Structural modifications and antimicrobial activity of N-cycloalkenyl-2- acylalkylidene-2,3-dihydro-1,3-benzothiazoles

Article abstract of DOI:10.1016/j.farmac.2005.01.010

A series of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 5a-j, N-cycloalkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 8a-e, and N-alkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 8f-h, were synthesized and tested for in vitro

Full text of DOI:10.1016/j.farmac.2005.01.010

Il Farmaco 60 (2005) 291–297
http://france.elsevier.com/direct/FARMAC/
Structural modifications and antimicrobial activity
of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles
Andrea Latrofa *, Massimo Franco, Angela Lopedota, Antonio Rosato,
Dora Carone, Cesare Vitali
Dipartimento Farmaco-Chimico, Facoltà di Farmacia, Università degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy
Received 12 July 2004; received in revised form 20 January 2005; accepted 27 January 2005
Abstract
A series of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 5a–j, N-cycloalkyl-2-acylalkylidene-2,3-dihydro-1,3-
benzothiazoles 8a–e, and N-alkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 8f–h, were synthesized and tested for in vitro antibacte-
rial and antifungal activities against four gram-positive and five gram-negative bacteria (Bacillus subtilis 6633, Enterococcus faecalis 29212,
Staphylococcus aureus 6538, Staphylococcus aureus 25923, Escherichia coli 25922, Acinetobacter calcoaceticus a1, A. calcoaceticus a2,
Pseudomonas aeruginosa 27835, Klebsiella oxytoca 49131), four yeast-like fungi and one fungus (Candida tropicalis 750, C. albicans
14053, C. albicans 10231, Criptococcus laurentii 18803, and Saccharomyces cerevisiae). Microdilution broth and agar dilution methods
were used for antimicrobial tests. The findings obtained showed that some of the tested compounds 5 and 8 were effective against some of the
bacterial strains used, whereas, only compounds 8b–g exhibited a moderate antifungal activity against the yeast strains evaluated.
© 2005 Elsevier SAS. All rights reserved.
Keywords: N-Cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles; N-Cycloalkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles; Antibacterial activ-
ity; Antifungal activity
1. Introduction
fungal activity. The preliminary structure–antibacterial activ-
ity relationship [11] suggested the lipophilicity as an important
property modulating the activity of the reported compounds,
together with the observation that the antibacterial activity
disappeared on replacing the hydrogen atom of the structure
A (R′ = H) with an alkyl or acyl group.
The small and simple benzothiazole nucleus is present in
compounds involved in research aimed at evaluating new
products that possess interesting biological activities: antitu-
moral [1,2], antimicrobial [3], antitubercular [4], and antima-
larial [5]. As part of our program aimed at the synthesis and
biological evaluation of azasulfurated heterocyclic com-
pounds [6–9], we have recently reported that also
N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-
benzothiazoles A (Fig. 1), obtained by reaction of N-acyl-2,3-
dihydro-1,3-benzothiazoles with carboxylic anhydrides [10],
showed interesting activity against some gram-positive and
gram-negative bacteria. Some of the evaluated compounds
[11] exhibited a minimal inhibitory concentration (MIC) lower
than the reference compounds chloramphenicol and gentami-
cine, whereas all the tested compounds proved devoid of anti-
* Corresponding author. Dipartimento Farmaco-Chimico, Via Orabona 4,
70125 Bari, Italy.
Fig.
1
.
Structural modifications (- - -
) of the N-cycloalkenyl-2-
→
E-mail address: alatrofa@farmchim.uniba.it (A. Latrofa).
acylalkylidene-2,3-dihydro-1,3-benzothiazole.
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.01.010

292
A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
Consistent with the previous studies, and in view of the
acyl chloride with N-benzyl-2-methyl-benzothiazole bro-
mide (Scheme 2) obtained by a modified reported method
[14]. This last modified method [14] failed in the synthesis of
the other compounds 8, which alternatively were prepared
according to the previously reported procedures, by ring cleav-
age of the 2,3-dihydro-1,3-benzothiazole compounds 3
(Scheme 1) with sodium borohydride to yield the correspond-
ing N,N′-dialkyldithiodianiline 6 [15]. The reaction of com-
pounds 6 with the appropriate b-ketoester 7 led to com-
pounds 8 (and by products of reaction 9) [9] in roughly
equimolar amount, as in Scheme 1.
