Synthesis and anti-microbial activity of isothiosemicarbazones and cyclic analogues.

Article abstract of DOI:10.1016/S0014-827X(02)01288-0

It is known that some derivatives of both thiourea and thiosemicarbazide exhibit potent anti-microbial activity. In order to investigate the effects on the biological properties of structural modifications of such structures, we have synthesised and studi

Full text of DOI:10.1016/S0014-827X(02)01288-0

Il Farmaco 57 (2002) 809Á817
/
www.elsevier.com/locate/farmac
Synthesis and anti-microbial activity of isothiosemicarbazones and
cyclic analogues
E. Maccioni a,*, M.C. Cardia a, L. Bonsignore a, A. Plumitallo a, M.L. Pellerano b,
A. De Logu b
a Dipartimento farmaco chimico tecnologico, Via Ospedale, 72, 09124 Cagliari, Italy
b Dipartimento di scienze chirurgiche e trapianti d’organo, Via Palabanda, 14, 09123 Cagliari, Italy
Received 9 January 2002; accepted 9 June 2002
Abstract
It is known that some derivatives of both thiourea and thiosemicarbazide exhibit potent anti-microbial activity. In order to
investigate the effects on the biological properties of structural modifications of such structures, we have synthesised and studied
some arylidenisothiosemicarbazones. In this paper we report on the synthesis and structureÁactivity relationships of some
/
isothiosemicarbazones, where the arylidene group has been replaced with a cycloalkyl group and the sulfur atom has been either
differently substituted or enclosed in a thiazole ring.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Isothiosemicarbazones; Thiazole; Biological activity
1. Introduction
Our efforts were therefore concentrated on the
introduction of different subtituents on the sulfur
atom, thus leading to some interesting results. We
observed that the introduction of a benzyl group bearing
chlorine atoms on the aromatic ring always led to the
most potent compounds. Surprisingly the same activity
was not observed on replacing chlorine with other
halogens. Moreover, a chlorine position-depending
influence was observed, the para-substituted compounds
being always more active than the meta and ortho ones.
In the light of the above we now wish to investigate
the influence of structural modifications on these kinds
of structures further. In particular the arylidene group
will be replaced with a cycloalkyl group. This change
will lead to the elimination of the aromatic planar
lipophile centre, and to the introduction of an aliphatic
flexible cyclic system.
During the past years an increasing interest has been
devoted to the study of new and more selective anti-
microbial agents. Due to this, not only have new
synthetic methods been developed, but a greater amount
of interest has also been devoted to the comprehension
of their mechanisms of action and structureÁ
relationships.
/
activity
In a previous paper we reported on the synthesis and
biological properties of some arylidenisothiosemicarba-
zones [1].
In particular the influence of structural modifications
of both the sulfur and the nitrogen atom on the
biological properties of these molecules were investi-
gated (see Fig. 1).
The substitutions on the nitrogen atom (Rƒ in Fig. 1),
not only did not lead to a significant enhancement of the
anti-microbial activity, but, in some cases, led to a
complete loss of activity.
Two different kinds of changes will be operated on the
sulfur atom: the introduction of the substituents that led
to the highest activity in the case of the previously
studied arylidenisothiosemicarbazones, and the intro-
duction of the sulfur itself in a thiazole ring.
Furthermore we wish to investigate the structureÁ
/
activity relationships by measuring the lipophilic cha-
racter of the synthesised molecules and by relating it to
* Corresponding author
E-mail address: maccione@unica.it (E. Maccioni).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 2 8 8 - 0

810
E. Maccioni et al. / Il Farmaco 57 (2002) 809Á817
/
inclusion of both the sulfur and the nitrogen atom in a
thiazole ring. Compounds 1Á11 are reported in Fig. 2a,
while compounds 12Á17 are depicted in Fig. 2b.
The synthetic pathway to compounds 1Á
trated in Scheme 1.
