Synthesis and antimicrobial activity of novel bis-azaphenothiazines

Article abstract of DOI:10.1007/s00044-012-0454-7

N-alkylation of azaphenothiazines using dibromoalkanes or dibromoalkenes did not result in the formation of bis-azaphenothiazines under known conditions such as refluxing, for more than 100 h, with NaNH₂/xylene or NaH/toluene. However, when the same reaction was tried with NaH/DMF at -5 C to r.t., it yielded the desired product in 68-71 % yield. These novel bis-azaphenothiazines containing a suitable alkyl or alkenyl spacer were found to possess moderate to significant antimicrobial activities against three gram positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermis), four gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) as well as four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Candida albicans). Compounds 2b-d were found to exhibit strong antifungal activity, comparable to the standard drug miconazole against A. niger and A. fumigatus.

Full text of DOI:10.1007/s00044-012-0454-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4459–4467
DOI 10.1007/s00044-012-0454-7
ORIGINAL RESEARCH
Synthesis and antimicrobial activity of novel
bis-azaphenothiazines
•
Khushbu Kushwaha Rajeev Sakhuja
•
Subhash C. Jain
Received: 23 August 2012 / Accepted: 26 December 2012 / Published online: 10 January 2013
Ó Springer Science+Business Media New York 2013
Abstract N-alkylation of azaphenothiazines using dib-
romoalkanesordibromoalkenesdidnot resultintheformation
of bis-azaphenothiazines under known conditions such as
refluxing, for more than 100 h, with NaNH2/xylene or NaH/
toluene. However, when the same reaction was tried with
NaH/DMF at -5 °C to r.t., it yielded the desired product in
68–71 % yield. These novel bis-azaphenothiazines contain-
ing a suitable alkyl or alkenyl spacer were found to possess
moderate to significant antimicrobial activities against three
gram positive bacteria (Staphylococcus aureus, Bacillus
subtilis, and Staphylococcus epidermis), four gram negative
bacteria (Escherichia coli, Pseudomonas aeruginosa, Sal-
monella typhi, and Klebsiella pneumoniae) as well as four
fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus
flavus, and Candida albicans). Compounds 2b–d were found
to exhibit strong antifungal activity, comparable to the stan-
dard drug miconazole against A. niger and A. fumigatus.
et al., 1971), neuropharmacologic (Atkinson et al., 1971),
ganglioplegic (Giuliano et al., 1965), and antihypertensive
(Beschke and Schuler, 1964). Besides these, some 1-azaphe-
nothiazine derivatives and their mesoionic compounds also
showed antimicrobial activity (Swati et al., 1996) against
Aspergillus flavus, Aspergillus niger, Candida lunata, Esche-
richia coli, andStaphylococcus aureus. Despite these activities,
work on various other derivatives of azaphenothiazines did not
take off because of the problems associated with their synthesis
as well as alkylation. It required harsh reaction conditions
which normally lead to the formation of side products and
hence poor yield of the final product. The above biological
potential of azaphenothiazines prompted us to synthesize novel
bis-azaphenothiazines as number of reports, in the literature,
indicated the enhancement of biological activity of bis-
heterocycles containing a suitable spacer over simple hetero-
cyclic compounds (Brana et al., 1997; Hernandez et al., 1995;
Nitiss et al., 1998; Chaires et al., 1997; Vennerstrom et al.,
1998;Yanget al., 2011;Mayetal., 2003). Since no reportas yet
is available on the bis-azaphenothiazines, we, therefore, took up
their synthesis in order to understand the combined biological
effects of the azaphenothiazines moieties containing a suitable
alkyl or alkenyl spacer. We have now developed a facile and
elegant synthesis of novel bis-azaphenothiazines and evaluate
their minimum inhibitory concentration (MIC) and zone of
inhibition for antimicrobial activity against various strains of
bacteria and fungi, and the details are presented here.
Keywords Azaphenothiazines Á
10H-benzo[b]pyrido[2,3-e][1,4]thiazines Á
Alkyl/alkenyl linker Á Bis-azaphenothiazines Á
Antimicrobial activity
Introduction
Azaphenothiazines have been known to possess numerous
biological activities such as antidepressant (Clark et al., 1965),
antitumor (Krystian et al., 2010), antiemetic (Gloria, 1968;
Lenke etal., 1971), antitussive (Gloria, 1968), analgesic (Lenke
Results and discussion
Chemistry
K. Kushwaha Á R. Sakhuja Á S. C. Jain (&)
Department of Chemistry, University of Delhi,
Delhi 110 007, India
The synthesis of bis-azaphenothiazines was done by
introducing an alkyl or alkenyl spacer between two
e-mail: jainsc48@hotmail.com
123

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Med Chem Res (2013) 22:4459–4467
azaphenothiazine moieties using N-alkylation or N-alkeny-
lation reaction. Earlier we have reported the synthesis of
7-substituted azaphenothiazines (Gupta et al., 2007) and
have now adopted an improved methodology for the syn-
thesis of 7-bromo-10H-benzo[b]pyrido[2,3-e][1,4]thiazine
(3d) (Scheme 1), starting from the previously synthesized
intermediate 2a. Bromination of 2a gave exclusively 2d,
suggesting that the bromination had only occurred at para
position in 2a. Compound 2d in KOH/acetone undergoes
Smiles rearrangement (Yale and Sowinski, 1958) involving
simultaneously inversion, cyclization, and deacetylation to
give 3d characterized as 7-bromo-10H-benzo[b]pyrido
[2,3-e][1,4]thiazine on the basis of spectral studies.
