Synthesis and antimicrobial activity of 2-(acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5 (or -6 or -8)-monochloro,7-fluoro-substituted-4H-1,4- benzothiazines

Article abstract of DOI:10.1002/jhet.5570430536

A series of 2-(acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5 (or -6 or -8)-monochloro,7-fluoro-substituted-4H-1,4-benzothiazines 3a-x were prepared to investigate their potential biological activity. In this work the 3a-w structures are supported with ph

Full text of DOI:10.1002/jhet.5570430536

Sep-Oct 2006
1371
Synthesis and Antimicrobial Activity of 2-(Acyl or carboxyalkyl)-3-
(H or alkyl or aryl)-5 (or -6 or -8)-monochloro,7-fluoro-substituted-
4H-1,4-benzothiazines
Domenico Armenise, Nicolino De Laurentis, Antonio Rosato and Flaviano Morlacchi*
Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università degli Studi di Bari,
Via E. Orabona 4, 70125 Bari (Italy)
Received November 4, 2005
R1 R2
C
C
DMSO
reflux
F
S
R2
R3
R4 R3
F
SH
+
or
R2
C
or
100°C
N
H
NH2
.
NH2
-NH
2
H
2O
Cl
Cl
C
R3
A series of 2-(acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5 (or -6 or -8)-monochloro,7-fluoro-
substituted-4H-1,4-benzothiazines 3a-x were prepared to investigate their potential biological activity. In
this work the 3a-w structures are supported with physical and analytical data and the results of their in vitro
antimicrobial activity against some strains Gram positive, Gram negative, and Fungi are reported. It was
found that compounds 3a, 3d displayed interesting antibacterial activity, whereas compound 3f displayed
interesting antifungal activity.
J. Heterocyclic Chem., 43, 1371 (2006).
Introduction.
R2
R1
R4
R3
S
The 4H-1,4-benzothiazine heterocyclic nucleus, even
when part of complex molecules [1a-c], possesses a wide
spectrum of pharmacological and biological activities [1a-
c,2] similar to phenothiazines. For example, a large
number of type 1 and 2 (Figure 1) compounds, endowed
with innumerable biological activities [3], including
antibacterial and antifungal, have been known for some
time. Recently, in addition, some dihydro1,4-benzo-
thiazin-3-one compounds of type 1b (Figure 1) were
described as histamine H1-receptor antagonists with anti-
pruritic activities [4] and inhibitory effects on xanthine
oxidase [5].
a) R1, R4 = H; R2, R3 = H, alkyl, etc.
b) R3 + R4 = O
N
H
1
Cpd
R2
R3
S
R2
(Z)
2a[1b]
2d[1b]
2g[1b]
2j[1d]
2m[1a]
2p[1b]
2s[1b]
2v[1e]
COCH3
COCH3
COCH3
COOCH3
COOCH3
COOC2H5
COOC2H5
COOC2H5
H
CH3
C6H5
H
CH3
H
N
H
R3
2
CH3
C6H5
Moreover, some dihalogenated benzothiazines
3
(Figure 1) have been cited for their activities. In
particular, 1-(5-chloro-7-fluoro-3-methyl-4H-1,4-benzo-
thiazine-2-yl)ethanone (3d) and ethyl 5-chloro-7-fluoro-3-
methyl-4H-1,4-benzothiazine-2-carboxylate (3s) have
been mentioned as potential anticancer agents [2].
Furthermore, the corresponding acid of methyl 6-chloro-
7-fluoro-4H-1,4-benzothiazine-2-carboxylate (3k) [6], has
also been tested because of its analogy to the antibacterial
quinolones.
R2
R3
F
S
R2
(Z)
3d[2] 5-Cl
3k[6] 6-Cl
COCH3
COOCH3
COOC2H5
CH3
H
CH3
N
H
R3
Cl
3s[2]
5-Cl
3
S
N
F
F
COR2
R1
F
S
N
(Z)
NH CO
(E)
Cl
Continuing our studies aimed at synthesizing aza and
thiaza ethero bi- and tri-cyclic potentially bio-active
compounds [1b,c], as an enlargement of our researches on
the dehalogenated benzothiazoles type 4 (Figure 1)
activity [7a], we recently reported the synthesis and
(CH2)n
5
4
Figure 1

1372
D. Armenise, N. De Laurentis, A. Rosato and F. Morlacchi
Vol 43
antifungal activities of a series of 6-fluoro-4(5 or 7)-
chloro-2-(difluorobenzoyl) aminobenzothiazoles type 5
(Figure 1) [7b].
Taking into account these findings, and as an extension
of our syntheses of non-halogenated benzothiazine
compounds [1b] type 2 (Figure 1), with the intention of
enlarging the number of compounds analogous to the
three already known compounds [2,6] of type 3 (Figure
1), we decided to synthesize a large number of
dihalogenated benzothiazines variously substituted with
appropriate pharmacophoric groups.
