Synthesis, reactions, and biological activity of 1,4-benzothiazine derivatives

Article abstract of DOI:10.1007/s00706-010-0312-6

6,7-Dimethoxy-2H-1,4-benzothiazin-3(4H)-one reacts with dimethylformamide dimethylacetal (DMF-DMA) to give the novel enaminone 2-(dimethylaminomethylene)- 6,7-dimethoxy-2H-1,4-benzothiazin-3(4H)-one. The reaction of the latter with various active methylen

Full text of DOI:10.1007/s00706-010-0312-6

Monatsh Chem (2010) 141:661–667
DOI 10.1007/s00706-010-0312-6
ORIGINAL PAPER
Synthesis, reactions, and biological activity of 1,4-benzothiazine
derivatives
•
Eman M. H. Abbas Thoraya A. Farghaly
Received: 30 August 2009 / Accepted: 5 April 2010 / Published online: 28 April 2010
Ó Springer-Verlag 2010
Abstract 6,7-Dimethoxy-2H-1,4-benzothiazin-3(4H)-one
reacts with dimethylformamide dimethylacetal (DMF-DMA)
to give the novel enaminone 2-(dimethylaminomethylene)-
6,7-dimethoxy-2H-1,4-benzothiazin-3(4H)-one. The reaction
of the latter with various active methylene compounds
afforded pyrido[3,2-b][1,4]benzothiazines. Also, coupling of
the enaminone with diazotized aniline derivatives gave
2-(arylhydrazono)-6,7-dimethoxy-2H-1,4-benzothiazin-3
(4H)-ones. Spectral data indicated that the latter compounds
exist predominantly in the hydrazone tautomeric form. In
addition, coupling of the enaminone with diazotized het-
erocyclic amines afforded tetra- and pentaheterocyclic ring
systems. The antitumor and antimicrobial activity of some
of the synthesized compounds was screened.
heterocycles, as many of them are useful in the field of
material sciences and theoretical chemistry [15, 16].
In addition to these applications, azo compounds are
used as photosensitive species in photographic or electro-
photographic systems and are the dominant organic
photoconductive materials in commercial copiers [17].
Benzothiazines are biologically interesting molecules with
established utility in the pharmaceutical and agrochemical
industries. Compounds with these ring systems have
diverse pharmacological activity such as anticancer
[18–20], antiproliferative [18–20], antimicrobial [21, 22],
and antifungal activity [23, 24]. Herein we report synthesis
of a new enaminone as a precursor for synthesis of several
fused benzothiazines and hydrazones which are expected to
be biologically active.
Keywords Enaminone ꢀ 2H-1,4-Benzothiazin-3(4H)-one ꢀ
Cytotoxic activity ꢀ Antimicrobial activity
Results and discussion
Introduction
Concurrent reduction and dehydrative cyclization of
compound 1 with tin in hydrochloric acid afforded
6,7-dimethoxy-2H-1,4-benzothiazin-3(4H)-one (2). Con-
version of the latter into the required enaminone 3 was
carried out by a procedure differing from that reported for
synthesis of other 2-(dimethylaminomethylene)-1,4-benzo-
thiazines [25]. Thus, treatment of 2 with dimethylformamide
dimethylacetal (DMF-DMA) under solvent-free conditions
furnished a single product [examined by thin-layer
chromatography (TLC)] that was identified as 2-(dimethyl-
aminomethylene)-6,7-dimethoxy-2H-1,4-benzothiazin-3(4H)-
one (3) (Scheme 1). Elemental analysis and spectral data
were in complete accordance with the assigned structure 3;
Enaminones are versatile reagents, and their utility in
heterocyclic synthesis has recently earned considerable
attention [1–4]. In our previous papers [5–14], we were
interested in the azo-hydrazone tautomerism of the arylazo
E. M. H. Abbas
Department of Chemistry of Natural and Microbial Products,
National Research Center, Dokki, Cairo, Egypt
T. A. Farghaly (&)
1
for example, the H nuclear magnetic resonance (NMR)
Department of Chemistry, Faculty of Science,
University of Cairo, Giza, Egypt
e-mail: thoraya-f@hotmail.com
spectrum of compound 3 revealed two singlet signals at d =
3.09 and 7.26 ppm, characteristic for N,N-dimethylamino and
123

662
E. M. H. Abbas, T. A. Farghaly
Scheme 1
H
H3CO
H3CO
N
O
H3CO
H3CO
NO2
S
Sn / HCl
CH2-COOH
S
2
1
DMF-DMA
COR'
H
H
N
H2C
H3CO
N
O
H3CO
H3CO
O
4
COR'
R
C
H
H3CO
S
H
C
CH-N(Me)2
R
S
3
H
5
N(Me)2
NH3
-NH(Me)2
H
N
R'
R
H3CO
N
H3CO
S
6
R/ R' : a, COCH3/CH3; b, COOEt/CH3; c, COOEt/Ph; d, COPh/ Ph
the (E)-configuration of exocyclic C=CH protons, respec-
tively [26, 27].
