Synthesis of imidazoline and imidazo[2,1-c][1,2,4]triazole aryl derivatives containing the methylthio group as possible antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2006.01.019

1-Arylimidazolidine-2-thiones (1a-g) were synthesized by the condensation reaction of N-arylethylenediamines with carbon disulfide in xylene medium. Their further alkylation with methyl iodide led to the formation of some biologically active 1-aryl-2-meth

Full text of DOI:10.1016/j.bmc.2006.01.019

Bioorganic & Medicinal Chemistry 14 (2006) 3635–3642
Synthesis of imidazoline and imidazo[2,1-c][1,2,4]triazole
aryl derivatives containing the methylthio group as
possible antibacterial agents
b
c
Krzysztof Sztanke,a, Kazimierz Pasternak, Anna Sidor-Wojtowicz,
*
´
c
d
´
´ ´
Janina Truchlinska and Krzysztof Jozwiak
aChair and Department of Synthesis and Chemical Technology of Pharmaceutical Substances, Medical University,
6 Staszica Str, 20-081 Lublin, Poland
bDepartment of General Chemistry, Medical University, 4 Staszica Str, 20-081 Lublin, Poland
cDepartment of Medical Microbiology, Medical University, 1 Chodz´ki Str, 20-093 Lublin, Poland
dDepartment of Chemistry, Medical University, 6 Staszica Str, 20-081 Lublin, Poland
Received 23 November 2005; revised 8 January 2006; accepted 10 January 2006
Available online 7 February 2006
Abstract—1-Arylimidazolidine-2-thiones (1a–g) were synthesized by the condensation reaction of N-arylethylenediamines with car-
bon disulfide in xylene medium. Their further alkylation with methyl iodide led to the formation of some biologically active 1-aryl-2-
methylthio-imidazolines (2a–g). The 7-(4-methylphenyl)-3-methylthio-5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazole (4b) was obtained
by the alkylation of the respective 7-(4-methylphenyl)-2,5,6,7-tetrahydroimidazo[2,1-c][1,2,4]triazol-3(H)-thione (3b) with methyl
iodide. Antimicrobial activities of 1-aryl-2-methylthio-imidazolines (2a–g) and the 7-(4-methylphenyl)-3- methylthio-5H-6,7-dihy-
droimidazo[2,1-c][1,2,4]triazole (4b) are presented. All tested compounds showed MIC in the range of 11.0–89.2 lM. Compounds
2a,e were found to be equipotent to chloramphenicol in vitro, whereas 2a,c,e–g and 4b showed superior activity (MIC) to ampicillin.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
It has been shown that the alkylthio group bound to an
electron-deficient carbon atom in various heterocycles
could be considered to be the pharmacophore of antimy-
Pharmacophores are defined as specific spatial maps of
structural properties common to compounds, which
exhibit particular pharmacological activities. The idea
of pharmacophores forms a new direction of pharma-
ceutical thinking, as substances from different groups
of the system of organic chemistry may contain the same
pharmacophore. The present paper is based on this idea.
cobacterial and/or antimycotic activity. This hypothesis
4–6
ˇ
was formulated and proved by Klimesova et al. and
Waisser et al.7,8 for 6-amino-2-alkylthiobenzothiazoles,
1-aryl-5-alkylthio-1,2,3,4-tetrazoles, substituted 4-alkyl-
thiopyridine-4-carbothioamides and 4-carbonitriles and
substituted 2-alkylthiopyridine-4-carbothioamides and
4-carbonitriles, bioactive molecules from different
heterocyclic systems but with the same pharmacophore.
Antimycobacterial activity of the compounds containing
the alkylthio group bound to an electron-deficient car-
bon atom is already well known. For instance, Ditophal
(S,S0-diethyl-dithioisophthalate), containing this struc-
tural moiety, found application in the treatment of
lepromatous leprosy.1–3
Moreover,ontheotherhandfromtheliteraturedataitfol-
lows that depending on the type of substituent certain
derivatives of imidazoline may also show antimicrobial
properties.9–11Thedefinitederivativesofimidazoline,aro-
matic diimidazolines related to pentamidine, were proved
tobeactiveagainstAIDSrelatedopportunisticpathogens,
such as Candida albicans and Candida neoformans.9
Keywords: Imidazolines; Imidazo[2,1-c][1,2,4]triazole; The methylthio
group; Antibacterial activity; In vitro study; MIC.
