s, 3H, CH3), 2.73 (m, 2H, CH2), 4.23 (m, 2H, NCH2), 7.10
(d, 1H, H-7), 7.11 (d, 1H, H-8), 7.29 (t, 1H, H-2), 7.45 (d, 1H, H-10),
7.52 (t, 1H, H-3), 7.63 (m, 2H, H-1, H-4), 7.94 (s, 1H, H-12). FAB MS
4.2.5.3. 6-Acetylaminopropyl-9-methylthioquinobenzothiazine (14c).
m/z: 354 (M
þ
1, 100), 337 (M
þ
1
ꢁ
NH3, 40), 296
(0.34 g, 86%), m.p. 138e139 ꢂC. 1H NMR (CDCl3)
d: 1.26 (t, 3H, CH3),
(M þ 1 ꢁ C3H6NH2, 20). Anal. calcd. for C19H19N3S2: C 64.56, H
2.07 (m, 2H, CH2), 2.45 (s, 3H, SCH3), 3.36 (m, 2H, CH2), 4.10 (m, 2H,
CH2), 4.34 (m, 2H, NCH2), 6.88 (d, 1H, H-7), 6.99 (d, 1H, H-10), 7.08
(dd, 1H, H-8), 7.28 (t, 1H, H-2), 7.51 (m, 2H, H-1, H-3), 7.63 (s, 1H, H-
12), 7.93 (m, 1H, H-4). FAB MS m/z: 426 (M þ 1, 100), 296
(M þ 1 ꢁ C3H6NHCO2C2H5, 40). Anal. calcd. for C21H21N3OS2: C
63.77, H 5.35, N 10.62. Found: C 63.55, H 5.37, N 10.47.
5.42, N 11.89. Found: C 64.46, H 5.45, N 11.61.
4.2.4.4. 6-Aminobutylquinobenzothiazine (13d). (0.27 g, 84%), m.p.
158e159 ꢂC. 1H NMR (CDCl3)
d: 1.66 (m, 2H, CH2), 1.92 (m, 2H, CH2),
2.28 (t, 2H, NCH2), 4.24 (t, 2H, NCH2), 6.90 (t, 1H, H-9), 6.91 (d, 1H,
H-7), 7.06 (d, 1H, H-10), 7.15 (t, 1H, H-8), 7.23 (t, 1H, H-2), 7.47 (t, 1H,
H-3), 7.48 (d, 1H, H-1), 7.58 (s, 1H, H-12), 7.69 (d, 1H, H-4). 13C NMR
4.2.5.4. 6-Acetylaminobutylquinobenzothiazine (14d). (0.33 g, 91%),
(75 MHz, CDCl3)
d
: 27.96, 29.72, 38.95, 42.75, 115.75, 118.41, 120.09,
m.p. 132e133 ꢂC. 1H NMR (CDCl3)
d: 1.70 (m, 2H, CH2), 1.91 (m, 2H,
122.77, 124.25, 125.94, 126.09, 126.71, 126.93, 127.72, 129.22, 131.64,
140.91, 145.35, 152.07. FAB MS m/z: 322 (M þ 1, 100), 263
(M þ 1 ꢁ C3H7NH2, 30), 250 (M þ 1 ꢁ C4H8NH2, 75). Anal. calcd. for
CH2), 1.93 (s, 3H, CH3), 3.36 (m, 2H, NCH2), 4.29 (m, 2H, NCH2), 6.93
(m, 2H, H-7, H-9), 7.09 (d, 1H, H-10), 7.17 (t, 1H, H-8), 7.25 (t, 1H, H-
2), 7.50 (t, 1H, H-3), 7.51 (d, 1H, H-1), 7.63 (s, 1H, H-12), 7.75 (m, 1H,
H-4). FAB MS m/z: 364 (M þ 1, 100), 277 (M þ 1 ꢁ C2H5NHCOCH3,
15), 263 (M þ 1 ꢁ C3H7NHCOCH3, 20), 250 (M þ 1 ꢁ C4H8NHCOCH3,
50). Anal. calcd. for C21H21N3OS: C 69.39, H 5.82, N 11.56. Found: C
69.22, H 5.85, N 11.43.
C
19H19N3S: C 71.00, H 5.96, N 13.07. Found: C 7.89, H 5.91, N 12.88.
4.2.4.5. 6-Aminobutyl-9-chloroquinobenzothiazine (13e). (0.25 g,
70%), m.p. 95e96 ꢂC. 1H NMR (DMSO-d6)
: 1.68 (m, 2H, CH2), 1.77
d
(m, 2H, CH2), 2.83 (m, 2H, NCH2), 4.19 (m, 2H, NCH2), 7.10 (d, 2H, H-
7), 7.22 (d,1H, H-10), 7.30 (m, 2H, H-8, H-2), 7.53 (t,1H, H-3), 7.64 (m,
2H, H-1, H-4), 7.97 (s, 1H, H-12). FAB MS m/z: 356 (M þ 1, 100), 284
(M þ 1 ꢁ C4H8NH2, 35). Anal. calcd. for C19H18ClN3S: C 64.12, H 5.10,
N 11.81. Found: C 64.00, H 5.11, N 11.59.
4.2.5.5. 6-Acetylaminobutyl-9-chloroquinobenzothiazine
(14e).
(0.34 g, 86%), m.p. 144e145 ꢂC. 1H NMR (CDCl3)
d
: 1.72 (m, 2H,
CH2), 1.91 (m, 2H, CH2), 1.96 (s, 3H, CH3), 3.34 (m, 2H, NCH2), 4.40
(m, 2H, NCH2), 6.90 (d, 1H, H-7), 7.10 (m, 1H, H-8), 7.16 (d, 1H, H-
10), 7.25 (m, 1H, H-2), 7.34 (t, 1H, H-3), 7.56 (m, 2H, H-1, H-4),
4.2.4.6. 6-Aminobutyl-9-methylthioquinobenzothiazine
(13f).
