Synthesis and antibacterial activity of novel fused 1,3-thiazoles and 1,3-thiazines incorporating a 2,4-dihydroxyphenyl residue

Article abstract of DOI:10.1002/ardp.201100251

In an attempt to find a new class of antimicrobial agents, a series of benzothiazoles, 1,3-thiazolo[5,4-b]pyridines, 4H-3,1-benzothiazines, naphtho[2,3-d][1,3]thiazole-4,9-diones and other related compounds containing a 2,4-dihydroxyphenyl moiety were prepared. They were obtained via the reaction of aryl-modified sulfinyl[bis(2,4-dihydroxyphenylmethanethione)]s with appropriate commercial chemical reagents in the endocyclization processes. The MIC values of the compounds towards eight reference bacterial strains were assessed by the two-fold serial micro-dilution broth method. They exhibited inhibitory effects against the Gram-positive strains tested opposite to Gram-negative ones. Some compounds were more effective than the reference drug. 4-(6-Chloro-4H-3,1- benzothiazin-2-yl)-6-methylbenzene-1,3-diol (5b) due to its very good activity (MIC from 1.56 to 3.13 μg/mL) and low cytotoxicity (IC₅₀ > 50 μg/mL) may be regarded as a promising precursor for the development of novel antibacterial agents. A series of benzothiazoles, 1,3-thiazolo[5,4-b]pyridines, 4H-3,1-benzothiazines, naphtho[2,3-d][1,3]thiazole-4,9-diones and other related compounds containing a 2,4-dihydroxyphenyl moiety were prepared and their MIC values towards eight reference bacterial strains were determined. 4-(6-Chloro-4H-3,1-benzothiazin-2-yl)-6-methylbenzene-1,3-diol may be regarded as a promising precursor for the development of novel antibacterial agents.

Full text of DOI:10.1002/ardp.201100251

302
Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Full Paper
Synthesis and Antibacterial Activity of Novel Fused
1,3-Thiazoles and 1,3-Thiazines Incorporating
a 2,4-Dihydroxyphenyl Residue
1
2
2
3
1
´
Joanna Matysiak , Renata Los , Anna Malm , Monika M. Karpinska , Urszula Głaszcz ,
Barbara Rajtar4, Małgorzata Polz-Dacewicz4, Marta Trojanowska-Wesołowska5, and
Andrzej Niewiadomy1,3
1 Department of Chemistry, University of Life Sciences, Lublin, Poland
2 Department of Pharmaceutical Microbiology, Medical University of Lublin, Lublin, Poland
3 Institute of Industrial Organic Chemistry, Warszawa, Poland
4 Department of Virology, Medical University of Lublin, Lublin, Poland
5 Department of Biotechnology, University of Life Sciences, Lublin, Poland
In an attempt to find a new class of antimicrobial agents, a series of benzothiazoles, 1,3-thiazolo[5,4-
b]pyridines, 4H-3,1-benzothiazines, naphtho[2,3-d][1,3]thiazole-4,9-diones and other related compounds
containing a 2,4-dihydroxyphenyl moiety were prepared. They were obtained via the reaction of aryl-
modified sulfinyl[bis(2,4-dihydroxyphenylmethanethione)]s with appropriate commercial chemical
reagents in the endocyclization processes. The MIC values of the compounds towards eight reference
bacterial strains were assessed by the two-fold serial micro-dilution broth method. They exhibited
inhibitory effects against the Gram-positive strains tested opposite to Gram-negative ones. Some
compounds were more effective than the reference drug. 4-(6-Chloro-4H-3,1-benzothiazin-2-yl)-6-
methylbenzene-1,3-diol (5b) due to its very good activity (MIC from 1.56 to 3.13 mg/mL) and low
cytotoxicity (IC50 > 50 mg/mL) may be regarded as a promising precursor for the development of
novel antibacterial agents.
Keywords: Antibacterial activity / Cytotoxicity / Fused thiazines / Fused thiazoles / Synthesis
Received: July 14, 2011; Revised: September 8, 2011; Accepted: September 20, 2011
DOI 10.1002/ardp.201100251
Introduction
chlobenthiazone and TCMTB have been widely used in agricul-
ture [12]. Recently a promising selective fungal N-myristoyl-
transferase inhibitor from the benzothiazole group with a
strong in vivo antifungal effect has been described [13–15].
2-Methoxy-2,6-difluorobenzamide derivatives of thiazo-
lo[4,5]pyridine are potent antistaphylococcal compounds with
suboptimal drug-like properties [16]. They act as inhibitors of
the bacterial cell division protein FtsZ. The others are described
as useful antimicrobial agents effective against a variety of
human and veterinary pathogens including, among others,
Gram-positive and Gram-negative aerobic and anaerobic bac-
teria as well as mycobacteria [17]. Naphthothiazole derivatives
are also known as antibacterial agents [18].
Fused heterocycles incorporating 1,3-thiazole and 1,3-thiazine
rings have attracted a lot of attention from medicinal and
agricultural chemists due to their broad-spectrum antifungal
and antibacterial activities as well as action mechanism [1–4].
For example, benzothiazole derivatives are commonly known
as antimycotic agents [5–9]. 6-Amino-2-pentylthiobenzothiazole
(APB), is a compound characterized by in vitro and in vivo anti-
fungal activities. An especially strong effect was observed in the
combination with other commonly applied antimycotics
[10, 11]. Some of these types of compounds like bentaluron,
4-(4H-3,1-Benzothiazin-2-yl)benzene-l,3-diols obtained in
our laboratory show a strong antifungal effect against
various strains of moulds, yeasts and dermatophytes on
the level of nystatin studied comparatively [19]. Similar prop-
erties for some benzothiazole derivatives have been found
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: þ4881 5333549
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Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Synthesis and Antibacterial Activity of Fused Heterocycles
303
–
[20]. Extending the research in this area, we designed and
obtained new fused heterocycles incorporating a 1,3-thiazole
or 1,3-thiazine ring with a 2,4-dihydroxyphenyl residue. To
evaluate the potential antibacterial efficacy of the com-
pounds we determined their in vitro activity against the panel
of reference bacterial strains. Additionally, the most active
compounds were tested for their cytotoxic potential. The
structure-activity relationship has been discussed.
O–H and C N respectively. The mass spectra (EI) of the com-
–
pounds gave molecular ion peaks, however, with different
intensities.
Antibacterial activity
The newly synthesized (1–4, 6–9) and previously described (5)
[23] compounds (Table 1) were screened for their antibacterial
properties against the panel of the reference Gram-positive and
Gram-negative bacteria. First, the power of activity was assessed
by the determination of the minimal inhibitory concentration
(MIC) values towards both groups of strains. The obtained data
are presented in Table 2. Generally, the tested compounds exert
different activities against the Gram-positive bacteria (MIC
ranged 0.78 – 200 mg/mL). They depend clearly on the type
of fused azole (azine) ring and the presence of the additional
substituents in the benzenediol moiety. In contrast, the growth
of Gram-negative bacteria has not been inhibited even at the
highest concentration of compounds (MIC >200 mg/mL). The
selective antibacterial activity of the compounds is due to
the difference in the structure of bacterial cell wall, and
Gram-negative bacteria are generally more resistant to anti-
bacterial agents than the Gram-positive ones [24].