In particular, the synthesis of 2-(acyl or carboxyalkyl)-
3-(H or alkyl or aryl)-5 (or -6 or -8)-monochloro,7-fluoro-
substituted 4H-1,4-benzothiazine compounds 3 (Figure 1)
Scheme 1
F
F
S
SH
NH2
Reflux: 50% NaOH for 24h
N
6 a-c
Cl
NH2
Cl
7 a-c
a = 4 Cl
b = 5 Cl
c = 7 Cl
a = 3 Cl
b = 4 Cl
c = 6 Cl
R
R
R
R
1
2
3
C
F
S
N
R
F
2
3
SH
NH2
(Z)
C
4
R
Cl
Cl
7 a-c
H
3 a-x
a) DMSO reflux or b)
NH2NH2H20 at 100°
a =3 Cl
b =4 Cl
c =6 Cl
a, d, g, j, m, p, s, v =5Cl
b, e, h, k, n, q, t, w =6Cl
c, f, i, l, o, r, u, x =8Cl
Cpd
Cpd
[method]
3a[a]
Yield %
Yield
5
6
8
R2
R3
R1
R4
av.[*]
3-Butyn-2-one
CH CO CH
7a
7b
7c
47.7
29.2
63.5
Cl
COCH3
COCH3
COCH3
H
H
H
—
—
—
I
3b[a]
46,8
Cl
3
C
3c[a]
Cl
Cl
7a
7b
7c
3d[a]
3e[a]
3f[a]
32.2
48.6
68.5
Cl
COCH3
COCH3
COCH3
CH3
CH3
CH3
H
H
H
OH
OH
OH
2,4-Pentanedione (enol-form)
49.8
Cl
II
CH3
CO CH
C
OH
CH3
7a
7b
7c
3g[b]
3h[b]
3i[a]
2.1
5.0
5.5
Cl
Cl
Cl
COCH3
COCH3
COCH3
C6H5
C6H5
C6H5
—
—
—
4-Phenyl-3-Butyn-2-one
CH CO C6H5
4.2
Cl
Cl
Cl
III
Cl
Cl
Cl
3
C
C
7a
7b
7c
3j[a]
3k[a]
3l[b]
17.7
71.2
6.4
COOCH3
COOCH3
COOCH3
H
H
H
—
—
—
Methyl-propiolate
CH -O-CO C C H
31.8
46.9
IV
3
7a
7b
7c
3m[a]
3n[a]
3o[a]
26.0
84.4
30.2
COOCH3
COOCH3
COOCH3
CH3
CH3
CH3
H
H
H
OH
OH
OH
Methyl-acetoacetate (enol form)
V
CH3-O-CO
CH
C
CH3
OH
7a
7b
7c
3p[a]
3q[a]
3r[b]
47.9
42.5
5.0
Cl
Cl
COOC2H5
COOC2H5
COOC2H5
H
H
H
—
—
—
Ethyl propiolate
O CO
31.8
47.2
Cl
Cl
VI
Cl
Cl
C2H5
C
CH
7a
7b
7c
3s[a]
3t[a]
3u[a]
49.0
43.2
49.4
COOC2H5
COOC2H5
COOC2H5
CH3
CH3
CH3
H
H
H
OH
OH
OH
Ethyl acetoacetate (enol form)
VII
C2H5
O
CO CH
C
CH3
OH
7a
7b
7c
3v[a]
3w[a]
3x[b]
14.8
35.9
0
Cl
COOC2H5
COOC2H5
COOC2H5
C6H5
C6H5
C6H5
—
—
—
Phenyl-ethyl--propiolate
16.9
Cl
VIII
Cl
C2H5-O-CO
C
C C6H5
(*) Average yield of three isomers. For example, for 3a-c; (47.7+29.2+63.5)/3 = 46.8.

Sep-Oct 2006 Synthesis and Antimicrobial Activity of 2-(Acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5
1373
is part of our current research program aimed at
discovering new aza-sulfurated antimicrobial agents [2,7].
It is also, from an SAR viewpoint, an attempt to evaluate
the relationship between the antimicrobial activity of
derivatives 3a-w and their substituent groups.
Scheme 1 allows two interpretations. In the first of these it
is possible to discern the influence of the chloro
substituent. Thus, selecting the highest yield of each of
eight groups of isomers (3a-c, d-f,...),the results indicate
that there is no straightforward dependency between Cl-
position and the yields. In fact, with each of the eight
reagents, the highest yield of 3 compounds often occurred
for different positions (5 or 6 or 8) of the chloro
substituent (**). The second interpretation of Scheme 1 is
based on the consideration that the combination of the
yields of various three isomers can remove the effect of
the dependency between the yields and Cl-position. This
way, considering, for each reagent, the average yields of
each of the three isomers, the yields obtained by each
reagent can be compared without regard to the chloro
position (***).
Results and Discussion.
Chemistry.
As shown in Scheme 1, the desired compounds 3a-x
were prepared following a two-step procedure involving
the synthesis of the appropriate 3(4 or 6)-monochloro-5-
fluoro-2-amino benzenthioles (7a-c) by hydrolytic basic
cleavage [8] of 4(5 or 7)-monochloro-6-fluoro-2-
aminobenzothiazoles (6a-c) [7b]. Next, all the eight series
of three possible isomers, namely the twenty four
compounds 3a-x, were prepared by cyclization of
compounds 7a-c with the eight suitable unsaturated
(enolic or acetylenic) reagents as shown in Scheme 1,
using two different procedures a and b. Briefly, these
were as follows, procedure a: refluxing in dimethyl
sulfoxide for three hours, procedure b: using hydrazine
monohydrate as catalyst at 100°C for ten minute. Further
details of these procedures are described below in the
section Experimental Chemistry.
In short, from a synthetic point of view, the results
indicate that, with regard to reactivity, the reagents
themselves are more influential than the position of the
chloro substituent in the benzene ring. Moreover, among
the reagents, the enol forms are more reactive than
acetylenic compounds. The latter are, in fact, inactive
when a phenyl group is present ꢀ to the ethyne group
(see: 3x, yield=0%).
Generally, procedure
a gave the better yields.
Microbiology.
Nevertheless, in cases where it failed (3g,h,l,r,x),
procedure b had the merit of producing the desired
compound (except for 3x), albeit in low yields (max.
5.2% for 3l).