H
N
H
N
H3CO
H3CO
O
H3CO
H3CO
O
Ar-N=N-Cl
N=N-Ar
Reaction of enaminone 3 with active methylene com-
pounds acetylacetone (4a), ethyl acetoacetate (4b), ethyl
benzoylacetate (4c), and dibenzoylmethane (4d) in glacial
acetic acid in presence of ammonium acetate gave the
corresponding pyrido[3,2-b][1,4]benzothiazine derivatives
6a–6d (Scheme 1). The structures of these products were
S
S
CH-N(Me)2
3
N
H3C
CH3
H2O
1
H
N
assigned based on their H NMR spectra, which showed
H3CO
O
Ar
H
N
H3CO
O
N
characteristic singlet signals at d = 7.98–8.14 ppm char-
acteristic for the pyridine-4H [28].
N
N
H
N
-DMF
H3CO
S
O
H3CO
S
Ar
In conjunction with our interest in the azo-hydrazone
tautomerism [5–14], we report here on the coupling reac-
tion of the enaminone 3 with diazotized anilines, giving the
respective arylazo derivatives 7a–7f (Scheme 2). On the
H
7
N
H3C
CH3
Ar = C6H4X
X: a, 4-OCH3; b, H; c, 4-Cl; d, 4-Br; e, 4-NO2; f, 4-COCH3
1
basis of elemental analyses, infrared (IR), H NMR, and
Scheme 2
ultraviolet (UV) spectra (see Experimental), the isolated
products were assigned structure 7.
[5, 6, 9], the electronic absorption spectra of 7 in dioxane
revealed in each case two characteristic absorption bands in
the regions 395–371 and 316–299 nm (Table 1), and the
spectra of compound 7b (taken as a typical example of the
series prepared) in different solvents exhibit little, if any,
solvent dependence (Table 1). Also, their IR revealed in
each case two NH and carbonyl carbon absorption bands in the
Compounds 7 can exist in one or more of five tautomeric
forms: the hydroxy-azo form 7A, the keto-hydrazone form
7B, hydroxy-hydrazone 7C, CH-keto-azo form 7D, and
CH-hydroxy-azo tautomeric form 7E (Fig. 1). Of these five
forms, the tautomeric form 7B seems to be the form of
choice for the studied compounds, as it is consistent with
1
their electronic absorption spectra and H NMR spectra.
