Synthesis and anticancer activity of new 2-aryl-4h-3,1-benzothiazines

Article abstract of DOI:10.1002/ardp.201000228

New compounds of 2-aryl-4H-3,1-benzothiazine set were synthesized and tested for their antiproliferative activity as part of our research in the antitumor field. The title compounds were obtained by the reaction of aryl-modified sulfinylbis((2,4-dihydroxyphenyl)methanethione) with 2-aminobenzyl alcohols. The reaction proceeded through thiobenzanilide intermediates, which were converted to the 4H-3,1-benzothiazine fused ring by an endocyclization process. The structures of compounds were identified from elemental, IR, ¹H-NMR, ¹³C-NMR, and MS spectra analyses. The cytotoxicity in vitro against four human cancer cell lines was determined. The antiproliferative properties of some compounds were more beneficial than cisplatin studied comparatively.

Full text of DOI:10.1002/ardp.201000228

224
Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Full Paper
Synthesis and Anticancer Activity of New
2-Aryl-4H-3,1-benzothiazines
2
1
Andrzej Niewiadomy1,2, Joanna Matysiak , and Monika M. Karpinska
´
1 Institute of Industrial Organic Chemistry, Warszawa, Poland
2 Department of Chemistry, University of Life Sciences, Lublin, Poland
New compounds of 2-aryl-4H-3,1-benzothiazine set were synthesized and tested for their
antiproliferative activity as part of our research in the antitumor field. The title compounds were
obtained by the reaction of aryl-modified sulfinylbis((2,4-dihydroxyphenyl)methanethione) with 2-
aminobenzyl alcohols. The reaction proceeded through thiobenzanilide intermediates, which were
converted to the 4H-3,1-benzothiazine fused ring by an endocyclization process. The structures of
compounds were identified from elemental, IR, 1H-NMR, 13C-NMR, and MS spectra analyses. The
cytotoxicity in vitro against four human cancer cell lines was determined. The antiproliferative
properties of some compounds were more beneficial than cisplatin studied comparatively.
Keywords: Antiproliferative activity / 4H-3,1-Benzothiazines / Sulfinylbis((2,4-dihydroxyphenyl)methanethione)
Received: August 4, 2010; Revised: November 4, 2010; Accepted: November 12, 2010
DOI 10.1002/ardp.201000228
Introduction
result of an electrophilic attack of the benzhydryl carbocation
on the sulfur atom. Tandem nucleophilic addition/epoxy ring-
opening cyclization reactions of 2-(3-phenyloxiranyl)phenyl
isothiocyanates to produce 4-hydroxy(phenyl)methyl-4H-3,1-
benzothiazines were investigated by Otani and others [17].
Recently, a series of 4H-3,1-benzothiazines has been suc-
cessfully synthesized in a three-step sequence starting from
commercially available 2-iodoaniline. The key step consists of
the cyclization via an intramolecular S-conjugate addition
[18]. An efficient path to enantiopure 1,3-benzothiazines has
been elaborated by the reaction of cyclic sulfenamides and
alkylpropiolates or tosylacetylenes catalyzed by cesium
fluoride [19]. Direct thionation of quinazoline or benzoxazine
derivatives has also been described [20, 21]. Other approaches
to 4H-3,1-benzothiazines are confined to a restricted range of
products [22–24].
Several synthetic approaches to 4H-1,3-benzothiazines have
been elaborated but there is very little information about
their biological activity. The most common route involves the
condensation between o-aminobenzyl chlorides and different
thiocarboxamides, benzopyran-2-thiones or thiourea [1–3]. A
related process uses 2-aminobenzyl or 2-(N-acylamino)benzyl
alcohols as starting materials [4, 5]. In the cyclization reac-
tion leading to 4H-3,1-benzothiazin-4-ones 2-thioureido
derivatives of benzoates, benzoic acids, benzamides, and
dithiocarbamates were applied [6, 7]. Another strategy
involves the cycloaddition of thiones to conveniently func-
tionalized ketenimines [8–10].
Moreover, various isothiocyanates have been used as starting
materials to obtain this heterocycle through other cyclization
processes [11–17]. Based on the rearrangement of 2-isothio-
cyano triarylmethanes in the presence of AlCl3 Abaev and
co-workers synthesized 2,4-diaryl-4H-3,1-benzothiazines [12].
An aryl ring is found to migrate to the carbon atom of an
isothiocyano group followed by intramolecular cyclization as a
Biological activities of 4H-3,1-benzothiazines and hetero-
cyclic-fused analogues have been investigated less extensively
[25]. There has been identified 2-amino- and 2-alkylthio-4H-
3,1-benzothiazin-4-ones as selective inhibitors of human cath-
epsin L and human leukocyte elastase [7]. The other examples
include 6-thiaoxanosines, imidazo[1,5-a][1,3]thiazin-7(3H)-
one ribosides with strong antiviral and anticancer properties
[26], 2-arylamino substituted thieno[1,3]thiazin-4-ones, and
analogues of [1,3]thiazino[5,4-b]indole-4-one as inhibitors of
HLE [27, 28].
