Synthesis and antimicrobial activity of new diazoimidazole derivatives containing an N-acylpyrrolidine ring.

Article abstract of DOI:10.1016/S0014-827X(01)01154-5

A series of 4-diazoimidazole-5-carboxamides bearing in position 2 lipophilic substituents was synthesized and their antimicrobial activity was evaluated in vitro against pathogenic Gram-positive, Gram-negative bacteria and fungi. Some compounds presented

Full text of DOI:10.1016/S0014-827X(01)01154-5

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 885–890
Synthesis and antimicrobial activity of new diazoimidazole
derivatives containing an N-acylpyrrolidine ring
Lucilla Varoli *, Silvia Burnelli, Laura Garuti, Beatrice Vitali
Dipartimento di Scienze Farmaceutiche, Uni6ersita` degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 25 May 2001; accepted 27 July 2001
Abstract
A series of 4-diazoimidazole-5-carboxamides bearing in position 2 lipophilic substituents was synthesized and their antimicro-
bial activity was evaluated in vitro against pathogenic Gram-positive, Gram-negative bacteria and fungi. Some compounds
presented antifungal activity, particularly two derivatives (1g and 1h) showed good MIC values (10–50 mg/ml) against both
moulds and yeasts. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Diazoimidazoles; Antifungal activity; Synthesis; SAR
1. Introduction
2. Chemistry
Diazoazoles have various types of biological activity
[1], probably due to their high reactivity towards amine,
hydroxyl and sulfydryl groups. They have inhibitory
action against several tumor cell lines and enzymes, and
their antimicrobial activity has also been demonstrated
[2].
All the compounds 1a–p were synthesized by stan-
dard procedures as reported in Scheme 1. The thioimi-
date salts 3a–p were prepared from the appropriate
nitriles 2a–p, commercially available, and benzylmer-
captan in anhydrous ether saturated with dry hydro-
chloric acid. The reaction of 3a–p with the amide 6
according to Rose and Ainsworth [4] afforded the
2-substituted-4-aminoimidazoles 7a–p. Compound 6
was prepared by reduction with amalgamated alu-
minum of ethyl cyanoglyoxylate-2-oxime 4 to amine 5
[5] and subsequent reaction with pyrrolidine, at 0 °C.
Finally, diazotization of the amino derivatives 7a–p
with sodium nitrite in dilute hydrochloric acid followed
by neutralization of the acid solution gave the desired
4-diazoimidazoles 1a–p.
In a previous work, we reported the synthesis and the
antimicrobial activity of a series of 2-substituted-4-dia-
zoimidazole-5-carboxamides, bearing different sub-
stituents at C-2 and on the 5-carboxamido moiety [3].
Among the synthesized compounds, a moderate anti-
fungal activity against yeasts was observed for those
compounds bearing small groups on the carboxamido
moiety and no hydrophilic substituent at the 2 position.
The obtained results prompted us to continue our
research in order to achieve additional data for a
structure–activity relationship. Therefore, a series of 16
new diazoimidazoles 1a–p has been prepared. These
compounds have a pyrrolidine ring in the carboxamido
moiety and in C-2 position either an alkyl of different
length and steric hindrance, or a phenyl or a substi-
tuted-phenyl substituent. Our aim was to evaluate the
effect of the increased lipophilicity on the biological
activity.
3. Microbiology
All the compounds 1a–p were evaluated in vitro, for
antimicrobial activity, by the agar dilution method [6]
against representative human pathogenic fungi (Can-
dida albicans, Candida lypolitica, Cryptococcus neofor-
mans, Aspergillus niger), Gram-positive (Staphylococcus
aureus, Enterococcus faecalis) and Gram-negative (Es-
cherichia coli, Proteus mirabilis) bacteria. The minimal
* Corresponding author.
E-mail address: quark@alma.unibo.it (L. Varoli).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01154-5