regions 3,437–3,310, 3,270–3,182, and 1,671–1,652 cm-1
,
This conclusion is consistent with other literature reports
on the tautomerism of analogous arylazo derivatives of
benzothiazine [29, 30]. For example, like typical hydrazones
respectively. The 1H NMR spectra are characterized by
two singlet signals assignable to two NH protons at
123

Synthesis, reactions, and biological activity of 1,4-benzothiazine derivatives
663
N
N
N
H
N
H
N
H
N
H3CO
H3CO
O
N
H3CO
H3CO
O
N
N=N-SO4H
11
H3CO
H3CO
O
H
N
H
N
N=N-Cl
H
N
H
N
H
8
S
S
Ar
Ar
S
CH-N(Me)2
7B
7A
3
H
N
O
H3CO
H3CO
N
H
H3CO
H3CO
H
O
H
N
H
H
H3CO
N
OH
N
H3CO
N
O
N
H
N
S
N
N
N
H
S
N
N
N
N
9
H3CO
S
H3CO
S
12
Ar
N
N
7C
7D
Ar
N
N
H
H3CO
H3CO
N
OH
H
N
H3CO
H3CO
N
N
N
H3CO
H3CO
N
N
H
N
N
S
N
S
N
N
S
10
13
Ar
7E
Scheme 3
Fig. 1 Possible tautomeric forms of compounds 7
Table 2 Cytotoxic activity of tested compounds
Table 1 UV spectral data of compounds 7a–7f in dioxane
Compound
IC50 (lg/cm3)
Compound
kmax (nm) (log e)
HCT-116
HEPG2
MCF7
7a
7ba
7c
7d
7e
7f
389 (4.47), 300 (4.91)
377 (4.20), 299 (4.44)
371 (4.13), 300 (4.59)
379 (4.48), 306 (4.28)
382 (4.57), 304 (4.54)
395 (4.73), 316 (4.21)
6a
7c
7e
4.19
4.79
3.74
4.97
6.69
5.79
–
–
10
IC50, 50% inhibition concentration
In a similar manner, enaminone 3 coupled readily with
the diazonium salt of 2-aminobenzimidazole (11) under the
same experimental conditions to afford a single product 13
according to TLC (Scheme 3). The structure of the isolated
product is based on the elemental analysis and spectral data
(see ‘‘Experimental’’).
a
Solvent kmax (log e): acetic acid 376 (4.21), 300 (4.35); ethanol 375
(4.51), 301 (4.02); acetone 374 (4.00), 299 (4.27); DMF 375 (3.99),
301 (4.12)
d = 10.90–10.69 and 10.20–9.74 ppm. The formation of
the products 7a–7f is assumed to take place via Japp–
Klingemann-type cleavage of dimethylformamide to afford
the product 7, as illustrated in Scheme 2. This finding
indicated that the isolated product was found in tautomeric
form 7B.
Antitumor screening test
The cytotoxic effects of three products 6a, 7c, and 7e were
tested against colon cancer cell line HCT-116, liver car-
cinoma cell line HEPG2-1, and human breast cell line
MCF-7. They were evaluated in the National Institute of
Cancer, Cairo, Egypt. As shown in Table 2, the analysis of
the data obtained indicated that all tested compounds
showed reactivity against HCT-116 cell line more than
HEPG2 and MCF.
The foregoing results prompted us to investigate the
behavior of enaminone 3 towards some diazotized het-
erocyclic amines. Thus, coupling reaction of enaminone 3
with the diazonium salt of 3-amino-1,2,4-triazole (8) in
pyridine afforded the corresponding hydrazone 9, which
undergoes in situ intramolecular cyclization to afford
8,9-dimethoxy-11H-[1,2,4]triazolo[30,40:3,4][1,2,4]triazino
[6,5-b][1,4]benzothiazine (10) (Scheme 3). The structure
of the isolated product 10 was established by its spec-
troscopic [IR, 1H NMR, and mass spectrometry (MS)]
data and elemental analyses (see Experimental). Its
mass spectrum revealed the molecular ion peak at
m/z = 302, and its 1H NMR spectrum showed a char-
acteristic signal at d = 8.13 ppm assignable to the
triazole CH proton.