Correspondence: Joanna Matysiak, Department of Chemistry, University
of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland,
E-mail: joanna.matysiak@up.lublin.pl
Fax: þ48 81 5333549
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Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Anticancer Activity of 4H-3,1-Benzothiazines
225
In a previous paper we reported the synthesis method of
4H-3,1-benzothiazine skeleton by the reaction of sulfinyl-
bis((2,4-dihydroxyphenyl)methanethione) with o-substituted
anilines bearing an activated methylene group, o-aminobenz-
anilides or 2-aminobenzophenones [29]. The compounds
showed antiproliferative properties against the cells of a
human breast cancer T47D line similar to cisplatin. To extend
our research in this field we have obtained and studied a new
series of 2-phenyl-4H-3,1-benzothiazines modified in both
aryl rings by substituents of various electronic and hydro-
phobic effects.
benzylhydroxynium cation formation. The presence of a
positive charge on the oxygen atom enhances polarization
þ
of the –C–OH2 bond and in view of immobilization of the
neighboring substituent, the approach of the nucleophilic
amine residue is facilitated. Closure of the system into a ring
takes place in the transient stage after formation of a new C–S
bond. The system stabilizes by the cleavage of the C–O bond
and release of a water molecule. Cyclization reaction takes
place in one cycle (protonation is very fast and does not affect
the resultant reaction rate and that of the transition stage
formation) and has a dominant effect on the course of syn-
thesis. Two charged groups of the transient compound take
part in it but the kinetic dependences can be adjusted first
of all by the capability of –SH group ionization (Scheme 2).
The purity of the compounds was examined by RP-18 HPLC
chromatography using the methanol/water mobile phase
at pH 4.
Results and discussion
Chemistry
Compounds from the 2-aryl-4H-3,1-benzothiazine group
have been obtained in the reaction of 2-aminobenzyl alcohols
with
a
series of aryl-modified thioacylating reagents
The 1H-NMR spectra of these compounds show, as expected,
a singlet in the range d 4.04–4.20 ppm for the protons of the
–CH2 – moiety of the benzothiazine ring [1]. In the range of
low fields, as a rule, there are registered two signals corre-
sponding to the protons of -OH groups in the resorcinol
moiety. The resonance bands of protons of a hydroxy group
in the position ortho- appear in the range of about 14.5 ppm. It
may suggest formation of the intramolecular hydrogen bond
with a nitrogen atom of the benzothiazine ring.
(Scheme 1) [30, 31]. The reaction of endogenous cyclization
proceeded with the thioamide (thiol-imide) transformation
processes, forming a thiazine ring according to the nucleo-
philic substitution mechanism. Its course is facilitated by the
induction interactions of –X- and –Y-substituents affecting
basicity of –CH2OH group and capability of tautomeric re-
arrangement (–SH group nucleophilicity) respectively. These
conditions are responsible for thiol dissociation and
STB
2, 7
Y
Y
S
R3
R1
OH
NH2
SETB
1, 10
3 8
R2
N
S3MTB
S5MTB
,
X
X
HO
OH
4 9
,
1-12
a: X, Y= -H
b: X= -CH3, Y=-H
SClTB
S3TTB
S6TTB
5 11
,
1: X=Y= -H; R2= -CH2CH3
7: Y= -Cl
c
: Y=-Cl, X= -H
1
2
: X= -CH3
8
9
: Y= -Cl; R = -CH3
6
1
: Y= -Cl; R2= -CH3
3
: X= -CH3, R = -CH3
4: X= -CH3, R2= -CH3
10: Y= -Cl; R2= -CH2CH3
12
2
2
5
: X= -CH3, R = -Cl
11
: Y=R = -Cl
6: X= -CH3, R1= -OH
12: Y= -Cl; R3= -OH
OH
R2
S
S
OH
R1
R1
OH
STB: R1=R2=R3=-H
SETB: R2=-Et, R1=R3=-H
SClTB: R2=-Cl, R1=R3=-H
S3TTB: R1=-OH, R2=R3=-H
S6TTB: R3=-OH, R1=R2=-H
S
O
S3MTB: R1=-Me, R2=R3=-H
S5MTB: R2=-Me, R1=R3=-H
HO
R3 R3
R2
STB: sulfinylbis((2,4-dihydroxyphenyl)methanethione), S3MTB: sulfinylbis((2,4-dihydroxy-3-methylphenyl)methanethione),
S5MTB: sulfinylbis((2,4-dihydroxy-5-methylphenyl)methanethione), SETB: sulfinylbis((5-ethyl-2,4-
dihydroxyphenyl)methanethione), SClTB: sulfinylbis((5-chloro-2,4-dihydroxyphenyl)methanethione), S3TTB: sulfinylbis((2,3,4-
trihydroxyphenyl)methanethione), S6TTB:sulfinylbis((2,4,6-trihydroxyphenyl)methanethione)
Scheme 1. Synthesis scheme of 2-aryl-4H-3,1-benzothiazines.
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A. Niewiadomy et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Y
Y
Y
Y
OH2
OH
OH
NH2
OH
NH
E
N
S
N
SH
OH
R
H
X
X
X
X
OH
S
R
R
OH
HO
OH
OH
H
S
OH2
OH2
H
Y
Y
H
S
OH
S
OH
OH
- H2O
N
N
N
R
X
R
X
X
OH
R
OH
OH
X, Y= -H, -CH3, -Cl
R= -H, -CH3, -C2H5, -Cl, -OH
E: STB, S3MTB, S5MTB, SETB, SClTB, S3TTB, S6TTB
R
R
HO
OH
O
S
OH
S
S
OH
Scheme 2. Mechanism synthesis of 2-aryl-4H-3,1-benzothiazines.
Antiproliferative activity
cells. Cisplatin was used as a reference drug. The results of
screening are summarized in Table 1.