7.73 (s, 1H, H12). FAB MS m/z: 398 (M
þ
1, 100), 297
(0.30 g, 83%), m.p. 102e103 ꢂC. 1H NMR (DMSO-d6)
d
: 1.50 (m, 2H,
(M þ 1 ꢁ C3H7NHCOCH3, 35), 284 (M þ 1 ꢁ C4H8NHCOCH3, 80).
Anal. calcd. for C21H20ClN3OS: C 63.39, H 5.07, N 10.56. Found: C
63.26 H 5.05, N 10.38.
CH2), 1.73 (m, 2H, CH2), 2.46 (s, 3H, SCH3), 3.29 (m, 2H, CH2), 4.16
(m, 2H, NCH2), 7.03 (d, 2H, H-7), 7.07 (m, 2H, H-9, H-10), 7.27 (t,
1H, H-2) 7.50 (t, 1H, H-3), 7.58 (d, 1H, H-1), 7.26 (d, 1H, H-4), 7.92
(s, 1H, H-12). FAB MS m/z: 368 (M þ 1, 100), 351 (M þ 1 ꢁ NH3,
50), 337 (M ꢁ CH2NH2, 30), 309 (M ꢁ C3H6NH2, 50), 296
(M þ 1 ꢁ C4H8NH2, 90). Anal. calcd. for C20H21N3S2: C 65.36, H
5.76, N 11.43. Found: C 65.16, H 5.71, N 11.28.
4.2.5.6. 6-Acetylaminobutyl-9-methylthioquinobenzothiazine (14f).
(0.345 g, 86%), m.p. 135e136 ꢂC. 1H NMR (CDCl3)
d: 1.75 (t, 2H, CH2),
1.96 (t, 2H, CH2), 1.98 (s, 3H, CH3), 2.47 (s, 3H, SCH3), 3.34 (m, 2H,
CH2), 4.54 (m, 2H, NCH2), 6.97 (d, 1H, H-7), 7.03 (d, 1H, H-10), 7.12
(d, 1H, H-8), 7.40 (t, 1H, H-2), 7.58 (d, 1H, H-1), 7.63 (t, 1H, H-3), 7.80
(s, 1H, H-1, H-12), 8.49 (d, 1H, H-4). FAB MS m/z: 410 (M þ 1, 100),
296 (M þ 1 ꢁ C4H8NHCOCH3, 45). Anal. calcd. for C22H23N3OS2: C
64.52, H 5.66, N 10.26. Found: C 64.38, H 5.63, N 10.05.
4.2.5. Synthesis of 9-substituted 6-acetylaminoalkylquinobenzo-
thiazines 14
To a suspension of aminoalkylquinothiazines 13aef (1 mmol) in
pyridine (5 mL) acetic anhydride (6 mL, 64 mmol) was added and
the mixture was stirred at rt for 24 h. The reaction mixture was
poured into water (20 mL) and the resulting solid was filtered off,
washed with water, air-dried and purified by column chromatog-
raphy (Al2O3, CHCl3) to give compounds 14.
4.2.6. Synthesis of 9-substituted 6-etoxycarbonylaminoalkylquino-
benzothiazines 15
To a stirred solution of aminoalkylquinobenzothiazines 13aef
(1 mmol) in a mixture of CH2Cl2 (5 mL) and 10% Na2CO3 solution
(5 mL), a solution of ethyl chloroformate (1.3 mL, 1.3 mmol) in
CH2Cl2 (3 mL) was added. The mixture was stirred at rt for 24 h. The
organic phase was separated and aqueous phase was extracted
with CH2Cl2 (2 ꢀ 5 mL). The combined extracts were washed with
water (2 ꢀ 10 mL) and dried over Na2SO4. The drying agent was
filtered off and filtrate was evaporated. The resulting residue was
purified by column chromatography (Al2O3, CHCl3) to give com-
pounds 15:
4.2.5.1. 6-Acetylaminopropylquinobenzothiazine (14a). (0.31 g, 89%),
m.p. 114e115 ꢂC. 1H NMR (CDCl3)
d: 1.99 (s, 3H, CH3), 2.09 (m, 2H,
CH2), 3.43 (m, 2H, NCH2), 4.39 (m, 2H, NCH2), 6.28 (s, 1H, NH), 6.96
(m, 2H, H-7, H-9), 7.09 (d, 1H, H-10), 7.16 (t, 1H, H-8), 7.28 (t, 1H, H-
2), 7.58 (m, 2H, H-1, H-3), 7.64 (s, 1H, H-12), 7.72 (m, 1H, H-4). 13
C
NMR (75 MHz, CDCl3)
d: 23.41, 26.44, 37.54, 42.34, 115.82, 118.61,
120.42, 122.97, 124.37, 126.11, 126.32, 126.79, 126.94, 127.81, 129.31,

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453
4.2.6.1. 6-Ethoxycarbonylaminopropylquinobenzothiazine (15a).
(0.30 g, 79%), m.p. 82e83 ꢂC. 1H NMR (CDCl3)
: 1.28 (t, 3H, CH3),
and at rt for 24 h. After evaporation of EtOH in vaccuo the residue
was purified by column chromatography (Al2O3, CHCl3) to give
compounds 16.
d
2.10 (m, 2H, CH2), 3.38 (m, 2H, NCH2), 4.13 (m, 2H, CH2), 4.32 (m,
2H, NCH2), 6.92 (t, 1H, H-9), 6.94 (d, 1H, H-7), 7.06 (d, 1H, H-10),
7.15 (t, 1H, H-8), 7.27 (t, 1H, H-2), 7.50 (m, 2H, H-1, H-3), 7.60 (s,
1H, H-12), 7.85 (m, 1H, H-4). FAB MS m/z: 380 (M þ 1, 100), 277
(M þ 1 ꢁ CH2NHCO2C2H5, 15), 263 (M þ 1 ꢁ C2H5NHCO2C2H5, 20),
250 (M þ 1 ꢁ C3H6NCO2C2H5, 35). Anal. calcd. for C21H21N3O2S: C
66.47, H 5.58, N 11.07. Found: C 66.40, H 5.51, N 10.88.