Antimicrobial activity
The products 3, 6a, 6d, 7a, 7c–7e, 9, and 11 were screened
for their antibacterial activity (in nutrient agar broth) and
antifungal activity (in Dox’s medium and Sabouraud’s
agar) by agar diffusion method [31, 32] at concentration of
20 mg/cm3 using dimethyl sulfoxide (DMSO) as solvent
and blank. Compounds were tested for activity against
123

664
E. M. H. Abbas, T. A. Farghaly
Experimental
Table 3 Antimicrobial activity of the tested compounds
Compound
Inhibition zone diameter (mm/mg sample)
All melting points were determined by using an electro-
thermal Gallenkamp apparatus. Solvents were generally
distilled and dried by standard literature procedures prior to
use. IR spectra were measured using a Pye-Unicam SP300
E. coli
S. aureus
A. flavus
C. albicans
3
16
12
12
13
15
14
14
15
13
30
–
17
13
13
13
15
14
13
13
13
30
–
15
–
14
–
6a
1
instrument in potassium bromide discs. H and 13C NMR
6d
–
–
spectra were recorded using a Varian Mercury VXR-300
spectrometer (300 or 400 MHz for 1H NMR and 125 MHz
for 13C NMR), and chemical shifts were related to that of the
solvent DMSO-d6. Mass spectra were recorded using
GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spec-
trometers, with ionizing voltage of 70 eV. Elemental
analyses were carried out by the Microanalytical Center of
Cairo University, Giza, Egypt. 2-(4,5-Dimethoxy-2-
nitrophenylthio)acetic acid (1) and 6,7-dimethoxy-2H-1,4-
benzothiazin-3(4H)-one (2) were prepared as previously
described [33, 34].
7a
–
13
12
12
12
13
13
–
7c
–
7d
–
7e
–
9
–
11
–
Tetracycline
Amphotericin
–
18
21
Solution concentration of 20.0 mg/cm3 was tested
E. coli: Escherichia coli, Gram-negative bacteria
S. aureus: Staphylococcus aureus, Gram-positive bacteria
A. flavus: Aspergillus flavus, fungus
2-(Dimethylaminomethylene)-6,7-dimethoxy-2H-1,4-
benzothiazin-3(4H)-one (3, C13H16N2O3S)
C. albicans: Candida albicans, fungus
A mixture of 2.25 g compound 2 (10 mmol) and 2 cm3
DMF-DMA was heated under reflux for 10 h. The reaction
mixture was triturated with ethanol to give a solid product
that was collected by filtration and crystallized from
ethanol to give compound 3 as orange crystals. Yield
2.52 g (90%); m.p.: 218–220 °C; 1H NMR (300 MHz,
DMSO-d6): d = 3.09 (s, 6H, 2CH3), 3.65 (s, 6H, 2 OCH3),
6.57 (s, 1H, ArH), 6.71 (s, 1H, ArH), 7.26 (s, 1H, = CH),
9.49 (s, 1H, NH) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 43.08, 56.12, 56.53, 81.63, 101.74, 108.96, 110.02,
132.01, 144.68, 145.39, 146.72, 148.37, 167.30 ppm; IR
Gram-positive bacteria (Staphylococcus aureus) and Gram-
negative bacteria (Escherichia coli), in addition to the
pathogenic fungi Aspergillus flavus and Candida albicans.
The antimicrobial screening results were measured by the
average diameter of the inhibition zones, expressed in mm,
and are depicted in Table 3. The results showed that all
tested compounds displayed significant activities against
E. coli and S. aureus. Compounds 3, 7a, 7c–7e, 9, and 11
showed moderate activity against C. albicans, but only one
compound, enaminone 3, showed a high degree of activity
against A. flavus.
(KBr): m = 3,159 (NH), 1,652 (C=O) cm-1; MS: m/z
ꢀ
(%) = 281 (M? ? 1, 33), 280 (M?, 83), 266 (15), 265
(58), 100 (61), 85 (30), 77 (40), 68 (100).
Conclusion
Reaction of enaminone 3 with active methylene
compounds 4a–4d
The novel enaminone 3 was proved to be a valuable syn-
thon for synthesis of fused 1,4-thiazines, such as pyrido
[3,2-b][1,4]benzothiazine derivatives, 2-(arylhydrazono)-6,
7-dimethoxy-2H-1,4-benzothiazin-3(4H)-ones, 8,9-dime-
thoxy-11H-[1,2,4]triazolo[30,40:3,4][1,2,4]triazino[6,5-b][1,4]
benzothiazine (10), and 10,11-dimethoxy-13H-benzimi-
dazo[20,10:3,4][1,2,4]triazino[6,5-b][1,4]benzothiazine (13).