The compounds of the structure presented in Scheme 1 have
been evaluated for their antiproliferative activity against the
human bladder cancer HCV29T cells. The cytotoxic activity
in vitro was expressed as ID50 [mg mLꢀ1], the concentration of
the compound inhibiting the proliferation rate of the
tumour cells by 50% as compared to the control untreated
Activity of compounds is various and depends clearly on
the type of substitution of both aryl rings. Significantly the
highest effect against the HCV29T cells is exhibited by com-
pounds 7–11 (Table 1) with a chlorine atom in the benzothia-
zine ring. Derivatives without the additional substituent or
Table 1. Antiproliferative activity of 2-aryl-4H-3,1-benzothiazines against the cells of human cancer lines: HCV29T, SW707, A549, and
T47D expressed as ID50 [mg mLS1]a).
ID50 [mg mLS1
]
No.
HCV29T
T47D
A 549
SW 707
b)
b)
b)
1
2
3
4
5
6
7
8
9
10
11
12
19.36 ꢁ 2.72
36.07 ꢁ 1.81
32.13 ꢁ 2.10
19.36 ꢁ 2.72
17.21 ꢁ 3.48
38.89 ꢁ 12.20
5.16 ꢁ 1.96
4.14 ꢁ 0.67
3.30 ꢁ 0.12
3.71 ꢁ 0.86
3.75 ꢁ 0.74
14.53 ꢁ 4.63
1.67 ꢁ 0.45
–
–
–
–
–
–
–
–
b)
b)
b)
–
–
b)
b)
b)
–
–
b)
b)
b)
–
–
b)
b)
–
b)
–
b)
b)
b)
–
–
2.21 ꢁ 0.64
4.66 ꢁ 0.45
3.12 ꢁ 0.18
4.17 ꢁ 0.23
3.13 ꢁ 0.29
6.41 ꢁ 2.78
3.59 ꢁ 0.77
3.85 ꢁ 0.39
4.82 ꢁ 1.44
3.57 ꢁ 0.36
12.31 ꢁ 6.28
3.51 ꢁ 1.21
3.39 ꢁ 1.78
3.68 ꢁ 1.35
3.26 ꢁ 0.09
b)
b)
–
b)
–
–
Cisplatin
3.96 ꢁ 0.87
5.63 ꢁ 2.00
4.37 ꢁ 1.44
ID50 [mg mLꢀ1], indicates the compound concentration that inhibits the proliferation rate of tumor cells by 50% as compared to the
control untreated cells. The values are the means ꢁ SD of 9 independent experiments
a)
b)
– experiment was not performed
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Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Anticancer Activity of 4H-3,1-Benzothiazines
227
with the methyl one exhibit weaker activity. The lowest
antiproliferative effect is exhibited by the compound with
the third hydroxyl group in the resorcinol moiety (6). The
derivatives did not reveal cytotoxic activity against the
HCV29T cells below the concentration characteristic of
cisplatin.
interesting. Therefore, further studies including design, syn-
thesis, and analysis of subsequent derivatives as well as
extension of anticancer activity investigations will be carried
out.
Experimental
The selected compounds of the highest activity against
HCV29T were also tested against A549 (human non-small cell
lung carcinoma), T47D (human breast cancer), SW707
(human rectal adenocarcinoma) cells (Table 1). An antiproli-
ferative effect of these compounds is on the level of cisplatin.
Only compound 7 shows slightly poorer activity against the
SW707. Additionally, its activity is various contrary to the
others that inhibit all cell lines on the same level (about
4 mg mLꢀ1). At the same time, compounds 9 and 11, as the
most active derivatives from the set of tested 2-aryl-4H-3,1-
benzothiazines, meet the cytotoxic activity criterion against
all human cancer cell lines for potentially new antiprolifer-
ative drugs (ID50 ꢂ 4 mg mLꢀ1) [32]. The other compounds
inhibit cells growth of only some cell lines below this
concentration.
Chemistry
The melting point (m.p.) was determined using a BUCHI B-540
(Switzerland) melting point apparatus. The elemental analysis
was performed in order to determine C, H, and N contents
(Perkin-Elmer 2400). The analyses were within ꢁ0.4% of the
theoretical values. The vibrational spectra were recorded with
a Perkin-Elmer FT-IR 1725X spectrophotometer (in potassium
bromide). The spectra were made in the range of 600–
4000 cmꢀ1. NMR spectra were recorded in DMSO-d6 or CDCl3
using a Bruker DRX 500 instrument. Chemical shifts (d, ppm)
were given in relation to tetramethylsilane (TMS). The MS spectra
(EI, 70 eV) were recorded using the apparatus AMD-604.
The chromatographic measurements were performed by
the liquid chromatograph Knauer with a dual pump, a 20-mL
simple injection valve and a UV-VIS detector 330 nm. The
ACE AQ (3 mm, 75 ꢃ 2.1 mm) column was used as the stationary
phase. The mobile phase consisted of different contents of
methanol and acetate buffer (pH 4, 20 nM) as the aqueous phase.
The flow rate was 0.35 mL minꢀ1 at room temperature. The
retention time of an unretained solute (to) was determined by
the injection of a small amount of acetone dissolved in water.
The logk values for 80% of methanol (v/v) in the mobile phase are
presented.