4.2.7.1. 6-Chloroethylureidopropylquinobenzothiazine
(16a).
(0.27 g, 66%), m.p. 177e178 ꢂC. 1H NMR (CDCl3)
d
: 1.25 (m, 2H, CH2),
2.09 (m, 2H, CH2), 3.49 (m, 2H, NCH2), 3.50 (m, 2H, CH2), 3.57 (m,
2H, NCH2), 4.44 (broad s, 2H, 2NH), 6.99 (m, 2H, H-7, H-9), 7.12
(d, 1H, H-10), 7.20 (t, 1H, H-8), 7.31 (t, 1H, H-2), 7.54 (m, 2H, H-1,
H-3), 7.69 (m, 1H, H-4) 7.93 (s, 1H, H-12). FAB MS m/z: 413 (M þ 1,
40), 377 (M þ 1 ꢁ HCl, 40), 277 (M þ 1 ꢁ CH2NHCONHCH2CH2Cl,
4.2.6.2. 9-Chloro-6-ethoxycarbonylaminopropylquinobenzothiazine
(15b). (0.32 g, 77%), m.p. 121e122 ꢂC. 1H NMR (CDCl3)
d: 1.24 (t, 3H,
35), 263 (M
þ
1
ꢁ
C2H5NHCONHCH2CH2Cl, 45), 250
CH3), 2.06 (m, 2H, CH2), 3.37 (m, 2H, NCH2), 4.07 (m, 2H, NCH2),
4.39 (t, 2H, NCH2), 6.89 (d, 1H, H-7), 7.08 (d, 1H, H-10), 7.13 (dd, 1H,
H-8), 7.32 (t, 1H, H-2), 7.53 (m, 2H, H-1, H-3), 7.68 (s, 1H, H-12), 8.05
(M ꢁ C3H6NHCONHCH2CH2Cl, 70). Anal. calcd. for C21H21ClN4OS: C
61.08, H 5.13, N 13.57. Found: C 60.88, H 5.12, N 13.39.
(m, 1H, H-4). FAB MS m/z: 414 (M
þ
1, 100), 311
4.2.7.2. 6-Chloroethylureidopropyl-9-chloroquinobenzothiazine
(M þ 1 ꢁ CH3NHCO2C2H5, 40), 297 (M þ 1 ꢁ C2H5NHCO2C2H5, 45),
284 (M þ 1-C3H6NHCO2C2H5, 65). Anal. calcd. for C21H20ClN3O2S: C
60.94, H 4.87, N 10.15. Found: C 60.73, H 4.875 N 10.01.
(16b). (0.31 g, 69%), m.p. 151e152 ꢂC. 1H NMR (CDCl3)
d: 1.25 (m, 2H,
CH2), 2.09 (m, 2H, CH2), 3.43 (m, 2H, NCH2), 3.52 (m, 2H, NCH2),
3.59(broad s, 2H, 2NH), 4.59(t, 2H, NCH2), 7.10 (d,1H, H-7), 7.21 (d,1H,
H-10), 7.29 (d, 1H, H-8), 7.55 (t, 1H, H-2), 7.66 (d, 1H, H-1), 7.75
(m, 2H, H-3, H-4), 7.97 (s, 1H, H-12). FAB MS m/z: 448 (M þ 1, 21)
411 (M þ 1 ꢁ HCl, 100), 368 (M ꢁ NH2CH2CH2Cl, 22), 325
(M ꢁ NH2CONHCH2CH2Cl, 38), 284 (M þ 1 ꢁ C3H6NHCONHCH2CH2Cl,
43). Anal. calcd. for C21H20Cl2N4OS: C 56.38, H 4.51, N 12.52. Found: C
56.19, H 4.53, N 12.37.
4.2.6.3. 6-Ethoxycarbonylaminopropyl-9-methylthioquinobenzo-
thiazine (15c). (0.36 g, 85%), m.p. 125e126 ꢂC. 1H NMR (CDCl3)
d:
1.26 (t, 3H, CH3), 2.07 (m, 2H, CH2), 2.45 (s, 3H, SCH3), 3.36 (m, 2H,
CH2), 4.10 (m, 2H, CH2), 4.34 (m, 2H, NCH2), 6.88 (d,1H, H-7), 6.99 (d,
1H, H-10), 7.08 (dd, 1H, H-8), 7.28 (t,1H, H-2), 7.51 (m, 2H, H-1, H-3),
7.63 (s, 1H, H-12), 7.93 (m, 1H, H-4). FAB MS m/z: 426 (M þ 1, 100),
296 (M þ 1 ꢁ C3H6NHCO2C2H5, 40). Anal. calcd. for C22H23N3O2S2: C
62.09, H 5.45, N 9.87. Found: C 61.87, H 5.46, N 9.63.
4.2.7.3. 6-Chloroethylureidopropyl-9-methylthioquinobenzothiazine
(16c). (0.30 g, 66%), m.p.167e168 ꢂC.1H NMR (CDCl3)
d: 1.25 (m, 2H,
CH2), 2.06 (m, 2H, CH2), 2.46 (s, 3H, SCH3), 3.48 (m, 2H, CH2), 3.49 (m,
2H, CH2), 3.55 (t, 2H, NCH2), 4.50 (broad s, 2H, 2NH), 6.92 (d, 1H, H-
7), 7.02 (d,1H, H-10), 7.08 (d,1H, H-8), 7.34 (t,1H, H-2), 7.54 (d,1H, H-
1), 7.58 (t,1H, H-3), 7.72 (s,1H, H-12), 8.14 (m,1H, H-4). FAB MS m/z:
458 (Mþ, 100), 459 (M þ 1, 90), 380 (M ꢁ NHC2H4Cl, 30), 323
(M ꢁ CH2NHCONHC2H4Cl, 10), 310 (M þ 1 ꢁ C2H4NHCONHC2H4Cl,
20), 296 (M þ 1 ꢁ C3H6NHCONHC2H4Cl, 40). Anal. calcd. for
4.2.6.4. 6-Ethoxycarbonylaminobutylquinobenzothiazine (15d).