Results and discussion
Chemistry
The structures of the compounds under consideration are
presented in Table 1. They were obtained via the reaction of
sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB) or its
aryl-modified analogs: sulfinylbis[(2,4-dihydroxy-3-methyl-
phenyl)methanethione] (S3MTB), sulfinylbis[(2,4-dihydroxy-
5-methylphenyl)methanethione] (S5MTB), sulfinylbis[(5-ethyl-
2,4-dihydroxyphenyl)methanethione] (SETB): sulfinylbis[(5-
chloro-2,4-dihydroxyphenyl)methanethione] (SClTB), sulfi-
nylbis[(2,3,4-trihydroxyphenyl)methanethione] (S3TTB) with
the appropriate commercial chemical reagents containing
the amine group (Table 1, Schemes 1–6). In the case of com-
pounds 1, 2 and 7 cyclization proceeded by the elimination of
H2S, HCl or NH3 (phenylamine) respectively (Schemes 1 and
2). Compounds 5 and 6 were obtained as a result of the
elimination of H2O (Scheme 3). The intramolecular cycliza-
tion process proceeded according to the mechanism of
nucleophilic addition to the carbonyl groups in the case of
compounds 3, 4, 8 and 9 (Schemes 4, 5 and 6). The synthesis
mechanism of 7 and 8 was described previously [19].
Generally, cyclization processes are connected with thi-
olimine isomerization and ionization capabilities of the -SH
group. The reaction promotes a tendency of the compounds
with the thioamide moiety toward transition into the thi-
olimine form compared with the analogs with an oxygen atom
[21]. Purity of compounds was monitored by the reversed-
phase (RP-18) HPLC chromatography with methanol/water as
a mobile phase. STB and its analogues were obtained accord-
ing to the method described previously i.e. by treatment of
the corresponding dithioic acids with SOCl2 [22].
According to our data (Table 2), all tested naphtho[2,3-
d][1,3]thiazole-4,9-diones (2) exert an inhibitory effect on the
Gram-positive bacteria at promising concentrations. They
are effective at the MIC 0.78–6.25 mg/mL independently of
the substitution type of benzenediol residue. Significant differ-
ences in susceptibility of Gram-positive strains to the
particular 4H-3,1-benzothiazine derivatives (5) are observed
(MIC from 1.56 to 200 mg/mL). The most favourable is the
presence of the Me or Et group in position 5 of the resorcinol
moiety (5b, 5c). Compound 5a without the additional substitu-
ent exhibits the weakest potency. In the all screened groups,
compounds 2a and 2d (naphtho[2,3-d][1,3]thiazole-4,9-diones)
are found to be the most active (MIC ¼ 0.78–1.56 mg/mL),
however, the following compounds: 2b, 5b, 5c (4H-3,1-benzo-
thiazines) and 8a (4-hydroxy-4H-3,1-benzothiazine) exhibit
comparably high potency of bacterial growth inhibition with
the MICs ranging from 0.78 to 3.13 mg/mL. As can be seen in
Table 2, compounds 2a and 2d appear to be more effective (MIC
0.78–1.56 mg/mL) against all Gram-positive bacteria tested
than ampicillin (MIC 3.13–6.25 mg/mL). Moreover, compounds
2b, 3b, 5b, 5c, and 8a show a higher or similar activity
compared to that of ampicillin depending on the strain.
Good activity is also revealed by both derivatives from the
1,3-thiazolo[5,4-d]pyrimidin-5(4H)-one group (3).
The spectroscopic data of new derivatives are in agreement
with the proposed structures. In the lH NMR spectrum in the
range of low fields as a rule two signals corresponding to the
protons of the -OH groups in the resorcinol moiety are regis-
tered. OH and NH protons are sometimes invisible in the
background on the base line (3b, 6b, 8b, 8c).
Generally, the structure-activity analysis of the compounds
under consideration reveals that antibacterial potency of
bezenediols is enhanced by the naphtho[2,3-d][1,3]thiazole-
4,9-dione residue. However, modification of their structure
by two fused five-membered rings of 7a-hydroxy-7,7a-dihy-
dro[1,3]thiazolo[4,3-b][1,3,4]thiadiazole-5-thione proves to be
The resonance signals of resorcinol moiety in the case of
additional substitution in position 5 appear as characteristic
two singlets in the range about 7.9 and 6.6 ppm. In the IR
spectrum, there are strong bands in the region about 3400–
3100 and 1635–1600 cmꢀ1 corresponding to the vibrations of
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Table 1. The structures of compounds 1–9 and reagents applied for their syntheses
No.
Substituents
Structure of compounds
Reagents
Nucleophile
Electrophile
SH
HO
F3C
N
S
1
STB
OH
OH
F3C
NH2
O
R1
O
O
2a
2b
2c
2d
R1 ¼ R2 ¼ H
R1 ¼ Me, R2 ¼ H
R1 ¼ H, R2 ¼ Et
R1 ¼ H, R2 ¼ Cl
STB
S3MTB
SETB
HO
NH2
Cl
N
S
SClTB
R2
O
R3'
O
R2
R2'
O
NH2
R3'
R1'
O
3a
3b
R1 ¼ H, R2 ¼ Cl
SClTB
N
N
N
0
0
R1 ¼ R2 ¼ Me
OH
0
R3 ¼ NH2
N
S
N
R1'
OH
HO
R1
R2'
1’ 2’
R , R = H, Me;
0
0
0
R1 ¼ Me, R2 ¼ R1 ¼ R2 ¼ R3 ¼ H
S3MTB
3’
R
= H, NH
2
HO
O
OH
S
S
OH
N
NH2
4
SClTB
N
S
N
Cl
S
S
Cl
Cl
S
S
OH
OH
NH2
5a
5b
5c
5d
R ¼ H
R ¼ Me
R ¼ Et
R ¼ Cl
STB
S5MTB
SETB
N
N
SClTB
OH
R
O
R1'
R2'
0
0
R1'
R2'
6a
6b
R ¼ R1 ¼ R2 ¼ H
STB
OH
CH3
R
NH2
0
0
R ¼ Me, R1 ¼ R2 ¼ OMe
S3MTB
R1’= R2’= H, OMe
OH
O
N
O
R ¼ OH, R0 ¼ H
R ¼ H, R0 ¼ Cl
S3TTB
R'
R'
7a
7b
S
OH
NH
X
R
NH2
X= H, Ph; R’= H, Cl
OH
STB
continued
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Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Table 1. (continued )
Synthesis and Antibacterial Activity of Fused Heterocycles
305
No.
Substituents
Structure of compounds
Reagents
Nucleophile
Electrophile
R1'
OH
O
NH2
0
0
0
8a
R ¼ Et, R1 ¼ R3 ¼ Cl, R2 ¼ H
SETB
HO
S
R1'
R2'
OH
R3'
R3'
R2'
N
1’ 3’
R
R , R = H, Cl;
0
0
0
8b
R ¼ Cl, R1 ¼ R3 ¼ H, R2 ¼ Me
SClTB
SClTB
2’
R
= H, Me
OH
NH2
O
O
NH2
COOH
8c
OH
S
NH2
OH
Cl
N
OH
O
NH2
HO
Cl
N
S
9
SClTB
OH
OH
Scheme 1. Reaction mechanism of the formation of 4-(1,3-benzothiazol-2-yl)benzene-1,3-diol.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Scheme 2. Reaction mechanism of the formation of 2-(2,4-dihydroxyphenyl)naphtho[2,3-d][1,3]thiazole-4,9-diones.