each integrating for one proton. The compound showed
(M??2) peak at m/z 369 and M? peak at m/z 367 corre-
sponding to molecular formula C13H10N3O3SBr indicating
that the monobromination has taken place. Thus compound
2d was identified as 2-acetylamino-5-bromophenyl-
(3-nitro-2-pyridyl)sulfide. The next step of the methodol-
ogy was the inversion, cyclization, and deacetylation of 2d
in KOH-acetone via Smiles rearrangement to obtain 3d.
The structure of 3d was assigned on the basis of detailed
spectral studies. Its 1H NMR spectra showed a mark
deviation in the chemical shift values than those observed
for compound 2d along with the absence of the acetyl
protons in the upfield region. It further showed a D2O
exchangeable peak for the NH proton at d 7.09. The mass
spectrum showed molecular ion peak M? at m/z 279 along
with M??2 peak at m/z 281 corresponding to molecular
formula C11H7BrN2S. Thus on the basis of above studies,
compound 3d was characterized 7-bromo-10H-benzo[b]
pyrido[2,3-e][1,4]thiazine.
Compound 2d was purified as yellow colored solid in
70 % yield. Its IR spectrum showed absorption band at
1,688 cm-1 for acetyl carbonyl and characteristic absorp-
tion bands at 1,557 and 1,435 cm-1 for the presence of
nitro group in the compound. The presence of acetyl group
was also confirmed by a singlet at d 2.09 for three protons
1
in the H NMR spectrum. This was further confirmed by
The next step of the synthesis involved alkylation at
10th position in 3a–d. Earlier N-alkylation of 1-azaphe-
nothiazine with different alkylbromides has been carried by
heating the reactants with either NaNH2 in xylene or NaH
in toluene for 30–40 h (Ledochowski et al., 1963; Beschke
and Schuler, 1964). However, when we tried similar con-
ditions for the synthesis of bis-azaphenothiazines using
1-azaphenothiazine and dibromoalkane or dibromoalkene
in 2:1 ratio, we did not obtain the desired product. The bis-
compounds could not also be obtained by varying reaction
conditions such as using K2CO3 in acetone/THF/DMF or
the presence of peak at d 168.9 for the carbonyl carbon and
at d 22.9 for the methyl group attached to the carbonyl
carbon. A broad singlet at d 7.96 (D2O exchangeable)
confirmed the presence of NH group. Multiplet at d
8.56–8.53 integrating for two protons and doublet at d 8.39
integrating for one proton were assigned to the protons of
the pyridyl nucleus. The bromination that had occured in
the phenyl ring was confirmed by the presence of three
aromatic protons of the phenyl ring that appeared as sin-
glet, doublet, and multiplet at d 7.67, 7.65, and 7.32–7.30
O
CH3
O2N
Scheme 1 Synthesis of
7-substituted azaphenothiazines
H
N
N
NH2
NH
NH2
O2N
b
a
S
R
N
R
S
R
N
R
S
3a R = H
3b R = Cl
3c R = F
2a R = H
2b R = Cl
2c R = F
1a R = H
1b R = Cl
1c R = F
R = H, Cl, F
O
CH3
O2N
O
CH3
NH
O2N
NH
c
N
S
Br
N
S
2d
2a
d
1
H
N
9
10
N
8
2
3
S
5
7
Br
6
4
a = Acetic anhydride/rt
3d
b,d = KOH, acetone, reflux
c
= Br2/CH3COOH/rt
123

Med Chem Res (2013) 22:4459–4467
4461
Et3N in DMF/THF. Finally, N-alkylation of 1-azapheno-
thiazines 3a–d using 1,6-dibromohexane proceeded in the
presence of NaH at -5 °C in DMF and the desired bis-
compounds 4a–d were obtained in good yield. Using
similar conditions, we have also synthesized compounds
5a–d and 6a–b using different alkyl/alkenyl dibromides
(Scheme 2). All the synthesized compounds were charac-
terized on the basis of their spectral studies. Compounds
1a–c, 2a–c, and 3a–c were obtained by the literature pro-
cedures and characterized by comparing their m.p. and
spectral data from those reported in the literature (Gupta
et al., 2007).
at d 45.1 in 13C NMR confirmed that the two nuclei have
been linked in 4a via 1,6 hexyl spacer. The rest of the
methylene protons of hexyl spacer were observed as two
multiplets at d 1.81–1.77 and d 1.56–1.49 each integrating
for four protons. The aromatic protons were observed as
double doublets, doublets at d 7.98–7.96 (J = 4.8 and
1.5 Hz), d 7.23–7.20 (J = 7.4 and 1.4 Hz), d 7.03
(J = 7.6 Hz), and d 6.82 (J = 8.1 Hz), respectively, each
integrating for two protons; multiplet and triplet at d
6.74–6.70 and 6.89 (J = 7.2 Hz) also integrating for two
protons clearly showing that 4a has been formed. The final
evidence came from its EIMS mass spectrum that showed
the molecular ion peak M? at m/z 482 corresponding to
molecular formula C28H28N4S2. Thus on the basis of above
studies 4a has been assigned structure 1,6-bis(10H-
benzo[b]pyrido[2,3-e][1,4]thiazin-10-yl]hexane.