continuous interest for new antimicrobial agents, we deemed
it worthwhile to investigate other N-substituted-2-
acylalkylidene-2,3-dihydro-1,3-benzothiazoles to better
evaluate the main structural requirements needful to improve
and extend antimicrobial activity. We have therefore enlarged
our investigations to determine if the increasing of the lipo-
philic character of the previously evaluated compounds [11],
together with some structural modifications of A might influ-
ence the antimicrobial activity of the N-cycloalkenyl-2-
acylalkylidene-2,3-dihydro-1,3-benzothiazoles. In this work,
we have designed and synthesized a new series of 1,3-
benzothiazole compounds to evaluate the effects of their struc-
tural modifications on activity against some gram-positive and
gram-negative bacteria, as well as against some fungi. The
structural changes of compound A comprised: (i) the ring
enlargement of the N-cycloalkenyl moiety (5a–d); (ii) the
introduction of different substituents on the benzene ring (5f–
j); (iii) the variation in the length of acyl chain (5e); (iv) reduc-
tion of the N-cycloalkenyl moiety 8a–e, including a cyclic
acyl chain 8d, or a N-alkyl moiety 8h, containing also an het-
erocyclic acyl chain 8g. Antimicrobial activity of the new
compounds and of the known compounds 8c,e,f [9] was evalu-
ated using both the microdilution broth and agar dilution
methods.
2.1.1. General procedure for the synthesis
of N-cycloalkenyl-compounds 5a–j
A mixture of 3-acetyl-2,3-dihydro-1,3-benzothiazole 4
(10 mmol) and the appropriate carboxylic anhydride (10 ml)
was refluxed until the disappearance of the starting material
monitored by TLC. Then, the excess of the anhydride was
evaporated under reduced pressure, and the residue was puri-
fied by column chromatography. Physical and spectral data
of the new compounds 5 are reported below.
2.1.1.1. 2-(Acetylmethylene)-N-(cyclododec-1-enyl)-2,3-
dihydro-1,3-benzothiazole (5a). Yield: 75%; m.p. 177–
178 °C; Anal (C, H, N) C22H29NOS. IR (cm–1) 1610 (C=O);
1H NMR (CDCl3) d (ppm): 1.20–1.95 (m, 18H, CH2), 2.20
(s, 3H, CH3), 2.32–2.70 (m, 4H, CH2), 5.70–5.90 (m, 1H,
=CH–CH2), 5.92 (s, 1H, =CH), 7.0–7.7 (m, 4H, CHAr); m/z
355 (M+ 31), 312 (M+ –(CH3CO), base).
2. Experimental
2.1. Chemistry
2.1.1.2. 2-(Propionylmethylene)-N-(cyclododec-1-enyl)-2,3-
dihydro-1,3-benzothiazole (5b). Yield: 35%, m.p. 171–
172 °C; Anal (C, H, N) C23 H31NOS. IR (cm–1) 1610 (C=O);
1H NMR (CDCl3) d (ppm): 1.10 (t, 3H, CH3), 1.20–1.95 (m,
18H, CH2), 2.25–2.70 (m, 4H, CH2), 5.7–5.9 (m, 1H, =CH–
CH2), 5.93 (s, 1H, =CH), 7.0–7.7 (m, 4H, CHAr); m/z 369
(M+, 29), 312 (M+ –(CH3CH2CO), base).
Melting points were determined on a Büchi apparatus and
are uncorrected. IR spectra were recorded on a Perkin Elmer
257 Spectrophotometer as KBr disks or liquid films. 1H NMR
spectra were obtained on aVarian EM 390 spectrometer using
TMS as internal standard (chemical shifts are expressed in d
units). Elemental analyses (C, H, N) were performed on Carlo
Erba 1106 analyser, and the results agreed with the theoreti-
cal values 0.4%. GC–MS analysis was carried out on a
Hewlet–Packard 5995C-GC–MS instrument. Column chro-
matography was performed using silica gel (Merck 70–
235 mesh) as stationary phase and light petroleum ether/ethyl
acetate 8:2 v/v and 9:1 v/v as eluent for compounds 5 and 8,
respectively. The progress of the reactions was monitored by
thin-layer chromatography (TLC) with F254 silica gel pre-
coated sheets (Merck) using light petroleum ether–ethyl
acetate (85:15 v/v) as the mobile phase.