Scheme 3. Reaction mechanism of the formation of 4-(4-methylidene-4H-3,1-benzothiazin-2-yl)benzene-1,3-diols.
unfavourable. In the group of 4H-3,1-benzothiazines the
strongest antibacterial potency is exhibited by the analogs
with unmodified C(4) of fused ring or with the hydroxyl and
phenyl substituents at this position. Analyzing an effect of
the substitution of the benzenediol moiety it can be found
that Et group enhances antibacterial activity of 4H-3,1-ben-
zothiazines. It causes the opposite effect in the case of naph-
tho[2,3-d][1,3]thiazole-4,9-dione derivatives.
determination of the minimal bactericidal concentration
(MBC) and compared with the MIC of a particular compound.
It is worth emphasizing that the low values of the MBC/MIC
ratio (ꢁ 4) indicate a bactericidal agent. According to our data
(Table 2) such potency against all strains is possessed only by
the compounds from the 4-substituted-4H-3,1-benzothiazine
group 8c and 6a. Other benzothiazines 8a and 5d exhibit the
same effect against 2 strains (S. epidermidis, S. aureus and
S. epidermidis, M. luteus, respectively). Furthermore, compound
9 (fluoreno[1,9-de][1,3]thiazine) exhibits bactericidal activity
Moreover, the activity of the tested compounds against
Gram-positive bacteria was examined in more detail by the
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Synthesis and Antibacterial Activity of Fused Heterocycles
307
Scheme 4. Reaction mechanism of the formation of 2-(2,4-dihydroxyphenyl)-3a-hydroxy-3a,6-dihydro[1,3]thiazolo[5,4-d]pyrimidin-
5(6H)-ones.
Conclusion
Summing up, a series of novel fused heterocycles incorporat-
ing the 1,3-thiazole or 1,3-thiazine ring and the 2,4-
dihydroxyphenyl substituent has been designed and
synthesized. The compounds inhibit in vitro growth of the
Gram-positive strains opposite to the Gram-negative ones.
Very good activity against the Gram-positive bacteria and
the low cytotoxicity profile of compound 5b make them
promising for further optimization in the development
of novel antibacterial agents. The results of biological
studies of compounds 2a, 2b, 2d, 5c, and 8a as the inhibitors
Scheme 5. Reaction mechanism of the formation of 2-(5-chloro-
of Gram-positive strains are certainly encouraging, but
2,4-dihydroxyphenyl)-7a-hydroxy-7,7a-dihydro[1,3]thiazolo[4,3-
b][1,3,4]thiadiazole-5-thione.
their cytotoxicity can confine the potential therapeutic
applications.
Experimental protocol
against 3 strains (S. epidermidis, B. subtilis, M. luteus) (Table 2).
The remaining compounds show bacteriostatic activity.
Analytical studies
Melting points (m.p.) were determined using a BUCHI B-540
¨
Cytotoxicity
(Switzerland) melting point apparatus. Elemental analyses
(C, H, N) were conducted using a Perkin-Elmer 2400 instrument;
their results were found to be in good agreement (ꢂ0.4%)
with the calculated values. IR spectra were recorded with a
Perkin-Elmer FT-IR 1725X spectrophotometer (in KBr) in the
range of 600–4000 cmꢀ1. 1H and 13C-NMR spectra were recorded
in DMSO-d6 by means of a Bruker DRX 500 instrument. Chemical
shifts (d, ppm) were given in relation to tetramethylsilane (TMS).
The mass spectra (EI, 70 eV) were recorded with an AMD-604
instrument.
The purity of the compounds was examined by a liquid
chromatograph Knauer with a dual pump, a 20 mL simple injec-
tion valve and a UV-visible detector at 280 nm. The Hypersil Gold
C18 (3 mm, 100 ꢃ 3 mm) column was used as the stationary
Cytotoxicity is one of the most important characteristics of a
compound intended for biomedical applications. In our
study, compounds 2a, 2b, 2d, 5b, 5c, and 8a, exhibiting
promising activity against the Gram-positive bacteria, were
included in the cytotoxicity test. The results in Table 3
reveal that the tested compounds show different levels of
cytotoxicity effect. The highest cytotoxicity is observed
for compounds 5c, 8a, and 2b (IC50 >6.25 mg/mL,
IC50 >6.25 mg/mL, and IC50 >12.5 mg/mL, respectively).
Compound 5b is found to possess the lowest cytotoxicity
effect (IC50 >50 mg/mL).
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Scheme 6. Reaction mechanism of the formation of 4-chloro-6-(10b-hydroxy-10bH-fluoreno[1,9-de][1,3]thiazin-2-yl)benzene-1,3-diol.
Table 2. The minimal inhibitory concentrations (MICs) and the minimal bactericidal concentrations (MBCs) of the tested compounds against
the reference Gram-positive bacteria
Compound
Antibacterial activity [mg/ml]
S. aureus ATCC 25923 B. subtilis ATCC 6633
S. epidermidis ATCCa 12228
M. luteus ATCC 10240
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1d
12.5
0.78
1.56
12.5
1.56
12.5
6.25
25
100
3.13
1.56
25
12.5
12.5
6.25
6.25
1.56
25
6.25
12.5
6.25
>200
100
100
>200
200
200
50
>200
nd b
50
12.5
100
50
200
200
200
6.25
200
25
12.5
0.78
1.56
6.25
1.56
6.25
3.13
12.5
200
1.56
3.13
25
12.5
6.25
6.25
12.5
3.13
25
>200
200
100
200
200
200
>200
>200
nd b
200
25
12.5
0.78
1.56
6.25
0.78
3.13
3.13
25
200
3.13
1.56
25
6.25
6.25
3.13
12.5
3.13
50
200
25
25
100
50
6.25
1.56
3.13
6.25
1.56
3.13
3.13
25
200
25
50
200
25
50
50
200
nd b
12.5
12.5
100
12.5
50
50
25
6.25
100
25
2ac
2bc
2cd
2dc
3ad
3bc
4e
200
200
>200
nd b
25
12.5
200
25
200
200
200
50
100
25
5ae
5bc
5cc
50
1.56
1.56
25
5de
6ad
6bd
7ac
7bd
8ac
8be
8cd
9e
200
25
1.56
6.25
6.25
6.25
0.78
12.5
6.25
6.25
6.25
200
200
200
6.25
200
25
6.25
12.5
3.13
12.5
25
3.13
50
nd b
100
nd b
50
nd b
25
nd b
Ampicillin
a
ATCC ¼ American Type Culture Collection
nd ¼ not determined
- very good activity
- good activity
- moderate activity
b
c
d
e
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Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Synthesis and Antibacterial Activity of Fused Heterocycles
309
Table 3. Effect of the tested compounds on the cell viability in the Vero cell line
Conc.