We have also tried to repeat the simple N-alkylation of
1-azaphenothiazines under above mild conditions using
various alkyl/alkenyl bromides and obtained the desired
N-alkylated azaphenothiazines in good yield, without the
formation of any side product. So novel method involving
mild reaction conditions (instead of earlier harsh condi-
tions) have been developed by us to obtain not only
N-alkylated azaphenothiazines, but also bis-azaphenothia-
zines containing a suitable spacer of choice, in good yield.
Compounds 3a–d were separately subjected to alkyl-
ation using 1,6-dibromohexane and 1,4-dibromobutane in
the presence of NaH at -5 °C to obtain 4a–d and 5a–d,
respectively, which were characterized on the basis of their
complete spectral studies. The main evidence of the for-
Compounds 3a and 3d were also reacted with trans-1,
4-dibromobut-2-ene to yield 6a–b. The main evidence of the
formation of compound 6a came from its 1H NMR spectrum
as it showed the presence of multiplet at d 4.69–4.66, inte-
grating for four protons for the methylenes attached to the
two nitrogen atoms. The corresponding peak in 13C NMR
appeared at d 50.3 that revealed that the two nuclei have been
linked in 6a via trans-1,4-but-2-enyl spacer. The olefinic
protons of the linker were observed as multiplet at d
5.93–5.91 integrating for two protons. The aromatic protons
were observed as double doublets at d 7.96–7.95 (J = 4.5
and 1.2 Hz) for two protons, doublets at d 7.21 (J = 7.2 Hz),
d 6.99 (J = 7.4 Hz), and d 6.94 (J = 7.7 Hz) each inte-
grating for two protons, and multiplets at d 6.87–6.82 and
1
mation of compound 4a came from its H NMR spectrum
that showed the presence of a triplet at d 4.06, integrating
for four protons for the methylenes attached to the two
nitrogen atoms of the compound. The corresponding peak
H
Scheme 2 Synthesis of 7,
7-disubstituted bis-
azaphenothiazines
N
N
3a R = H
3b R = Cl
3c R = F
3d R = Br
R
a
S
a
Br
Br
Br
Br
a
Br
Br
R
S
R
S
N
N
N
R
S
N
N
N
N
N
N
S
N
S
N
S
N
R
R
6a R = H
6b R = 7-Br
R
4a R = H
4b R = Cl
4c R = F
4d R = Br
5a R = H
5b R = Cl
5c R = F
5d R = Br
a = NaH/DMF/-5 oC-rt
123

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Med Chem Res (2013) 22:4459–4467
d 6.74–6.69 integrating for four and two protons, respec-
tively. Its TOF mass spectrum showed the M??1 at m/z 453
corresponding to its molecular formula C26H20N4S2. Thus
on the basis of above studies compound 6a has been assigned
structure 1,4-bis(10H-benzo[b]pyrido[2,3-e][1,4]thiazin-
10-yl)but-2E-ene. Synthesized compounds 4a–d, 5a–d, and
6a–b were found to be completely soluble in chloroform.
solvent. Clinically antimicrobial drugs ciprofloxacin and
miconazole were used as the positive control and DMSO was
used for blank.
MIC is the minimum concentration of the compound,
which inhibits the visible growth of bacteria or fungi. For
the determination of zone of inhibition, inoculated MH
agar for bacteria and SD agar for fungi were separately
poured onto the sterilized petri dishes. The poured material
was allowed to set and thereafter the ‘‘CUPS’’ (08 mm
diameter) were made by punching into the agar surface
with a sterile cork borer and scooping out the punched part
of the agar. The test compound solution was added into
these cups with the help of a sterile syringe. The plates
were then incubated at 37 °C for 16 h for bacteria and
28–30 h for fungi. Ciprofloxacin and miconazole were
used as standard drugs for bacteria and fungi, respectively.
The experiments were repeated three times to reduce the
error and the average value of antibacterial and antifungal
activities are represented in Tables 3 and 4.