Analyzing the structure of compounds and their activity it
was found that the presence of a methyl substituent in
position 6 of the benzothiazine ring reduces antiproliferative
activity. Antiproliferative properties of 2-(2,4-dihydroxy-
phenyl)-4H-3,1-benzothiazine (unsubstituted additionally)
against the T47D cells were reported previously as
31.1 mg mLꢀ1 [29]. A chlorine atom in position 8 has a
positive effect on proliferation inhibition. It also exhibits
evident influence on the activity of the resorcinol moiety
substitution. The presence of a chlorine atom in position 5 of
the resorcinol ring has a beneficial effect. Similar action is
observed for methyl and ethyl substituents in this position
but their influence on the increase of the activity is somehow
smaller. The CH3- group in the meta-position also improves
antiproliferative effect but to a bit smaller extent than in
position 5 (compounds 3 and 4 or 8 and 9). This analysis shows
that the hydrophobic substituent (p > 0) in the resorcinol
ring increases the antiproliferative activity (electronic effects
of Cl- and alkyl group are opposite). The derivatives without
the additional substituent or with the third hydroxyl group
(p < 0) (2, 6, 12) show weaker activity than the analogues
without or with other substituents. It confirms the earlier
finding that the presence of a chlorine atom or methyl (ethyl,
isopropyl) substituents in position 5 of resorcinol moiety of
resorcylazoles improves their anticancer properties [33–35].
The presented results indicate high level of antiprolifera-
tive activity of the studied compounds of 2-aryl-4H-3,1-benzo-
thiazine set. Taking into account the low toxicity of the
compounds with 2,4-dihydroxyphenyl substituent and the
low exploration of 3,1-benzothiazine in the biological
activity area this group of compounds seems to be very
Preparation of compounds
2-(5-Ethyl-2,4-dihydroxyphenyl)-4H-3,1-benzothiazine 1
A mixture of 2-aminobenzyl alcohol (Merck) (0.002 mol) and
SETB [31] (0.002 mol) in methanol (50 mL) was refluxed for
3 h. The hot reaction mixture was filtered and the filtrate was
concentrated. The precipitated product was dissolved in 5%
NaOH (5 mL). The mixture was filtered and 5% HCl (5 mL) was
added to the filtrate. The formed solid was filtered off and
recrystallized from methanol (5 mL).
Yield: 78%, m.p.: 147–1518C, HPLC: logk ¼ 0.838; IR (KBr, cmꢀ1):
–
3389 (HO), 2960 (CH ), 2930 (CH ), 2880 (CH ), 1626 (C N), 1507
–
–
(C C), 1470, 1383, 1320, 1273, 1244, 1180 (C–OH), 1040, 1005, 895,
3
2
3
–
1
0
846, 794; H-NMR (500 MHz, DMSO-d6, d): 14.39 (s, 1H, C2 -OHꢄꢄꢄN),
0
0
13.63 (s, 1H, C4 -OH), 7.44 (s, 1H, C6 -H), 7.21 (m, 1H, CBT-H), 7.17 (m,
0
1H, CBT-H), 7.09 (m, 2H, CBT-H), 6.08 (s, 1H, C3 -H), 4.04 (s, 2H, CH2),
2.29 (q, J ¼ 7.5 Hz, 2H, CH2CH3), 1.16 (t, J ¼ 7.5 Hz, 3H, CH3); MS
(EI, m/z, B): 285 (Mþ, 7), 284 (33), 268 (10), 250 (11), 226 (4), 120 (100),
91 (9), 77 (8), 65 (4), 40 (11), 120 (16); anal. calcd.
for C16H15NO2S (285.36): C, 67.34; H, 5.30; N, 4.91. Found: C,
67.56; H, 5.28; N, 4.93.
2-(2,4-Dihydroxyphenyl)-8-methyl-4H-3,1-benzothiazine 2
A
mixture of 2-amino-3-methylbenzyl alcohol (Aldrich)
(0.002 mol) and STB [31] (0.0055 mol) in methanol (25 mL) was
refluxed for 3 h. The hot reaction mixture was filtered. The
filtrate was concentrated. The precipitated product was dissolved
in 5% NaOH (5 mL). The mixture was filtered and 5% HCl (5 mL)
was added to the filtrate. The formed solid was filtered off and
recrystallized from methanol (7.5 mL).
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A. Niewiadomy et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Yield: 73%, m.p.: 191–1948C, HPLC: logk ¼ 0.075; IR (KBr, cmꢀ1):
1214, 1194 (C–OH), 1120, 1078, 965, 958, 879, 774; 1H-NMR
2-(5-Chloro-2,4-dihydroxyphenyl)-8-methyl-
4H-3,1-benzothiazine 5
mixture of 2-amino-3-methylbenzyl alcohol (Aldrich)
(0.0035 mol) and SClTB [31] (0.0025 mol) in methanol (20 mL)
was refluxed for 3 h. The hot reaction mixture was filtered. The
filtrate was concentrated, the formed solid was filtered off and
recrystallized from methanol (10 mL).
–
–
–
3425 (HO), 2989 (CH ), 1607 (C N), 1531 (C C), 1483, 1337, 1278,
–
3
A
0
(500 MHz, DMSO-d6, d): 14.36 (s, 1H, C2 -OHꢄꢄꢄN), 11.36 (s, 1H,
0
0
C4 -OH), 7.74 (d, J ¼ 8.7 Hz, 1H, C6 -H), 7.28 (m, 1H, CBT-H), 7.20
0
(m, 1H, CBT-H), 6.98 (m, 1H, CBT-H), 6.56 (d, J ¼ 2.3 Hz, C3 -H), 6.43
0
(dd, J ¼ 8.7 and 2.3 Hz, C5 -H), 4.17 (s, 2H, CH2), 2.34 (s, 3H, CH3);
13C-NMR: 163.15, 161.10, 158.22, 141.81, 130.12, 129.78, 129.17,
124.77, 123.46, 120.33, 114.78, 103.89, 103.67, 27.63 (CH2), 18.27
(CH3); MS (EI, m/z, B): 271 (Mþ, 100), 242 (5), 238 (57), 223 (13), 210
(26), 167 (14), 153 (5), 138 (18), 136 (8), 120 (16), 91 (27), 77 (19), 65
(14), 51 (14), 45 (10), 44 (25), 40 (21); anal. calcd.
for C15H13NO2S (271.33): C, 66.40; H, 4.83; N, 5.16. Found: 66.64;
H, 4.81; N, 5.18.