[mg/mL]
Cell viability [%]a
2a
2b
2d
5b
5c
8a
100
50
25
12.5
6.25
3.13
1.56
0.78
0.39
0 (control)
12.5 ꢂ 0.5
21.1 ꢂ 6.8
54.5 ꢂ 5.5
67.3 ꢂ 7.1
86.1 ꢂ 1.0
98.0 ꢂ 2.0
99.0 ꢂ 1.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
8.7 ꢂ 1.0
9.4 ꢂ 0.3
19.5 ꢂ 4.5
61.5 ꢂ 1.5
72.5 ꢂ 2.5
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
5.7 ꢂ 0.8
16.7 ꢂ 2.5
55.7 ꢂ 3.4
85.0 ꢂ 6.1
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
18.0 ꢂ 4.0
65.6 ꢂ 3.6
88.3 ꢂ 3.7
92.1 ꢂ 0.1
98.1 ꢂ 1.9
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
8.1 ꢂ 1.9
8.2 ꢂ 1.8
8.3 ꢂ 1.7
29.7 ꢂ 0.3
82.1 ꢂ 5.1
88.0 ꢂ 7.7
98.6 ꢂ 0.4
99.5 ꢂ 0.4
100 ꢂ 0.0
100 ꢂ 0.0
3.7 ꢂ 1.8
4.1 ꢂ 2.0
2.9 ꢂ 1.4
3.3 ꢂ 1.6
87.5 ꢂ 2.5
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
100 ꢂ 0.0
a
The results estimated as the percentage of viability of Vero cells plus compound tested to the control cell culture
– –
(C C), 1510 (C C), 1478, 1459, 1417, 1385, 1322, 1304, 1275,
– –
phase. The mobile phase included different contents of MeOH
and acetate buffer (pH 4, 20 nM) as the aqueous phase. The flow
rate was 0.5 mL/min 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. Log k values for 70%
of MeOH (v/v) in the mobile phase are presented.
1221, 1174, 1125, 1058, 1017, 1005, 978, 918, 852, 818, 785,
760, 721; 1H-NMR d: 11.13 (s, 1H, OH), 10.19 (s, 1H, OH), 8.35
(d, J ¼ 8.4 Hz, 1H, Ar-H), 8.27 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.99
(d, J ¼ 8.8 Hz, 1H, Ar-H), 7.75 (t, J ¼ 7.5 Hz, 1H, Ar-H), 7.3
(t, J ¼ 7.2 Hz, 1H, Ar-H), 6.57 (dd, J ¼ 8.4 and 2.4 Hz, 1H, Ar-H),
6.51 (d, J ¼ 2.1 Hz, 1H, Ar-H); 13C-NMR d: 181.0, 179.2, 175.1, 147.0,
134.8 (2C), 132.6 (2C), 132.3, 129.8, 126.1 (2C), 125.8 (2C), 117.9,
108.8, 102.4; EI-MS m/z (%): 323 (Mþ, 100), 292 (28), 264 (3), 235 (6),
184 (36), 153 (8), 136 (10), 129 (9), 124 (11), 101 (6), 69 (4), 36 (10),
85 (23), 80 (7), 69 (17), 63 (7), 51 (10), 44 (48), 40 (39), 36 (22). Anal.
Calcd (C17H9NO4S): C. 63.15; H, 2.81; N, 4.33. Found: C, 63.28; H,
2.79; N, 4.31.
Synthesis of compounds
4-[5-(Trifluoromethyl)-1,3-benzothiazol-2-yl]-
benzene-1,3-diol 1
A mixture of 2-amino-4-(trifluoromethyl)benzenethiol (0.0011 mol)
and STB (0.0011 mol) in MeOH (5.5 mL) was treated to reflux for
3 h. The hot mixture was filtered via a Bu¨chner funnel. The
formed solid was combined with that removed after the concen-
tration of the filtrate and the compound was recrystallized from
MeOH (4 mL).
2-(2,4-Dihydroxy-3-methylphenyl)naphtho[2,3-d][1,3]-
thiazole-4,9-dione 2b
A mixture of 2-amino-3-chloronaphthalene-1,4-dione (0.0024 mol)
and S3MTB (0.0048 mol) in MeOH (12 mL) was treated to reflux
for 3 h. The hot mixture was filtered via a Bu¨chner funnel and the
filtrate was concentrated. The formed solid was recrystallized
from MeOH (5 mL).
Yield: 66%; m.p.: 235–2368C; HPLC: log k ¼ 0.551; IR (KBr): 3411
–
–
–
(OH), 3032 (Ar-H), 2922 (CH), 2837 (CH), 1619 (C N), 1582 (C C),
–
–
–
1543 (C C), 1500 (C C), 1483, 1459, 1398, 1334, 1263, 1258,
–
–
1215, 1177, 1136, 1080, 1057, 1005, 987, 962, 863, 815, 774,
737, 713; 1H-NMR d: 11.48 (s, 1H, OH), 10.33 (s, 1H, OH), 8.31
(d, J ¼ 8.3 Hz, 1H, Ar-H), 8.28 (t, J ¼ 0.8 Hz, 1H, Ar-H), 8.07
(d, J ¼ 8.7 Hz, 1H, Ar-H), 7.68 (dd, J ¼ 8.4 and 1.2 Hz, 1H,
Ar-H), 6.55 (d, J ¼ 2.3 Hz, 1H, Ar-H), 6.49 (dd, J ¼ 8.8 and
2.3 Hz, 1H, Ar-H); 13C-NMR d: 167.6, 162.2, 158.4, 151.0, 138.1,
130.1, 127.2, 125.5, 123.35, 120.1, 117.9, 110.2, 108.6, 102.7;
EI-MS m/z (%): 311 (Mþ, 100), 292 (3), 282 (3), 254 (31), 222 (4),
157 (3), 142 (5). Anal. Calcd (C14H8F3NO2S): C, 54.02; H, 2.59;
N, 4.50. Found: C, 54.23; H, 2.61; N, 4.48.
Yield: 71%; m.p.: 197–2008C; HPLC: log k ¼ 0.515; IR (KBr):
3413 (OH), 3305 (OH), 3120 (OH), 2920 (CH), 2843 (CH), 1685
–
–
–
–
–
–
(C O), 1638 (C N), 1610 (C N), 1578 (C C), 1560 (C C), 1480,
–
–
–
–
1458, 1386, 1334, 1306, 1276, 1228, 1177, 1103, 1040, 1006,
1
963, 852, 820, 783, 719; H-NMR d: 12.30 (s, 1H, OH), 9.53 (s, 1H,
OH), 8.10–8.08 (m, 2H, Ar-H), 7.53 (d, J ¼ 8.8, 1H, Ar-H), 7.49–7.46
(m, 1H, Ar-H), 7.40–7.36 (m, 1H, Ar-H), 6.53 (d, J ¼ 8.7, 1H, Ar-H),
2.01 (s, 3H, CH3); EI-MS m/z (%): 337 (Mþ, 65), 323 (45), 315 (100), 307
(44), 298 (46), 286 (18), 270 (19), 258 (12), 240 (7), 209 (17), 182 (15),
172 (42), 167 (9), 160 (22), 151 (37), 131 (13), 124 (11), 105 (54), 89
(22), 76 (32), 51 (12), 44 (14), 36 (82), 44 (48), 40 (39), 36 (22). Anal.
Calcd (C18H11NO4S): C, 64.09; H, 3.29; N, 4.15. Found: C, 64.31;
H, 3.27; N 4.13.
2-(2,4-Dihydroxyphenyl)naphtho[2,3-d][1,3]-
thiazole-4,9-dione 2a
A mixture of 2-amino-3-chloronaphthalene-1,4-dione (0.0024 mol)
and STB (0.0048 mol) in MeOH (12 mL) was treated to reflux for
3 h. The hot mixture was filtered via a Bu¨chner funnel and the
filtrate was left at room temperature (24 h). The formed solid
was inserted into CHCl3 (25 mL) and refluxed for 2 h. The hot
mixture was filtered and the product was recrystallized from
MeOH/H2O (2:1, 15 mL).
2-(5-Ethyl-2,4-dihydroxyphenyl)naphtho[2,3-d][1,3]-
thiazole-4,9-dione 2c
A mixture of 2-amino-3-chloronaphthalene-l,4-dione (0.0024 mol)
and SETB (0.0048 mol) in MeOH (12 mL) was treated to reflux
for 3 h. The hot mixture was filtered via a Bu¨chner funnel.