(0.35 g, 89%), m.p. 90e91 ꢂC. 1H NMR (CDCl3)
d: 1.26 (t, 3H, CH3), 1.70
(m, 2H, CH2), 1.92 (m, 2H, CH2), 3.29 (m, 2H, NCH2), 4.10 (m, 2H, CH2),
4.26(m, 2H, NCH2), 6.91(m, 2H, H-7, H-9), 7.08(d,1H, H-10), 7.16(t,1H,
H-8), 7.25 (t,1H, H-2), 7.49 (t,1H, H-3), 7.50 (d,1H, H-1), 7.60 (s,1H, H-
12), 7.74 (m,1H, H-4). 13C NMR (75 MHz, CDCl3)
d: 14.68, 23.48, 27.36,
40.49, 44.80, 60.70, 115.74, 118.68, 120.48, 122.62, 124.13, 126.03,
126.13, 126.86, 127.27, 127.65, 129.06, 131.71, 141.34, 145.72, 152.45,
156.73. FAB MS m/z: 394 (M þ 1, 100), 277 (M þ 1 ꢁ C2H5NHCO2C2H5,
20), 250 (M þ 1 ꢁ C4H8NCO2C2H5, 35). Anal. calcd. for C22H23N3O2S: C
67.15, H 5.89, N 10.68. Found: C 67.01, H 5.88, N 10.41.
C22H23ClN4OS2: C 57.57, H 5.05, N 12.21. Found: C 57.38, H 5.07, N
12.00.
4.2.7.4. 6-Chloroethylureidobutylquinobenzothiazine
(16d).
(0.28 g, 66%), m.p. 132e133 ꢂC. 1H NMR (CDCl3)
d: 1.67 (m, 2H, CH2),
1.89 (m, 2H, CH2), 3.27 (m, 2H, CH2), 3.47 (m, 2H, CH2), 3.56 (m, 2H,
NCH2), 4.23 (m, 2H, NCH2), 4.96 (broad s, 2H, 2NH), 6.91 (m, 2H, H-
7, H-9), 7.06 (m, 1H, H-10), 7.15 (t, 1H, H-8), 7.24 (t, 1H, H-2), 7.47 (t,
1H, H-3), 7.48 (d, 1H, H-1), 7.58 (s, 1H, H-12), 7.69 (m, 1H, H-4). FAB
MS m/z: 427 (M þ 1, 100), 391 (M þ 1 ꢁ HCl, 35), 263 (M þ 1 ꢁ
C3H7NHCONHCH2CH2Cl, 30), 250 (M þ 1 ꢁ C4H8NHCONHCH2
CH2Cl, 80). Anal. calcd. for C22H23ClN4OS: C 61.89, H 5.43, N 13.12.
Found: C 61.62, H 5.44, N 12.87.
4.2.6.5. 9-Chloro-6-ethoxycarbonylaminobutylquinobenzothiazine
(15e). (0.34 g, 80%), m.p. 130e131 ꢂC. 1H NMR (CDCl3)
d: 1.21 (m,
5H, CH3, CH2), 1.69 (m, 2H, CH2), 1.89 (m, 2H, NCH2), 3.25 (m, 2H,
NCH2), 4.06 (m, 2H, NCH2), 6.98 (d, 1H, H-7), 7.15 (d, 1H, H-10), 7.20
(dd, 1H, H-8), 7.26 (t, 1H, H-2), 7.40 (t, 1H, H-3), 7.59 (d, 1H, H-1),
7.63 (d, 1H, H-4), 7.82 (s, 1H, H-12). FAB MS m/z: 428 (M þ 1, 100),
297 (M þ 1 ꢁ C3H7NHCO2C2H5, 30), 284 (M þ 1 ꢁ C4H8NHCO2C2H5,
62). Anal. calcd. for C22H22ClN3O2S: C 61.75, H 5.18, N 9.82. Found: C
61.47, H 5.20, N 9.60.
4.2.7.5. 6-Chloroethylureidobutyl-9-chloroquinobenzothiazine (16e).
(0.29 g, 63%), m.p. 164e165 ꢂC. 1H NMR (CDCl3)
d: 1.25 (m, 2H, CH2),
4.2.6.6. 6-Ethoxycarbonylaminobutyl-9-methylthioquinobenzo-
1.88 (m, 2H, CH2), 1.99 (m, 4H, 2CH2), 3.50 (m, 2H, NCH2), 3.71 (m,
2H, NCH2), 4.75 (broad s, 2H, 2NH), 7.10 (d, 1H, H-7), 7.35 (dd, 1H, H-
8), 7.59 (m, 2H, H-2, H-10), 7.71 (d, 1H, H-1), 7.82 (m, 2H, H-3, H-4),
8.06 (s, 1H, H-12). FAB MS m/z: 462 (M þ 1, 3), 425 (M ꢁ HCl, 100),
382 (M ꢁ NH2CH2CH2Cl, 6), 297 (M þ 1 ꢁ C3H7NHCONHCH2CH2Cl,
16), 284 (M þ 1 ꢁ C4H8NHCONHCH2CH2Cl, 38). Anal. calcd. for
thiazine (15f). (0.37 g, 84%), m.p. 95e96 ꢂC. 1H NMR (CDCl3)
d: 1.22
(t, 3H, CH3), 1.69 (t, 2H, CH2), 1.90 (t, 2H, CH2), 2.46 (s, 3H, CH3), 3.28
(m, 2H, CH2), 4.09 (m, 2H, CH2), 4.36 (n, 2H, NCH2), 6.89 (d,1H, H-7),
7.02 (d,1H, H-10), 7.09 (d,1H, H-8), 7.30 (t,1H, H-2), 7.52 (d,1H, H-1),
7.53 (t,1H, H-3), 7.67 (s,1H, H-12), 7.96 (d,1H, H-4). FAB MS m/z: 440
(M þ 1, 100), 296 (M þ 1 ꢁ C4H8NHCO2C2H5, 35). Anal. calcd. for
C
22H22Cl2N4OS: C 57.27, H 4.81, N 12.14. Found: C 57.20, H 4.82, N
C
23H25N3O2S2: C 62.84, H 5.73, N 9.56. Found: C 62.61, H 5.76, N 9.39.