Benzo[1,4]thiazine compounds are known to exhibit a
wide range of pharmacological properties including
antidepressant, anxiolytic, and calcium antagonist activity
[3]. In our search, devoted, as it was, to exploring the
antimicrobial properties of 1,4-benzothiazine compounds,
all the synthesized compounds were tested in vitro against
some Gram-positive and Gram-negative bacteria and the
fungus Candida albicans. The results are summarized in
Table 5 in which only the most significant findings are
listed (i.e. compounds showing MIC ꢀ 250 μg/ml).
In Scheme 1, both cyclization procedures, the yields of
compounds 3a-x and the eight average yields of all the
three possible isomers of compounds 3 (i.e. 8 “three
possible isomers”: 3a-c, 3d-f,…) are also presented. The
scheme also reports the structures of compounds 6a-c [7b]
and 7a-c, those of eight unsaturated (enolic or acetylenic)
reagents employed, as well as of the compounds 2-(acyl
or carboxyalkyl)-3-(H or alkyl or aryl)-5(6 or 8)-
monochloro,7-fluoro-4H-1,4-benzothiazines (3a-x).
The structures of the new compounds are fully
supported by microanalytical and spectral (ir, 1H nmr, and
mass) data. Compounds 3d [2], 3k [6] and 3s [2] show
spectral data consistent with those reported in the
literature. Tables 1-4 show the IUPAC names, the
elemental analyses, the main physical properties and
spectral data for compounds 7a-c and 3a-w.
As shown in Table 5, as regards Gram-positive bacteria,
the compounds 3a and 3d displayed an interesting
antibacterial activity against strains of Bacillus subtilis
and Bacillus cereus (MIC 15.6 μg/ml) and a moderate
activity (MIC 62.5 μg/ml) against two strains of
Staphylococcus aureus and against the Enterococcus
faecalis.
Moderate activity (MIC 62.5 μg/ml) was
displayed respectively by compound 3w against four
strains (Bacillus subtilis, Bacillus cereus and two strains
of Staphylococcus aureus), by compound 3r against three
strains (Enterococcus faecalis and two strains of
Staphylococcus aureus), and finally by compound 3p
against the two strains of Staphylococcus aureus.
There are no compounds with any significant
antibacterial activity against the Gram-negative strains
tested.
In connection with the yields, we note that they can
alter depending on the position of the chloro substituent in
the benzene ring and on the chemical structure of the
reagent employed. The highest yields were observed with
procedure a for compounds 3n (84.4%), 3k (71.2%), 3f
(68.5%), 3c (63.5%). Considering that each of the eight
reagents yielded three isomeric benzothiazines (3),

1374
D. Armenise, N. De Laurentis, A. Rosato and F. Morlacchi
Vol 43
0.4% of the theoretical values. Column chromatography on
silica gel 60 (Merck 70-230 mesh) was carried out by using light
petroleum ether (bp 40-70°)-ethyl acetate (7:3 v/v) as eluent.
Thin layer chromatography was performed using light petroleum
ether-ethyl acetate (7:3 v/v) or hexane-ethyl acetate (7:3 v/v) as
eluent, in each case specified.
As regards the fungi, the compound 3f displayed an
interesting antifungal activity (MIC 31.2–15.6 μg/ml)
against the four strains of Candida albicans, and the
compounds 3h, 3m, 3n displayed against the same strains
a moderate activity (MIC 62.5 μg/ml). Actually, the above
compounds, together with the compounds 3a and 3d
(MIC 125 μg/ml), exhibit an antifungal activity between
those of our standards fluconazole (MIC:1–0.5 μg/ml) and
sorbic acid (MIC:500–250 μg/ml).
In conclusion, from an SAR standpoint, we observe
that, of all compounds tested (3a-w of Table 5), the
highest antimicrobial activity occurs in compounds 3a, 3d
when the molecule 7-fluoro-4H-1,4-benzothiazine
possesses the acetyl substituent in position 2 and the
chloro in position 5 (Scheme 1).
Concerning the activity against the strain Bacillus
subtilis ATCC 6633, it is interesting to compare the
activities of the two more active compounds 3a and 3d
(type 3, Figure 1) (MIC 15.6 μg/ml) with the activities of
two analogous non-halogenated compounds 1-(4H-1,4-
benzothiazin-2-yl)ethanone (MIC 62.5 μg/ml [1b]) and 1-
(3-methyl-4H-1,4-benzothiazin-2-yl)ethanone (MIC >250
μg/ml [1b]) (2a and 2d, Figure 1). The comparison
reveals that the 5-chloro position (and not the 6 or the 8
position) causes a remarkable increase in the activities.
General Procedure for the Preparation of Compounds 7 [8]
A solution of 2-amino-(4 or 5 or 7)-chloro-6-fluorobenzo-
thiazolo (6a or 6b or 6c) (5 g, 24.7 mmoles) in 82.80 ml of 50%
NaOH was refluxed vigorously until the evolution of ammonia
ceased (about 24 h). After cooling, the reaction mixture was
diluted with distilled water (30 ml) and extracted with ethyl
acetate (3x100). The combined organic layers were washed with
water, dried (anhydrous sodium sulfate) and evaporated under
reduced pressure. The crude product was separated by column-
chromatography on silica gel (eluent: n-hexane-ethyl acetate 7:3
v/v) to obtain the pure compound 7a (3.27 g) or 7b (3.80 g) or
7c (3.13 g), respectively. Each of the three isomers showed m/z
177 (M+, base), 142, 132. The yields and the physical and
analytical data are reported in Tables 1 and 2.
General Procedure for the Preparation of Compounds 3.
Method a [2] for 3 a-f, i-k, m-q, s-w.