The filtrate was concentrated, CHCl3 (20 mL) was added to
Yield: 69%; m.p.: 172–1738C; HPLC: log k ¼ 0.529; IR (KBr): 3460
–
–
–
(OH), 3318 (OH), 2922 (CH), 2843 (CH), 1682 (C O), 1617 (C N), 1578
–
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310
J. Matysiak et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
the mixture and refluxed for 0.5 h. The formed solid was recrys-
tallized from MeOH (5 mL).
filtered via a Bu¨chner funnel. The filtrate was concentrated, the
formed solid was filtered off and recrystallized from methanol (4 mL).
Yield: 69%; m.p.: 244–2468C; HPLC: log k ¼ ꢀ0.731; IR (KBr):
Yield: 59%; m.p.: 163–1658C; HPLC: log k ¼ 0.520; IR (KBr): 3447
–
–
–
– –
3436, 3310 (OH), 2957 (CH), 1691 (C O), 1618 (C N), 1552 (C C),
– –
–
(OH), 2923 (CH), 2942 (CH), 1687 (C O), 1638 (C N), 1591 (C C),
–
–
–
–
–
1513 (C C), 1460, 1438, 1387, 1331, 1291, 1286, 1217, 1158, 1136,
–
1500, 1482, 1427, 1367, 1388, 1303, 1270, 1214, 1182, 1119, 1079,
1067, 1003, 963, 892, 818, 780, 762, 704; 1H-NMR d: 11.51 (s, 1H,
OH), 11.33 (s, 1H, OH), 10.97 (s, 1H, OH), 7.74 (d, J ¼ 8.6 Hz, 1H,
Ar-H), 6.55 (d, J ¼ 8.6 Hz, 1H, Ar-H), 6.48 (s, 1H, CH), 2.05 (s, 3H,
CH3); 13C-NMR d: 193.7, 167.5, 160.8, 160.0, 157.0, 150.2, 136.5,
127.5, 115.3, 114.2, 111.4, 107.0; EI-MS m/z (%): 293 (Mþ, 49), 275
(39), 260 (38), 232 (14), 217 (18), 181 (11), 167 (78), 151 (100), 144
(11), 122 (8), 85 (8), 77 (6), 65 (5), 39 (4). Anal. Calcd (C12H11N3O4S):
C, 49.14; H, 3.79; N, 14.33. Found: C, 49.22; H, 3.77; N, 14.29.
1107, 1018, 936, 857, 755, 718; 1H-NMR d: 11.45 (s, 1H, OH), 10.38
(s, 1H, OH), 8.12–8.10 (m, 1H, Ar-H), 7.99–7.97 (m, 1H, Ar-H), 7.91–
7.88 (m, 1H, Ar-H), 7.50–7.48 (m, 1H, Ar-H), 7.01 (s, 1H, Ar-H), 6.61
(s, 1H, Ar-H), 2.45 (m, J ¼ 7.0 Hz, 2H, CH2CH3), 1.17 (t, J ¼ 7.2 Hz,
3H, CH2CH3); EI-MS m/z (%): 351 (Mþ, 43), 336 (Mþ–CH3, 92), 322 (19),
266 (9), 173 (19), 155 (10), 146 (8), 138 (42), 123 (100), 105 (19), 89
(13), 77 (15), 69 (11), 64 (34), 48 (11), 40 (14), 38 (17), 36 (51).
Anal. Calcd (C19H13NO4S): C, 64.95; H, 3.73; N, 3.98. Found: C,
65.18; H, 3.71; N, 3.96.
2-(5-Chloro-2,4-dihydroxyphenyl)-7a-hydroxy-7,7a-
2-(5-Chloro-2,4-dihydroxyphenyl)naphtho[2,3-d][1,3]-
thiazole-4,9-dione 2d
dihydro[1,3]thiazolo[4,3-b][1,3,4]thiadiazole-5-thione 4
A mixture of 3-amino-2-thioxothiazolidin-4-one (0.0017 mol) and
SClTB (0.0017 mol) in MeOH (8.5 mL) was treated to reflux for
3 h. The hot mixture was filtered via a Bu¨chner funnel and the
filtrate was left at room temperature (24 h). The formed solid was
combined with that removed after the concentration of the
filtrate and the compound was recrystallized from MeOH (4 mL).
Yield: 78%; m.p.: 235–2378C; HPLC: log k ¼ ꢀ0.508; IR (KBr): 3421
A mixture of 2-amino-3-chloronaphthalene-1,4-dione (0.0024 mol)
and SClTB (0.0048 mol) in MeOH (12 mL) was treated to reflux for
3 h, left at room temperature (24 h) and filtrated. CHCl3 (40 mL)
was added to the precipitate and treated to reflux for 1 h.
The reaction mixture was filtered off and the formed solid was
recrystallized from MeOH (15 mL).
– –
(OH), 2923 (CH), 2836 (CH), 1604 (C N), 1508 (C C), 1438, 1417,
– –
Yield: 63%; m.p.: 181–1838C; HPLC: log k ¼ 0.347; IR (KBr): 3419
–
–
–
(OH), 3257 (OH), 2921 (CH), 2844 (CH), 1654 (C O), 1622 (C N), 1587
1396, 1298, 1273, 1220, 1194, 1182, 1089, 1004, 888, 876, 809,
775, 732; 1H-NMR d: 11.98 (s, 1H, OH), 11.39 (s, 1H, OH), 10.87
(s, 1H, OH), 8.03 (s, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 3.68 (s, 2H, CH2);
13C-NMR d: 168.5, 162.7, 162.0, 156.3, 154.5, 127.3, 111.8, 109.1,
103.5, 52.5; EI-MS m/z (%): 334 (Mþ, 42), 333 (15), 332 (100), 300
(98), 272 (33), 218 (7), 196 (14), 170 (10), 169 (19), 109 (6), 69 (5),
45 (5), 36 (2). Anal. Calcd (C10H7ClN2O3S3): C, 35.87; H, 2.11; N, 8.36.
Found: C, 36.00; H, 2.10; N, 8.33.
Compounds: 4-(6-chloro-4H-3,1-benzothiazin-2-yl)benzene-1,3-diol 5a, 4-
(6-chloro-4H-3,1-benzothiazin-2-yl)-6-methylbenzene-1,3-diol 5b, 4-(6-chloro-4H-
3,1-benzothiazin-2-yl)-6-ethylbenzene-1,3-diol 5c, 4-chloro-6- (6-chloro-4H-3,1-
benzothiazin-2-yl)benzene-1,3-diol 5d were described previously [23].
–
–
–
–
(C C), 1514 (C C), 1480, 1393, 1373, 1298, 1262, 1093, 951, 883,
–
864, 842, 742, 718; 1H-NMR d: 11.93 (s, 1H, OH), 11.15 (s, 1H, OH),
8.21 (s, 1H, Ar-H), 8.19–8.17 (m, 1H, Ar-H), 8.12–8.10 (m, 1H, Ar-H),
7.92–7.88 (m, 2H, Ar-H), 6.78 (s, 1H, Ar-H); EI-MS m/z (%): 357
(Mþ, 100), 323 (6), 294 (5), 266 (13), 188 (27), 160 (11), 137 (16),
104 (28), 89 (7), 76 (20), 55 (5), 44 (17), 40 (12), 36 (6), 69 (17), 63 (7), 51
(10), 44 (48), 40 (39), 36 (22). Anal. Calcd (C17H8ClNO4S): C, 57.07;
H, 2.25; N, 3.92. Found: C, 57.21; H, 2.26; N, 3.94.