Yield: 81%, m.p.: 199–2028C; HPLC: logk ¼ 0.322; IR (KBr,
cmꢀ1): 3415 (HO), 2725 (CH), 1610 (C N), 1557 (C C), 1514
–
–
–
–
–
(C C), 1472, 1416, 1381, 1326, 1250, 1222, 1188 (C-OH), 1114,
–
1095 (C–Cl), 1053, 1030, 969, 916, 876, 846; 1H-NMR (500 MHz,
0
0
DMSO-d6, d): 14.58 (s, 1H, C2 -OHꢄꢄꢄN), 11.36 (s, 1H, C4 -OH), 7.78 (s,
0
1H, C6 -H), 7.29 (d, 1H, J ¼ 7.1 Hz, CBT-H), 7.21 (t, J ¼ 7.2 Hz,
0
1H, CBT-H), 7.17 (m, J ¼ 7.1 Hz, 1H, CBT-H), 6.65 (s, 1H, C3 -H),
4.17 (s, 2H, CH2), 2.34 (s, 3H, CH3); 13C-NMR: 163.78, 160.89,
158.32, 140.31, 132.12, 130.51, 129.89, 127.77, 125.71, 122.73,
120.82, 112.71, 104.51, 27.52 (CH2), 18.54 (CH3); MS (EI, m/z, B):
305 (Mþ, 100), 289 (37), 272 (24), 254 (11), 244 (12), 237 (12), 212
(9), 171 (15), 135 (12), 119 (18), 91 (24), 77 (11), 65 (9), 51 (11), 39 (9);
anal. calcd. for C15H12ClNO2S (305.78): C, 58.92; H, 3.96; N, 4.58.
Found: C, 59.17; H, 3.95; N, 4.56.
2-(2,4-Dihydroxy-3-methylphenyl)-8-methyl-
4H-3,1-benzothiazine 3
A
mixture of 2-amino-3-methylbenzyl alcohol (Aldrich)
(0.002 mol) and S3MTB [31] (0.002 mol) in methanol (10 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated and the precipitated product
was dissolved in 5% NaOH (5 mL). The mixture was filtered
and 5% HCl (5 mL) was added to the filtrate. The formed solid
was filtered off and recrystallized from methanol (5 mL).
Yield: 78%, m.p.: 150–1538C, HPLC: logk ¼ 0.208; IR (KBr, cmꢀ1):
8-Methyl-2-(2,3,4-trihydroxyphenyl)-4H-3,1-benzothiazine
6
A
mixture of 2-amino-3-methylbenzyl alcohol (Aldrich)
–
–
–
3426 (HO), 2932 (CH ), 2895 (CH ), 1622 (C N), 1560 (C C), 1438,
–
2
3
(0.002 mol) and S3TTB [31] (0.002 mol) in methanol (15 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated. The precipitated product was
dissolved in 5% NaOH (5 mL). The mixture was filtered and 5%
HCl (5 mL) was added to the filtrate. The formed solid was filtered
off and recrystallized from methanol (5 mL).
1411, 1367, 1344, 1262, 1243, 1168 (C–OH), 1091, 1045, 984, 846,
0
1
782; H-NMR (500 MHz, DMSO-d6, d): 12.29 (s, 1H, C2 -OH), 10.32
0
0
(s, 1H, C4 -OH), 7.74 (d, 1H, J ¼ 9.1 Hz, C6 -H), 7.28 (m, 1H, CBT-H),
7.19 (m, 1H, CBT-H), 6.98 (m, 1H, CBT-H), 6.52 (d, 1H, J ¼ 9.1 Hz,
C5 -H), 4.19 (s, 2H, CH2), 2.38 (s, 3H, CH3), 2.05 (s, 3H, CH3); 13C-NMR:
0
162.38, 161.21, 159.02, 140.09, 131.52, 129.78, 125.87, 125.17,
113.71, 110.88, 107.61, 106.87, 102.51, 27.09 (CH2), 18.17 (CH3),
8.17 (CH3); MS (EI, m/z, B): 285 (Mþ, 100), 271 (11), 252 (47), 238 (8),
206 (16), 198 (12), 156 (16), 151 (33), 107 (5), 91 (8), 65 (71), 47 (8);
anal. calcd. for C16H15NO2S (285.36): C, 67.34; H, 5.30; N, 4.91.
Found: C, 67.48; H, 5.28; N, 4.92.
Yield: 62%, m.p.: 230–2328C, HPLC: logk ¼ –0.219; IR (KBr, cmꢀ1):
–
–
–
3435 (HO), 2960 (CH ), 2830 (CH ), 1618 (C N), 1560 (C C), 1472,
–
3
2
1382, 1340, 1320, 1270, 1240, 1160 (C-OH), 1080 (C–Cl), 1053,
1050, 920, 916, 880, 840, 776; 1H-NMR (500 MHz, DMSO-d6,
d): OH group bands are not visible in the background on the
0
base line, 7.45 (d, J ¼ 8.7 Hz, 1H, C6 -H), 7.28 (m, 1H, CBT-H), 7.17
0
(m, 2H, CBT-H), 6.47 (d, 1H, J ¼ 8.7 Hz, C5 -H), 4.19 (s, 2H, CH2),
2-(2,4-Dihydroxy-5-methylphenyl)-8-methyl-
4H-3,1-benzothiazine 4
2.38 (s, 3H, CH3); MS (EI, m/z, B): 287 (Mþ, 50), 271 (39), 254 (11),
226 (15), 200 (65), 168 (100), 152 (76), 126 (18), 120 (42), 112 (17),
106 (9), 91 (12), 68 (8), 51 (10), 33 (14); anal. calcd.
for C15H13NO3S (287.33): C, 62.70; H, 4.56; N, 4.87. Found: C,
62.95; H, 4.54; N, 4.89.