To a well-stirred solution of 7 (2.8 mmoles) in dimethyl
sulfoxide (10 ml) was added the appropriate enolic or acetylenic
reagent (2.8 mmoles) at room temperature. The mixture was
refluxed for 3 h. The crude mixture was purified by column
chromatography on silica gel using light petroleum ether (bp 40-
70°)-ethyl acetate (7:3 v/v) as eluent. The yield, the elemental
analyses and the chemical and physical properties are reported in
Tables 3 and 4.
EXPERIMENTAL
Chemistry.
Melting points were determined in open capillary tubes with a
Büchi apparatus and are uncorrected. Ir spectra were obtained on
a Perkin-Elmer 283 spectrophotometer (nujol mull). 1H nmr
spectra were recorded on Varian-Mercury instrument operating
at 300 MHz. Chemical shifts are given in ꢀ values (ppm) and the
coupling constants are expressed in J values (Hz) downfield
from tetramethylsilane as internal standard. Mass spectra were
recorded on a Hewlett-Packard 5995c GC-MS low resolution
spectrometer. All compounds showed appropriate ir, 1H nmr and
mass spectra. Elemental analyses were carried out with
Eurovector EuroEA 3000 analyzer and the results were within ±
Method b [1a] for 3 g, h, l, r,x.
A well stirred mixture of 7 (0.5 g, 2.8 mmoles) and hydrazine
monohydrate (14 mg, 0.28 mmoles) was heated at 100°C for 2-3
minutes before introducing the appropriate enolic or acetylenic
reagent (2.8 mmol) and warming the reaction to 100°C for a
further 10 minutes. After cooling to room temperature, the crude
mixture was purified by column chromatography on silica gel
using light petroleum ether (bp 40-70°)-ethyl acetate (7:3 v/v) as
eluent. The yield, the elemental analyses and the chemical and
physical properties are reported in Tables 3 and 4.
Table 1
Yield, Physical and Analytical Data for the Benzenethiol Compounds 7
N°
[a]
Compound
Yield
%
State
[b]
Mp (°C)
Molecular Formula
(Molecular Weight)
Analysis (%)
Calcd/Found
Rf
[c]
C
H
N
7a[2] 2-amino-3-chloro-5-
fluorobenzenethiol
7b[8] 2-amino-4-chloro-5-
fluorobenzenethiol
74.65
86.75
71.46
Y
G
G
100-106
121-124
124-128
C6H5ClFNS
177.628
40.57
40.20
40.57
40.22
40.57
40.90
2.84
2.76
2.84
2.85
2.84
2.64
7.89
7.82
7.89
7.56
7.89
7.57
0.88
0.30
0.52
7c[8]
2-amino-6-chloro-5-
fluorobenzenethiol
[a] compounds mentioned in references have been characterized more completely;[b] Y= yellow solid, G = green solid; [c] Eluant:
n-hexane-ethyl acetate 7:3 v/v (TLC).

Sep-Oct 2006 Synthesis and Antimicrobial Activity of 2-(Acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5
1375
Table 2
Physical and Analytical Data for the Benzothiazine Compounds 3
N°
Compound
State
[a]
Mp (°C)
Molecular Formula
(Molecular Weight)
Analysis (%)
Calcd/Found
H
Rf
[b]
C
N
3a
3b
3c
1-(5-chloro-7-fluoro-4H-1,4-benzothiazin-2-
yl)ethanone
1-(6-chloro-7-fluoro-4H-1,4-benzothiazin-2-
yl)ethanone
O
O
O
O
O
O
O
R
B
Y
B
B
B
Y
O
Y
O
O
B
B
B
B’
O
251-253
276-277
236-239
51-55
C10H7ClFNOS
(243.686)
49.29
48.92
49.29
49.55
49.29
49.24
51.27
50.94
51.27
50.89
51.27
51.50
60.09
59.88
60.09
60.12
60.09
59.83
46.25
46.60
46.25
46.46
46.25
46.53
48.27
48.33
48.27
47.98
48.27
48.08
48.27
47.90
48.27
48.51
48.27
48.27
50.09
49.69
50.09
49.97
50.09
49.78
2.90
3.30
2.90
3.24
2.90
3.29
3.52
3.82
3.52
3.58
3.52
3.90
3.47
3.79
3.47
3.70
3.47
3.12
2.72
3.07
2.72
3.02
2.72
3.00
3.31
3.43
3.31
3.42
3.31
3.30
3.31
3.70
3.31
3.44
3.31
3.63
3.85
3.71
3.85
3.72
3.85
3.76
5.75
5.37
5.75
6.02
5.75
5.55
5.44
5.83
5.44
5.33
5.44
5.05
4.38
4.05
4.38
4.28
4.38
4.00
5.39
5.79
5.39
5.01
5.39
5.32
5.12
5.09
5.12
5.07
5.12
5.06
5.12
4.97
5.12
4.80
5.12
4.89
4.87
4.99
4.87
4.84
4.87
5.25
0.45
0.09
0.03
0.68
0.50
0.13
0.71
0.62
0.60
0.70
0.49