The synthesis of compounds 1Á
/
/
/
17 is illus-
Fig. 1.
/
17 was accomplished
their biological properties. It has been reported that
pharmaceutical properties are strongly related to the
by functionalisation of the thiosemicarbazide moiety.
In the case of compounds 1Á11 the thiosemicarbazide
/
lipophilic character [2Á5]. In particular Boyce and
/
was reacted, under reflux in isopropyl alcohol, with the
appropriate benzyl chlorides.
The obtained isothiosemicarbazides were then re-
fluxed in isopropyl alcohol in the presence of the
required cycloketones and catalytic amounts of acetic
acid.
These conditions proved to be the best, especially
because the required product directly precipitates from
the reaction mixture.
Only in the case of compounds 5, 9 and 11, where the
cyclopentanone is reacted with benzylisothiosemicarba-
zides, are some precautions required. In particular the
Milborrow introduced the chromatographic value RM
[6,7], which can be related to the partition coefficient
and calculated from the equation:
RM ꢀlog(1=Rf ꢁ1)
Many different methods to determine the values of
RM have been reported in the literature by either
reversed or non-reversed (straight) phase systems and
also by paper or thin layer chromatographic methods
[8Á10].
/
RM calculation method to obtain an index of the
lipophilic character of a drug presents some advantages
with respect to direct partition methods, e.g. the
simultaneous analysis of a large number of samples,
which also enables the direct comparison of the obtained
results, the fact that a very little quantity of products is
required (very important when biological-originating
molecules are under investigation), and the fact that
there is no need for a quantitative analysis of the solute
[11].
temperature should not be higher than 50Á
avoid the formation of undesired side products.
In the case of compounds 12Á17 the cycloketone is
/
60 8C to
/
directly reacted with thiosemicarbazide.
The obtained cycloalkylthiosemicarbazones are then
reacted with v-bromoacetophenone to achieve the
formation of the 4-phenylthiazole ring. Also in this
case, isopropyl alcohol proved to be the most appro-
priate solvent for our purpose. In fact, the reaction
products can easily be separated as a white foam, which
after cooling down can be filtered and purified by
crystallisation from ethanol or water/ethanol. All the
synthesised products have been fully characterised by
means of analytical methods, 1H NMR, and Mass
Spectrometry in electron ionisation (e.i.) conditions.
As an example the fragmentation pathway of compound
6 is depicted in Scheme 2.
2. Results and discussion
2.1. Chemistry
Several new isothiosemicarbazone derivatives have
been synthesised with the aim of obtaining, not only
effective anti-microbial agents, but also structureÁacti-
/
vity relationship data that may make up the basis for the
design of new and more efficient derivatives.
2.2. Microbiology
Two different lines of products have been prepared
and tested against a number of microbial species, the
first bearing a substituted benzyl group on the sulfur
All the synthesised products have been tested to
evaluate their anti-microbial activity against several
microbial species.
atom (1Á
/
11), while the second is characterised by the
In the case of compounds (1Á11), as well as for the
/
previously reported compounds [1], almost no activity
was found against fungi. The MFC (Minimum Fungi-
cidal Concentration) values where too high for all
compounds 1Á11 (higher than 200 mg/ml). Surprisingly,
/
while in the case of arylidene derivatives [1] the activity
observed against Staphylococcus aureus, S. epidermidis,
B. subtilis, and B. thurigensis, was higher than that
against Streptococcus agalactiae and S. faecalis, in the
case of the cycloalkyl derivatives 1Á11 an almost
/
selective activity against S. agalactiae was observed. In
order to obtain further information on the influence of
the structural modifications on biological activity, the
Fig. 2.

E. Maccioni et al. / Il Farmaco 57 (2002) 809Á
/817
811
Scheme 1.
RM values of compounds 1Á
/
11 have been measured and
the benzyl group, which is necessary to increase the
lipophilic character of the molecule. In fact the need for
chlorine atoms becomes less important on increasing the
number of carbon atoms.
related to their biological activity against S. agalactiae.