A
mixture of 2-amino-3-methylbenzyl alcohol (Aldrich)
(0.002 mol) and S5MTB [31] (0.002 mol) in methanol (10 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated and the precipitated product
was dissolved in 5% NaOH (5 mL). The mixture was filtered
and 5% HCl (5 mL) was added to the filtrate. The formed solid
was filtered off and recrystallized from methanol (7.5 mL).
Yield: 83%, m.p.: 152–1558C, HPLC: logk ¼ 0.492; IR (KBr,
6-Chloro-2-(2,4-dihydroxyphenyl)-4H-3,1-benzothiazine 7
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
(0.002 mol) and STB [31] (0.0015 mol) in methanol (15 mL) was
refluxed for 3 h. The hot reaction mixture was filtered. The
filtrate was concentrated and the formed solid was recrystallized
from methanol (5 mL).
cmꢀ1): 3389 (HO), 2924 (CH ), 2853 (CH ), 1624 (C N), 1578
–
–
2
2
–
–
–
–
(C C), 1521 (C C), 1478, 1410, 1420, 1383, 1352, 1265, 1222
(C–OH), 1141, 1080, 1037, 1009, 976, 843, 775; 1H-NMR
0
(500 MHz, DMSO-d6, d): 14.36 (s, 1H, C2 -OHꢄꢄꢄN), 11.76
Yield: 69%, m.p.: 124–1268C, HPLC: logk ¼ 0.593; IR (KBr,
1337, 1285, 1188 (C–OH), 1081 (C–Cl), 953, 797, 746; 1H-NMR
cmꢀ1): 3403 (HO), 2923 (CH ), 1619 (C N), 1539 (C C), 1478,
–
–
2
–
–
0
(s, 1H, C4 -OH), 7.59 (s, 1H, C6 -H), 7.28 (m, 1H, CBT-H), 7.18
(m, 1H, CBT-H), 7.14 (m, 1H, CBT-H), 6.44 (s, 1H, C3 -H), 4.18
0
0
(s, 2H, CH2), 2.33 (s, 3H, CH3), 2.10 (s, 3H, CH3); 13C-NMR:
164.13, 160.88, 160.22, 140.19, 131.42, 130.51, 129.82, 126.67,
125.21, 120.33, 115.88, 111.07, 102.21, 27.00 (CH2), 18.14 (CH3),
15.17 (CH3); MS (EI, m/z, B): 285 (Mþ, 100), 269 (23), 252 (37), 237
(7), 198 (19), 167 (13), 120 (5), 91 (4), 77 (13); anal. calcd.
for C16H15NO2S (285.36): C, 67.34; H, 5.30; N, 4.91. Found: C,
67.09; H, 5.32; N, 4.89.
(500 MHz, DMSO-d6, d): 11.82 (s, 1H, C2 -OHꢄꢄꢄN), 10.55
0
0
0
(s, 1H, C4 -OH), 7.69 (d, J ¼ 8.7 Hz, 1H, C6 -H), 7.56 (m, 1H, CBT-H),
0
7.45 (m, 2H, CBT-H), 6.40 (d, J ¼ 2.3 Hz, 1H, C3 -H), 6.35 (dd,
J ¼ 8.7 and 2.3 Hz, 1H, C5 -H), 4.19 (s, 2H, CH2); 13C-NMR: 166.01,
0
162.48, 162.32, 140.81, 131.42, 131.31, 128.82, 127.68, 126.91,
122.88, 111.68, 108.47, 103.31, 26.82 (CH2); MS (EI, m/z, B): 291
(Mþ, 100), 275 (9), 260 (6), 223 (61), 167 (11), 155 (11), 137 (8), 121
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
Anticancer Activity of 4H-3,1-Benzothiazines
229
0
(7), 69 (12), 63 (5), 45 (10); anal. calcd. for C14H10ClNO2S (291.75):
C, 57.63; H, 3.45; N, 4.80. Found: C, 57.40; H, 3.46; N, 4.78.
1H, CBT(5)-H), 7.29 (d, J ¼ 8.2 Hz, 1H, CBT(8)-H), 6.45 (s, 1H, C3 -H),
4.19 (s, 2H, CH2), 2.50 (q, J ¼ 7.4 Hz, 2H, CH2CH3); 1.13
(t, J ¼ 7.4 Hz, 3H, CH2CH3); 13C-NMR: 165.97, 161.23, 159.95,
140.09, 130.90, 129.34, 123.36, 127.20, 126.16, 122.47 (2C),
110.68, 102.56, 26.48 (CH2), 22.09 (CH2), 14.18 (CH3); MS (EI,
m/z, B): 319 (Mþ, 62), 306 (38), 304 (100), 290 (4), 271 (5), 251
(5), 155 (4), 135 (4), 121 (4), 89 (5), 77 (7), 69 (13), 45 (6), 39 (5); anal.
calcd. for C16H14ClNO2S (319.81): C, 60.09; H, 4.41; N, 4.38. Found:
C, 59.83; H, 4.43; N, 3.37.