Previous studies concerning bridgehead nitrogen-hetero-
cyclic compounds obtained by fusion of 4,5-dihydroimi-
*
Corresponding author. E-mail: krzysztof.sztanke@am.lublin.pl
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.019

3636
K. Sztanke et al. / Bioorg. Med. Chem. 14 (2006) 3635–3642
dazole and 1,2,4-triazole nuclei carried out in the Depart-
ment of Synthesis and Chemical Technology of Pharma-
ceutical Substances have identified one compound having
a sulfanyl group at position 3 and a 4-chlorophenyl substi-
tuent at position 7 (i.e., 7-(4-chlorophenyl)-3-thiolo-5H-
6,7-dihydroimidazo[2,1-c][1,2,4]triazole) with a signif-
icant antifungal activity.12 It is of interest that some
derivatives having the same heterocyclic skeleton—7-
aryl-5-methyl-3-thiolo-imidazo[2,1-c][1,2,4]triazol-6-
ones described in the literature have been evaluated for
their general pharmacological activities and have been
found to possess high antifungal activity. These
compounds exhibited fungicidal action almost equivalent
to that of mancozeb (Dithane M-45) at 1000 ppm concen-
tration and inhibited growth of Aspergillus niger and
Fusarium oxysporum by more than 48% and 47% even
at 10 ppm concentration.13
1a,c–g with 1 equiv methyl iodide afforded the
1-aryl-2-methylthio-imidazolines (2a,c–g) in 75–85%
yields. Use of the methyl iodide over dimethyl sulfate
for S-methylation was experimentally found to be
superior in terms of yield. Biologically active 1-aryl-
2-methylthio-imidazolines (2a,c–g) and their starting
materials (1a,c–g) were prepared by patent pending
according to Sztanke. The majority of obtained com-
pounds, with the exception of 1a, 2a,g22–24 and 1b,
2b,25 are new and their synthesis, physicochemical,
spectral and biological activity data have not been
reported in the literature as yet. The 7-(4-methylphe-
nyl)-3-methylthio-5H-6,7-dihydroimidazo[2,1-c][1,2,4]tri-
azole (4b) was obtained in good yield (78%) from the
respective 7-(4-methylphenyl)-2,5,6,7-tetrahydroimidazo-
[2,1-c][1,2,4]triazol-3(H)-thione25 (3b) by alkylation
with methyl iodide. The methylation reaction was
carried out in ambient temperature for 24 h and then
in boiling solvent for 5 h. Substrates were solved in
anhydrous ethanol prior to mixing them together in
a molar ratio of 1:1. The reaction conditions were
established experimentally.
The following antifungal 1,2,4-triazole derivatives are
applicable in medicine: fluconazole, itraconazole and
terconazole. Moreover, from the literature data, it
follows that depending on the type of substituent deriva-
tives of 1,2,4-triazole show antimicrobial properties.14–17
In view of continuous and widespread interest in the
design and synthesis of novel heterocyclic derivatives
containing the methylthio group as an expected pharma-
cophore, the synthetic approach leading to the forma-
tion of methylthio derivatives of imidazoline and
imidazo-triazole, described by us, might be considered
as a useful method for preparation of these biologically
active compounds because of the affordability of the
starting materials, good yields obtained and straightfor-
ward product isolation.
Prompted by these reports and in attempt to prepare
heterocyclic compounds of biological interest,12,18 it
seemed worthwhile to synthesize some of the hitherto
unknown derivatives of 1-aryl-imidazoline and 7-aryl-
5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazole containing
the methylthio group as an expected pharmacophore
of antimicrobial activity. In the present communication,
we would like to report the results on the antimicrobial
activity investigation of seven representatives of 1-aryl-
2-methylthio-imidazolines (2a–g) and one imidazo-tria-
zole derivative, that is, 7-(4-methylphenyl)-3-methyl-
All new compounds reported here were characterized on
the basis of complementary spectroscopic (1H NMR, IR
and MS) and analytical data. The physicochemical
properties and spectral data of synthesized compounds
are presented in Table 1.
thio-5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazole
(4b).