Particular effort has been placed in the measurements of
the RM values in order to avoid adsorption mechanisms.
For this purpose the measurements were achieved on
thin layer chromatography, where the stationary phase
was previously saturated with liquid paraffin, and the
mobile phase was a water/acetone mixture. With this
procedure, reproducible results were obtained by using
either Al2O3 or silica gel thin layers pre-impregnated
with paraffin. This means that the stationary phase,
either Al2O3 or silica gel, is acting only as a support for
the non-aqueous solvent, and it cannot adsorb the
compounds under investigation.
In the case of the cyclic compounds 12Á17, quite a
/
different behaviour was observed. All the compounds
showed almost no activity against all the tested bacteria.
Cyclisation completely suppresses the anti-bacterial
activity.
On the contrary their activity against fungi is more
than encouraging.
In particular their activity against C. albicans and C.
krusei is very interesting (Table 1). The MNTD (maxi-
mum non-toxic dose) varied between 62.5 mg/ml for
compound 15 and 500 mg/ml for compound 12. In
particular the most active compounds showed the lowest
toxicity.
The relations between the RM values and the anti-
microbial activity of the most active compounds (1, 2, 5,
6, 8, 9, 11) are reported in Fig. 3.
In the case of C. albicans most of the compounds
exhibit an activity comparable or higher than that of the
reference compounds. This data is even more interesting
if we consider that although many Candida species have
been identified as etiologic agents of opportunistic
infections, C. albicans and C. krusei are the most
involved pathogens in these infections [12]. In the case
of compounds 16 and 17 a chiral centre is present. In
this study only the racemic mixture has been employed
in the biologic tests.
A higher anti-microbial activity was generally ob-
served for those compounds where the number of
carbon atoms on the cycloalkyl ring is odd.
Moreover, as can be seen in Fig. 3, the higher the RM
value, the lower the MIC’s, meaning that an increase in
the lipophilic character leads to an increase in anti-
microbial activity. However, the measured RM values
are not absolute data but can only be related to these
series of compounds.
Unlike in the other odd cycloalkyl compounds, the
activity observed in compound 11 was rather low. This
is reasonably due to the absence of a chlorine atom on
In the light of these data some SAR should be taken
into consideration. In the case of C. albicans, com-

812
E. Maccioni et al. / Il Farmaco 57 (2002) 809Á
/817
Scheme 2.
pounds 12 and 16 exhibited the highest fungicidal
activity (measured as MFC). These two compounds
differ from the cycloalkyl ring. Compound 12 bears a
cyclopentyl, while compound 16 bears a cyclohexyl, but
both showed a comparable antifungal activity.
Surprisingly, also in the case of compound 13, a
cyclohexane ring is present, but the absence of the
methyl group leads to a dramatic decrease in the
fungicidal activity. On comparing the biological proper-
ties of the two isomers 16 and 17, in which the methyl
group is in position 2 and 4, respectively, it can be
observed that the position of the methyl group on the
cyclohexane ring is important for fungicidal activity. In
fact, the MFC values shown by compound 17 are much
higher than those shown by its isomer 16.
Along the series 12, 13, 14, and 15, two different
behaviours can be observed. On the one hand fungicidal
activity decreases on increasing the size of the cycloalkyl
ring, while on the other fungicidal activity is higher for
the odd carbon number cycles, indicating that the higher
the molecular symmetry the lower the fungicidal acti-
vity.
Also in the case of compounds 16 and 17, a higher
molecular symmetry seems to lead to a decrease in
fungicidal activity.
3. Conclusions
An easy and economic synthetic pathway to both
antibacterial and antifungal agents has been set up. In

E. Maccioni et al. / Il Farmaco 57 (2002) 809Á
/817
813
Fig. 3. Relationships between the MIC values and the measured RM values of the seven most acive isothiosemicarbazones listed in order of
increasing RM values. Compounds: 11, 9, 8, 2, 6, 5, 1.
the case of compounds 1Á
/
11, the structureÁ/activity data
4.2. Chemistry
suggests that a higher lipophilic character will probably
lead to more effective products.