The structure of the newly synthesized compounds was
To a solution of 1.40 g 3 (5 mmol) in 15 cm3 glacial acetic
acid in the presence of 0.5 g ammonium acetate was added
acetylacetone, ethyl acetoacetate, ethyl benzoylacetate, or
dibenzoylmethane (5 mmol). The reaction mixture was
heated under reflux for several hours. The reaction was
followed by TLC. The solvent was evaporated under
reduced pressure, and the solid precipitate was collected by
filtration and washed with ethanol to give the respective
product 6a–6d.
1
established on the basis of mass, IR, H NMR, and ele-
mental analyses. Also, the tautomeric structure of the
hydrazone compounds was discussed. The antitumor
activity of selected products showed that these compounds
are reactive against HCT-116 cell line more than HEPG2
and MCF. In addition, the antimicrobial activity of some
selected products showed significant activities against
E. coli, S. aureus, and A. flavus.
3-Acetyl-7,8-dimethoxy-2-methyl-10H-pyrido-
[3,2-b][1,4]benzothiazine (6a, C16H16N2O3S)
1
Yellow solid, yield 0.95 g (60%); m.p.: 298–300 °C; H
NMR (300 MHz, DMSO-d6): d = 2.32 (s, 3H, CH3), 2.67
123

Synthesis, reactions, and biological activity of 1,4-benzothiazine derivatives
665
(s, 3H, COCH3), 3.08 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
6.60 (s, 1H, ArH), 6.75 (s, 1H, ArH), 8.14 (s, 1H, pyridine-
H), 10.30 (s, 1H, NH) ppm; 13C NMR (125 MHz, DMSO-
d6): d = 14.26, 21.61, 56.27, 56.69, 102.90, 109.21,
111.30, 127.55, 128.34, 133.26, 140.69, 144.64, 148.32,
added dropwise over a period of 20 min a solution of the
appropriate arenediazonium chloride, prepared as usual by
diazotizing the respective aniline (5 mmol) in 3 cm3
hydrochloric acid (6 M). The whole mixture was then left in
a refrigerator overnight. The precipitated solid was col-
lected, washed with water, and finally crystallized from the
appropriate solvent to give the respective hydrazone 7a–7f.
ꢀ
160.37, 164.80, 199.0 ppm; IR (KBr): m = 3,162 (NH),
1,710 (C=O) cm-1; MS: m/z (%) = 317 (M? ? 1, 1), 316
(M?, 2), 192 (9), 110 (12), 83 (5).
6,7-Dimethoxy-2-(4-methoxyphenylhydrazono)-2H-1,4-
benzothiazin-3(4H)-one (7a, C17H17N3O4S)
Ethyl 7,8-dimethoxy-2-methyl-10H-pyrido[3,2-b][1,4]-
benzothiazine-3-carboxylate (6b, C17H18N2O4S)
Pale yellow solid, yield 75%; m.p.: 352 °C (ethanol/
1
Pale yellow solid, yield 1.07 g (62%); m.p.: 170–172 °C;
1H NMR (300 MHz, DMSO-d6): d = 1.28 (t, J = 7 Hz,
3H, CH3), 2.70 (s, 3H, CH3), 3.66 (s, 3H, OCH3), 3.67 (s,
3H, OCH3), 4.30 (q, J = 7 Hz, 2H, CH2), 6.61 (s, 1H,
ArH), 6.83 (s, 1H, ArH), 8.48 (s, 1H, pyridine-H), 10.19 (s,
1H, NH) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 14.56, 25.00, 56.14, 56.50, 61.76, 102.82, 109.59,
111.53, 123.20, 125.90, 131.56, 140.42, 145.12, 148.57,
dioxane); H NMR (300 MHz, DMSO-d6): d = 3.50 (s,
3H, OCH3), 3.62 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 6.69
(s, 1H, ArH), 6.89 (s, 1H, ArH), 7.42 (d, J = 8 Hz, 2H,
ArH), 7.90 (d, J = 8 Hz, 2H, ArH), 10.20 (s, 1H, NH),
10.90 (s, 1H, NH) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 56.30, 56.64, 57.47, 102.20, 109.22, 115.85, 120.11,
123.53, 129.27, 129.89, 142.10, 145.18, 147.34, 156.21,
ꢀ
163.10 ppm; IR (KBr): m = 3,426, 3,200 (2NH), 1,652
(C=O) cm-1; MS: m/z (%) = 360 (M? ? 1, 28), 359 (M?,
28), 265 (88), 122 (96), 108 (64), 77 (92), 57 (100).