For the estimation of potential activity in vitro the influ-
ence of tested compounds in examined concentrations of
100 and 200 lg mLꢀ1 on the growth of Gram-positive
and Gram-negative bacterial strains, mould and yeast-
like fungi strains and the MIC values against two refer-
ence strains of Gram-positive (Staphylococcus aureus
ATCC 25923) and Gram-negative (Escherichia coli
ATCC 25922) bacteria were determined.
In the case of compounds 1a–g, the existence of thiol–
thione tautomerism is possible. In their H NMR spec-
1
tra, a slightly broadened singlet signal in the range
9.14–9.58 ppm and corresponding to the –NH–C@S
group was observed. From this signal chemical shift val-
ue it can be concluded that the tautomeric equilibrium in
solution should be moved towards 2-thione form rather
than the 2-thiol one.
2. Chemistry
The synthetic pathway for compounds described was
achieved by a sequence of reactions starting from the
respective anilines and is illustrated in Scheme 1.
1
In the H NMR spectra of compounds 2a–g, a single
absorption band at about 2.9 ppm corresponding to
the S–CH3 group and a single absorption band at about
9.9 ppm, characterizing the presence of N+H, were
seen, while the signals belonging to –NH–C@S group
disappeared.
In the first step, commercially available anilines were
converted into N-arylethylenediamines by the
Lehmann method19 or by the classical Knoevenagel
and Mercklin method with Takeda modification.20,21
Their further condensation with carbon disulfide in
the xylene medium led to the formation of intermedi-
ates—dithiocarbaminic acid derivatives, which could
easily be cyclized in boiling solvent under reaction
conditions to the 1-arylimidazolidine-2-thiones (com-
pounds 1a,c–g) with concomitant liberation of a
hydrogen sulfide molecule. Alkylation of compounds
NMR spectral characteristic of representative of the
heterocyclic dihydroimidazo-triazole system (4b) re-
vealed in its 1H NMR spectrum the two double-
doublet signals of the H5 and H6 at 4.21 and
4.51 ppm,
with
the
coupling
constants
of
J = 7.4 Hz, J0 = 6.1 Hz and J = 7.5 Hz, J0 = 6.1 Hz,
respectively. The difference between chemical shifts

K. Sztanke et al. / Bioorg. Med. Chem. 14 (2006) 3635–3642
3637
R
R
R
R
a
SH
S
NH
NH
N
NH
NH2
S
d
NH2
NH
1a-g
b
R
c
NH
R
CN
R
N
N
N
SCH3
HI
e
SH
N
2a-g
CH3
CH3
CH3
N
N
N
N
N
f
N
N
N
N
N
N
NH
S
SH
SCH3
4b
3b
a : R = H; b : R = 4-CH3; c : R = 2-CH3O; d : R = 4-CH3O; e : R = 3-Cl; f : R= 2,6-Cl2
g : R = 3,4-Cl2,
Scheme 1. Synthetic route to obtained compounds. Reagents and conditions: (a) aziridine, AlCl3, dry toulene; (b) HCHO, Na2S2O5, NaCN, water,
reflux; (c) H2, NiRa, methanol/NH3, 100 ꢁC; (d) CS2, xylene, rt, 20 min, reflux, 7 h; (e) CH3I/MeOH, rt, 48 h, reflux, 6 h; (f) CH3I, abs EtOH, rt, 24 h,
reflux, 5 h, Na2CO3.
of both signals can suggest an important difference
in the acidity of both hydrogen atoms. Moreover,
the two additional singlet signals derived from the
CH3 and the SCH3 groups were observed at 2.28
and 2.89 ppm, integrating for three proton and three
proton, respectively.