Analytical data of compounds 1Á/17 are reported in
However, steric and electronic factors could also play
an important role in determine biological properties,
and they will be considered in the design of future
molecules.
Table 2.
4.3. Synthesis of starting isothiosemicarbazides
In the case of thiazole derivatives, a potent antifungal
activity, comparable with commercially available com-
pounds, has been observed, in particular against C.
albicans and C. krusei.
Starting isothiosemicarbazides have been synthesised
according to previously reported methods [1]: 1 equiv. of
thiosemicarbazide is refluxed in isopropyl alcohol with 1
equiv. of the appropriate benzyl chloride. The mixture is
These results are very encouraging to place greater
efforts in this research.
refluxed until complete dissolution (30Á90 min) and
/
then allowed to cool down to room temperature (r.t.).
The required isothiosemicarbazide precipitates. After
filtration the product is crystallised from isopropyl
alcohol.
4. Experimental
General method for the synthesis of compounds 1Á
/
11.
4.1. Materials and methods
4.4. Synthesis of cycloheptyliden-S-(2,4-
dichlorobenzyl)-isothiosemicarbazone (1)
M.p. are uncorrected and were determined on a
Reichert Kofler thermopan apparatus. IR spectra were
In a two-necked flask, equipped with a reflux
condenser, a mixture of cycloheptanone 2 g (0.018
mol) and S-(2,4-dichlorobenzyl)-isothiosemicarbazide
(0.018 mol) are reacted in 80 ml of isopropylalcohol in
the presence of a catalytic amount of AcOH. The
recorded on a PerkinÁElmer 1640 FT spectrometer
/
1
(KBr discs, in cmꢁ1). H NMR spectra were recorded
on a Bruker AMX (300 MHz) using tetramethylsilane
(TMS) as internal standard (chemical shifts in d values).
Electron ionisation (EI) mass spectra were obtained by a
Fisons QMD 1000 mass spectrometer (70 eV, 200 mA,
ion source temperature 200 8C). The samples were
introduced directly into the ion source. Elemental
suspension is refluxed until complete dissolution (30Á
/
90 min) and then allowed to cool down to r.t. The
product is obtained as a crystalline solid.
1H NMR (CDCl3): d 1.60Á
Cꢁ
S); 7.19Á
/
1.74 (m, 8H, c-CH2,); 2.47
N); 2.62 (t, 2H, c-CH2ꢀCꢁN); 4.59 (s,
7.59 (m, 3H, phenylꢀH); 8.92, 9.93
analyses were obtained on a PerkinÁ/Elmer 240 B
(t, 2H, c-CH2ꢀ
/
/
/
/
microanalyser.