0.21
0.74
0.66
0.38
0.70
0.50
0.37
0.81
0.45
0.28
0.75
0.82
1-(8-chloro-7-fluoro-4H-1,4-benzothiazin-2-
yl)ethanone
3d[2] 1-(5-chloro-7-fluoro-3-methyl-4H-1,4-
C11H9ClFNOS
(257.712)
benzothiazin-2-yl) ethanone
1-(6-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazin-2-yl)ethanone
1-(8-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazin-2-yl)ethanone
1-(5-chloro-7-fluoro-3-phenyl-4H-1,4-
benzothiazin-2-yl)ethanone
1-(6-chloro-7-fluoro-3-phenyl-4H-1,4-
benzothiazin-2-yl)ethanone
1-(8-chloro-7-fluoro-3-phenyl-4H-1,4-
benzothiazin-2-yl)ethanone
3e
3f
3g
3h
3i
262-265
274-275
131-135
173-177
172-180
157-162
206-209
222-226
110-113
230-235
225-230
107-112
196-199
178-181
66-70
C16H11ClFNOS
(319.782)
3j
methyl 5-chloro-7-fluoro-4H-1,4-
benzothiazine-2-carboxylate
3k[6] methyl 6-chloro-7-fluoro-4H-1,4-
C10H7ClFNO2S
(259.685)
benzothiazine-2-carboxylate
methyl 8-chloro-7-fluoro-4H-1,4-
benzothiazine-2-carboxylate
methyl 5-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazine-2-carboxylate
methyl 6-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazine-2-carboxylate
methyl 8-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazine-2-carboxylate
ethyl 5-chloro-7-fluoro-4H-1,4-
benzothiazine-2-carboxylate
ethyl 6-chloro-7-fluoro-4H-1,4-
benzothiazine-2-carboxylate
3l
3m
3n
3o
3p
3q
3r
C11H9ClFNO2S
(273,712)
C11H9ClFNO2S
(273,712)
ethyl 8-chloro-7-fluoro-4H-1,4-
benzothiazine-2-carboxylate
3s[2] ethyl 5-chloro-7-fluoro-3-methyl-4H-1,4-
C12H11ClFNO2S
(287.738)
benzothiazine-2-carboxylate
ethyl 6-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazine-2-carboxylate
ethyl 8-chloro-7-fluoro-3-methyl-4H-1,4-
benzothiazine-2-carboxylate
ethyl 5-chloro-7-fluoro-3-phenyl-4H-1,4-
benzothiazine-2-carboxylate
3t
3u
3v
3w
171-174
218-220
oil
C17H13ClFNO2S
(349.808)
ethyl 6-chloro-7-fluoro-3-phenyl-4H-1,4-
benzothiazine-2-carboxylate
60
58.37
58.75
3.75
4.10
4.00
3.88
[a] O = orange solid; Y = yellow solid; R = red solid; B = brown solid; B’ = brown oil; [b] Eluant: petrol ether-ethyl acetate 7:3 v/v (TLC).
Microbiology.
negative bacteria (Escherichia coli ATCC 8739, Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853) and
fungal strains too (Candida albicans ATCC 10231, Candida
albicans ATCC 14053, Candida albicans NRRL y 869 and
Candida albicans y NRRL 12983).
Determination of Antibacterial Activity.
Organisms.
For bacterial strains the MICs were determined by the
microdilution method, the inocula were prepared as follows:
colonies derived from fresh mature strains on MHA (Mueller
Hinton agar) were suspended in saline solutions (0.85%) and
suspensions were adjusted to 108 cfu ml-1 (0.5 MacFarland) as
determined by viable count.
The synthesised compounds 3a-w were tested for
a variety of Gram-positive
(Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778,
Staphylococcus aureus ATCC 29213, Staphylococcus aureus
ATCC 6538p, Enterococcus faecalis ATCC 29212) and Gram-
antimicrobial activity against

1376
D. Armenise, N. De Laurentis, A. Rosato and F. Morlacchi
Vol 43
Table 3
Spectral Data for the Benzenethiol Compounds 7
IR (nujol mull) (cm-1)
NH2
1
N°
[a]
H nmr ꢀ (ppm)
[b]
7a[2]
3489 and 3392
(CDCl3) ꢀ 4.56 (s, broad, 3H, SH + NH2), 6.83 and 6.86 (dd, 1H, J = 2.5 Hz, H6), 7.04 and 7.07 (dd, 1H, J
= 2.5 Hz, H4)
7b[8]
7c[8]
3470 and 3380
3449 and 3352
(CDCl3) ꢀ 4.52 (s, broad, 3H, SH + NH2), 6.72 (d, 1H, J = 6.0 Hz, H3), 6.95 (d, 1H, J = 9.0 Hz, H6)
(DMSO-d6) ꢀ 4.30 (s, broad, 3H, SH + NH2), 6.50 and 6.53 (dd, 1H, J = 5.0 Hz, H3), 6.95 (t, 1H, J = 8.0
Hz, H4)
[a] compounds mentioned in references have been characterized more fully; [b]= All NH2 signals disappear with D2O.