6-Chloro-2-(2,4-dihydroxy-3-methylphenyl)-
4H-3,1-benzothiazine 8
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
(0.0015 mol) and S3MTB [31] (0.0015 mol) in methanol (15 mL)
was refluxed for 3 h. The hot reaction mixture was filtered. The
filtrate was concentrated and the formed solid was recrystallized
from methanol-water (3:1) (5 mL).
6-Chloro-2-(5-chloro-2,4-dihydroxyphenyl)-
4H-3,1-benzothiazine 11
Yield: 83%, m.p.: 195–1978C, HPLC: logk ¼ 0.315; IR (KBr, cmꢀ1):
–
3279 (HO), 2930 (CH), 2920 (CH2), 1609 (C N), 1542
–
–
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
(C C), 1499, 1455, 1365, 1320, 1240, 1205 (C–OH), 1168, 1081
–
(C–Cl), 936, 862, 843, 796, 758; 1H-NMR (500 MHz, DMSO-d6,
(0.0015 mol) and SClTB [31] (0.001 mol) in methanol (15 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated and the formed solid was crystal-
lized from methanol (5 mL) and recrystallized from methanol/
water (3:1) (5 mL).
0
0
d): 14.55 (s, 1H, C2 -OHꢄꢄꢄN), 10.31 (s, 1H, C4 -OH), 7.59 (d, 1H,
0
J ¼ 8.8 Hz, C6 -H), 7.43 (d, 1H, C7BT-H), 7.41 (d, 1H, C5BT-H), 7.33
0
(m, 1H, C8BT-H), 6.51 (d, 1H, J ¼ 8.8 Hz, C5 -H), 4.17 (s, 2H, CH2),
2.03 (s, 3H, CH3); 13C-NMR: 166.23, 161.09, 160.32, 140.94, 131.28,
128.81, 127.92, 127.63, 126.90, 122.98, 111.44, 110.97, 107.50,
26.87 (CH2), 8.54 (CH3); MS (EI, m/z, B): 305 (Mþ, 100), 289 (8), 272
(14), 237 (34), 209 (4), 153 (6), 77 (5); anal. calcd.
for C15H12ClNO2S (305.78): C, 58.92; H, 3.96; N, 4.58; Found: C,
59.15; H, 3.97; N, 4.56.
Yield: 64%, m.p.: 213–2168C, HPLC: logk ¼ 0.393; IR (KBr,
cmꢀ1): 3492 (HO), 3084 (CAR-H), 2930 (CH ), 1615 (C N), 1528
–
–
2
–
(C C), 1491, 1480, 1428, 1376, 1326, 1280, 1245, 1192 (C-OH),
–
1080 (C-Cl), 953, 898, 836; 1H-NMR (500 MHz, DMSO-d6, d): 14.49
0
0
0
(s, 1H, C2 -OHꢄꢄꢄN), 11.27 (s, 1H, C4 -OH), 7.78 (s, 1H, C6 -H), 7.47–
7.33 (m, 3H, CBT-H), 6.65 (s, 1H, C3 -H), 4.18 (s, 2H, CH2); 13C-NMR:
0
164.93, 160.68, 158.52, 140.71, 131.82, 130.11, 128.89, 127.67,
127.11, 122.93, 112.58, 111.63, 104.51, 26.81 (CH2); MS (EI, m/z, B):
327 (68), 325 (Mþ, 100), 309 (6), 290 (22), 257 (71), 201 (6), 155 (12),
121 (7), 111 (6), 89 (5), 77 (10), 69 (11), 51 (7); anal. calcd.
for C14H9Cl2NO2S (326.20): C, 51.55; H, 2.78; N, 4.29. Found: C,
51.72; H, 2.77; N, 4.31.
6-Chloro-2-(2,4-dihydroxy-5-methylphenyl)-
4H-3,1-benzothiazine 9
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
(0.002 mol) and S5MTB [31] (0.0015 mol) in methanol (15 mL)
was refluxed for 3 h. The hot reaction mixture was filtered. The
filtrate was concentrated and the formed solid was recrystallized
from methanol/water (4:1) (7.5 mL).
2-(2,4,6-Trihydroxyphenyl)-6-chloro-4H-3,1-benzothiazine
12
Yield: 83%, m.p.: 229–2328C, HPLC: logk ¼ 0.344; IR (KBr, cmꢀ1):
–
3435 (HO), 3066 (CAR-H), 2960 (CH ), 2920 (CH ), 1612 (C N), 1585
–
–
(C C), 1548 (C C), 1520, 1478, 1440, 1420, 1338, 1285, 1250,
3
2
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
–
–
–
(0.002 mol) and S6TTB [31] (0.0015 mol) in methanol (20 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated and the formed solid was recrystal-
lized from methanol/water (3:1) (7.5 mL).
1220, 1200 (C-OH), 1152, 1080 (C-Cl), 1050, 1018, 960, 900, 860,
829, 750, 730; 1H-NMR (500 MHz, DMSO-d6, d): OH group
bands are not visible in the background on the base line,
0
7.56 (s, 1H, C6 -H), 7.43 (m, 1H, CBT(7)-H), 7.41 (d, J ¼ 2.4 Hz,
Yield: 74%, m.p.: 151–1548C, HPLC: logk ¼ –0.560; IR (KBr, cmꢀ1):
0
1H, CBT(5)-H), 7.31 (d, J ¼ 8.2 Hz 1H, CBT(8)-H), 6.50 (s, 1H, C3 -H),
–
–
–
3395 (HO), 3144, 1620 (C N), 1540 (C C), 1484, 1415, 1380, 1300,
–
4.20 (s, 2H, CH2), 2.09 (s, 3H, CH3); 13C-NMR: 165.97, 161.55,
159.95, 140.05, 130.92, 130.81, 128.72, 127.20, 126.14, 122.46,
116.32, 110.07, 102.41, 26.45 (CH2), 15.22 (CH3); MS (EI, m/z, B):
305 (Mþ, 100), 272 (14), 237 (23), 209 (3), 69 (5), 45 (5), 39 (5); anal.
calcd. for C15H12ClNO2S (305.78): C, 58.92; H, 3.96; N, 4.58. Found:
C, 59.13; H, 3.97; N, 4.56.