3. Pharmacology
Determination of in vitro antimicrobial activity of the
compounds tested was performed using the microdilu-
tion method, according to the National Committee for
Clinical Laboratory Standards (NCCLS)26–28 and the
disc-diffusion method by Kirby–Bauer.29–31
It is interesting to note that compounds 1a–g are present
in the solid state in the C@S form as indicated by their
IR spectra showing the presence of two absorption
bands at 1268–1284 and 1470–1479 cmꢀ1 and the
absence of absorption in the region 2500–2650 cmꢀ1
for SH stretching.
The in vitro activities of eight compounds (2a–g and 4b)
against pathogenic bacteria, yeast-like fungi and
moulds were compared. The following microorganisms
were used: S. aureus ATCC 25923, Staphylococcus
epidermidis, Streptococcus pyogenes, Streptococcus
agalactiae (Gram-positive bacteria), E. coli ATCC
25922, Pseudomonas aeruginosa, Proteus vulgaris,
Klebsiella pneumoniae, Enterobacter aerogenes (Gram-
negative bacteria), C. albicans and Aspergillus spp.
The majority of strains under study were clinical
isolates, identified with conventional morphological
and biochemical methods. The microdilution
method for estimation of MIC value (the lowest
concentration of compound required to inhibit the
growth of the tested microorganism) was applied to
The compounds 2a–g and 4b could be recognized by the
presence of specific absorption band of the S–CH3 bond
in the range of 1309–1325 cmꢀ1. The C@N absorption
band was present in the IR spectra of compounds
2a–g and 4b in the region 1548–1561 cmꢀ1
.
The mass spectra of obtained compounds showed
molecular ion peaks which were consistent with their
molecular formulae.

Table 1. Physicochemical and spectral properties of the synthesized compounds
Compound
R
H
Formula, mw
Mp (ꢁC)
Yield (%)
71
IR (cmꢀ1
)
1H NMR (CDCl3 as a solvent),a d (ppm); EIMS: m/z
1a
C9H10N2S, 178.26
170–2
1275, 1474 ˜C@S
3.75–4.2 (m, 4H, 2CH2); 7.02–7.65 (m, 5H, arom.) 9.22
(br s, 1H, NH); m/z: 178 [M+]
—
1b
1c
4-CH3
2-CH3O
C10H12N2S, 192.28
C10H12N2OS, 208.28
Ref. 25
243–4
—
67
—
1280, 1476 ˜C@S
3.83 (s, 3H, OCH3); 3.75–4.17 (m, 4H, 2CH2); 7.15–7.70
(m, 4H, arom.); 9.14 (br s, 1H, NH); m/z: 208 [M+]
3.84 (s, 3H, OCH3); 3.67–4.19 (m, 4H, 2CH2); 6.86–7.49
(m, 4H arom.); 9.21 (br s, 1H, NH); m/z: 208 [M+]
3.74–4.3 (m, 4H, 2CH2); 7.26–7.50 (m, 4H, arom.); 9.51
(br s, 1H, NH); m/z: 212 [ M+]
1d
1e
1f
4-CH3O
3-Cl
C10H12N2OS, 208.28
C9H9ClN2S, 212.70
C9H8Cl2N2S, 247.15
C9H8Cl2N2S, 247.15
C10H13IN2S, 320.20
156–8
181–3
226–8