ꢀ
161.80, 165.40, 165.69 ppm; IR (KBr): m = 3,202 (NH),
1,720 (C=O) cm-1; MS: m/z (%) = 347 (M? ? 1, 7), 346
(M?, 24), 137 (25), 75 (100).
6,7-Dimethoxy-2-phenylhydrazono-2H-1,4-benzothiazin-
3(4H)-one (7b, C16H15N3O3S)
Ethyl 7,8-dimethoxy-2-phenyl-10H-pyrido[3,2-b][1,4]-
benzothiazine-3-carboxylate (6c, C22H20N2O4S)
Yellow solid, yield 80%; m.p. [ 320 °C (ethanol/dioxane);
1H NMR (300 MHz, DMSO-d6): d = 3.72 (s, 3H, OCH3),
3.78 (s, 3H, OCH3), 6.84 (s, 1H, ArH), 6.88 (s, 1H, ArH),
7.22–7.36 (m, 5H, ArH), 9.74 (s, 1H, NH), 10.81 (s, 1H,
NH) ppm; 13C NMR (125 MHz, DMSO-d6): d = 56.36,
56.58, 103.94, 109.0, 118.13, 119.24, 119.78, 132.24,
142.57, 144.25, 145.61, 147.27, 154.01, 162.24 ppm; IR
1
Yellow solid, yield 1.16 g (57%); m.p.: 154–156 °C; H
NMR (300 MHz, DMSO-d6): d = 1.20 (t, J = 7 Hz, 3H,
CH3), 3.70 (s, 6H, 2OCH3), 4.39 (q, J = 7 Hz, 2H, CH2),
6.72 (s, 1H, ArH), 6.92 (s, 1H, ArH), 7.20–7.78 (m, 5H,
ArH), 8.06 (s, 1H, pyridine-H), 10.82 (s, 1H, NH) ppm; 13
C
NMR (125 MHz, DMSO-d6): d = 14.54, 56.11, 56.70,
62.05, 103.66, 110.34, 112.46, 119.60, 124.65, 127.91,
129.34, 129.56, 130.71, 133.15, 137.21, 139.47, 145.20,
(KBr): m = 3,310, 3,212 (2NH), 1,662 (C=O) cm-1; MS:
ꢀ
m/z (%) = 329 (M?, 31), 224 (52), 196 (32), 152 (23), 105
(45), 95 (34), 93 (87), 77 (100).
ꢀ
160.97, 164.28, 165.23 ppm; IR (KBr): m = 1,728 (C=O)
cm-1; MS: m/z (%) = 409 (M? ? 1, 6), 408 (M?, 25), 331
(61), 91 (83), 77 (100).
2-(4-Chlorophenylhydrazono)-6,7-dimethoxy-2H-1,4-
benzothiazin-3(4H)-one (7c, C16H14ClN3O3S)
Orange solid, yield 90%; m.p.: 300 °C (ethanol); 1H NMR
(300 MHz, DMSO-d6): d = 3.62 (s, 3H, OCH3), 3.74 (s,
3H, OCH3), 6.62 (s, 1H, ArH), 6.82 (s, 1H, ArH), 7.20 (d,
J = 8 Hz, 2H, ArH), 8.01 (d, J = 8 Hz, 2H, ArH), 9.86 (s,
1H, NH), 10.54 (s, 1H, NH) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 56.35, 56.59, 104.28, 110.05, 116.31,
118.0, 121.08, 129.14, 132.35, 143.11, 145.08, 148.34,
3-Benzoyl-7,8-dimethoxy-2-phenyl-10H-pyrido-
[3,2-b][1,4]benzothiazine (6d, C26H20N2O3S)
1
Pale yellow solid, yield 62%; m.p.: 222–224 °C; H NMR
(300 MHz, DMSO-d6): d = 3.69 (s, 3H, OCH3), 3.76 (s,
3H, OCH3), 6.68 (s, 1H, ArH), 6.86 (s, 1H, ArH), 7.10–
7.74 (m, 10H, ArH), 7.98 (s, 1H, pyridine-H), 10.11 (s, 1H,
-1
ꢀ
NH) ppm; IR (KBr): m = 3,223 (NH), 1,689 (C=O) cm
;
ꢀ
154.07, 165.24 ppm; IR (KBr): m = 3,327, 3,185 (2NH),
MS: m/z (%) = 441 (M? ? 1, 10), 440 (M?, 25), 103 (43),
91 (100), 77 (62).