Biological activity
Compounds 2a–d, 4a–d, 5a–d, and 6a–b were screened for
their invitro antimicrobial activities to determine zone of
inhibition at 100 lg/ml against three gram positive bacteria
(Staphylococcus aureus MTCC 096, Bacillus subtilis MTCC
441, and Staphylococcus epidermis MTCC 435), four gram
negative bacteria (Escherichia coli MTCC 443, Pseudomo-
nas aeruginosa MTCC 424, Salmonella typhi MTCC 733,
and Klebsiella pneumonia MTCC 432) as well as four fungi
(Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC
343, Aspergillus flavus MTCC 277, and Candida albicans
MTCC 227) using cup plate method (Chuickshank et al.,
1975). Inoculated Muller–Hilton agar for bacteria and sab-
ouraud dextrose agar for fungi were poured separately into
the sterilized petridishes (25–30 ml: each petri dish). The
poured material was allowed to set (30 min) and thereafter
the ‘‘CUPS’’ (08 mm diameter) were made by punching into
the agar surface with a sterile cork borer and scooping out the
punched part of the agar. The test compound solution
(0.1 ml) was placed with the help of a micro pipette into
these cups. The plates were incubated at 37 °C for 16–18 h
for bacteria and 28–30 h for fungi, and the results were
recorded. The test solution was prepared using DMSO as
The results depicted in Tables 1, 2, 3, and 4 showed that
most of the compounds possessed moderate activity against
the bacterial strains. However, monoacetates 2b–d have
shown very good activity against P. aeruginosa which
suggested that halogen substitution in the ring increases the
biological activity. Among halo substituted compounds,
bromo compound 2c is the most active against A. niger and
A. fumigatus and its antifungal activity was comparable to
the standard drug miconazole at 100 lg/ml. Compound 4d
was found to possess significant activity against S. epi-
dermis. It was also seen that azaphenothiazines 4a–d which
were separated via –(CH2)6– linker gave better zone of
inhibition as compared to the compounds 5a–d and 6a–b
where azaphenothiazines were linked via –(CH2)4– or –
Table 1 Antibacterial
Compound
S. aureus B. subtilis S. epidermis E. coli P. aeruginosa S. typhi K. pneumoniae
evaluation of 2a–d, 4a–d, 5a–d,
and 6a–b (zone of inhibition in
mm)
2a
2b
2c
2d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
11
12
13
12
13
12
12
12
11
12
11
12
12
12
12
12
12
12
12
12
12
13
10
11
12
11
10
11
18
11
13
12
13
13
10
12
14
12
12
12
11
10
11
16
10
12
14
11
12
11
12
12
11
12
11
12
11
12
20
12
14
14
14
11
12
13
13
13
11
10
12
12
10
18
12
12
12
11
12
12
12
11
12
12
12
10
11
11
19
11
13
11
13
12
11
11
12
12
11
11
11
11
11
17
Bold values represent the zone
of inhibition of compounds
showing good to promising
activity
Ciprofloxacin 19
Zone of inhibition at 100 lg/ml
123

Med Chem Res (2013) 22:4459–4467
4463
Table 2 Antifungal evaluation of 2a–d, 4a–d, 5a–d, and 6a–b (Zone
of inhibition in mm)
Table 4 Antifungal evaluation of 2a–d, 4a–d, 5a–d, and 6a–b (MIC
in mm)
Compound
A. niger
A. fumigatus
A. flavus
C. albicans
Compound
A. niger
A. fumigatus
A. flavus
C. albicans
2a
–
14
18
16
11
–
–
12
16
14
10
12
11
11
–
12
12
10
12
12
10
12
12
–
12
14
14
13
12
12
13
11
–
2a
[50
6.25
6.25
6.25
25
[50
12.5
6.25
6.25
[50
12.5
25
12.5
25
12.5
6.25
6.25
6.25
12.5
12.5
6.25
25
2b
2b
2c
2c
[50
12.5
25
2d
2d
4a
4a
4b
4b
[50
50
[50
12.5
12.5
[50
12.5
25
4c
10
11
–
4c
4d
4d
[50
[50
[50
50
25
5a
5a
[50
50
[50
25
5b
10
11
11
12
–
11
10
12
13
12
15
12
12
10
12
11
17
11
10
11
12
10
19
5b
5c
5c
[50
12.5
6.25
12.5
6.25
[50
50
5d
5d
50
[50
12.5
[50
12.5
6a
6a
12.5
[50
6.25
12.5
[50
6.25
6b
6b
Miconazole
18
Miconazole
Bold values represent the zone of inhibition of compounds showing
good to promising activity
– Compounds which does not show any activity, zone of inhibition at
100 lg/ml
activity against C. albicans. Among the final compounds,
6a–b showed good activity against A. fumigatus.
(CH2–CH=CH–CH2)– linker, when tested on most of the
bacterial strains. So optimum lipophilic chain length
required for activity could be C6 alkyl chain.
Against the fungal strains, monoacetates were found to
possess even better activity as compared to bis-compounds.