Table 4
Spectral Data for the Benzothiazine Compounds 3
IR (nujol mull) (cm-1)
NH , CO
1
N°
H nmr ꢀ (ppm)
[a]
3a
3b
3c
3d
3e
3f
3247, 1590
3244, 1554
3240, 1568
3330, 1590
3238, 1564
3286, 1560
3406, 1603
3264, 1565
3260, 1576
3333, 1635
3285, 1633
3278, 1629
3415, 1620
3324, 1640
3328, 1639
3331, 1632
3291, 1635
3297, 1632
3335, 1635
3331, 1622
3333, 1627
3357, 1622
3304, 1613
(CDCl3) 2.19 (s, 3H, COCH3), 6.1 (s, br, 1H, NH), 6.43 and 6.46 (dd, 1H, J = 2.0 Hz, H8), 6.62 and 6.65 (dd, 1H,
J = 2.0 Hz, H6), 7.02 (d, 1H, J = 7.5 Hz, H3)
(CDCl3) 2.10 (s, 3H, COCH3), 6.57 (d, 1H, J = 7.0 Hz, H5), 6.82 (d, 1H, J = 9.0 Hz, H8), 7.24 (d, 1H, J = 7.0 Hz,
H3), 8.9 (s, br, 1H, NH)
(CDCl3) 1.6 (s, br, 1H, NH), 2.11 (s, 3H, COCH3), 6.80 and 7.64 (dd, 1H, J = 3.4 Hz, H3), 7.1-7.2 (m, 1H, H5),
7.50 (t, 1H, J = 8.4 Hz, H6)
(CDCl3) 1.8 (s, br, 1H, NH), 2.30 (s, 3H, CH3), 2.38 (s, 3H, COCH3), 6.62 and 6.66 (dd, 1H, J = 2.7 Hz, H8), 6.78
and 6.82 (dd, 1H, J = 2.7 Hz, H6)
(Acetone deut.) 2.86 (s, 3H, CH3), 2.88 (s, 3H, COCH3), 6.76 and 6.79 (dd, 1H, J = 2.2 Hz, H5), 6.88 and 6.92
(dd, 1H, J = 2.2 Hz, H8), 8.1 (s, br, 1H, NH)
(DMSO-d6) 1.5 (s, br, 1H, NH), 2.18 (s, 3H, CH3), 2.23 (s, 3H, COCH3), 6.6-6.4 (m, 1H, H5), 6.89 (t, 1H, J = 8.0
Hz, H6)
(CDCl3) 2.10 (s, 3H, COCH3), 6.25 (s, br, 1H, NH), 6.61 and 6.63 (dd, 1H, J = 2.75 Hz, H8), 6.73 and 6.76 (dd,
1H, J = 2.75 Hz, H6), 7.4-7.6 (m, 5H, aromatic-H)
(CDCl3) 2.10 (s, 3H, COCH3), 7.19 (d, 1H, J = 7.5 Hz, H5), 7.43 (t, 3H, J = 7.5 Hz, o-p aromatic-H), 7.51 (1H, d,
J = 6.75, H8), 7.58-7.60 (m, 2H, m aromatic-H), 11.61 (s, br, 1H, NH)
(CDCl3) 2.62 (s, 3H, COCH3), 6.26 and 6.29 (dd, 1H, J = 5.05 Hz , H5), 6.70 (t, 1H, J = 7.58 Hz, H6,), 7.3-7.6 (m,
5H, aromatic-H), 11.48 (s, br, 1H, NH)
(CDCl3) 3.73 (s, 3H, COOCH3), 6.06 (s, br, 1H, NH), 6.42 and 6.44 (dd, 1H, J = 2.7 Hz, H8), 6.66 and 6.67 (dd,
1H, J = 3.3 Hz, H6), 7.06 (d, 1H, J = 5.9 Hz, H3)
(CDCl3) 3.73 (s, 3H, COOCH3), 5.59 (s, br, 1H, NH), 6.29 (d, 1H, J = 5.9 Hz, H5), 6.55 (d, 1H, J = 8.3 Hz, H8),
7.06 (d, 1H, J = 5.9 Hz, H3)
(CDCl3) 3.73 (s, 3H, COOCH3), 5.45 (s, br, 1H, NH), 6.00 and 6.03 (dd, 1H, J = 3.8 Hz, H5), 6.57 (t, 1H, J = 8.5
Hz, H6), 6.95 (d, 1H, J = 6.3 Hz, H3)
(CDCl3) 2.25 (s, 3H, CH3), 3.70 (s, 3H, COOCH3), 6.0 (s, br, 1H, NH), 6.59 and 6.52 (dd, 1H, J = 2.9 Hz, H8),
6.77 and 6.70 (dd, 1H, J = 2.9 Hz, H6)
(CDCl3) 2.30 (s, 3H, CH3), 3.73 (s, 3H, COOCH3), 5.52 (s, br, 1H, NH), 6.42 (d, 1H, J = 5.8 Hz, H5), 6.67 (d, 1H,
J = 8.3 Hz, H8)
(CDCl3) 2.25 (s, 3H, CH3), 3.73 (s, 3H, COOCH3), 5.4 (s, br, 1H, NH), 6.11 and 6.14 (1H, dd, J = 3.8 Hz, H5),
6.25 (t, 1H, J = 8.3 Hz, H6)
(DMSO-d6) 1.18 (t, 3H, J = 7.0 Hz, CH2CH3), 4.07 (q, 2H, J = 7.0 Hz, CH2CH3), 6.70 and 6.73 (dd, 1H, J = 3..0
Hz, H8), 6.95 (d, 1H, J = 7.0 Hz, H3), 6.98 and 7.00 (dd, 1H, J = 3.0 Hz, H6), 8.28 (d, 1H, J = 7.0 Hz, NH)
(DMSO-d6) 1.15 (t, 3H, J = 7.0 Hz, CH2CH3), 4.05 (q, 2H, J = 7.0 Hz, CH2CH3), 6.57 (d, 1H, J = 7.0 Hz, H5),
6.88 (d, 1H, J = 10 Hz, H8), 7.03 (d, 1H, J = 7.0 Hz, H3) , 8.72 (d, 1H, J = 7.0 Hz, NH)
(CDCl3) 1.15 (t, 3H, J = 7.0 Hz, CH2CH3), 4.05 (q, 2H, J = 7.0 Hz, CH2CH3), 5.5 (s, br, 1H, NH), 6.00 (d, 1H, J =
9.0 Hz, H6), 6.55 (d, 1H, J = 7.0 Hz, H5), 6.93 (d, 1H, J = 7.0 Hz, H3)
(DMSO-d6) 1.17 (t, 3H, J = 7.0 Hz, CH2CH3), 2.27 (s, 3H, CH3), 4.05 (q, 2H, J = 7.0 Hz, CH2CH3), 6.05 (d, 1H, J
= 8.5, H6), 7.05 (d, 1H, J = 8.5 Hz, H8), 7.8 (s, br, 1H, NH)
(CDCl3) 1.30 (t, 3H, J = 7.0 Hz, CH2CH3), 2.28 (s, 3H, CH3), 4.19 (q, 2H, J = 7.0 Hz, CH2CH3), 5.56 (s, br, 1H,
NH), 6.42 (d, 1H, J = 6.5 Hz, H5), 6.68 (d, 1H, J = 9.5 Hz, H8)
(DMSO-d6) 1.18 (t, 3H, J = 7.0 Hz, CH2CH3), 2.1 (s, 3H, CH3), 4.03 (q, 2H, J = 6.0 Hz, CH2CH3), 6.4-6.5 (m, 1H,
H6), 6.82 (d, 1H, J = 8.0 Hz, H5), 8.7 (s, br, 1H, NH)
(CDCl3) 1.18 (t, 3H, J = 6.7 Hz, CH2CH3), 4.18 (q, 2H, J = 6.7 Hz, CH2CH3), 6.98 (d, 1H, J = 9.0 Hz, H6), 7.30 (s,
1H, H8), 7.35-7.53 (m, 5H, aromatic-H), 8.00 (d, 1H, J = 8.0 Hz, NH)
(CDCl3) 1.09 (t, 3H, J = 7.0 Hz, CH2CH3), 4.08 (q, 2H, J = 7.0 Hz, CH2CH3), 6.0 (s, br, 1H, NH), 7.1-7.6 (m, 5H,
aromatic-H), 7.12 (d, 1H, J = 7.0 Hz, H5), 8.00 (d, 1H, J = 7.0 Hz, H8)
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
[a]= All NH signals disappear with D2O.