1,671 (C=O) cm-1; MS: m/z (%) = 366 (M? ? 2, 6), 365
(M? ? 1, 25), 364 (M?, 45), 363 (63), 224 (100), 196 (17),
127 (45), 111 (36), 99 (27), 75 (28).
Preparation of 2-(arylhydrazono)-6,7-dimethoxy-2H-
1,4-benzothiazin-3(4H)-ones 7a–7f
2-(4-Bromophenylhydrazono)-6,7-dimethoxy-2H-1,4-
benzothiazin-3(4H)-one (7d, C16H14BrN3O3S)
To a stirred solution of 1.40 g enaminone 3 (5 mmol) in
20 cm3 ethanol was added 0.7 g sodium acetate trihydrate
(0.005 mol), and the mixture was cooled in an ice bath to
0–5 °C. To the resulting solution, while being stirred, was
Orange solid, yield 89%; m.p.: 324–326 °C (ethanol/dioxane);
1H NMR (300 MHz, DMSO-d6): d = 3.60 (s, 3H, OCH3),
3.78 (s, 3H, OCH3), 6.65 (s, 1H, ArH), 6.78 (s, 1H, ArH), 7.80
(d, J = 9 Hz, 2H, ArH), 8.21 (d, J = 9 Hz, 2H, ArH), 9.89
123

666
E. M. H. Abbas, T. A. Farghaly
(s, 1H, NH), 10.69 (s, 1H, NH) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 56.10, 56.42, 102.51, 108.66, 116.18,
119.28, 120.36, 132.08, 136.47, 142.12, 145.70, 146.94,
156.21, 164.97 ppm; IR (KBr): ꢀm = 3,429, 3,182 (2NH),
1,669 (C=O) cm-1; MS: m/z (%) = 410 (M? ? 2, 2), 409
(M? ? 1, 38), 408 (M?, 11), 407 (28), 224 (100), 211 (12),
171 (20), 108 (13), 91 (46), 85 (16), 64 (30).
2.82 (s, 3H, COCH3), 6.90 (s, 2H, ArH), 8.13 (s, 1H,
triazole-H), 11.01 (s, 1H, NH) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 56.48, 56.50, 107.05, 112.38, 120.59,
122.87, 128.94, 130.22, 132.35, 145.55, 149.24,
156.00 ppm; IR (KBr): m = 3,325 (NH) cm-1; MS: m/z
ꢀ
(%) = 302 (M?, 16), 301 (26), 196 (45), 164 (50), 108
(58), 84 (45), 81 (34), 77 (37), 52 (100).
6,7-Dimethoxy-2-(4-nitrophenylhydrazono)-2H-1,4-
benzothiazin-3(4H)-one (7e, C16H14N4O5S)
10,11-Dimethoxy-13H-benzimidazo[20,10:3,4][1,2,4]-
triazino[6,5-b][1,4]benzothiazine (13, C17H13N5O2S)
Red solid, yield 89%; m.p.: 306–308 °C (dioxane); 1H
NMR (300 MHz, DMSO-d6): d = 3.59 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 6.81 (s, 1H, ArH), 6.93 (s, 1H, ArH),
7.92 (d, J = 9 Hz, 2H, ArH), 8.10 (d, J = 9 Hz, 2H, ArH),
10.02 (s, 1H, NH), 10.80 (s, 1H, NH) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 56.30, 56.62, 102.18, 112.24,
116.64, 117.08, 125.18, 130.29, 132.40, 145.27, 145.11,
Yellow solid, yield 92%; m.p. 246–248 °C (dioxane); H
NMR (300 MHz, DMSO-d6): d = 3.46 (s, 3H, OCH3),
3.65 (s, 3H, OCH3), 6.92 (s, 1H, ArH), 7.02 (s, 1H, ArH),
1
-1
ꢀ
10.55 (s, 1H, NH) ppm; IR (KBr): m = 3,210 (NH) cm
;
MS: m/z (%) = 351 (M?, 100), 91 (43), 77 (97).