165–7
163–5
65
67
64
62
82
1284, 1479 ˜C@S
1276, 1476 ˜C@S
1272, 1472 ˜C@S
1268, 1470 ˜C@S
2,6-Cl2
3,4-Cl2
H
3.86–4.15 (m, 4H, 2CH2); 7.21–7.51 (m, 3H, arom.); 9.31
(br s, 1H, NH); m/z: 247 [M+]
1g
2a
3.88–4.22 (m, 4H, 2CH2); 7.33–7.64 (m, 3H, arom.); 9.58
(br s, 1H, NH); m/z: 247 [M+]
1312 S–CH3 1511
N+H 1550 C@N
—
2.84 (s, 3H, SCH3); 4.08–4.28 (m, 4H, 2CH2); 7.15–7.74
(m, 5H, arom.); 9.85 (s, N+H); m/z: 320 [M+]
—
2b
2c
4-CH3
2-CH3O
C11H15IN2S, 334.22
C11H15IN2OS, 350.22
Ref. 25
181–3
—
78
1322 S–CH3 1507
N+H 1556 C@N
2.93 (s, 3H, SCH3); 3.83 (s, 3H, OCH3); 4.24–4.38 (m, 4H,
2CH2); 7.18–7.79 (m, 4H, arom.); 9.85 (s, N+H); m/z: 350
[M+]
2d
4-CH3O
C11H15IN2OS, 350.22
165–7
79
1325 S–CH3 1505
N+H 1557 C@N
2.95 (s, 3H, SCH3); 3.84 (s, 3H, OCH3); 4.31–4.42 (m, 4H,
2CH2); 6.93–7.41 (m, 4H, arom.); 9.9 (s, N+H); m/z: 350
[M+]
2e
2f
3-Cl
C
10H12ClIN2S, 354.64
175–7
164–6
177–9
85
79
75
1310 S–CH31510
N+H 1551 C@N
1314 S–CH3 1512
N+H 1561 C@N
1316 S–CH31515
N+H 1557 C@N
—
2.89 (s, 3H, SCH3); 4.28–4.44 (m, 4H, 2CH2); 7.19–7.56
(m, 4H, arom.); 9.70 (s, N+H); m/z: 354 [M+]
2.91 (s, 3H, SCH3); 4.31–4.47 (m, 4H, 2CH2); 7.11–7.44
(m, 3H, arom.); 10.1 (s, N+H); m/z: 389 [M+]
2.96 (s, 3H, SCH3); 4.17–4.32 (m, 4H, 2CH2); 7.26–7.64
(m, 3H, arom.); 10.3 (s, N+H); m/z: 389 [M+]
—
2,6-Cl2
3,4-Cl2
C10H11Cl2IN2S, 389.09
C10H11Cl2IN2S, 389.09
2g
3b
4b
4-CH3
4-CH3
C11H12N4S, 232.31
C12H14N4S, 246.33
Ref. 25
214–16
—
78
1309 S–CH31548 C@N
2.28 (s, 3H,CH3); 2.89 (s, 3H, SCH3); 4.21 (J = 7.4 Hz,
J0 = 6.1 Hz, dd, 2H, CH2); 4.51 (J = 7.5 Hz, J0 = 6.1 Hz,
dd, 2H, CH2); 7.11–7.55 (m, 4H, arom.); m/z: 246 [M+]
a Except for compound 4b: DMSO-d6.

K. Sztanke et al. / Bioorg. Med. Chem. 14 (2006) 3635–3642
3639
evaluate the antibacterial activity. In this method, two
reference strains of bacteria—S. aureus ATCC 25923
and E. coli ATCC 25922, were included in
this study. The antibacterial potency of the com-
pounds under conditions was compared with the
activity of topical antibacterial drugs—ampicillin and
chloramphenicol.
relation to typical Gram-positive (S. aureus ATCC
25923) and Gram-negative (E. coli ATCC 25922) bacte-
rial strains with MIC values from 11.0 to 89.2 lM. On
the whole, the most potent in vitro antibacterial effect
was demonstrated by compounds 2e and 2a in relation
to typical Gram-positive S. aureus ATCC 25923 refer-
ence bacterial strain and Gram-negative E. coli ATCC
25922 standard bacterial strain, with minimal inhibitory
concentration values of 11.0 and 12.2 lM, respectively.