The starting 5-substituted-2-aminobenzenethiols1f–j were
obtained from the corresponding 6-substituted-2-amino-
benzothiazoles (Scheme 1), according to the reported proce-
dure [12]. The N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-
1,3-benzothiazoles 5 were prepared according to the literature
procedure [10] by treatment of the N-acetyl-2,3-dihydro-1,3-
benzothiazole compounds 4 with appropriate carboxylic anhy-
dride as in Scheme 1. Compound 8h was prepared, according
to a known procedure [13], by reaction of the appropriate
2.1.1.3. 2-(Butyrylmethylene)-N-(cyclododec-1-enyl)-2,3-
dihydro-1,3-benzothiazole (5c). Yield: 65%, m.p. 167–
168 °C; Anal (C, H, N) C24H33NOS. IR (cm–1) 1610 (C=O);
1H NMR (CDCl3) d (ppm): 0.95 (t, 3H, CH3), 1.2–1.9 (m,
18H, CH2), 2.42–2.70 (m, 6H, CH2), 5.70–5.90 (m, 1H, =CH–
CH2), 5.92 (s, 1H, =CH), 7.0–7.70 (m, 4H, CHAr); m/z 383
(M+, 30), 312 (M+ –(CH3CH2CH2CO), base).
2.1.1.4. 2-(Isobutyrylmethylene)-N-(cyclododec-1-enyl)-2,3-
dihydro-1,3-benzothiazole (5d). Yield: 42%, m.p. 162–
163 °C; Anal (C, H, N) C24H33NOS. IR (cm–1) 1610 (C=O);
1H NMR (CDCl3) d (ppm): 1.20 (d, 6H, CH3), 1.22–1.90 (m,
18H, CH2), 2.24–2.80 (m, 5H, CH + CH2), 5.70–5.90 (m,
1H, =CH–CH2), 6.0 (s, 1H, =CH), 7.0–7.7 (m, 4H, CHAr);
m/z 383 (M+, 30), 312 (M+ –((CH3)2CHCO), base).
2.1.1.5. 2-(Heptanoylmethylene)-N-(cyclohept-1-enyl)-2,3-
dihydro-1,3-benzothiazole (5e). Yield: 55%, oil; Anal (C, H,
N) C22H29NOS. IR (cm–1) 1610 (C=O); 1H NMR (CDCl3) d

A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
293
Scheme 1. Synthesis of compounds 5,8 and 9.
(ppm): 0.85 (t, 3H, CH3), 1.05–1.45 (m, 6H, CH2), 1.50–
1.90 (m, 8H, CH2), 2.23–2.60 (m, 6H, CH2), 5.85 (s, 1H,
=CH), 6.0–6.20 (m, 1H, =CH–CH2), 7.0–7.70 (m, 4H, CHAr);
m/z 355 (M+, 20), 242 (M+ –(CH3(CH2)5CO), base).
2.1.1.7. 2-(Butyrylmethylene)-N-(cyclohept-1-enyl)-6 meth-
oxy-2,3-dihydro-1,3-benzothiazole (5g). Yield: 35%, oil;Anal
1
(C, H, N) C20H25NO2S. IR (cm–1) 1600 (C=O); H NMR
(CDCl3) d (ppm): 0.95 (t, 3H, CH3), 1.50–2.0 (m, 8H, CH2),
2.30–2.63 (m, 6H, CH2), 3.90 (s, 3H, CH3), 5.80 (s, 1H, =CH),
6.10 (t, 1H, =CH–CH2), 6.80–7.20 (m, 3H, CHAr); m/z 343
(M+, 19), 272 (M+ –(CH3CH2CH2CO), base).
2.1.1.6. 2-(Butyrylmethylene)-N-(cyclohept-1-enyl)-6 fluoro-
2,3-dihydro-1,3-benzothiazole (5f). Yield: 30%, m.p. 88–
89 °C; Anal (C, H, N) C19H22NOSF. IR (cm–1) 1590 (C=O);
1H NMR (CDCl3) d (ppm): 0.95 (t, 3H, CH3), 1.50–2.02 (m,
8H, CH2), 2.30–2.63 (m, 6H, CH2), 5.83 (s, 1H, =CH), 6.10
(t, 1H, =CH–CH2), 6.90–7.33 (m, 3H, CHAr); m/z 331 (M+,
18), 260 (M+ –(CH3CH2CH2CO), base).
2.1.1.8. 2-(Isobutyrylmethylene)-N-(cyclohept-1-enyl)-
6 methoxy-2,3-dihydro-1,3-benzothiazole (5h). Yield: 40%,
m.p. 95–96 °C; Anal (C, H, N) C20H25NO2S. IR (cm–1) 1600
(C=O); 1H NMR (CDCl3) d (ppm): 1.20 (d, 6H, CH3), 1.50–

294
A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
Scheme 2. Synthesis of compound 8h.