1275, 1222, 1198 (C-OH), 1064, 1018, 958, 900, 823, 746; 1H-NMR
0
(500 MHz, DMSO-d6, d): 12.88 (s, 1H, C2 -OH), 11.49 (s, 1H, C6 -OH),
0
0
10.02 (s, 1H, C4 -OH), 7.44 (m, 1H, CBT-H), 7.38 (m, 1H, CBT-H), 7.20
(m, 1H, CBT-H), 5.82 (m, 2H, C3 ,5 -H), 4.09 (s, 2H, CH2); 13C-NMR:
162.53, 160.88, 158.12, 138.71, 130.82, 128.11, 126.81, 125.22,
122.81, 102.23, 94.78, 94.60, 94.00, 26.73 (CH2); MS (EI, m/z, B): 307
(Mþ, 54), 291 (7), 265 (6), 237 (10), 223 (7), 200 (12), 159 (100), 140
(73), 130 (28), 111 (9), 103 (8), 77 (11), 69 (10), 36 (68); anal. calcd.
for C14H10ClNO3S (307.75): C, 54.64; H, 3.28; N, 4.55. Found: C,
54.70; H, 3.27; N, 4.53.
0
0
6-Chloro-2-(5-ethyl-2,4-dihydroxyphenyl)-
4H-3,1-benzothiazine 10
A
mixture of 2-amino-5-chlorobenzyl alcohol (Alfa Aesar)
(0.0015 mol) and SETB [31] (0.001 mol) in methanol (15 mL)
was refluxed for 3 h. The hot reaction mixture was filtered.
The filtrate was concentrated and the formed solid was recrystal-
lized from methanol/water (3:1) (5 mL).
Antiproliferative assay in vitro
There were applied the following human cell lines applied estab-
lished in vitro: T47D (breast cancer), SW707 (rectal adenocarci-
noma), A549 (non-small cell lung carcinoma) from the American
Type Culture Collection (Rockville, Maryland, USA), and HCV29T
(bladder cancer) from the Fibiger Institute, Copenhagen,
Denmark. Twenty-four hours before the addition of the tested
agents, the cells were plated in 96-well plates (Sarstedt, USA) at a
density of 104 cells/well. All cell lines were maintained in the
Yield: 71%, m.p.: 129–1328C, HPLC: logk ¼ 0.499; IR (KBr, cmꢀ1):
–
–
–
3418 (HO), 2962 (CH ), 2921 (CH ), 1613 (C N), 1551 (C C),
–
3
2
–
1521 (C C), 1478, 1412, 1324, 1279, 1215, 1192 (C-OH), 1138,
–
1084 (C-Cl), 1056, 949, 898, 847, 821, 746; 1H-NMR (500 MHz,
0
0
DMSO-d6, d): 13.85 (s, 1H, C2 -OHꢄꢄꢄN), 10.55 (s, 1H, C4 -OH),
0
7.54 (s, 1H, C6 -H), 7.43 (m, 1H, CBT(7)-H), 7.40 (d, J ¼ 2.4 Hz,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

230
A. Niewiadomy et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 224–230
opti-MEM medium supplement with 2 mM glutamine (Gibco,
Warsaw, Poland), streptomycin (50 mg mLꢀ1), penicillin
(50 U mLꢀ1) (Polfa, Tarchomin, Poland) and 5% fetal calf serum
(Gibco, Grand Island, USA). The cells were incubated at 378C in
the humid atmosphere saturated with 5% CO2. The solutions of
compounds (1 mg mLꢀ1) were prepared ex tempore by dissolving
the substance in 100 mL of DMSO completed with 900 mL of
tissue culture medium. Afterwards, the compounds were diluted
in the culture medium to reach the final concentrations ranging
from 0.1 to 100 mg mLꢀ1. The solvent (DMSO) in the highest
concentration used in the test did not reveal any cytotoxic
activity. Cisplatin was applied as a test referential agent. The
cytotoxicity assay was performed after 72 h exposure of the
cultured cells with the concentration ranging from 0.1 to
100 mg mLꢀ1 of the tested agents. The SRB test measuring the
cell proliferation inhibition in the in-vitro culture was
applied [36]. The cells attached to the plastic were fixed with
cold 50% TCA (trichloroacetic acid, Aldrich-Chemie, Germany)
added on the top of the culture medium in each well. The plates
were incubated at 48C for 1 h and then washed five times with
tap water. The background optical density was measured in
the wells filled with the culture medium without the cells.
The cellular material fixed with TCA was stained with 0.4%
sulforhodamine B (SRB, Sigma, Germany) dissolved in 1% acetic
acid (POCh, Gliwice, Poland) for 30 min. The unbound dye was
removed by rinsing (four times) with 1% acetic acid, and the
protein-bound dye was extracted with 10 mM unbuffered Tris
base (tris (hydroxymethyl) aminomethane, POCh, Gliwice,
Poland) for determination of optical density (at 540 nm) in a
computer-interfaced, 96-well microtiter plate reader Uniskan II
(Labsystems, Helsinki, Finland). The compounds were tested in
triplicates per experiment. The experiments were repeated at
least 3 times.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com