7-Amino-2-(5-chloro-2,4-dihydroxyphenyl)-3a-hydroxy-
4,6-dimethyl-3a,6-dihydro[1,3]thiazolo[5,4-d]-
pyrimidin-5(4H)-one 3a
A mixture of 5,6-diamino-1,3-dimethylpyrimidine-2,4(1H,3H)-
dione (0.0015 mol) and SClTB (0.0015 mol) in MeOH (7.5 mL)
was treated to reflux for 3 h. The hot mixture was filtered via
a Bu¨chner funnel. The formed solid was combined with that
removed after the concentration of the filtrate and the com-
pound was recrystallized from MeOH (4 mL).
4-(4-Methylidene-4H-3,1-benzothiazin-2-yl)-
benzene-1,3-diol 6a
A mixture of o-amino(acetophenone) (0.00185 mol) and STB
(0.0014 mol) in MeOH (15 mL) was treated to reflux for 3 h.
The hot mixture was filtered via a Bu¨chner funnel. The filtrate
was concentrated, the formed solid was filtered off and recrystal-
lized from MeOH (5 mL).
Yield: 63%; m.p. 328–3308C; HPLC: log k ¼ ꢀ1.111; IR (KBr):
3539 (NH), 3297 (OH), 3190 (OH), 2962 (CH), 2837 (CH), 1690
–
Yield: 71%; m.p.: 206–2088C; HPLC: log k ¼ 1.235; IR (KBr): 3067
–
–
–
–
– –
(OH), 1611 (C N), 1542 (C C), 1509 (C C), 1452, 1376, 1326, 1292,
– –
–
(C O), 1638 (C N), 1611 (C N), 1564 (C C), 1516 (C C), 1468,
–
–
–
–
–
1437, 1413, 1383, 1361, 1300, 1246, 1228, 1203, 1135, 1077,
–
1261, 1196 (C-OH), 1132, 1035, 981, 943, 843, 805, 760; 1H-NMR d:
14.38 (s, 1H, OH), 10.42 (s, 1H, OH), 7.65 (d, J ¼ 8.6 Hz, 1H, Ar-H),
7.54–7.39 (m, 4H, Ar-H), 6.74 (s, 1H, CH), 6.34 (d, J ¼ 2.3 Hz, 1H,
Ar-H), 6.28 (m, 2H, Ar-H, CH); 13C-NMR d: 162.8, 159.8, 139.6,
129.2, 128.1, 127.0, 126.7, 124.6, 120.2, 111.2, 110.2, 108.1, 107.9,
102.9, 47.7; EI-MS m/z (%): 269 (Mþ, 100), 236 (34), 225 (11), 212
(16), 180 (11), 167 (8), 154 (3), 135 (3), 108 (4), 89 (11), 76 (5), 63 (5),
44 (8). Anal. Calcd (C15H11NO2S) C, 66.89; H, 4.12; N, 5.20. Found:
C, 66.72; H, 4.13; N, 5.18.
1
1043, 980, 941, 915, 864, 770, 759; H-NMR d: 11.96 (s, 1H, OH),
10.90 (s, 1H, OH), 10.54 (s, 1H, OH), 8.06 (s, 1H, Ar-H), 6.78 (br. s,
2H, NH2), 6.57 (s, 1H, Ar-H), 3.37 (s, 3H, CH3), 3.34 (s, 3H, CH3); EI-MS
m/z (%): 356 (Mþ, 5), 339 (100), 322 (31), 306 (3), 282 (61), 265 (15),
255 (5), 248 (12), 224 (9), 219 (6), 208 (6), 187 (84), 170 (34), 153 (25),
131 (9), 126 (5), 85 (23), 80 (7), 69 (17), 63 (7), 51 (10), 44 (48), 40 (39),
36 (22). Anal. Calcd (C13H13ClN4O4S): C, 43.76; H, 3.67; N, 15.70.
Found: C, 43.69; H, 3.69; N, 15.64.
2-(2,4-Dihydroxy-3-methylphenyl)-3a-hydroxy-3a,
4-(6,7-Dimethoxy-4-methylidene-4H-3,1-benzothiazin-
2-yl)-2-methylbenzene-1,3-diol 6b
A mixture of 20-amino-40,50-dimethoxyacetophenone (0.0013 mol)
and S3MTB (0.0013 mol) in MeOH (12 mL) was treated to reflux
6-dihydro[1,3]thiazolo[5,4-d]pyrimidin-5(4H)-one 3b
A mixture of 5-aminouracil (0.002 mol) and S3MTB (0.002 mol) in
methanol (10 mL) was refluxed for 3 h. The hot reaction mixture was
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Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
Synthesis and Antibacterial Activity of Fused Heterocycles
311
for 3 h. The hot mixture was filtered via a Bu¨chner funnel. The
filtrate was concentrated and the formed solid was recrystallized
from MeOH/H2O (3:1, 4 mL).
filtrate was concentrated, the formed solid was filtered off and
recrystallized from MeOH (4 mL).
Yield: 62%; m.p.: 188–1908C; HPLC: log k ¼ 1.141; IR (KBr): 3438
Yield 68%; m.p.: 195–1978C; HPLC: log k ¼ 1.216; IR (KBr, cmꢀ1):
(OH), 3197 (OH), 2969 (CH), 1610 (C N), 1582 (C C), 1546 (C C),
–
–
–
–
–
–
–
–
–
–
3435 (OH), 2922 (CH), 1614 (C N), 1502 (C C), 1480, 1463, 1338,
1521 (C C), 1493, 1466, 1402, 1377, 1338, 1267, 1219, 1198,
–
–
1314, 1272, 1218, 1089, 1064, 1058, 1026, 942, 877, 822, 780;
1H-NMR d: 10.25 (br.s, 1H, OH), 7.80 (d, J ¼ 8.8 Hz, 1H, Ar-H),
7.25 (s, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 6.65 (s, 1H, CH), 6.55
(d, J ¼ 8.7 Hz, 1H, Ar-H), 6.40 (s, 1H, CH), 3.91–3.80 (m, 6H,
OCH3), 1.18 (s, 3H, CH3); EI-MS m/z (%): 343 (Mþ, 43), 321 (17),
297 (17), 277 (31), 249 (22), 222 (36), 162 (43), 151 (100), 114
(15), 77 (13), 57 (61), 40 (60). Anal. Calcd (C18H17NO4S) C, 62.96;
H, 4.99; N, 4.08. Found: C, 63.28; H, 5.01; N, 4.06.
1153, 1088, 1057, 989, 951, 880, 828, 758, 732; 1H-NMR d:
13.66 (s, 1H, OH), 11.50 (s, 1H, OH), 10.47 (s, 1H, OH), 8.14
(d, J ¼ 8.7 Hz, 1H, Ar-H), 7.56–7.54 (m, 3H, Ar-H), 7.52–7.50
(m, 2H, Ar-H), 7.46 (s, 1H, Ar-H), 6.81–6.79 (m, 1H, Ar-H), 6.42
(s, 1H, Ar-H), 2.46–2.44 (m, J ¼ 7.5 Hz, 2H, CH2CH3), 1.09
(t, J ¼ 7.5 Hz, 3H, CH2CH3); EI-MS m/z (%): 445 (6), 427 (53), 414
(22), 394 (4), 306 (7), 296 (6), 261 (7), 246 (3), 230 (4), 212 (6), 195 (5),
186 (4), 164 (6), 139 (43), 111 (10), 91 (3), 69 (7), 45 (44), 36 (100).