2.0 (m, 6H, CH2), 2.20–2.50 (m, 4H, CH2), 2.50–2.80 (m,
1H, CH), 3.82 (s, 3H, CH3), 5.80 (s, 1H, =CH), 6.10 (t, 1H,
=CH–CH2), 6.80–7.20 (m, 3H, CHAr); m/z 343 (M+, 22),
272 (M+ –((CH3)2CHCO), base).
1H NMR (CDCl3) d (ppm): 1.40–1.90 (m, 14H, CH2), 3.40–
3.60 (m, 1H, CH), 4.90 (bs, 1H, NH), 6.40–6.60 (m, 2H,
CHAr), 7.10–7.30 (m, 2H, CHAr); m/z 436 (M+, 88), 202
(base).
2.1.1.9. 2-(Isobutyrylmethylene)-N-(cyclohept-1-enyl)-
6 methyl-2,3-dihydro-1,3-benzothiazole (5i). Yield: 70%, m.p.
108–110 °C; Anal (C, H, N) C20H25NOS. IR (cm–1) 1610
(C=O); 1H NMR (CDCl3) d (ppm): 1.20 (d, 6H, CH3), 1.60–
2.03 (m, 6H, CH2), 2.20–2.80 (m, 8H, CH2 + CH3 + CH),
5.80 (s, 1H, =CH), 6.10 (t, 1H, =CH–CH2), 6.80–7.40 (m,
3H, CHAr); m/z 327 (M+, 21), 256 (M+ –((CH3)2CHCO),
base).
2.1.3. General procedure for the synthesis
of N-alkyl-1,3-benzothiazole compounds 8
A solution of the N,N′-dialkyl-dithiodianiline 6 (10 mmol)
and the appropriate b-ketoester 7 (22 mmol) in toluene con-
taining catalytic amounts of p-toluenesulfonic acid, was
refluxed by stirring, whilst monitoring the reaction progress
by TLC. The solvent was then evaporated under reduced pres-
sure, and the resulting crude was purified by column chroma-
tography on silica gel to obtain the by products of the reac-
tion 9 and compounds 8 in the order given. Physical and
spectral data of the new compounds 8a,b,d,g and 9a,b,d,g
are reported below, while compounds 8c,e,f and 9c,e,f have
been reported previously [9].
2.1.1.10. 2-(Valerylmethylene)-N-(cyclohept-1-enyl)-6 methyl-
2,3-dihydro-1,3-benzothiazole (5j). Yield: 80%, m.p. 72–
73 °C; Anal (C, H, N) C21H27NOS. IR (cm–1) 1610 (C=O);
1H NMR (CDCl3) d (ppm): 0.95 (t, 3H, CH3), 1.20–2.03 (m,
12H, CH2), 2.30–2.60 (m, 7H, CH2 + CH3), 5.80 (s, 1H,
=CH), 6.10 (t, 1H, =CH–CH2), 6.80–7.40 (m, 3H, CHAr);
m/z 341 (M+, 21), 256 (M+ –(CH3(CH2)3CO), base).
2.1.3.1. 1-(3-Cyclo-octyl-1,3-benzothiazole-2(3H)-ylidene)bu-
tan-2-one (8a). Yield: 20%, m.p. 110–111 °C; Anal (C, H,
N) C19H25NOS. IR (cm–1) 1615 (C=O); 1H NMR (CDCl3) d
(ppm): 1.20 (t, 3H, CH3), 1.50–2.13 (m, 12H, CH2), 2.30–
2.70 (m, 4H, CH2), 4.4–4.9 (m, 1H, CH), 5.90 (s, 1H, =CH),
7.0–7.7 (m, 4H, CHAr); m/z 315 (M+, 20), 149 (base).
2.1.2. General one-flask procedure for the synthesis
of N,N′-dialkyldithiodianilines 6
A solution of 2-aminobenzenethiol 1 (10 mmol), appro-
priate carbonyl compound 2 (10.1 mmol) and catalytic amount
of p-toluenesulfonic acid in toluene (60 ml), was refluxed for
3 h under nitrogen, while the water formed was continuously
separated. The solvent was then evaporated and methanol
(80 ml) was added to the residue. Then, the methanolic solu-
tion was treated with sodium borohydride (50 mmol) portion-
wise under nitrogen, monitoring the progress of the reaction
by TLC. Removal of the solvent was followed by dilution
with ice water (100 ml), acidification with acetic acid, and
extraction with ether (3 × 50 ml). The combined extracts were
washed with water (3 × 30 ml), dried with sodium sulfate,
stirred with exposure to air overnight and concentrated. The
residual yellow oil was purified by column chromatography
on silica gel 60, using light petroleum ether/ethyl acetate
95:5 v/v as eluent to give pure compound 6. Physical and
spectral data of the new compound 6a are reported below.