886
L. Varoli et al. / Il Farmaco 56 (2001) 885–890
inhibitory concentrations (MICs, mg/ml) of the synthe-
sized compounds 1a–p investigated against yeasts (C.
albicans, C. lypolitica, C. neoformans) and a filamentous
fungus (A. niger) are presented in Table 1. The corre-
sponding data of the previously published unsubstituted
derivative 1 [3], amphotericin B and fluconazole are also
shown. All the compounds showed a MIC value \100
mg/ml against bacteria, with the exception of compound
1j, which exhibited a MIC=10 mg/ml against S. aureus.
tal analysis was performed for compounds 1a–p and the
results were within 90.4% of the theoretical values.
Infrared spectra (IR) in Nujol were recorded in a
Perkin–Elmer 683 or a Nicolet Avatar 320 spectropho-
1
tometer (wmax in cm−1). H and 13C NMR spectra were
registered in a Varian VXR 300 spectrometer, peak
positions are given in parts per million (l) relative to the
standard chemical shift of the solvent. Coupling con-
stants are reported in Hertz. Merck silica gel 60 (230–
400 mesh) was used for column chromatography.
Thin-layer chromatography (Merck silica gel 60 F254
analytical plates) was used to monitor reactions. Ether
(Et2O) and tetrahydrofuran (THF) were dried by distil-
lation under nitrogen from sodium metal. The term
‘dried’ refers to the use of anhydrous sodium sulfate.
4. Experimental
Melting points were recorded in a Electrothermal
open capillary apparatus and are uncorrected. Elemen-
Scheme 1. Reagents: (a) benzylmercaptan/HCl; (b) amalgamated aluminum; (c) pyrrolidine; (d) EtOH, reflux; (e) NaNO2/HCl.
Table 1
Antifungal activity in vitro of compounds 1a–p (MIC a; mg/ml)
Comp.
R
Candida albicans
Candida lypolitica
Cryptococcus neoformans
Aspergillus niger
1 b
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
H
CH3
C2H5
n-C3H7
i-C3H7
n-C4H9
C6H5
p-FꢀC6H4
p-ClꢀC6H4
p-CH3ꢀC6H4
p-NO2ꢀC6H4
m-FꢀC6H4
m-ClꢀC6H4
o-FꢀC6H4
o-ClꢀC6H4
o,p-F2ꢀC6H3
o,p-Cl2ꢀC6H3
100
200
200
200
\200
200
100
50
100
40
50
100
100
100
100
10
25
25
50
100
100
200
50
200
50
50
50
50
50
100
50
50
10
25
50
200
100
200
\200
\200
\200
\200
25
25
100
200
100
\200
\200
50
\200
100
25
25
100
100
200
\200
50
200
100
2.5
50
100
100
200
50
200
50
1p
Amphotericin B
Fluconazole
20
20
10
5
20
50
2.5
a Minimum inhibitory concentration.
b Ref. [3].