These derivatives were equipotent to chloramphenicol
and 3-fold more potent to ampicillin (Table 3). Com-
pound 2a was also found to inhibit growth of P. vulgaris
and S. epidermidis in a concentrations of 100 lg mLꢀ1
(312.3 lM) and 200 lg mLꢀ1 (624.6 lM), K. pneumoniae
and E. aerogenes in a concentration of 200 lg mLꢀ1
(624.6 lM) in the disc-diffusion assay. The growth of
S. aureus ATCC 25923 was inhibited by compound 2e,
that is, 1-(3-chlorophenyl)-2-methylthio-imidazoline
hydroiodide at 11.0 lM and its derivative 2d (1-(4-meth-
oxyphenyl)-2-methylthio-imidazoline hydroiodide) at
89.2 lM. It is noteworthy in this case that the replace-
ment of 3-chlorophenyl substituent in 2e with 4-meth-
oxy substituent as in 2d led to 8-fold decrease in
activity against S. aureus ATCC 25923. Compound 2e
also significantly inhibited growth of S. epidermidis in
a concentration of 200 lg mLꢀ1 (564.0 lM). This bacte-
rial strain was found to be intermediate susceptible to
that compound in a concentration of 100 lg mLꢀ1
(282.0 lM). Compound 2d showed in vitro effectiveness
against P. aeruginosa in a concentration of 200 lg mLꢀ1
4. Results and discussion
The antimicrobial activities of obtained compounds
against bacterial, moulds and yeast-like fungi strains
and the MIC values against two reference strains
(S. aureus ATCC 25923 and E. coli ATCC 25922) were
tested by using the disc-diffusion and the microdilution
assays. The results from experiments were compared
with those of ampicillin and chloramphenicol as the
standards for antibacterial agents. The minimum
inhibitory concentrations (MICs) of ampicillin and
chloramphenicol for S. aureus and E. coli are 35.8,
35.8 lM and 12.1, 12.1 lM, respectively. MIC values
of the examined compounds are listed in Table 3.
Eight compounds tested in the present study were found
to have highly significant antibacterial activities against
the microorganisms listed in Tables 2 and 3, but no anti-
fungal activities. All examined compounds were also
inactive against moulds and yeast-like fungi. 1-Aryl-2-
methylthio-imidazolines (2a–g) showed high activity in
Table 2. Antimicrobial activities of eight evaluated compounds against the tested bacterial and fungal isolates using the disc-diffusion method
Microorganisms
Compound
2e
Standard
2a
2b
2c
2d
2f
2g
4b
I
II
I
II
I
II
I
II
I
II
I
II
I
II
I
II
Escherichia coli (ATCC)
Pseudomonas aeruginosa
Proteus vulgaris
++
ꢀ
++
ꢀ
++
+
+
+
+
++
ꢀ
ꢀ
ꢀ
+
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
+
ꢀ
+
++
ꢀ
+
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
+
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
+
++
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
Klebsiella pneumoniae
Enterobacter aerogenes
Staphylococcus aureus (ATCC)
Staphylococcus epidermidis
Streptococcus pyogenes
Streptococcus agalactiae
Candida albicans
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
++
ꢀ
+++
++
ꢀ
++
+
++
+
++
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Aspergillus spp.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Zones of growth inhibition in: ꢀ, +, ++, +++.
ꢀ: 0–10 mm (R, resistant); +: 11–16 mm (I, intermediate susceptible); ++: 17–25 mm (S, susceptible); +++: >25 mm (S, susceptible); Standard:
ampicillin in a concentration of 200 lg mLꢀ1 (572.4 lM).
I: concentration of 100 lg mLꢀ1, which correspond to concentration ranges of 257.0–406.0 lM depending on molecular weight of the examined
compound.
II: concentration of 200 lg mLꢀ1, which correspond to concentration ranges of 514.0–812.0 lM depending on molecular weight of the examined
compound.
Table 3. Antibacterial activity expressed as MIC (lM) of selected compounds
Microorganisms/code
Compound
2a
2b
2c
2d
2e
2f
2g
4b
A
C
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 25923
12.2
NT
46.7
NT
22.3
22.3
NT
89.2
NT
11.0
20.1
20.1
20.1
20.1
NT
31.7
35.8
35.8
12.1
12.1
NT: not tested due to inactivity in the disc-diffusion assay. Standards: A—ampicillin; C—chloramphenicol.

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K. Sztanke et al. / Bioorg. Med. Chem. 14 (2006) 3635–3642
(571.1 lM) and against P. vulgaris and S. epidermidis in
concentrations of 100 lg mLꢀ1 (285.5 lM) and
200 lg mLꢀ1 (571.1 lM) in the disc-diffusion assay.