Anal. Calcd (C22H17Cl2NO3S): C, 59.20; H, 3.84; N, 3.14; found: C,
59.28; H, 3.82; N, 3.12.
2-(2,3,4-Trihydroxyphenyl)-4H-3,1-benzothiazin-4-one 7a
A mixture of 2-amino-N-phenylbenzamide (0.0012 mol) and
S3TTB (0.0017 mol) in MeOH (6 mL) was treated to reflux for
3 h. The hot mixture was filtered via a Bu¨chner funnel. The
obtained product was combined with that removed after the
concentration of the filtrate and the compound was recrystal-
lized from MeOH (4 mL).
4-Chloro-6-(4-hydroxy-7-methyl-4-phenyl-4H-3,1-
benzothiazin-2-yl)benzene-1,3-diol 8b
A mixture of 2-amino-4-methylbenzphenone (0.0012 mol) and
SClTB (0.002 mol) in MeOH (6 mL) was refluxed for 3 h. The
hot reaction mixture was filtered via a Bu¨chner funnel. The
filtrate was concentrated, NaOH solution (5%) was added
(5 mL) and the mixture was filtrated. HCl solution (5%) was
added to the filtrate to afford the product. The compound was
recrystallized from MeOH (4 mL).
Yield: 74%; m.p.: 246–2488C; HPLC: log k ¼ 0.061; IR (KBr):
–
–
–
3559 (OH), 3068 (Ar-H), 2856 (CH), 1705 (C O), 1645 (C N), 1598
–
–
–
–
(C C),1543 (C C), 1504 (C C), 1474, 1442, 1363, 1322, 1282, 1261,
–
–
–
1215, 1181, 1116, 1091, 995, 931, 916, 894, 865, 812, 792, 765, 747,
720, 709; 1H-NMR d: 13.35 (s, 1H, OH), 10.07 (s, 1H, OH), 8.67 (s, 1H,
OH), 8.12–8.11 (m, 1H, Ar-H), 7.95–7.93 (m, 1H, Ar-H), 7.81
(d, J ¼ 8.7 Hz, 1H, Ar-H), 7.62–7.61 (m, 1H, Ar-H), 7.16–7.15
(m, 1H, Ar-H), 6.51 (d, J ¼ 8.6 Hz, 1H, Ar-H); EI-MS m/z (%): 287
(Mþ, 100), 259 (3), 227 (59), 202 (12), 140 (4), 153 (3), 143 (5), 136 (2),
115 (3), 104 (2), 77 (18), 65 (3), 51 (4), 39 (4). Anal. Calcd (C14H9NO4S):
C, 58.53; H, 3.16; N, 4.88. Found: C, 58.60; H, 3.14; N, 4.86.
Yield: 64%; m.p.: 154–1568C; HPLC: log k ¼ 0.488; IR (KBr): 3432
–
–
–
(OH), 2922 (CH), 2843 (CH), 1617 (C N), 1534 (C C), 1490, 1441,
–
1339, 1298, 1257, 1207, 1159, 1078, 1043, 964, 923, 882, 845, 778,
1
742; H-NMR d: 14.35 (s, 1H, OH), 11.72 (s, 1H, OH), 11.35 (s, 1H,
OH), 7.94 (s, 1H, Ar-H), 7.69–7.67 (m, 1H, Ar-H), 7.57–7.52 (m, 3H,
Ar-H), 7.43–7.40 (m, 2H, Ar-H), 6.99–6.96 (m, 1H, Ar-H), 6.76 (s, 1H,
Ar-H), 6.57–6.53 (m, 1H, Ar-H), 1.75 (s, 3H, CH3); 13C-NMR d: 160.8,
160.5, 159.2, 158.1, 140.2, 139.4, 135.9, 129.9, 129.2, 128.6, 128.2,
127.3, 126.6, 119.7, 115.0, 112.1, 111.5, 111.3, 104.4, 58.3, 19.5
(CH3); EI-MS m/z (%): 397 (Mþ, 5), 381 (100), 348 (6), 313 (5), 304 (9),
218 (53), 186 (46), 142 (35), 130 (15), 114 (9), 107 (5), 87 (6), 79 (11),
77 (7), 53 (11), 51 (20), 44 (15), 39 (9). Anal. Calcd (C21H16ClNO3S):
C, 63.39; H, 4.05; N, 3.52. Found: C, 63.13; H, 4.03; N, 3.54.
6-Chloro-2-(2,4-dihydroxyphenyl)-4H-3,1-benzothiazin-
4-one 7b
A mixture of 2-amino-5-chlorobenzamide (0.0015 mol) and STB
(0.0015 mol) in MeOH (7.5 mL) was treated to reflux for 3 h. The
hot mixture was filtered via a Bu¨chner funnel. The obtained
product was combined with that removed after the concentration
of the filtrate and the compound was recrystallized from MeOH
(4 mL).
2-Amino-3-[2-(5-chloro-2,4-dihydroxyphenyl)-4-hydroxy-
4H-3,1-benzothiazin-4-yl]propanoic acid 8c
A
mixture of 2-amino-4-(2-aminophenyl)-4-oxobutanoic acid
Yield: 78%; m.p.: 249–2508C; HPLC: log k ¼ 0.347; IR (KBr): 3398
(0.0012 mol) and SClTB (0.0012 mol) in MeOH (6 mL) was
refluxed for 3 h. The reaction mixture was left at room tempera-
ture (24 h) and the formed solid was filtered off. The obtained
product was combined with that removed after the filtrate
concentration. The compound was recrystallized from MeOH/
H2O (1:1) (4 mL).
–
–
–
–
(OH), 3082 (Ar-H), 2843 (CH), 1703 (C O), 1631 (C N), 1598 (C C),
–
–
–
–
–
1562 (C C), 1538 (C C), 1516 (C C), 1470, 1448, 1403, 1382,
–
–
–
1326, 1314, 1247, 1194, 1135, 1069, 980, 937, 870, 835, 796,
745, 704; 1H-NMR d: 12.93 (s, 1H, OH), 10.59 (s, 1H, OH), 8.06
(d, J ¼ 2.5 Hz, 1H, Ar-H), 7.99–7.97 (m, 1H, Ar-H), 7.93 (m, 1H,
Ar-H), 7.67 (d, J ¼ 8.8 Hz, 1H, Ar-H), 6.47 (dd, J ¼ 8.8 and 2.4 Hz,
1H, Ar-H), 6.38 (d, J ¼ 2.3 Hz, 1H, Ar-H); 13C-NMR d: 178.4, 177.5,
167.4, 157.3, 156.5, 152.3, 139.7, 134.3, 134.0, 132.9, 128.5, 126.0,
111.8, 103.4; EI-MS m/z (%): 305 (Mþ, 100), 277 (4), 245 (60), 217
(13), 182 (7), 154 (11), 108 (16), 75 (6), 69 (5), 63 (5), 52 (4), 39 (5).
Anal. Calcd (C14H8ClNO3S): C, 55.00; H, 2.64; N, 4.58. Found: C,
54.86; H, 2.65; N, 4.60.