2H, CH2ꢀ
/
/
/

Table 1
Activity of compounds 12Á
/
17 against different fungi in comparison with Miconazole and Clortrimazole
Compound activity
12
13
14
15
16
17
Miconazole
Clotrimazole
MIC
MFC
MIC
MFC
12.5
MIC
MFC
6.25
MIC
MFC
MIC
MFC
MIC
MFC
25
MIC
MFC
MIC
MFC
C. albicans
C. glabrata
C. krusei
3.12
25
3.12
ꢀ100
6.25
3.12
ꢀ100
6.25
0.78
50
3.12
ꢀ100
3.12
3.12
ꢀ100
25
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
3.12
25
3.12
ꢀ100
12.5
3.12
ꢀ100
3.12
0.39
50
3.12 12.5
6.25 25
0.78 50
0.09 25
3.12
6.25
0.09
0.04
12.5
3.12
12.5
25
ꢀ100
100
ꢀ100
100
100
ꢀ100
50
50
0.78
0.78
12.5
0.78
1.56
0.78
50
1.56
0.78
25
1.56
1.56
50
1.56
3.12
50
25
C. parapsilosis
S. cerevisiae
C. tropicalis
ꢀ100
ꢀ100
100
ꢀ100
ꢀ100
ꢀ100
50
12.5 50
0.39 25
0.78
1.56
3.12

E. Maccioni et al. / Il Farmaco 57 (2002) 809Á
/817
815
Table 2
Analytical and physicochemical properties of compounds 1Á
/17
a
a
a
Compound
M.p. (8C)
Crystal/solvent
Formula
C%
H%
N%
m/z
Yield
1
2
184Á
191Á
175Á
199Á
193Á
151Á
147Á
192Á
186Á
200Á
186Á
215
189Á
208Á
177Á
160Á
191Á
/
187
192
177
200
195
153
149
193
189
202
189
Water/ethanol
Water/ethanol
Water/ethanol
Ethanol
C15H19Cl2N3S
C15H20ClN3S
C14H17Cl2N3S
C14H18ClN3S
C13H15Cl2N3S
C15H20ClN3S
C14H18ClN3S
C15H21N3S
52.33 (52.52)
58.15 (57.91)
50.91 (50.79)
56.84 (57.00)
49.37 (49.17)
58.15 (58.25)
56.84 (57.02)
65.42 (65.60)
55.41 (55.28)
64.33 (64.18)
63.12 (62.93)
65.34 (65.13)
66.39 (66.25)
67.33 (67.17)
68.19 (67.95)
67.33 (67.21)
67.33 (67.17)
5.56 (5.58)
6.51 (6.49)
5.19 (5.21)
6.13 (6.15)
4.78 (4.80)
6.51 (6.53)
6.13 (6.14)
7.69 (7.71)
5.72 (5.69)
7.33 (7.30)
6.93 (6.90)
5.88 (5.90)
6.32 (6.29)
6.71 (6.69)
7.07 (7.05)
6.71 (6.73)
6.71 (6.70)
12.20 (12.16)
13.56 (13.59)
12.72 (12.69)
14.20 (14.17)
13.29 (13.31)
13.56 (13.59)
14.20 (14.17)
15.26 (15.30)
14.91 (14.88)
16.08 (16.04)
16.99 (17.02)
16.33 (16.31)
15.48 (15.51)
14.72 (14.75)
14.03 (14.07)
14.72 (14.68)
14.72 (14.69)
344
309
330
295
316
309
295
275
247
261
247
259
273
287
301
287
287
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
ꢀ95
/
3
/
4
/
5
/
Ethanol
6
/
Ethanol
7
/
Ethanol
8
/
Ethanol
9
/
Ethanol
Ethanol
C13H16ClN3S
C14H19N3S
10
11
12
13
14
15
16
17
/
/
Ethanol
C13H17N3S
Water/ethanol
Water/ethanol
Ethanol
C14H15N3S
C15H17N3S
C16H19N3S
/
190
209
179
162
192
/
/
Water/ethanol
Water/ethanol
Water/ethanol
C17H21N3S
C16H19N3S
C16H19N3S
/
/
a
Found values in parentheses.
(ds, 1H, Cꢀ
/
NH2, D-exch.); 12.08 (s, 1H, ꢁ
/
Cꢀ
/
N2H, D-
(ds, 1H, Cꢀ
/
NH2, D-exch.); 12.00 (s, 1H, ꢁ
/
Cꢀ
/
N2H, D-
exch.).
According to the same procedure, the following listed
compounds have been synthesised.
exch.).