L. Varoli et al. / Il Farmaco 56 (2001) 885–890
887
4.1. General procedure for preparation of thioimidic
acid benzyl ester hydrochlorides (3a–p)
170 °C; 1H NMR (DMSO-d6): l 4.76 (2H, s, CH2),
7.35–7.50 (7H, m, ArH), 7.69–7.77 (2H, m, ArH). 3n
[10]: yield 5%; m.p. 170 °C; H NMR (DMSO-d6): l
1
A stirred solution of the appropriate nitrile derivative
2a–p (20 mmol) and benzylmercaptan (4.1 g, 33 mmol)
in anhydrous ether (10 ml) or THF (15 ml) (3j) was
saturated with dry hydrogen chloride and stirring was
continued at room temperature (r.t.) for 24 h (3a–l) or
5 days (3m–p). Anhydrous ether was added to the
solution until precipitation began. The white precipitate
was collected and washed with dry ether, giving the
thioimidate salts 3a–p, pure enough for use in the next
stage.
4.75 (2H, s, CH2), 7.38–7.51 (5H, m, ArH), 7.70 (2H,
d, ArH), 7.93 (2H, d, ArH). 3o: yield 46%; m.p. 159–
160 °C; 1H NMR (DMSO-d6): l 4.73 (2H, s, CH2),
7.33–7.60 (7H, m, ArH), 7.79–7.84 (1H, m, ArH). 3p:
yield 7%; m.p. 160 °C; H NMR (DMSO-d6): l 4.59
(2H, s, CH2), 7.30–7.43 (6H, m, ArH), 7.61 (1H, s,
1
ArH), 7.86 (1H, d, ArH).
4.2. 2-Amino-3-oxo-3-(1-pyrrolidinyl)-propionitrile (6)
3a: yield 90%; m.p. 158–159 °C (lit. [7]: 153–
155 °C); H NMR (DMSO-d6): l 2.66 (3H, s, CH3),
Ethyl cyanoglyoxylate-2-oxime (4) (1.42 g, 10 mmol)
was reduced with amalgamated aluminum to ethyl
aminocyanoacetate (5). To a solution of the crude
amine 5 in ether (20 ml), cooled to 0 °C, was added
dropwise pyrrolidine (1.42 g, 20 mmol). The solution
was stirred for 15 min, the solvent evaporated and the
oily residue was purified on a silica gel column eluting
with 8:2 chloroform–methanol, to give the amide 6
(35%). Oil; IR: 3360–3280 (NH2), 2220 (CN), 1650
(CꢁO); 1H NMR (DMSO-d6): l 1.75–1.92 (4H, m,
pyrrolidine), 2.58 (2H, brs, NH2, exch. D2O), 3.25–3.40
(2H, m, pyrrolidine), 3.55–3.66 (2H, m, pyrrolidine),
4.77 (1H, s, CH); 13C NMR (DMSO-d6): l 23.74, 25.60,
45.85, 45.96, 46.31, 119.41, 163.71.
1
4.67 (2H, s, CH2), 7.37–7.40 (5H, m, ArH), 12.47 (1H,
brs, NH). 3b [4]: yield 88%; m.p. 116–118 °C; 1H
NMR (DMSO-d6): l 1.23 (3H, t, J=8 Hz, CH3), 2.87
(2H, q, J=8 Hz, CH3CH2), 4.68 (2H, s, SCH2Ph),
7.28–7.51 (5H, m, ArH), 12.50 (1H, brs, NH). 3c [4]:
1
yield 97%; m.p. 135–136 °C; H NMR (DMSO-d6): l
0.89 (3H, t, J=8 Hz, CH3), 1.68 (2H, hex, J=8 Hz,
CH3CH2CH2), 2.80 (2H, t, J=8 Hz, CH3CH2CH2),
4.66 (2H, s, SCH2Ph), 7.35–7.49 (5H, m, ArH), 12.39
(1H, brs, NH). 3d: yield 54%; m.p. 164–165 °C (lit. [8]:
1
165–166 °C (dec.)); H NMR (DMSO-d6): l 1.28 (6H,
d, J=6.9 Hz, 2×CH3), 3.19 (1H, hept, J=6.9 Hz,
CH), 4.65 (2H, s, CH2), 7.38–7.51 (5H, m, ArH), 12.35
1
(1H, brs, NH). 3e [4]: yield 99%; m.p. 140–141 °C; H
NMR (DMSO-d6): l 0.87 (3H, t, J=7.4 Hz, CH3), 1.31
(2H, hex, J=7.6 Hz, CH3CH2CH2), 1.63 (2H, p, J=