11.93.
4.2.7. Synthesis of 9-substituted 6-chloroethylureidoalkylquinobenzo-
thiazines 16
4.2.7.6. 6-Chloroethylureidobutyl-9-methylthioquinobenzothiazine
(16f). (0.32 g, 68%), m.p. 129e130 ꢂC. 1H NMR (CDCl3)
d: 1.25 (m,
To a stirred solution of 6-aminoalkyldiquinothiazines 13aef
(1 mmol) in ethanol (25 mL) at 0 ꢂC 2-chloroethyl isocyanate
(0.16 mL, 2 mmol) was added. The mixture was stirred at 0 ꢂC for 1 h
2H, CH2), 1.71 (m, 2H, CH2), 1.92 (m, 2H, CH2), 2.46 (s, 3H, CH3), 3.32
(m, 2H, NCH2), 3.50 (m, 2H, CH2), 3.58 (m, 2H, NCH2), 4.40 (broad s,
2H, 2NH), 6.92 (d, 1H, H-7), 7.03 (d, 1H, H-10), 7.12 (d, 1H, H-8), 7.27

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454
(t, 1H, H-2), 7.39 (t, 1H, H-3), 7.55 (m, 2H, H-1, H-4), 7.72 (s, 1H, H-
12). FAB MS m/z: 473 (M þ 1, 98), 472 (Mþ, 100), 437 (M þ 1 ꢁ HCl),
394 (M ꢁ NHCH2CH2Cl, 35), 309 (M þ 1 ꢁ C3H7NHCONHCH2CH2Cl,
40), 296 (M þ 1 ꢁ C4H8NHCONHCH2CH2Cl, 100). Anal. calcd. for
4.70 (m, 2H, NCH2), 7.06 (d, 1H, H-7), 7.18 (d, 1H, H-10), 7.47 (m, 2H,
H-2, H-8), 7.62 (d,1H, H-1), 7.70 (m, 2H, H-3, H-4), 7.90 (s,1H, H-12).
FAB MS m/z: 434 (M þ 1, 100), 297 (M ꢁ C3H6NHSO2CH3, 19), 284
(M þ 1 ꢁ C4H8NHSO2CH3, 43). Anal. calcd. for C20H20ClN3O2S2: 2
55.35, H 4.65, N 9.68. Found: C 55.21, H 4.65, N 9.49.
C
23H25ClN4OS2: C 58.40, H 5.33, N 11.84. Found: C 58.29, H 5.35, N
11.61.
4.2.8.6. 6-Methanesulfonylaminobutyl-9-methylthioquinobenzo-
4.2.8. Synthesis of 9-substituted 6-methanesulfonylaminoalkyl-
quinobenzothiazines 17
thiazine (17f). (0.34 g, 76%), m.p. 119e120 ꢂC. 1H NMR (CDCl3)
d:
1.78 (m, 2H, CH2), 1.94 (m, 2H, CH2), 2.46 (s, 3H, SCH3), 2.94 (s, 3H,
CH3), 3.25 (m, 2H, CH2), 4.32 (m, 2H, NCH2), 6.86 (d, 1H, H-7), 7.05
(d, 1H, H-10), 7.09 (d, 2H, H-8), 7.27 (t, 1H, H-2), 7.51 (m, 2H, H-1, H-
3), 7.65 (s, 1H, H-12), 7.78 (m, 1H, H-4). FAB MS m/z: 446 (M þ 1,
100), 309 (M ꢁ C3H6NHSO2CH3, 15), 296 (M þ 1 ꢁ C4H8NHSO2CH3,
40). Anal. calcd. for C21H23N3O2S3: C 56.60, H 5.20, N 9.43. Found: C
56.40, H 5.21, N 9.28.
To a stirred solution of aminoalkyldiquinothiazines 13aef
(1 mmol) in a mixture of CH2Cl2 (10 mL) and 10% Na2CO3 solution
(14 mL), a solution of methanesulfonyl chloride (0.12 mL, 1.5 mmol)
was added. The mixture was stirred at rt for 24 h. The organic phase
was separated and aqueous phase was extracted with CH2Cl2
(2 ꢀ 5 mL). The combined extracts were washed with water
(2 ꢀ 10 mL) and dried over Na2SO4. The drying agent was filtered off
and filtrate was evaporated. The resulting residue was purified by
column chromatography (Al2O3, CHCl3) to give compounds 17.