Pharmacology
ꢀ
150.43, 154.25, 165.61 ppm; IR (KBr): m = 3,430, 3,270
(2NH), 1,668 (C=O) cm-1; MS: m/z (%) = 375 (M? ? 1,
15), 374 (M?, 100), 225 (20), 224 (98), 210 (16), 196 (25),
150 (19), 108 (27), 91 (17), 85 (21), 76 (36), 64 (67).
Cytotoxic activity against human breast cancer (MCF-7)
in vitro
The method applied is similar to that reported by Skehan
and Storeng [35] using Sulfo-Rhodamine-B stain (SRB).
Cells were plated in a 96-multiwell plate (104 cells/well)
for 24 h before treatment with the test compound to allow
attachment of cells to the wall of the plate, then different
concentrations of the compound under test (0, 1.0, 2.5, 5,
and 10 lg/cm3) were added to the cell monolayer in trip-
licate wells per individual dose. The monolayer cells were
incubated with the compounds for 48 h at 37 °C in atmo-
sphere of 5% CO2. After 48 h, cells were fixed, washed,
and stained with SRB stain. Excess stain was washed with
acetic acid, attached stain was recovered with Tris–ethy-
lenediamine tetraacetic acid (EDTA) buffer, and color
intensity was measured in an enzyme-linked immunosor-
bent assay (ELISA) reader. The relation between surviving
fraction and drug concentration was plotted to obtain the
survival curve of tumor cell line, and the IC50 was calcu-
lated. The results are summarized in Table 2.
2-(4-Acetylphenylhydrazono)-6,7-dimethoxy-2H-1,4-
benzothiazin-3(4H)-one (7f, C18H17N3O4S)
Orange crystals, yield 89%; m.p.: 270 °C (ethanol/diox-
1
ane); H NMR (300 MHz, DMSO-d6): d = 2.60 (s, 3H,
COCH3), 3.69 (s, 6H, 2OCH3), 6.75 (s, 1H, ArH), 6.84 (s,
1H, ArH), 7.34 (d, J = 9 Hz, 2H, ArH), 7.86 (d, J = 9 Hz,
2H, ArH), 10.16 (s, 1H, NH), 10.90 (s, 1H, NH) ppm; 13C
NMR (125 MHz, DMSO-d6): d = 24.16, 56.21, 56.60,
102.24, 111.56, 115.27, 118.64, 128.02, 130.28, 131.84,
144.38, 146.0, 153.29, 155.34, 164.05, 198.28 ppm; IR
ꢀ
(KBr): m = 3,437, 3,220 (2NH), 1,710, 1,656 (2C=O)
cm-1; MS: m/z (%) = 372 (M? ? 1, 7), 371 (M?, 32), 311
(10), 224 (54), 211 (12), 120 (61), 105 (26), 91 (33), 84
(28), 77 (64), 55 (100).
Preparation of compounds 10 and 13
A stirred solution of 1.40 g enaminone 3 (5 mmol) in
20 cm3 pyridine was cooled in an ice bath to 0–5 °C. To the
resulting solution, while being stirred, was added dropwise
over a period of 20 min a solution of the appropriate het-
erocyclic diazonium salt, prepared as usual by diazotizing
the respective heterocyclic amine (5 mmol) in 3 cm3 nitric
acid. The whole mixture was then left in a refrigerator
overnight. The precipitated solid was collected, washed with
water, and finally crystallized from the appropriate solvent
to give the respective compounds 10 or 13.
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