Monoacetates 2b–d showed promising antifungal activity
against A. niger, A. fumigatus, and C. albicans. Compounds
2c and 2d showed antifungal activity, comparable to that of
standard drug miconazole, against A. niger and A. fumig-
atus whereas compounds 2b and 2c showed moderate
Conclusions
In summary, we have developed a novel methodology for
the N-alkylation of azaphenothiazines and used it to syn-
thesize ten novel bis-azaphenothiazines in good yield. The
structures of all the compounds were fully characterized on
the basis of their detailed spectral studies. Antimicrobial
Table 3 Antibacterial
evaluation of 2a–d, 4a–d, 5a–d,
and 6a–b (MIC in mm)
Compound
S. aureus B. subtilis S. epidermis E. coli P. aeruginosa S. typhi K. pneumoniae
2a
2b
2c
2d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
[50
25
25
25
25
[50
12.5
6.25
[50
12.5
50
12.5
6.25
6.25
6.25
50
12.5
12.5
12.5
50
50
6.25
[50
6.25
25
6.25
25
12.5
25
25
25
6.25
6.25
[50
12.5
6.25
12.5
6.25
12.5
25
6.25
25
25
12.5
12.5
25
25
25
50
25
25
12.5
6.25
[50
12.5
25
6.25
6.25
6.25
50
50
25
12.5
[50
50
50
25
50
12.5
25
25
25
[50
50
25
12.5
50
[50
12.5
12.5
[50
6.25
12.5
[50
[50
50
12.5
12.5
25
12.5
50
50
[50
25
[50
[50
6.25
50
12.5
6.25
50
Ciprofloxacin 6.25
6.25
6.25
6.25
123

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Med Chem Res (2013) 22:4459–4467
study on these compounds has indicated that compounds
4a–d, 5a–d, and 6a–b possessed moderate to significant
activities, while acetates 2b–d were found to exhibit strong
antifungal activity which was comparable to the standard
drug miconazole at 100 lg/ml against two strains A. niger
and A. fumigatus.
distilled off and 50 ml of water was added to obtain light
brown colored residue which was filtered and crystallized
from petrol/ethylacetate to give 3d as yellow solid. Yield
0.75 g (83 %); m.p. 204–206 °C; IR mmax 3229 (–NH),
3056, 2986 (aromatic –CH strechings), 1513, 1428 (–C=
C– strechings), 1313, 1283 (–CN), 1155, 1120, 1082, 959,
1
902, 851, 768 cm-1; H NMR (CDCl3, 300 MHz) d 7.82
(s, 1H), 7.17 (d, 1H, J = 7.1 Hz), 7.09 (brs, 1H, NH), 7.05
(s, 1H), 6.84 (s, 1H, D2O exchangeable), 6.72–6.70 (m,
1H), 6.42 (d, 1H, J = 7.8 Hz); 13C NMR (d, CDCl3,
75.47 MHz) 148.3, 145.9, 143.1, 134.9, 129.2, 128.3,
120.7, 118.8, 118.4, 115.4, 113.4; mass spectral data, EIMS
m/z (%): 281 (M??2, 89), 279 (M?, 92), 204 (20), 200
(90), 126 (23).
Experimental section
Melting points were determined on an electronic apparatus
and are uncorrected. Infrared (FTIR) spectra were deter-
mined on a Perkin Elmer-2000 Spectrophotometer instru-
ment. 1H NMR spectra were recorded on Bruker AC (300/
400 MHz), and 13C NMR spectra were recorded on Bruker
AC (75.47/100 MHz) using tetramethylsilane as an internal
standard in CDCl3. Elemental analyses were performed on
a Perkin Elmer series 11, CHNS/O analyzer 2400. Mass
spectra were recorded on Jeol-JMS-DX 303 mass spec-
trometer. Substituted azaphenothiazines were prepared by
literature procedures (Gupta et al., 2007) starting from the
corresponding anilines.
General procedure
Synthesis of bis-azaphenothiazines 4a–d, 5a–d, and 6a–b
To a stirred solution of NaH (6.36 mmol) in 10 ml of dry
DMF at -5 °C,
a
solution of azaphenothiazine
(2.12 mmol) in DMF was added dropwise under inert
atmosphere. To the deep brown colored suspension, a
solution of dibromoalkane/dibromoalkene (1.06 mmol) in
DMF was added dropwise at -5 °C. The reaction mixture
was stirred at -5 °C for 30 min and then at room tem-
perature for another 8–9 h. The course of the reaction was
monitored by TLC. After completion of the reaction, the
reaction mixture was quenched with ice cold water and
then extracted with diethyl ether. The organic layer was
separated, washed with water, and dried over anhydrous
sodium sulfate. The solvent was removed under reduced
pressure and the crude product was subjected to column
chromatography in petroleum ether. Elutions with petro-
leum ether/ethyl acetate yielded pure compounds as white
crystalline solids.
2-Acetylamino-5-bromophenyl-(3-nitro-2-pyridyl)
sulfide (2d)
2-Acetylaminophenyl-(3-nitro-2-pyridyl)sulfide 2a (1.00 g,
3.46 mmol) was dissolved in 20 ml of acetic acid under
anhydrous conditions at room temperature. Bromine
(0.5 ml) in 10 ml of acetic acid was added dropwise in
10 min. Reaction mixture was stirred for 30 min at the
same temperature. The product that precipitated was fil-
tered, washed with water, and dried and crystallized from
chloroform/methanol to obtain 2d in pure form as yellow
colored solid. Yield 0.90 g (70 %); m.p. 184–186 °C;
IR mmax 3370 (–NH), 2923, 2852 (aromatic –CH stretchings),
1688 (–COCH3), 1557 (–NO2), 1435 (–NO2), 1337, 1292
(–CN), 1122, 1067, 856 cm-1
;
1H NMR (CDCl3,
1,6-bis(10H-benzo[b]pyrido[2,3-e][1,4]thiazin-10-
300 MHz) d 8.56–8.53 (m, 2H), 8.39 (d, 1H, J = 7.9 Hz),
7.96 (brs, 1H, NH, D2O exchangeable), 7.67 (s, 1H), 7.65
(d, 1H, J = 8.7 Hz), 7.32–7.30 (m, 1H), 2.09 (s, 3H,
–COCH3); 13C NMR (CDCl3, 75.47 MHz) d 168.9, 155.1,
150.6, 144.0, 139.8, 132.9, 128.6, 127.7, 124.1, 123.7,
119.0, 22.9; Mass Spectral data, EIMS m/z (%) 369
(M??2, 29), 367 (M?, 30), 353 (19), 325 (10), 288 (100),
246 (20).