2.1.3.2. 1-(3-Cycloheptyl-1,3-benzothiazole-2(3H)-ylidene)-
butan-2-one (8b). Yield: 25%, m.p. 84–86 °C; Anal (C, H,
N) C18H23NOS. IR (cm–1) 1615 (C=O); 1H NMR (CDCl3) d
(ppm): 1.20 (t, 3H, CH3), 1.50–2.13 (m, 10H, CH2), 2.30–
2.70 (m, 4H, CH2), 4.3–4.7 (m, 1H, CH), 5.90 (s. 1H, =CH),
7.0–7.70 (m, 4H, CHAr); m/z 301 (M+, 17), 149 (base).
2.1.3.3. 2-(N-Cycloheptyl-1,3-benzothiazol-2(3H)-ylidene)-
cyclopentanone (8d). Yield: 15%, m.p. 155–156 °C; Anal
(C, H, N) C19H23NOS. IR (cm–1) 1630 (C=O); H NMR
1
(CDCl3) d (ppm): 1.50–2.10 (m, 12H, CH2), 2.2–2.6 (m, 4H,
CH2), 3.15 (t, 2H, CH2), 4.80–5.22 (m, 1H, CH), 7.0–7.7 (m,
4H, CHAr); m/z 313 (M+, 2), 162 (base).
2.1.3.4. 1-Benzyl-4-(3-butyl-1,3-benzothiazol-2(3H)-ylidene)-
piperidin-3-one (8g). Yield: 35%, oil; Anal (C, H, N)
1
2.1.2.1. N,N′-dicyclo-octyl-2,2′-dithiodianiline (6a). Yield:
C23H26N2OS. IR (cm–1) 1660 (C=O); H NMR (CDCl3) d
85%, oil; Anal (C, H, N) C28H40N2S2. IR (cm–1) 3390 (NH);
(ppm): 0.90 (t, 3H, CH3), 1.10–1.90 (m, 4H, CH2), 2.70 (t,

A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
295
2H, CH2), 3.0 (t, 2H, CH2), 3.30 (s, 2H, CH2), 3.60 (s, 2H,
2.2. Antimicrobial assays
CH2), 4.22 (m, 2H, CH2), 7.0–7.40 (m, 7H, CHAr), 7.50–
7.60 (m, 2H, CHAr); m/z 378 (M+, 26), 287 (M+ –CH2C6H5,
base).
Compounds 5 and 8 were tested to evaluate their antimi-
crobial activity against a variety of gram-positive (B. subtilis
6633, E. faecalis 29212, S. aureus 6538p, S. aureus 25923)
and gram-negative bacteria (E. coli 25922, A. calcoaceticus
a1, A. calcoaceticus a2, P. aeruginosa 27853, K. oxytoca
49131), as well as against the yeasts (C. tropicalis 750, C. albi-
cans 14053, C. albicans 10231, S. cerevisiae 2366, C. lau-
rentii 18803).
The MICs (µg/ml) of compounds 5 and 8 were determined
by both microdilution broth method (MBM) and agar dilu-
tion method (ADM), the latter being a useful standardized
reliable susceptibility testing that may be used as a reference
for the evaluation of insoluble compounds of a series. Muel-
ler Hinton Broth (MHB) and Mueller Hinton Agar (MHA)
were used for the testing methods for bacteria, andYeast Malt
Broth (YMB) and Yeast Malt Agar (YMA) for yeast strains,
respectively.
For bacteria and yeast strains, the inocula were prepared
as follows: colonies derived from fresh mature strain on MHA
for bacteria or potato dextrose agar (PDA) for yeast plates
were suspended in saline solutions (0.85%) and suspensions
were adjusted approximately to 108 cfu/ml (0.5 Mac Far-
land) [16] for bacteria and 106 cfu/ml for yeast strain, as deter-
mined by viable count.
In the ADM, a series of twofold dilution in dimethyl sul-
foxide (DMSO) for the bacteria, or ethanol for yeasts, of each
tested product were prepared in MHA plates for bacteria, or
YMA plates for yeasts. Final product concentrations in the
Petri dish ranged 100–1.56 µg/ml. After drying for 30 min,
plates were spot inoculated by pipetting the desired test micro-
organism (40 µl for bacteria and 10 µl for yeasts) from the
prepared inoculum onto the plates [17]. The MICs were
recorded as the lowest concentration of antimicrobial agent
that inhibited visible growth for the two types of micro-
organisms after incubation for 24 h at 37 °C for bacteria and
48 h at 30 °C for yeasts.