Sep-Oct 2006 Synthesis and Antimicrobial Activity of 2-(Acyl or carboxyalkyl)-3-(H or alkyl or aryl)-5
1377
Susceptibility Testing.
malt broth medium (Clinical and Laboratory Standard Institute,
USA -, m27-a2) to give an inoculum of 1•103 cfu/ml. [9, 12]
A series of two fold dilutions of each product were prepared
in DMSO at required dilutions (500, 250, 125, 62.5, 31.2, 15.6,
7.8 μg/ml) and placed in 96 wells microdilution trays,
containing broth media. Each well was inoculated with 20 μl of
a final inoculum concentration that approximately contained
5•104 cfu/ml [9-11]. A growth control well test was also
prepared; this control well contained the broth media with
DMSO at the working concentrations (5%). Under these
conditions DMSO was inactive against our strains. The trays
were incubated at 37° for 24 hours. Each strain was tested at
least three times in duplicate before selection of the modal value.
The MICs were determined as the lowest concentration of the
agent resulting in no growth. To determine the MIC, we
compared the growth in every well visually with that of the
growth control well [9-11]. Appropriate controls employed the
antibiotic norfloxacin; this was tested as international reference
standard in accordance with the procedures suggested by CLSI-
NCCLS (MS100-S13). The microdilution technique was based
on the procedures recommended by CLSI-NCCLS (M7A6). [12]
Susceptibility Testing.
The broth microdilution assay was based on that recommended
by the Clinical and Laboratory Standard Institute [12]. The stock
solutions of the compounds were adjusted in DMSO [9]. Final
concentration ranges for each compound were from 500 to 7.8
μg/ml. Fluconazole was dissolved in sterile distilled water and
used as positive controls. Solvent and media controls were also
included for reference. A series of twofold dilutions of each agent
were prepared in yeast malt broth in a 96 wells microdilution tray
using fluconazole as reference compound. Each well was
inoculated with a final concentration of approximately 1•103 to
3•103 cfu/ml as confirmed by viable counts. Microdilution trays
were incubated for 48 h at 30° and then the growth was monitored
visually. The minimum inhibitory concentration was defined as
the lowest concentration required to arrest the growth of the fungi
at the end of 48 h of incubation at 30°C. Each isolate was tested at
least twice in duplicate [12].
Acknowledgements.
Determination of Antifungal Activity.
Organisms.
This work was supported by a grant of the Ministero
dell'Istruzione, dell’Università e della Ricerca (M.I.U.R.), Rome.
Standard strains of Candida albicans ATCC 10231, Candida
albicans ATCC 14053, Candida albicans NRRL y 869 and Candida
albicans NRRL y 12983 was grown and maintained on sabouraud
agar slants. The fungal strains were grown in Sabouraud dextrose
agar. Five colonies were selected and the suspensions adjusted to
approximately 1•106 cfu/ml. They were diluted appropriately in yeast
REFERENCES AND NOTES
*
Author to whom correspondence should be addressed: Prof.
Flaviano Morlacchi Dipartimento Farmaco-chimico, Facoltà di
-
Table 5
Antifungal and Antibacterial Activity of Compounds 3 against Fungi, Gram-positive and Gram-negative Bacteria (MIC, μg/ml)
Bacteria
Fungi
Gram positive
B.subt.
ATCC
6633
15.6
125
Gram negative
N°
B.cer.
ATCC
11778
15.6
250
125
15.6
----
S.aur.
ATCC
29213
62.5
250
125
62.5
----
S.aur.
ATCC
6538p
62.5
62.5
62.5
62.5
----
----
125
125
125
62.5
62.5
125
125
125
62.5
62.5
62.5
125
125
125
E.faec.
ATCC
29212
62.5
250
125
62.5
----
E.coli
ATCC
8739
250
----
250
250
----
E.coli
ATCC
25922
P.aerug
ATCC
27853
250
----
250
250
----
----
----
250
----
250
----
----
----
----
----
----
----
C.alb
ATCC
10231
125
----
250
125
250
31.2
250
62.5
250
250
---
C.alb.