yl]hexane (4a)
Yield 0.85 g (71 %); m.p. 160–162 °C; IR mmax 2921,
2849, 2360, 2336 (aromatic –CH strechings), 1868, 1731,
1633, 1540 (–C=C– strechings), 1228 (–CN), 1148, 1047,
;
872, 818, 756 (C–S), 665 cm-1 1H NMR (CDCl3,
300 MHz) d 7.98–7.96 (dd, 2H, J = 4.8 and 1.5 Hz),
7.23–7.20 (dd, 2H, J = 7.4 and 1.4 Hz), 7.14–7.08 (m,
2H), 7.03 (d, 2H, J = 7.6 Hz), 6.89 (t, 2H, J = 7.2 Hz),
6.82 (d, 2H, J = 8.1 Hz), 6.74–6.70 (m, 2H), 4.06 (t, 4H,
2x [ N–CH2–), 1.81–1.77 (m, 4H, 2x–CH2–), 1.56–1.49
(m, 4H, 2x–CH2–); 13C NMR (CDCl3, 75.47 MHz) d
145.4, 134.4, 127.9, 127.4, 123.1, 118.0, 115.8, 45.1, 30.0,
27.1; mass spectral data, EIMS m/z (%): 482 (M?, 75), 283
7-Bromo-10H-benzo[b]pyrido[2,3-e][1,4]thiazine (3d)
To a stirred refluxing solution of 2d (1.20 g, 3.26 mmol) in
50 ml of distilled acetone, powdered KOH (0.55 g,
9.8 mmol) was added in small portions in about 10 min.
The mixture was refluxed for 4 h. The solvent was then
123

Med Chem Res (2013) 22:4459–4467
4465
(23), 267 (21), 259 (23), 239 (10), 225 (25), 213 (62), 200
(70), 136 (15); anal. calcd. for C28H26N4S2. Calculated: C
69.68 %, H 5.43 %, N 11.61 %, found: C 70.01 %, H
5.24 %, N 11.12 %.
75.47 MHz) d 146.2, 135.2, 131.1, 130.2, 128.3, 120.7,
118.9, 117.4, 45.6, 27.5, 27.2; TOF ES 1 MS m/z 640
(M??2); anal. calcd. for C28H24Br2N4S2. Calculated: C
52.51 %, H 3.78 %, N 8.75 %, found: C 54.02 %, H
3.07 %, N 8.67 %.
1,6-Bis(7-chloro-10H-benzo[b]pyrido[2,3-e][1,4]
thiazin-10-yl]hexane (4b)
1,4-Bis(10H-benzo[b]pyrido[2,3-e][1,4]thiazin-10-
yl]butane (5a)
Yield 0.82 g (70 %); m.p. 158–160 °C; IR mmax 2925,
2857(aromatic –CH strechings), 1560, 1474, 1442 (–C=
C– strechings), 1337, 1209 (–CN), 1159, 1126, 1113, 1047,
884, 798, 787 (C–S) cm-1; 1H NMR (CDCl3, 300 MHz) d
7.98–7.96 (dd, 2H, J = 4.8 and 1.5 Hz), 7.22–7.20 (dd,
2H, J = 7.4 and 1.3 Hz), 7.06–7.03 (dd, 2H, J = 8.7 and
2.3 Hz), 7.00 (d, 2H, J = 2.2 Hz), 6.74–6.73 (m, 2H), 6.71
(d, 2H, J = 8.4 Hz), 4.02 (t, 4H, J = 7.2 Hz, 2x [
N–CH2–), 1.77–1.72 (m, 4H, 2x–CH2–), 1.59–1.47 (m, 4H,
2x–CH2–); 13C NMR (CDCl3, 75.47 MHz) d 155.1, 145.6,
142.0, 134.6, 128.0, 127.6, 126.9, 123.6, 118.2, 116.7,
116.4, 45.1, 26.9, 26.7; mass spectral data, EIMS m/z (%):
554 (M??4, 9), 552 (M??2, 28), 550 (M?, 88), 481 (15),
289 (30), 261 (21), 247 (48), 234 (51), 154 (100), 138 (45),
137 (62), 136 (78); anal. calcd. for C28H24Cl2N4S2. Cal-
culated: C 60.97 %, H 4.39 %, N 10.16 %, found: C
61.47 %, H 4.31 %, N 10.59 %.