2.1.3.5. 4-(Cyclo-octyl)-2-propionyl-2H-1,4-benzothiazin-
3(4H)-one (9a). Yield: 25%, oil;Anal (C, H, N) C19H25NO2S.
IR (cm–1) 1670, 1730 (C=O); 1H NMR (CDCl3) d (ppm): 1.0
(t, 3H, CH3), 1.40–2.80 (m, 16H, CH2), 4.22 (s, 1H, CH),
4.40–4.80 (m, 1H, CH), 6.90–7.40 (m, 4H, CHAr); m/z 331
(M+, 12), 165 (base).
2.1.3.6. 4-(Cycloheptyl)-2-propionyl-2H-1,4-benzothiazin-
3(4H)-one (9b). Yield: 20%, oil;Anal (C, H, N) C18H23NO2S.
IR (cm–1) 1670, 1720 (C=O); 1H NMR (CDCl3) d (ppm): 1.0
(t, 3H, CH3), 1.40–2.80 (m, 14H, CH2), 4.22 (s, 1H, CH),
4,30–4.60 (m, 1H, CH) 6.90–7.40 (m, 4H, CHAr); m/z 317
(M+, 22), 165 (base).
2.1.3.7. 4-(Cycloheptyl)-2′H-spiro-[1,4-benzothiazin-2,1′-
cyclopentane]-2′,3(4H)-dione (9d). Yield: 35%, oil; Anal (C,
H, N) C19H23NO2S. IR (cm–1) 1650, 1730 (C=O); 1H NMR
(CDCl3) d (ppm): 1.3–3.0 (m, 18H, CH2), 4.20–4.70 (m, 1H,
CH), 6.80–7.50 (m, 4H, CHAr); m/z 329 (M+, 42), 177 (base).
2.1.3.8. 1′-Benzyl-4-butyl-3′H-spiro[1,4-benzothiazine-2,4′-
piperidin]-3,3′(4H)-dione (9g). Yield: 30%, m.p. 58–60 °C;
Anal (C, H, N) C23H26N2O2S. IR (cm–1) 1660, 1720 (C=O);
1H NMR (CDCl3) d (ppm): 0.90 (t, 3H, CH3), 1.20–2.10 (m,
6H, CH2), 2.70–3.10 (m, 4H, CH2), 3.50-3.70 (m, 2H, CH2),
3.90–4.10 (m, 2H, CH2), 6.90–7.40 (m, 9H, CHAr); m/z 394
(M+, 71), 91 (base).
2.1.4. Procedure for the synthesis of compound 8h
Benzyl bromide (12 mmol) was added under nitrogen to
2-methyl benzothiazole (10 mmol) with stirring at 60–70 °C
for 3 h. The mixture was then cooled, the precipitate filtered
off, and washed with ether to give 3-benzyl-2-methyl-
benzothiazolium bromide. To the suspension of 3-benzyl-2-
methylbenzothiazolium bromide (5 mmol) in pyridine (5 ml),
isobutyryl bromide (10 mmol) was added under nitrogen with
stirring for 30 min. Then, ice water and dilute HCl were added
to the reaction mixture and extracted with chloroform
(3 × 20 ml). The organic layer was washed with 10% aque-
ous sodium bicarbonate (3 × 30 ml) and with water
(3 × 30 ml), dried over sodium sulfate, filtered, and the sol-
vent evaporated to give a solid, which crystallized from
ligroine afforded compound 8h whose physical and spectral
data are reported below.
In the MBM, for bacteria, a series of twofold dilution of
each product were prepared in DMSO and put in 96-well
microdilution trays containing broth media. Each well was
inoculated with 20 µl of a final inoculum concentration that
approximately contained 2.5 × 106 cfu/ml. The trays were
incubated at 37 °C for 24 h for bacteria. For yeasts, 100 µl of
inoculum containing 103 cfu/ml was used, then incubated for
48 h at 30 °C. The MICs were determined as the lowest con-
centration 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 for bacteria, and for yeasts,
the visual comparison of growth (50% inhibition) with that
of growth control [17].