Both reference strains: S. aureus ATCC 25923 and
E. coli ATCC 25922 were inhibited by compounds 2c,f
and g at MIC values of 22.3, 20.1 and 20.1 lM, respec-
tively. These derivatives were found to be 1.6- to 1.8-fold
more potent than ampicillin and about 1.8 times less
potent than chloramphenicol in vitro. Moreover,
compounds 2f and g were found to inhibit growth of
S. epidermidis in concentrations of 100 lg mLꢀ1 (257.0
lM) and 200 lg mLꢀ1 (514.0 lM) in the disc-diffusion
assay. The activity of 2f was specific for S. pyogenes, as
was found in the disc-diffusion assay. None of the tested
compounds, with the exception of 2f, was found to inhibit
growth of those bacteria. E. coli ATCC 25922 was inhib-
ited by compounds 2a and b at 12.2 and 46.7 lM, respec-
tively. In this case, an introduction of methyl group in the
position 4 to phenyl ring as in 2b led to an almost 4-fold
decrease in activity against E. coli ATCC 25922. Exam-
ined 7-(4-methylphenyl)-3-methylthio-5H-6,7-dihydro-
imidazo[2,1-c][1,2,4]triazole (4b) was strongly active
against S. aureus ATCC 25923, with MIC results at
31.7 lM. Its antibacterial potency was hardly 1.1 times
higher than that of ampicillin and 2.6-fold lower in
comparison to chloramphenicol. This compound was
also found to exhibit activity against S. epidermidis in
concentrations of 100 lg mLꢀ1 (406.0 lM) and 200 lg
mLꢀ1 (812.0 lM) in the disc-diffusion assay.
in vivo efficacy and safety are being planned for their
further development.
6. Experimental protocols
6.1. Instrumentations and general materials
Chemicals (carbon disulfide and methyl iodide) were
purchased from Merck as ‘synthesis grade’ and used
without further purification. Melting points (mp) were
determined on a Boetius apparatus and are given uncor-
1
rected. H NMR spectra were recorded on a Tesla BS-
567A 100 MHz spectrometer in CDCl3 (1a–g and
2a–g) and on a Varian Gemini 200 MHz spectrometer
in DMSO-d6 (4b) with TMS as an external standard at
295 K. The IR spectra were measured as potassium bro-
mide pellets using a Perkin-Elmer 1725X spectrometer.
Mass spectroscopic analyses were performed on an
AMD-402 mass spectrometer for molecular ion peaks.
Thin-layer chromatography was carried out on commer-
cial Merck SiO2 60 F254 plates having fluorescence indi-
cator; the spots were visualized with UV light
k = 254 nm and by spraying with a 2% ethanol solution
of ninhydrin or charging reagent. Elemental analyses
were performed on a Perkin-Elmer analyzer and were
in the range of 0.4% for each element analyzed (C,
H, N, Cl, I and S). More detailed data for compounds
1b, 2b and 3b are given in Ref. 25.
According to the results obtained, compounds 2a,e were
found to be equipotent to chloramphenicol and superior
to ampicillin antibacterial in vitro activity with MIC val-
ues of 12.2 and 11.0 lM, respectively, against S. aureus
ATCC 25923 and E. coli ATCC 25922, respectively.
Compounds 2a,c,e–g and 4b were found to be more
potent than ampicillin in vitro.
6.1.1. General procedure for synthesis of 1-arylimidazoli-
dine-2-thiones (1a,c–g). Appropriate N-arylethylenedi-
amine (0.1 mol) was dissolved in 120 mL of xylene.
Then carbon disulfide (0.1 mol) in xylene (30 mL) was
added dropwise over the time of 20 min. During that
time precipitation of the intermediate solid started.
The reaction mixture was slowly brought to boil and re-
fluxed for 7 h, next left overnight at room temperature.
The crude product obtained upon cooling was filtered
and finally purified by crystallization from methanol.
Taking into account the significant antibacterial activi-
ties of the presented compounds, the research in this
field will be continued. This refers to the structure mod-
ification of the presented molecules and synthesis of the
analogous systems with alkylthio chain. The study thus
represents a further confirmation of hypothesis of the
alkylthio group bound to an electron-deficient carbon
atom being a pharmacophore of antibacterial activity
as the results fall well in line with it.