Yield 0.79 g (70 %); m.p. 133 °C; IR mmax 2929, 2852
(aromatic –CH strechings), 1589, 1448 (–C=C– strech-
ings), 1411, 1372, 1259 (–CN), 1180, 1043, 776, 755
(C–S), 641 cm-1
; d
1H NMR (CDCl3, 400 MHz)
7.98–7.97 (dd, 2H, J = 6.6 and 1.4 Hz), 7.23–7.21 (dd,
2H, J = 6.4 and 1.4 Hz), 7.07 (d, 2H, J = 7.6 Hz) 7.03 (d,
2H, J = 6.8 Hz), 6.90 (d, 2H, J = 6.4 Hz), 6.82 (d, 2H,
J = 7.6 Hz), 6.73 (m, 2H), 4.14 (t, 4H, J = 7.2 Hz,
2x [ N–CH2–), 1.87–1.83 (m, 4H, 2x–CH2); 13C NMR
(CDCl3, 100 MHz) d 155.1, 145.0, 142.8, 134.1, 127.5,
127.0, 122.8, 121.5, 117.7, 117.3, 115.5, 44.4, 24.0; TOF
ES ? MS m/z 455 (M??1). Anal. calcd. for C26H22N4S2.
Calculated: C 68.69 %, H 4.88 %, N 12.32 %, found: C
70.24 %, H 4.34 %, N 11.45 %.
1,4-Bis(7-chloro-10H-benzo[b]pyrido[2,3-e][1,4]
1,6-Bis(7-fluoro-10H-benzo[b]pyrido[2,3-e][1,4]
thiazin-10-yl]butane (5b)
thiazin-10-yl]hexane (4c)
Yield 0.75 g (68 %); m.p. 140–142 °C; IR mmax 2925,
2854(aromatic –CH strechings), 1587, 1560 (–C=C– str-
echings), 1415, 1262 (–CN), 1111, 782, 802, 758 (C–S)
Yield 0.80 g (68 %); m.p. 152–154 °C; IR mmax 2926,
2364, 2345(aromatic –CH strechings), 1576, 1418 (–C=
C– strechings), 1257 (–CN), 1158, 1198, 1108, 885, 788
1
cm-1; H NMR (CDCl3, 400 MHz): d 7.97–7.96 (dd, 2H,
1
(C–S) cm-1; H NMR (CDCl3, 300 MHz) d 7.99 (d, 2H,
J = 4.4 and 2.2 Hz), 7.23–7.21 (dd, 2H, J = 8.8 and
1.4 Hz), 7.17–7.15 (dd, 2H, J = 8.8 and 2.3 Hz), 7.12 (d,
2H, J = 2.2 Hz), 6.75–6.74 (m, 2H), 6.62 (d, 2H,
J = 8.8 Hz), 4.09 (t, 4H, J = 6.5 Hz, 2x–NCH2),
1.91–1.87 (m, 4H, 2x–CH2); 13C NMR (CDCl3, 100 MHz)
d 154.8, 145.2, 141.8, 134.4, 130.2, 129.3, 123.9, 118.0,
116.7, 115.0, 44.1, 23.5; TOF ES ? MS m/z 523 (M??2);
anal. calcd. for C26H20Cl2N4S2. Calculated: C 59.65 %,
H 3.85 %, N 10.70 %, found: C 61.84 %, H 2.97 %,
N 10.61 %.
J = 4.1 Hz), 7.23 (d, 2H, J = 7.7 Hz), 6.81–6.72 (m, 8H),
4.02 (t, 4H, J = 7.2 Hz, 2x [ N–CH2–), 1.77–1.72 (m, 4H,
2x–CH2–), 1.47–1.44 (m, 4H, 2x–CH2–); 13C NMR
(CDCl3, 75.47 MHz) d 148.3, 145.9, 139.0, 134.9, 120.7,
115.4, 115.0, 113.1, 47.4, 27.5, 27.0; TOF ES ? MS m/
z 519 (M??1); anal. calcd. for C28H24F2N4S2. Calculated:
C 64.84 %, H 4.66 %, N 10.80 %, found: C 63.89 %,
H 4.66 %, N 10.67 %.
1,6-Bis(7-bromo-10H-benzo[b]pyrido[2,3-e][1,4]
thiazin-10-yl]hexane (4d)
1,4-Bis(7-fluoro-10H-benzo[b]pyrido[2,3-e][1,4]
thiazin-10-yl]butane (5c)
Yield 0.78 g (69 %); m.p. 152–154 °C; IR mmax 2944,
2873, 2852 (aromatic –CH strechings), 1560, 1464, 1417
(–C=C– strechings), 1373, 1261, 1251, 1231(-CN),
1168 cm-1; 1H NMR (CDCl3, 300 MHz) d 7.98 (d, 2H,
J = 4.3 Hz), 7.22–7.20 (m, 2H), 7.18 (t, 2H, J = 5.1 Hz),
7.14 (s, 2H), 6.76–6.72 (m, 2H), 6.67–6.64 (m, 2H), 4.02 (t,
4H, J = 6.7 Hz, 2x [ N–CH2–), 1.76–1.72 (m, 4H, 2x–
CH2–), 1.47–1.44 (m, 4H, 2x–CH2–); 13C NMR (CDCl3,
Yield 0.77 g (69 %); m.p. 139–141 °C; IR mmax 2924
(aromatic –CH strechings), 2122, 1723, 1613 (–C=C–),
1398, 1265 (–CN), 1145, 1071, 847, 748, 705 (C–S) cm-1
1H NMR (CDCl3, 400 MHz) d 7.98–7.97 (dd, 2H, J = 6.6
and 1.4 Hz), 7.21–7.20 (dd, 2H, J = 7.3 and 1.4 Hz), 7.00
(m, 4H), 6.74–6.72 (m, 4H), 4.04 (t, 4H, J = 7.3 Hz,
;
123

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Med Chem Res (2013) 22:4459–4467
2x–NCH2), 1.79–1.75 (m, 4H, 2x–CH2); 13C NMR (CDCl3,
100 MHz) d 161.4, 160.4, 155.5, 152.7, 125.9, 114.5,
112.9, 112.7, 102.5, 56.3, 18.9; TOF ES ? MS m/z 491
(M??2); anal. calcd. for C26H20F2N4S2. Calculated: C
63.65 %, H 4.11 %, N 11.42 %, found: C 65.51 %, H
3.98 %, N 11.45 %.