4.2.9. Synthesis of 9-substituted 6-p-toluenesulfonylaminolkylquino-
benzothiazines 18
To a stirred solution of aminoalkyldiquinothiazines 13aef
(1 mmol) in a mixture of CH2Cl2 (6 mL) and 10% Na2CO3 solution
(7 mL), a solution of p-toluenesulfonyl chloride (0.28 g,1.5 mmol) in
CH2Cl2 (6 mL) was added. The mixture was stirred at rt for 24 h. The
organic phase was separated and aqueous phase was extracted
with CH2Cl2 (2 ꢀ 5 mL). The combined extracts were washed with
water (2 ꢀ 10 mL) and dried over Na2SO4. The drying agent was
filtered off and filtrate was evaporated. The resulting residue was
purified by column chromatography (Al2O3, CHCl3) to give: com-
pounds 18.
4.2.8.1. 6-Methanesulfonylaminopropylquinobenzothiazine
(17a).
(0.28 g, 73%), m.p. 117e118 ꢂC. 1H NMR (CDCl3)
d: 2.16 (m, 2H, CH2),
2.86 (s, 3H, CH3), 3.31 (m, 2H, NCH2), 4.53 (m, 2H, NCH2), 5.99 (s,1H,
NH), 6.97 (m, 2H, H-7, H-9), 7.07 (d, 1H, H-10), 7.17 (t, 1H, H-8), 7.30
(t,1H, H-2), 7.51 (d, 1H, H-1), 7.55 (t,1H, H-3), 7.63 (s, 1H, H-12), 7.92
(m, 1H, H-4). 13C NMR (75 MHz, CDCl3)
d: 27.35, 39.97, 40.86,
41.82, 115.50, 118.06, 119.90, 123.06, 124.59, 126.08, 126.20,
126.53, 126.82, 127.76, 129.69, 131.88, 140.55, 145.22, 151.96. FAB MS
m/z: 386 (M þ 1, 100), 277 (M þ 1 ꢁ CH2NHSO2CH3, 35), 250
(M þ 1 ꢁ C3H6NHSO2CH3, 60). Anal. calcd. for C19H19N3O2S2: C
59.20, H 4.97, N 10.90. Found: C 59.03, H 4.92, N 10.67.
4.2.9.1. 6-p-Toluenesulfonylaminopropylquinobenzothiazine (18a).
(0.36 g, 78%), m.p. 68e69 ꢂC. 1H NMR (CDCl3)
d: 2.02 (m, 2H, CH2),
4.2.8.2. 9-Chloro-6-methanesulfonylaminopropylquinobenzothiazine
2.35 (s, 3H, CH3), 3.11 (m, 2H, NCH2), 4.24 (m, 2H, NCH2), 6.86 (d,1H,
H-7), 6.91 (t,1H, H-9), 7.02 (d,1H, H-10), 7.10 (t,1H, H-8), 7.14 (d, 2H,
2Hbenz.), 7.29 (t, 1H, H-2), 7.49 (d, 1H, H-1), 7.56 (m, 2H, H-3, H-12),
7.61 (d, 2H, 2Hbenz.), 7.92 (m,1H, H-4). FAB MS m/z: 462 (M þ 1,100),
(17b). (0.32 g, 76%), m.p. 129e130 ꢂC. 1H NMR (CDCl3)
d: 2.14 (m,
2H, CH2), 2.87 (s, 3H, CH3), 3.30 (m, 2H, NCH2), 4.44 (t, 2H, NCH2),
6.88 (d, 1H, H-7), 7.06 (d, 1H, H-10), 7.11 (dd, 1H, H-8), 7.32 (t, 1H, H-
2), 7.53 (d, 1H, H-1), 7.56 (t, 1H, H-3), 7.65 (s, 1H, H-12), 7.97 (m, 1H,
H-4). FAB MS m/z: 420 (M þ 1, 100), 311 (M þ 1 ꢁ CH3NHSO2CH3,
25), 297 (M þ 1 ꢁ C2H5NHSO2CH3, 30), 284 (M þ 1-C3H6NHSO2CH3,
50). Anal. calcd. for C19H18ClN3O2S2: C 54.34, H 4.32, N 10.01. Found:
C 54.09, H 4.34, N 9.82.
277 (M
þ
1
ꢁ
CH2NHSO2C6H4CH3, 60), 263 (M
þ
1
ꢁ
C2H5NHSO2C6H4CH3, 70), 251 (M þ 1 ꢁ C3H5NHSO2C6H4CH3, 96),
250 (M C3H6NHSO2C6H4CH3, 90). Anal. calcd. for
25H23N3O2S2: C 65.05, H 5.02, N 9.10. Found: C 64.81, H 5.00, N
8.95.
þ
1
ꢁ
C
4.2.8.3. 6-Methanesulfonylaminopropyl-9-methylthioquinobenzo-
4.2.9.2. 9-Chloro-6-p-toluenesulfonylaminopropylquinobenzothia-
zine (18b). (0.38 g, 77%), m.p. 116e117 ꢂC. 1H NMR (CDCl3)
: 2.01
thiazine (17c). (0.33 g, 77%), m.p. 100e101 ꢂC. 1H NMR (CDCl3)
d:
d
2.13 (m, 2H, CH2), 2.45 (s, 3H, CH3), 2.86 (s, 3H, SCH3), 3.29 (m, 2H,
CH2), 4.41 (m, 2H, NCH2), 6.88 (d, 1H, H-7), 6.97 (d, 1H, H-10), 7.05
(d, 1H, H-8), 7.28 (t, 1H, H-2), 7.49 (d, 1H, H-1), 7.53 (t, 1H, H-3), 7.61
(s, 1H, H-12), 7.91 (m, 1H, H-4). FAB MS m/z: 432 (M þ 1, 100), 353
(M þ 1 ꢁ SO2CH3, 10), 323 (M ꢁ CH2NHSO2CH3, 25), 309
(M ꢁ C2H4NHSO2CH3, 30), 296 (M þ 1 ꢁ C3H6NHSO2CH3, 60). Anal.
calcd. for C20H21N3O2S3: C 55.66, H 4.90, N 9.74. Found: C 55.48, H
4.88, N 9.59.