2.1.4.1. 1-(3-Benzyl-1,3-benzothiazol-2(3H)-ylidene)-3-
methylbutan-2-one (8h). Yield: 55%, m.p. 133–135 °C; Anal
1
(C, H, N) C19H19NOS. IR (cm–1) 1610 (C=O); H NMR
Appropriate controls involved cefepime for bacteria and
fluconazole as international reference standards [18] for
yeasts. Also DMSO and ethanol alone were tested for their
activities against bacteria and yeasts, and consequently quan-
tities of these two solvents under 5% for DMSO and 2% for
(CDCl3) d (ppm): 1.1 (d, 6H, CH3), 2.60 (hept, 1H, CH), 5.20
(s, 2H, CH2), 5.92 (s, 1H, =CH), 7.0–7.40 (m, 8H, CHAr),
7.48–7.60 (m, 1H, CHAr); m/z 309 (M+, 44), 266 (M+;
–(CH3)2CH, 83), 91 (CH2C6H5, base).

296
A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
ethanol were used during the assays. Each species was tested
at least three times in duplicate. The MBM and ADM assays
for bacteria were based on the recommended procedures by
NCCLS [17], whereas the modified reference [18] of theADM
was used for yeasts.
(C. tropicalis 750, C. albicans 14053, C. albicans 10231, S.
cerevisiae 2366, C. laurentii 18803), independent from the
length of the acyl chain (see 8b and 8c), as well as the pres-
ence of the cyclic acyl chain 8d. The absence of the antifun-
gal activity for compound 8a (if other reasons are not
involved) seemed attributable probably to the most lipophilic
N-cyclo-octyl moiety.
Furthermore, a moderate antimicrobial activity was ob-
served for compound 8a,b particularly against the gram-
negative bacteria tested. This activity appeared related to the
length of the acyl chain. In fact, compound 8c in which the
acyl chain is limited to a methyl group is devoid of any anti-
bacterial activity, whereas the presence of the cyclic acyl chain
(compound 8d) resulted in weak activity against the gram-
positive and gram-negative bacteria, with surprisingly high
activity only against E. faecalis 29212.
3. Results and discussion
The two different series of compounds 5a–j and 8a–h were
tested by both the microdilution broth and agar dilution meth-
ods to evaluate their antimicrobial activity against some micro-
bial species.
The findings obtained, reported in Table 1, showed that
the N-cycloalkenyl compounds (5a–j) exhibited no activity
against fungi analogously to the previously reported com-
pounds [11]. Moreover, an enhancement in antimicrobial
activity was observed by increasing the length of the acyl
chain R1, particularly against three gram-positive (E. faeca-
lis 29212, S. aureus 6538p, S. aureus 25923) and one gram-
negative bacteria (E. coli 25922) (compare 5a,b with 5c,d).
Surprisingly, the most lipophilic cyclododecenyl moiety on
the nitrogen atom (5a–d), as well as the heptanoil chain (5e)
did not lead to more effective compounds than the
N-cycloheptyl and N-cyclo-octyl analogues previously
reported [11]. Besides, the presence of fluorine (5f) on the
benzene ring dramatically reduced the activity in comparison
with the analogue not fluorinated, whilst the presence of the
methoxy group (5g,h) led to effective compounds against all
the bacteria tested. Finally, the presence of methyl group on
the benzene ring increased the antibacterial activity only for
compound 5j more lipophilic than the 5i analogue. The
N-cycloalkyl compounds 8b–e as well as the N-alkyl com-
pounds 8f–g, differently from the N-cycloalkenyl analogue
compounds, showed a certain activity against the tested fungi
4. Conclusions
The structural modifications of the N-cycloalkenyl-2-
acylalkylidene-2,3-dihydro-1,3-benzothiazole A, indicate that
on increasing the ring size of the N-cycloalkenyl moiety, as
well as on increasing the length of the acyl chain, or on insert-
ing a substituent on the benzene ring, an increase in antibac-
terial potency in comparison with the previously reported data
[11] is not observed. In particular, the antibacterial activity of
the new N-cyclododecenyl compounds 5a–d is comparable
with the early reported activity for the N-cycloheptenyl and
N-cyclo-octenyl analogues [11]. Moreover, the length of the
acyl chain seems conclusive for the antibacterial activity of
the N-cycloalkenyl compounds 5, whereas it does not appear
influent on the antifungal activity of the N-cycloalkyl com-
pounds 8. The substitution of the N-cycloalkenyl moiety with
the N-cycloalkyl group causes a new weak antifungal activ-
Table 1
Antimicrobial activity of compounds 5 and 8
B.
E.
S.
S.
E. coli
A. calco- A. calco- P. aeru- K.
C.