130.0, 128.9, 123.2, 118.1, 117.2, 116.2, 115.1, 47.0
(N–CH2). TOF ES ? MS m/z 610 (M? ?1); anal. calcd.
for C26H18Br2N4S2. Calculated: C 51.16 %, H 2.97 %,
N 9.18 %, found: C 52.84 %, H 2.93 %, N 8.09 %.
Acknowledgments Author (K.K) and (R.S) are thankful to CSIR
(Council of Scientific and Industrial Research), New Delhi for
research fellowship; and to Dr. Siva S Panda and Mahesh Chand for
biological evaluation.
1,4-Bis(7-bromo-10H-benzo[b]pyrido[2,3-e][1,4]thiazin-
10-yl]butane (5d)
Yield 0.77 g (71 %); m.p. 120–122 °C; IR mmax 2924, 2854
(aromatic –CH strechings), 1413, 1262 (–CN), 1183, 1132,
1111, 801, 758 (C–S) cm-1; 1H NMR (CDCl3, 300 MHz) d
7.97–7.96 (dd, 2H, J = 4.8 and 1.5 Hz), 7.23–7.21
(dd, 2H, J = 7.2 and 1.5 Hz), 7.17 (d, 2H, J = 2.1 Hz),
7.14–7.12 (dd, 2H, J = 6.9z and 2.4 Hz), 6.77–6.73 (m,
2H), 6.63 (d, 2H, J = 8.7 Hz), 4.09 (t, 4H, J = 7.2 Hz,
2x–NCH2), 1.90–1.86 (m, 4H, 2x–CH2);13C NMR (CDCl3,
75.47 MHz) d 154.8, 145.2, 141.8, 134.4, 130.2, 129.3,
123.9, 118.0, 116.7, 115.0, 44.1, 23.5; TOF ES ? MS m/
z 612 (M??2); anal. calcd. for C26H20Br2N4S2. Calculated:
C 50.99 %, H 3.29 %, N 9.15 %, found: C 52.34 %,
H 3.21 %, N 9.04 %.
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2E-ene (6a)
Yield 0.76 g(68 %); m.p. 132–134 °C; IR mmax 2925,
2854(aromatic –CH strechings), 1580 (–C=C–), 1412,
1364, 1256, 1219 (–CN), 1129, 1047, 779 (C–S) cm-1; 1H
NMR (CDCl3, 300 MHz) d 7.96–7.95 (dd, 2H, J = 4.5 and
1.2 Hz), 7.21 (d, 2H, J = 7.2 Hz), 6.99 (d, 2H,
J = 7.4 Hz), 6.94 (d, 2H, J = 7.7 Hz), 6.87–6.82 (m, 4H),
6.74–6.69 (m, 2H), 5.93–5.91 (m, 2H, –CH=CH–),
4.69–4.66 (m, 4H, 2xN–CH2); 13C NMR (CDCl3,
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117.5, 115.4, 113.8, 113.4, 50.3. TOF ES ? MS m/z 453
(M? ?1); anal.calcd. for C26H20N4S2. Calculated: C
69.00 %, H 4.45 %, N 12.38 %, found: C 68.89 %, H
4.41 %, N 13.25 %.
1,4-Bis(7-bromo-10H-benzo[b]pyrido[2,3-e][1,4]thiazin-
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1357, 1263, 1219 (–CN), 1130, 867, 801, 757 (C–S) cm-1
;
1H NMR (CDCl3, 300 MHz) d 7.95 (d, 2H, J = 4.8 Hz),
7.24–7.21 (dd, 2H, J = 8.7 and 1.8 Hz), 7.12 (d, 2H,
J = 2.1 Hz), 7.04–7.01 (d, 2H, J = 8.9 and 1.8 Hz),
6.78–6.74 (m, 2H), 6.61 (d, 2H, J = 8.7 Hz), 5.89–5.85
(m, 2H, –CH=CH–), 4.65–4.62 (m, 4H, 2xN–CH2–); 13C
NMR (CDCl3, 75.47 MHz) d 154.2, 145.2, 141.9, 134.2,
123

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4467
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123