(m, 2H, CH2), 2.36 (s, 3H, CH3), 3.11 (m, 2H, NCH2), 4.29 (t, 2H,
NCH2), 6.86 (d, 1H, H-7), 7.04 (d, 1H, H-10), 7.10 (dd, 1H, H-8), 7.16
(d, 2H, 2Hbenz.), 7.34 (t, 1H, H-2), 7.52 (d, 1H, H-1), 7.59 (t, 1H, H-3),
7.63 (m, 3H, H-12, 2Hbenz.), 8.03 (d, 1H, H-4). FAB MS m/z:
496 (M þ 1, 100), 311 (M þ 1 ꢁ CH3NHSO2C6H4CH3, 35),
297 (M
þ
1
ꢁ
C2H5NHSO2C6H4CH3, 30), 284 (M
þ
1
ꢁ
C3H6NHSO2C6H4CH3, 45). Anal. calcd. for C25H22ClN3O2S2: C 60.53,
H 4.47, N 8.47. Found: C 60.45, H 4.42, N 8.33.
4.2.8.4. 6-Methanesulfonylaminobutylquinobenzothiazine (17d).
4.2.9.3. 9-Methylthio-6-p-toluenesulfonylaminopropylquinobenzo-
(0.27 g, 68%), m.p. 97e98 ꢂC. 1H NMR (CDCl3)
d
: 1.78 (m, 2H, CH2),
thiazine (18c). (0.40 g, 79%), m.p. 70e71 ꢂC. 1H NMR (CDCl3)
d: 2.00
1.96 (m, 2H, CH2), 2.93 (s, 3H, CH3), 3.25 (m, 2H, NCH2), 4.34 (m, 2H,
NCH2), 4.76 (m, 1H, NH), 6.95 (m, 2H, H-7, H-9), 7.10 (d, 1H, H-10),
7.18 (t, 1H, H-8), 7.28 (t, 1H, H-2), 7.51 (m, 2H, H-1, H-3), 7.64 (s, 1H,
H-12), 7.85 (m, 1H, H-4). FAB MS m/z: 400 (M þ 1, 100), 263
(M ꢁ C3H6NHSO2CH3, 25), 250 (M þ 1 ꢁ C4H8NHSO2CH3, 50). Anal.
calcd. for C20H21N3O2S2: C 60.13, H 5.30, N 10.52. Found: C 59.96, H
5.29, N 10.36.
(m, 2H, CH2), 2.35 (s, 3H, CH3), 2.44 (s, 3H, CH3), 3.10 (m, 2H, CH2),
4.29 (m, 2H, NCH2), 6.79 (d, 1H, H-7), 6.94 (d, 1H, H-10), 7.01 (d, 1H,
H-8), 7.14 (d, 2H, 2Hbenz.), 7.31 (t, 1H, H-2), 7.50 (d, 1H, H-1), 7.58 (m,
2H, H-3, H-12) 7.65 (d, 2H, 2Hbenz.), 7.93 (d, 1H, H-4). FAB MS m/z:
508 (M
þ
1, 100), 323 (M
ꢁ
CH2NHSO2C6H4CH3, 25), 309
(M ꢁ C2H4NHSO2C6H4CH3, 30), 296 (M þ 1 ꢁ C3H6NHSO2C6H4CH3,
40). Anal. calcd. for C26H25N3O2S3: C 61.51, H 4.96, N 8.28. Found: C
61.38, H 4.94, N 8.09.
4.2.8.5. 9-Chloro-6-methanesulfonylaminobutylquinobenzothiazine
(17e). (0.32 g, 74%), m.p. 163e164 ꢂC. 1H NMR (CDCl3)
2H, CH2), 2.10 (m, 2H, CH2), 2.96 (s, 3H, CH3), 3.27 (m, 2H, NCH2),
d
: 1.83 (m,
4.2.9.4. 6-p-Toluenesulfonylaminobutylquinobenzothiazine
(18d).
(0.35 g, 74%), m.p. 63e64 ꢂC. 1H NMR (CDCl3)
d: 1.66 (m, 2H, CH2),

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455
2.39 (m, 2H, CH2), 2.39 (s, 3H, CH3), 3.07 (m, 2H, NCH2), 4.19 (m, 2H,
NCH2), 4.65 (m, 1H, NH), 6.87 (d, 1H, H-7), 6.92 (t, 1H, H-9), 7.08 (d,
1H, H-10), 7.15 (t,1H, H-8), 7.25 (m, 3H, H-2, 2Hbenz.), 7.49 (m, 2H, H-
1, H-3), 7.61 (s, 1H, H-12), 7.71 (m, 3H, H-4, 2Hbenz.). 13C NMR
determined by MTTcolorimetric method [31]. The results are given in
percentage inhibition as compared with appropriate DMSO controls.
4.3.5. Lipopolysaccharide-induced TNF-a production in whole blood
cell culture
(75 MHz, CDCl3)
d: 21.53, 23.30, 26.96, 42.87, 44.48, 115.73,
118.62, 120.45, 122.69, 124.19, 126.01, 126.15, 126.86, 127.08,
127.22, 127.71, 129.14, 129.70, 131.74, 136.99, 141.20, 143.34,
145.64, 152.38. FAB MS m/z: 476 (M þ 1, 100), 369 (M þ 1 ꢁ
OC6H4CH3, 25), 277 (M þ 1 ꢁ C2H4NHSO2C6H4CH3, 20), 250
(M þ 1 ꢁ C4H8NHSO2C6H4CH3, 20). Anal. calcd. for C26H25N3O2S2: C
65.66, H 5.30, N 8.83. Found: C 65.46, H 5.26, N 8.59.