4.5.4. Cycloheptyliden-S-(3-chlorobenzyl)-
isothiosemicarbazone (6)
1H NMR (CDCl3): d 1.57Á
/
1.74 (m, 8H, c-CH2,); 2.47
N); 2.59 (t, 2H, c-CH2ꢀCꢁN); 4.44 (s,
S); 7.20Á7.44 (m, 4H, phenylꢀH); 8.69, 9.81
(ds, 1H, CꢀNH2, D-exch.); 12.08 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
4.5. Cycloheptyliden-S-(2-chlorobenzyl)-
isothiosemicarbazone (2)
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
2H, CH2ꢀ
/
/
/
1H NMR (CDCl3): d 1.59Á
(t, 2H, c-CH2ꢀCꢁN); 2.63 (t, 2H, c-CH2ꢀ
2H, CH2ꢀS); 7.21Á7.56 (m, 4H, phenylꢀH); 8.15, 10.15
(ds, 1H, CꢀNH2, D-exch.); 12.34 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
/1.76 (m, 8H, c-CH2,); 2.45
/
/
/
/
/
/
CꢁN); 4.51 (s,
/
/
/
/
/
/
/
4.5.5. Cyclohexyliden-S-(3-chlorobenzyl)-
isothiosemicarbazone (7)
1H NMR (CDCl3): d 1.60Á
/
1.70 (m, 6H, c-CH2,); 2.27
N); 2.55 (t, 2H, c-CH2ꢀCꢁN); 4.45 (s,
S); 7.22Á7.48 (m, 4H, phenylꢀH); 8.90, 9.83
(ds, 1H, CꢀNH2, D-exch.); 12.34 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
4.5.1. Cyclohexyliden-S-(2,4-dichlorobenzyl)-
isothiosemicarbazone (3)
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
2H, CH2ꢀ
/
/
/
1H NMR (CDCl3): d 1.56Á
/
1.70 (m, 6H, c-CH2,); 2.32
N); 2.59 (t, 2H, c-CH2ꢀCꢁN); 4.55 (s,
S); 7.19Á7.52 (m, 3H, phenylꢀH); 8.47, 9.81
(ds, 1H, CꢀNH2, D-exch.); 12.35 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
/
/
/
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
2H, CH2ꢀ
/
/
/
/
/
/
4.5.6. Cycloheptyliden-S-benzyl-isothiosemicarbazone
(8)
1H NMR (CDCl3): d 1.60Á
/
1.81 (m, 8H, c-CH2,); 2.46
N); 2.63 (t, 2H, c-CH2ꢀCꢁN); 4.47 (s,
S); 7.29Á7.51 (m, 5H, phenylꢀH); 8.49, 9.96
(ds, 1H, CꢀNH2, D-exch.); 12.16 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
4.5.2. Cyclohexyliden-S-(2-chlorobenzyl)-
isothiosemicarbazone (4)
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
1H NMR (CDCl3): d 1.59Á
/
1.69 (m, 6H, c-CH2,); 2.29
N); 2.57 (t, 2H, c-CH2ꢀCꢁN); 4.56 (s,
S); 7.21Á7.56 (m, 4H, phenylꢀH); 8.55, 9.82
(ds, 1H, CꢀNH2, D-exch.); 12.45 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
2H, CH2ꢀ
/
/
/
/
/
/
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
2H, CH2ꢀ
/
/
/
/
/
/
4.5.7. Cyclopentyliden-S-(2-chlorobenzyl)-
isothiosemicarbazone (9)
4.5.3. Cyclopentyliden-S-(2,4-dichlorobenzyl)-
isothiosemicarbazone (5)
1H NMR (CDCl3): d 1.77Á
/
1.90 (m, 4H, c-CH2,); 2.41
N); 2.58 (t, 2H, c-CH2ꢀCꢁN); 4.64 (s,
S); 7.21Á7.59 (m, 4H, phenylꢀH); 8.96, 9.69
(ds, 1H, CꢀNH2, D-exch.); 12.07 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
1H NMR (CDCl3): d 1.79Á
/
1.90 (m, 4H, c-CH2,); 2.45
N); 2.55 (t, 2H, c-CH2ꢀCꢁN); 4.62 (s,
S); 7.22Á7.57 (m, 3H, phenylꢀH); 9.11, 9.64
2H, CH2ꢀ
/
/
/
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
/
/
/
2H, CH2ꢀ
/
/
/

816
E. Maccioni et al. / Il Farmaco 57 (2002) 809Á817
/
4.5.8. Cyclohexyliden-S-benzyl-isothiosemicarbazone
(10)
4.6.4. 2-Cyclooctylidenhydrazo-4-phenyl-thiazole (15)
1H NMR (CDCl3): d 1.45Á
1.99 (m, 10H, c-CH2,);
2.49 (t, 2H, c-CH2ꢀCꢁN); 2.64 (t, 2H, c-CH2ꢀCꢁN);
6.71 (s, 1H, CHꢀthiaz.ꢀH5); 7.29Á7.76 (m, 5H, phenylꢀ
H); 12.40 (s, 1H, ꢁCꢀ
N2H, D-exch.).