ATCC
14053
125
----
250
125
250
15.6
250
62.5
250
250
---
C.alb.
NRRL
Y869
62.5
----
250
62.5
250
31.2
125
125
250
250
---
C.alb.
NRRL
Y12983
125
----
250
125
250
31.2
250
62.5
250
250
---
3a
3b
3c
3d
3e
3f
3g
3h
3i
----
250
125
15.6
----
----
125
125
125
125
125
250
125
125
125
125
125
----
----
----
----
250
250
----
125
125
250
----
250
125
125
125
250
125
125
62.5
0.25
125
125
125
125
125
125
125
125
62.5
125
62.5
125
125
125
62.5
1
250
125
250
125
125
----
----
250
----
125
62.5
----
250
125
125
1
250
----
----
250
250
----
----
----
----
----
----
----
250
----
125
2
250
----
250
250
125
250
----
250
----
----
----
----
250
----
125
3j
3k
3m
3n
3o
3p
3q
3r
3s
3t
62.5
62.5
---
----
----
62.5
62.5
---
----
----
62.5
62.5
---
----
----
62.5
62.5
---
----
---
----
----
----
----
250
125
125
62.5
0.5
----
250
----
250
2
250
125
----
125
125
----
125
125
----
125
125
----
3u
3w
a
62.5
0.5
----
----
----
----
b
c
0.5
250
0.5
500
1
500
1
250
Reference compounds: a = Norfloxacine, b = Fluconazole, c = Sorbic acid; “----“ = MIC > 250 μg/ml.

1378
D. Armenise, N. De Laurentis, A. Rosato and F. Morlacchi
Vol 43
Farmacia, Università degli Studi di Bari, Via E .Orabona 4, 70125-Bari,
Italy; e-mail: fmorlacchi@farmchim.uniba.it
[4] T. Takizawa, J. Matsumoto, T. Tohma, T. Kanke, Y. Wada,
M. Nagoo, N. Inogaki, H. Nagai, M. Zhang, H. Timmerman, Jpn. J.
Pharmacol., 86(1), 55-64, (2001).
[5] S. Y. Sheu, C. Y. Lin, J. D. Wu, H. C. Ching, Anticancer
Research, 119-19(1A), 23 (1999).
[6] V. Cecchetti, A. Fravolini, F. Schiaffella, O. Tabarrini,
Weicheng Zhou, J. Heterocyclic Chem., 29, 375-382 (1992).
[7a] G. Trapani, A. Latrofa, M. Franco, D. Armenise, F.
Morlacchi, Arzneim.- Forsch./Drug Res., 44 (II), 8, 969-971 (1994); [b]
D. Armenise, N. De Laurentis, A. Reho, A. Rosato, F. Morlacchi, J.
Heterocyclic Chem.,41, 771-775 (2004).
[8] V. Cecchetti, A. Fravolini, R. Fringuelli, G. Mascellani, P.
Pagella, M. Palmioli, G. Segre, and P, Terni, J. Med. Chem., 30, 465-473
(1987).
[9] Methods for Dilution Antimicrobial Susceptibility Test for
Bacteria that Grow Aerobically (6th ed.). Approved standard M7-A6.
Clinical and Laboratory Standard Institute, Wayne, PA: USA (2003).
[10] Methods for Dilution Antimicrobial Susceptibility Test for
Bacteria that Grow Aerobically (5th ed.). Suggested standard MIOO-S13.
Clinical and Laboratory Standard Institute Wayne, PA: USA (2003).
[11] Manual of Clinical Microbiology. P. R. Murray et al., 7th ed.,
American Society of Microbiology, (New York, USA) (1999).
[12] Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts; Approved Standard (2nd ed.). Approved
standard M27-A2. Clinical and Laboratory Standard Institute Wayne,
PA: USA (2002).
** For 8-Cl there are four compounds with the highest yield (i.e.
3c (63.5%), 3f (68.5%), 3i (5.5%), 3u (49.4%)) that are derived from
reagents I°, II°, III° and VII° respectively. For 6-Cl there are three
compounds with the highest yield (i.e. 3k (71.2%), 3n (84,4%), 3w
(35,9%)) that are derived from reagents IV°, V°, VIII°. For 5-Cl there is
one only compound with the highest yield (i.e. 3p (47,9%) that is
derived from reagent VI°.
*** The average yields into each of the three isomers is about
50% for reagent II°, 47-40% in decreasing order for reagents VII°, V°
and I°, 31-17% for reagents IV°, VI° and VIII°, and finally 4% for
reagent III°.
[1a] Shivanji B. Munde, Sandeep P. Bondge, Venkatesh E.
Bhingolikar, Ramrao A. Mane, Green Chemistry, 5, 278-279 (2003); [b]
D. Armenise, G. Trapani, V. Arrivo, E. Laraspata, F. Morlacchi, J.
Heterocyclic Chem., 37, 1611-1616 (2000); [c] D. Armenise, G. Trapani,
F. Stasi, F. Morlacchi, Arch. Pharm. Pharm. Med. Chem., 331, 54-58
(1998); [d] G. Scapini, F. Duro, G. Pappalardo, Annali di Chimica,
58(10), 1058-1074 (1968); [e] F. Duro, P. Condorelli, G. Scapini, G.
Pappalardo, Annali di Chimica, 60(5), 383-392 (1970).
[2] M. Y. Hamadi, R. Gupta, R. R. Gupta, J. of Fluorine Chem.,
94, 169-174 (1999).
[3] R. R. Gupta, Phenothiazines and 1,4-benzothiazines
Chemical and Biomedical Aspects, Elsevier, Amsterdam, Vol. 4, pp.163-
-
269 (1988).