6.1.2. General method for synthesis of 1-aryl-2-methyl-
thio-imidazoline hydroiodides (2a,c–g). Appropriate
1-arylimidazolidine-2-thione (0.1 mol) and methyl io-
dide (0.1 mol) in 50 mL of methanol were left at ambient
temperature in closed flask for 48 h. Then the mixture
was heated under reflux for 6 h. The solvent was re-
moved by evaporation, oily residue was cooled and trit-
urated with diethyl ether. The crude product was
collected, washed with cold ethanol and finally crystal-
lized from propan-2-ol.
5. Conclusion
In this report, an easy and useful method to synthesize
antibacterially active imidazoline and imidazo-triazole
aryl derivatives containing the methylthio group as an
expected pharmacophore has been presented. The iden-
tified analogues, in particular 1-aryl-2-methylthio-imi-
dazoline derivatives (2a and e), more potent than
ampicillin and equipotent to chloramphenicol, can serve
as novel templates for bacterial infection chemotherapy.
Further optimization of these identified chemical leads
can possible lead to more active molecules against bac-
terial infections. Since both of analogues (2a and e)
are showing promising results, studies to establish their
6.1.3. Synthesis of 7-(4-methylphenyl)-3-methylthio-5H-
6,7-dihydroimidazo[2,1-c][1,2,4]triazole (4b). 7-(4-Meth-
ylphenyl)-2,5,6,7-tetrahydroimidazo[2,1-c][1,2,4]triazol-
3(H)-thione (0.05 mol)25 and methyl iodide (0.05 mol) in
50 mL of absolute ethanol were allow to stand at the
ambient temperature for 24 h in closed flask. Then the
mixture was heated under reflux for 5 h, cooled and left
overnight. The solution was then evaporated to dryness,
the residue taken up in water (50 mL), basified with solid
sodium carbonate and the suspension was extracted
with chloroform (3· 25 mL). Evaporation of the dried

K. Sztanke et al. / Bioorg. Med. Chem. 14 (2006) 3635–3642
3641
chloroform extracts yielded a solid, which was heated
under reflux with methanol (40 mL) for 1 h. The excess
solvent was removed by evaporation and upon cooling
the precipitate was collected, washed 2· 20 mL cold
propan-2-ol and finally recrystallized from methanol.
ganisms was determined visually after incubation for
24 h at 37 ꢁC. The lowest concentration at which there
was no visible growth (turbidity) was taken as the MIC.
The minimal inhibitory concentration (MIC) values
were studied for reference bacterial strains: S. aureus
ATCC 25923 and E. coli ATCC 25922 towards eight
compounds determined in the disc-diffusion assay.
Ampicillin and chloramphenicol were used as standard
drugs for comparison in the antibacterial studies. Con-
trol experiments using dimethylsulfoxide were done for
antibacterial activity studies. The presented results were
obtained from three independent measurements. The
MIC values (in micromolars) are given in Table 3. The
investigations were carried out in the Department of
Medical Microbiology, Medical University, Lublin.
6.2. Microbiology
6.2.1. Disc diffusion assay. Assay of antimicrobial activity
in vitro. The synthesized compounds were tested for their
antimicrobial (antibacterial and antifungal) activities by
disc-diffusion method by Kirby–Bauer, using Mueller–
Hinton medium for bacteria and the same medium with
4% glucose for fungi. The majority of tested microorgan-
isms were isolated from clinical specimens of the Labora-
tory of Medical Microbiology Department, Medical
University of Lublin. The assayed collection included
the following microorganisms: S. epidermidis, S. pyoge-
nes, S. agalactiae (Gram-positive bacteria), P. aeruginosa,
P. vulgaris, K. pneumoniae, E. aerogenes (Gram-negative
bacteria), C. albicans and Aspergillus spp. Besides, two
reference strains of bacteria—S. aureus ATCC 25923
and E. coli ATCC 25922 were included in these studies.
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dimethylsulfoxide (DMSO) at concentrations of 100
and 200 lg mLꢀ1 were used. Discs containing DMSO
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spread over the following appropriate media: Mueller–
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