/
1H NMR (CDCl3): d 1.62Á
/
1.81 (m, 6H, c-CH2,); 2.31
N); 2.59 (t, 2H, c-CH2ꢀCꢁN); 4.47 (s,
S); 7.24Á7.47 (m, 5H, phenylꢀH); 8.40, 9.74
(ds, 1H, CꢀNH2, D-exch.); 12.44 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
/
/
/
/
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
/
/
/
/
2H, CH2ꢀ
/
/
/
/
/
/
/
/
4.6.5. 2-(2-Methylcyclohexyliden)hydrazo-4-
phenylthiazole (16)
4.5.9. Cyclopentyliden-S-benzyl-isothiosemicarbazone
(11)
1H NMR (CDCl3): d 1.16Á
/
1.18 (d, 3H, CH3), 1.59Á
/
2.49 (m, 6H, c-CH2,); 3.07 (t, 2H, c-CH2ꢀ
(m, 1H, c-CHꢀCꢁN); 6.73 (s, 1H, CHꢀ
7.29Á7.74 (m, 5H, phenylꢀH); 12.22 (s, 1H, ꢁ
D-exch.).
/
Cꢁ
/
N); 3.09
H5);
1H NMR (CDCl3): d 1.76Á
/
1.92 (m, 4H, c-CH2,); 2.44
N); 2.56 (t, 2H, c-CH2ꢀCꢁN); 4.63 (s,
S); 7.23Á7.52 (m, 5H, phenylꢀH); 9.01, 9.54
(ds, 1H, CꢀNH2, D-exch.); 12.03 (s, 1H, ꢁCꢀ
N2H, D-
exch.).
/
/
/
thiaz.ꢀ
/
(t, 2H, c-CH2ꢀ
/
Cꢁ
/
/
/
/
/
/
Cꢀ
/
N2H,
2H, CH2ꢀ
/
/
/
/
/
/
4.6.6. 2-(4-Methylcyclohexyliden)hydrazo-4-
phenylthiazole (17)
4.6. Synthesis of starting thiosemicarbazones
1H NMR (CDCl3): d 1.00Á
/
1.02 (d, 3H, CH3); 1.25Á
CH3); 3.12 (t, 2H, c-CH2ꢀCꢁ
CꢁN); 6.71 (s, 1H, CHꢀthiaz.ꢀ
H); 12.52 (s, 1H, ꢁCꢀ
/
2.55 (m, 5H, c-CH2, CHꢀ
/
/
/
The starting thiosemicarbazones have been prepared
by slightly modifing the procedures reported in the
literature [13]. In a flask equipped with a reflux
condenser, equimolar amounts of thiosemicarbazide
and of the appropriate ketone are reacted in isopropyl-
alcohol in the presence of a catalytic amount of AcOH.
The mixture is then refluxed for 1 h. and the obtained
solid is filtered and used without further purification.
N); 3.17 (t, 2H, c-CH2ꢀ
/
/
/
/
H5); 7.22Á
/
7.75 (m, 5H, phenylꢀ
/
/
/
N2H, D-exch.).