Venous blood from a single donor was diluted 10ꢀ with RPMI-
1640 medium and distributed in 1 mL aliquots in 24-well culture
plates. The cultures were stimulated by addition of 1
from Escherichia coli, O111:B4. The compounds were added to the
cultures at concentrations of 5 and 25 g/mL. Higher concentra-
tions of the compounds could not be used because of inhibitory
effects on TNF- production by corresponding DMSO (the solvent)
mg/mL of LPS
m
a
4.2.9.5. 9-Chloro-6-p-toluenesulfonylaminobutylquinobenzothiazine
dilutions. Appropriate dilutions of DMSO served as controls. After
overnight incubation in a cell culture incubator, the supernatants
were harvested and frozen at ꢁ20 ꢂC until cytokine determination
by a biological assay [32]. The results are given in percentage in-
hibition as compared with appropriate DMSO controls.
(18e). (0.38 g, 75%), m.p. 140e141 ꢂC. 1H NMR (CDCl3)
d: 1.68 (m,
2H, CH2), 1.88 (m, 2H, CH2), 2.40 (s, 3H, CH3), 3.05 (m, 2H, NCH2),
4.34 (m, 2H, NCH2), 6.86 (d, 1H, H-7), 7.09 (d, 1H, H-10), 7.14 (d, 1H,
H-8), 7.25 (m, 2H, 2Hbenz.), 7.34 (t, 1H, H-2), 7.55 (d, 1H, H-1), 7.56 (t,
1H, H-3), 7.72 (m, 3H, H-12, 2Hbenz.), 8.04 (d, 1H, H-4). FAB MS m/z:
510 (M þ 1, 100), 297 (M þ 1 ꢁ C3H7NHSO2C6H4CH3, 21), 284
(M þ 1 ꢁ C4H8NHSO2C6H4CH3, 50). Anal. calcd. for C26H24ClN3O2S2:
C 61.22, H 4.74, N 8.24. Found: C 61.01, H 4.71, N 8.07.
4.3.6. Growth inhibition of tumor cell lines
A-431 epidermal cell line, L-1210 lymphoma and SW-948 colon
tumor cell lines derived from the Collection of Cell Lines of The
Institute of Immunology and Experimental Therapy, Wroc1aw,
Poland. The lines were re-suspended in the culture medium and
distributed into 96-well flat-bottom plates. L-1210 was present at
1.5 ꢀ 104 cells/well while SW-948 and A-431 at 2.5 ꢀ 104 cells/well.
The preparations were added to the wells at the concentration range
4.2.9.6. 9-Methylthio-6-p-toluenesulfonylaminobutylquinobenzo-
thiazine (18f). (0.42 g, 81%), m.p. 119e120 ꢂC. 1H NMR (CDCl3)
d:
1.64 (m, 2H, CH2), 1.83 (m, 2H, CH2), 2.38 (s, 3H, CH3), 2.44 (s, 3H,
SCH3), 3.05 (m, 2H, CH2), 4.16 (m, 2H, NCH2), 6.78 (d, 1H, H-7), 6.99
(d, 1H, H-10), 7.06 (d, 1H, H-8), 7.25 (m, 3H, H-2, 2Hbenz.), 7.49 (m,
1H, H-1, H-3), 7.59 (s, 1H, H-12), 7.72 (m, 5H, H-12, 2Hbenz.). FAB MS
m/z: 522 (M þ 1, 100), 521 (Mþ, 55), 309 (M-C3H7NHSO2C6H4CH3,
40). Anal. calcd. for C27H27N3O2S3: C 62.16, H 5.22, N 8.05. Found: C
62.01, H 5.19, N 7.81.
of 0.1e50 mg/mL. Cisplatin was used as a reference drug in the same
concentrations. After 3-day incubation in a cell culture incubator, the
proliferation was determined using MTT colorimetric method. The
data are presented as a mean OD value from quadruplicate wells ꢃSE.
4.3.7. Statistics
4.3. Biological assays
The results are presented as mean values ꢃ standard error (SE)
or percentage inhibition ¼ [(control value ꢁ tested value)/control
value]*100. BrowneForsyth’s test was used to determine the ho-
mogeneity of variance between groups. When the variance was
homogenous, analysis of variance (One-way ANOVA) was applied,
followed by post hoc comparisons with the Tukey’s test to estimate
the significance of the difference between groups. Nonparametric
data were evaluated with the KruskaleWallis’ analysis of variance.
Significance was determined at p < 0.05. Statistical analysis was
performed using STATISTICA 6.1 for Windows.
4.3.1. Preparation of the compounds for biological assays
The compounds were dissolved in DMSO (10 mg/mL) and sub-
sequently diluted in RPMI-1640 cell culture medium (see below).
4.3.2. Isolation of the peripheral blood mononuclear cells (PBMC)
Venous blood from a single donor was withdrawn into hepa-
rinized syringes and diluted twice with PBS. PBMC were isolated by
centrifugation on Ficoll-uropoline gradient (density 1.077 g/mL)
and centrifuged at 800ꢀ g for 20 min at 4 ꢂC. The interphase cells,
consisting of lymphocytes (20%) and monocytes (80%) were then
washed three times with Hanks’ medium and re-suspended in a
culture medium, referred to below as the culture medium, con-
Acknowledgments
The work is supported by the National Centre of Science (grant N
N405 101739).
sisting of RPMI-1640, supplemented with 10% fetal calf serum, L-
glutamine, sodium pyruvate, 2-mercaptoethanol and antibiotics, at
density of 2 ꢀ 106 cells/mL.
Appendix A. Supplementary data
4.3.3. PHA-induced proliferation of human blood mononuclear cells
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.02.023.
The isolated PBMC were distributed into 96-well flat-bottom
plates in 100
concentration of 5
10 and 50
m
L aliquots (2 ꢀ 105cells/well). PHA was added at a
m
g/mL. The compounds were tested at doses of
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After a four-day incubation in a cell culture incubator, the prolif-
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MTT method [31]. The results are given in percentage inhibition as
compared with appropriate DMSO controls.
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