C.
C.
S. cere- C.
subtilis faecalis aureus
aureus
29213
12.5
12.5
12.5
12.5
6.2
25922
aceticus aceticus ginosa oxytoca tropicalis albicans albicans visiae
laurentii
18803
>100
>100
> 100
> 100
> 100
> 100
> 100
25
6633
100
29212
12.5
12.5
12.5
12.5
12.5
25
6538p
12.5
12.5
12.5
12.5
12.5
25
a1
a2
27853
100
49131
100
100
100
12.5
12.5
50
750
14053
> 100
> 100
> 100
> 100
> 100
> 100
> 100
25
10231
> 100
> 100
> 100
> 100
> 100
> 100
> 100
50
2366
>100
>100
> 100
> 100
> 100
> 100
> 100
25
5c
12.5
12.5
12.5
6.2
100
100
100
6.2
100
100
100
12.5
12.5
25
> 100
> 100
> 100
> 100
> 100
> 100
> 100
50
5d 100
5e 100
5g 6.2
5h 6.2
100
100
12.5
12.5
25
6.2
12.5
50
5j
12.5
50
12.5
25
8a 100
8b 100
100
100
100
25
25
25
25
100
100
100
50
50
25
100
50
8c
> 100
> 100
12.5
> 100
> 100
100
> 100
100
> 100
100
> 100
50
> 100
50
> 100
50
> 100
1500
> 100
> 100
> 100
1
> 100
50
50
25
50
25
25
8d 100
8e
8f
8g 100
Cef 1.4
Flu
100
50
100
50
25
> 100
> 100
> 100
> 100
100
> 100
> 100
> 100
3.7
> 100
> 100
100
0.1
> 100
> 100
> 100
2.8
> 100
> 100
> 100
2.8
> 100
> 100
100
0.2
25
50
50
50
25
100
50
50
50
25
25
50
100
100
50
2.3
3.7
2
0.25
0.25
8
4
The omitted compounds 5a,b,f,i, and 8h, showed a MIC against the reported bacteria and yeasts ≥ 100. The reported MIC values refer to the agar dilution
method.
Cef: cefepime; Flu: fluconazole.

A. Latrofa et al. / Il Farmaco 60 (2005) 291–297
297
ity for some of these compounds. Finally, both microdilution
broth and agar dilution methods used are suitable for testing
the antimicrobial activity of a series of hydrophilic–lipo-
philic compounds.
Elemental analyses
% Calculated value
Compound
Formula
Molecular weight
% Actual value
H
C
H
N
C
N
5a
5b
5c
5d
5e
5f
5g
5h
5i
C22H29NOS
C23H31NOS
C24H33NOS
C24H33NOS
C22H29NOS
C19H22NOSF
C20H25NO2S
C20H25NO2S
C20H25NOS
C21H27NOS
C28H40N2S2
C19H25NOS
C18H23NOS
C19H23NOS
C23H26N2OS
C19H19NOS
C19H25NO2S
C18H23NO2S
C19H23NO2S
C23H26N2O2S
355.46
369.49
383.51
383.51
355.46
331.38
343.41
343.41
327.41
341.43
468.62
315.40
301.37
313.38
378.45
309.34
331.40
317.38
329.38
394.45
74.33
74.76
75.16
75.16
74.33
68.88
69.95
69.95
73.36
73.87
71.76
72.35
71.73
72.82
72.99
73.76
68.86
68.12
69.28
70,03
8.22
8.46
8.67
8.67
8.22
5.74
7.33
7.33
7.70
7.97
8.60
7.99
7.69
7.40
6.93
6.15
7.60
7.30
7.04
6.64
3.94
3.79
3.65
3.65
3.94
4.20
4.08
4.08
4.28
4.10
5.98
4.44
4.65
4.47
7.40
4.52
4.23
4.41
4.25
7.10
74.88
75.01
75.30
74.85
74.65
68.55
70.12
69.75
73.05
73.73
71.45
72.45
71.52
72.71
72.80
73.68
68.63
68.04
69.05
70.20
8.50
8.55
8.84
8.68
8.41
6.01
7.42
7.39
7.59
7.95
8.38
8.16
7.83
7.55
6.81
6.24
7.73
7.43
7.15
6.85
3.97
3.72
3.61
3.57
3.86
4.14
4.02
3.90
4.15
4.08
6.03
4.25
4.51
4.33
7.55
4.72
4.20
4.53
4.11
7.21
5j
6a
8a
8b
8d
8g
8h
9a
9b
9d
9g
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