4.7. Microbiology
4.7.1. Compounds
Compounds for anti-microbial studies were dissolved
in DMSO at 10 mg/ml and stored at ꢁ20 8C.
General method for the synthesis of compounds 12Á
/
/
17.
The working solutions were prepared in the same
medium employed for the tests. To avoid interference
from the solvent [14], the highest DMSO concentration
was 1%.
Equimolar amounts of cycloalkylthiosemicarbazone
and a-halogen ketone are reacted under reflux in
isopropyl alcohol. The mixture is refluxed until com-
plete dissolution, and then after a period ranging
between 30 and 120 min, a foaming product is formed.
The mixture is then allowed to cool down and the solid
filtered. The product is crystallised from ethanol or
water/ethanol.
4.7.2. Bacteria
The anti-microbial activity of compounds 1Á17 was
/
evaluated against five Gram positive species (S. aureus,
S. epidermidis, S. agalactiae, S. faecalis, and B. subtilis),
and five Gram negative species (Escherichia coli, Pseu-
domonas aeruginosa, Salmonella typhi, Proteus mira-
bilis, and Klebsiella pneumoniae) isolated from clinical
specimens.
C. albicans, C. glabrata, C. krusei, C. parapsilosis,
Saccharomyces cerevisiae, and C. tropicalis, all isolated
from clinical specimens, were employed in the evalua-
tion of antifungal activity.
According to this method, the following listed com-
pounds have been synthesised.
4.6.1. 2-Cyclopentylidenhydrazo-4-phenyl-thiazole (12)
1H NMR (CDCl3): d 1.83Á
(t, 2H, c-CH2ꢀ
1H, CHꢀthiaz.ꢀ
12.22 (s, 1H, ꢁ
/
1.97 (m, 4H, c-CH2,); 2.53
N); 2.66 (t, 2H, c-CH2ꢀCꢁN); 6.73 (s,
H5); 7.29Á7.74 (m, 5H, phenylꢀH);
Cꢀ
N2H, D-exch.).
/
Cꢁ
/
/
/
/
/
/
/
/
/
4.6.2. 2-Cyclohexylidenhydrazo-4-phenyl-thiazole (13)
1H NMR (CDCl3): d 1.69Á
1.80 (m, 6H, c-CH2,); 2.41
(t, 2H, c-CH2ꢀCꢁN); 2.65 (t, 2H, c-CH2ꢀCꢁN); 6.73 (s,
1H, CHꢀthiaz.ꢀH5); 7.29Á7.74 (m, 5H, phenylꢀH);
12.51 (s, 1H, ꢁCꢀ
N2H, D-exch.).
4.7.3. Determination of MICs
/
The MICs of the compounds against Gram positive
bacteria, Gram negative bacteria, and fungi, were
determined by a standard broth macro dilution method
[15,16]. Tests with Gram positive and Gram negative
bacteria were carried out in Mueller Hunton broth
(Difco Laboratories, Detroit, MI, USA). Antifungal
activity was evaluated in Sabouraud Dextrose broth
(Difco Laboratories) [17]. In the case of fungi also MFC
values, or more commonly MLC (minimum lethal
concentration) values were measured.
/
/
/
/
/
/
/
/
/
/
4.6.3. 2-Cycloheptylidenhydrazo-4-phenyl-thiazole (14)
1H NMR (CDCl3): d 1.64Á
1.84 (m, 8H, c-CH2,); 2.55
(t, 2H, c-CH2ꢀCꢁN); 2.73 (t, 2H, c-CH2ꢀCꢁN); 6.75 (s,
1H, CHꢀthiaz.ꢀH5); 7.29Á7.74 (m, 5H, phenylꢀH);
12.26 (s, 1H, ꢁCꢀ
N2H, D-exch.).
/
/
/
/
/
/
/
/
/
/
/

E. Maccioni et al. / Il Farmaco 57 (2002) 809Á
/817
817
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/
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/
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/
/
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