Yield: 66%; m.p.: 189–1918C; HPLC: log k ¼ ꢀ0.568; IR (KBr):
–
–
–
–
3423 (NH), 3240 (OH), 2920 (CH), 1700 (C O), 1615 (C N), 1539
(C C), 1506 (C C), 1490, 1428, 1342, 1297, 1256, 1198, 1162,
–
–
–
–
1084, 1047, 1023, 992, 847, 762; 1H-NMR d: 7.72 (d, J ¼ 8.1 Hz,
1H, Ar-H), 7.31–7.24 (m, 3H, Ar-H), 7.21 (br.s, 2H, NH2), 6.79 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 6.56 (t, J ¼ 7.2 Hz, 1H, Ar-H), 4.46 (m, 1H,
CH), 3.59 (d, J ¼ 6.2 Hz, 2H, CH2); 13C-NMR d: 170.8, 160.5, 158.1,
155.6, 151.4, 148.5, 134.9, 132.0, 117.1, 114.7, 111.6, 110.9, 104.4,
104.2, 104.0, 58.3, 52.2; EI-MS m/z (%): 394 (Mþ, 5), 381 (100), 348
(6), 313 (5), 304 (9), 218 (53), 186 (46), 142 (35), 130 (15), 114 (9),
107 (5), 87 (6), 79 (11), 77 (7), 53 (11), 51 (20), 44 (15), 39 (9). Anal.
Calcd (C17H15ClN2O5S): C, 51.71; H, 3.83; N, 7.10. Found: C, 51.98;
H, 3.81; N, 7.13.
4-[6-Chloro-4-(2-chlorophenyl)-4-hydroxy-4H-3,1-
benzothiazin-2-yl]-6-ethylbenzene-1,3-diol 8a
A mixture of 2-amino-20,5-dichlorobenzphenone (0.0013 mol)
and SETB (0.0013 mol) in MeOH (9.5 mL) was refluxed for 3 h.
The hot reaction mixture was filtered via a Bu¨chner funnel. The
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312
J. Matysiak et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 302–313
the experiment. The media in the culture (Minimum Essential
Medium Eagle, Sigma) were supplemented with 10% foetal
bovine serum (FBS, Sigma), 100 U/mL of penicillin and 0.1 mg/
mL of streptomycin (Polfa-Tarchomin, Poland). The cell culture
was incubated at 378C in the 5% CO2 atmosphere. All the inves-
tigated compounds were dissolved in DMSO (Sigma) to obtain the
concentration of 10 mg/mL. 100 mL of the Vero cell culture pre-
pared were plated into 96-well plastic plates (NUNC) at the density
2 ꢃ 104 cells per well. After incubation at 378C for 24 h the media
were removed and the cells were treated with a solution of the
examined compounds diluted in the media with 2% of serum. The
following final concentrations were applied: 0.39, 0.78, 1.56, 3.13,
6.25, 12.5, 25, 50, and 100 mg/mL. At the same time the cytotox-
icity of solvents in compounds was examined. The control cell
culture was supplemented with media including 2% of serum
only. The culture cells were incubated for 48 h at 378C in the 5%
CO2 atmosphere. After 48-h incubation with the compounds, cell
cultures were supplemented with 10 mL of 5 mg/mL MTT solution
per well, and further incubated for 4 h at 378C. Afterwards 100 mL
of water solution, including 50% dimethylformamide and 20%
SDS per well, were added and after overnight incubation the
absorbance was measured by the 96-well plastic plate reader
(Organon Teknika) at the wavelengths of 540 and 620 nm. The
obtained results were presented as percentage of cell viability in
comparison to the control. The investigation was carried out in
triplicates. Cytotoxicity was assessed using standard procedure
ISO 10993-5 [27].
4-Chloro-6-(10b-hydroxy-10bH-fluoreno[1,9-de][1,3]-
thiazin-2-yl)benzene-1,3-diol 9
A mixture of 1-amino-9H-fluoren-9-one (0.0013 mol) and SClTB
(0.0022 mol) in MeOH (10.5 mL) was refluxed for 3 h. The hot
reaction mixture was filtered via a Bu¨chner funnel. The filtrate
was concentrated, NaOH solution (5%) was added (5 mL) and
the mixture was filtrated. HCl solution (5%) was added to the
filtrate to afford the product. The compound was recrystallized
from MeOH (4.5 mL).
Yield: 74%; m.p.: 126–1288C; HPLC: log k ¼ 0.851; IR (KBr): 3376
–
–
–
(OH), 3055 (Ar-H), 2924 (CH), 2853 (CH), 1648 (C N), 1611 (C N),
–
–
–
–
–
1576 (C C), 1506 (C C), 1490, 1452, 1420, 1343, 1298, 1257,
1193, 1162, 1045, 961, 928, 896, 848, 797, 755; 1H-NMR d:
13.70 (s, 1H, OH), 11.94 (s, 1H, OH), 10.57 (s, 1H, OH), 7.91
(s, 1H, Ar-H), 7.69–7.64 (m, 2H, Ar-H), 7.55–7.49 (m, 1H, Ar-H),
7.35–7.30 (m, 1H, Ar-H), 7.26–7.21 (m, 1H, Ar-H), 6.90
(d, J ¼ 7.0 Hz, 1H, Ar-H), 6.64 (d, J ¼ 7.3 Hz, 1H, Ar-H), 6.63
(s, 1H, Ar-H); 13C-NMR d: 193.3, 168.2, 160.6, 159.3, 148.3,
143.4, 142.7, 136.2, 134.5, 133.5, 130.6, 128.7, 122.5, 120.7,
117.8, 115.0, 112.6, 111.6, 108.77, 103.8; EI-MS m/z (%): 381
(Mþ, 43), 365 (MþꢀOH, 100), 330 (3), 211 (14), 168 (18), 152
(20), 140 (16), 139 (35), 83 (7), 51 (5), 44 (9), 36 (7). Anal. Calcd
(C20H12ClNO3S): C, 62.91; H, 3.17; N, 3.67. Found: C, 63.04; H, 3.15;
N, 3.65.
Antibacterial assay in vitro
MICs of all synthesized compounds for eight reference strains,
including the Gram-positive bacteria (Staphylococcus epidermidis
ATCC 12228, Staphylococcus aureus ATCC 25923, Bacillus subtilis
ATCC 6633, and Micrococcus luteus ATCC 10240) and Gram-
negative bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 13883, Pseudomonas aeruginosa ATCC 9027, and Proteus
mirabilis ATCC 12453) were assessed by the micro-dilution broth
method according to Skalicka et al [25]. Briefly, the stock
solutions of the synthesized compounds were made in DMSO
(Sigma). Then the series of two-fold dilutions of these stock
solutions, ranging from 0.39 to 200 mg/mL, were prepared in
the Mueller-Hinton broth (Biocorp, Poland) in 96-well microtiter
plates (NUNC). The wells were inoculated with the bacterial
suspension (the final inoculum size of 106 colony forming units
– CFU/mL) and incubated at 358C for 24 h. MIC was defined as the
lowest concentration of the compound that completely sup-
pressed visible growth. Ampicillin was used as the reference
compound. MBC of the tested compounds (except 5a) towards
Gram-positive bacteria was determined by subculturing 100 mL
from each well that showed complete bacterial growth inhi-
bition on Mueller-Hinton agar (Biocorp, Poland) plates. After
the incubation (358C for 24 h), the MBC value was defined as
the lowest concentration of the compound at which there
was no bacterial growth. The experiment was carried out in
triplicates.
The authors have declared no conflict of interest.
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