7.6 Hz, CH3CH2CH2), 2.84 (2H, t, J=7.6 Hz,
CH3CH2CH2CH2), 4.67 (2H, s, SCH2Ph), 7.35–7.50
(5H, m, ArH), 12.45 (1H, brs, NH). 3f: yield 88%; m.p.
174–176 °C (lit. [9]: 172–174 °C (dec.)); 1H NMR
(DMSO-d6): l 4.86 (2H, s, CH2), 7.37–7.46 (3H, m,
ArH), 7.55–7.66 (4H, m, ArH), 7.80 (1H, t, ArH), 7.94
(2H, d, ArH), 12.39 (1H, brs, NH). 3g [10]: yield 59%;
4.3. General procedure for preparation of 2-substituted
4-amino-5-(1-pyrrolidinyl)carbonylimidazoles (7a–p)
A solution of compound 6 (1.53 g, 10 mmol) in
ethanol (5 ml) was treated portionwise with the appro-
priate crude thioimidic acid benzyl ester hydrochloride
3a–p (10 mmol), and the mixture was heated under
reflux for 15 min and then cooled. The solvent was
evaporated in vacuo, the residue was purified by flash
chromatography eluting with chloroform containing 1–
5% methanol, giving 4-aminoimidazole derivatives 7a–
p·HCl, which were crystallized from a suitable solvent.
1
m.p. 180–182 °C; H NMR (DMSO-d6): l 4.85 (2H, s,
CH2), 7.39–7.57 (7H, m, ArH), 8.03–8.08 (2H, m,
ArH). 3h: yield 29%; m.p. 179–180 °C (lit. [9]: 180–
1
1
181 °C); H NMR (DMSO-d6): l 4.81 (2H, s, CH2),
7a: yield 21%; m.p. 199–200 °C (MeOH–Et2O); H
7.39 (3H, dd, ArH), 7.53 (2H, dd, ArH), 7.69 (2H, d,
ArH), 7.95 (2H, d, ArH). 3i: yield 70%; m.p. 168–
170 °C; 1H NMR (DMSO-d6): l 2.41 (3H, s, CH3),
4.81 (2H, s, CH2), 7.38–7.46 (5H, m, ArH), 7.54 (2H,
d, ArH), 7.84 (2H, d, ArH). 3j: yield 65%; m.p. 164–
NMR (DMSO-d6): l 1.76 (4H, m, pyrrolidine), 2.10
(3H, s, CH3), 3.47 (2H, m, pyrrolidine), 3.85 (2H, m,
pyrrolidine), 5.71 (2H, s, NH2, exch. D2O), 11.16 (1H,
brs, NH, exch. D2O). 7b: yield 32%; m.p. 190–192 °C
(EtOH–Et2O); 1H NMR (DMSO-d6): l 1.12 (3H, t,
J=7.7 Hz, CH3), 1.76 (4H, m, pyrrolidine), 2.44 (2H,
q, J=7.7 Hz, CH2), 3.47 (2H, m, pyrrolidine), 3.84
(2H, m, pyrrolidine), 5.67 (2H, s, NH2, exch. D2O),
11.18 (1H, brs, NH, exch. D2O). 7c: yield 35%; m.p.
175–176 °C (EtOH–Et2O); 1H NMR (DMSO-d6): l
0.89 (3H, t, J=7.3 Hz, CH3), 1.59 (2H, hex, J=7.3
Hz, CH3CH2CH2), 1.78 (4H, m, pyrrolidine), 2.41 (2H,
t, J=7.3 Hz, CH3CH2CH2), 3.49 (2H, m, pyrrolidine),
3.89 (2H, m, pyrrolidine), 5.68 (2H, s, NH2, exch.
1
165 °C (lit. [9]: 166–170 °C (dec.)); H NMR (DMSO-
d6): l 4.85 (2H, s, CH2), 7.35–7.48 (3H, m, ArH), 7.58
(2H, d, ArH), 8.15 (2H, d, ArH), 8.35 (2H, d, ArH). 3k
1
[10]: yield 78%; m.p. 159–160 °C; H NMR (DMSO-
d6): l 4.85 (2H, s, CH2), 7.29–7.41 (3H, m, ArH), 7.55
(2H, dd, ArH), 7.63–7.86 (4H, m, ArH). 3l [10]: yield
1
59%; m.p. 154–155 °C; H NMR (DMSO-d6): l 4.82
(2H, s, CH2), 7.30–7.50 (5H, m, ArH), 7.85–7.90 (3H,
m, ArH), 8.00 (1H, s, ArH). 3m [10]: yield 31%; m.p.

888
L. Varoli et al. / Il Farmaco 56 (2001) 885–890
D2O), 11.15 (1H, brs, NH, exch. D2O). 7d: yield 31%;
m.p. 169–171 °C (EtOH–Et2O); 1H NMR (DMSO-
d6): l 1.16 (6H, d, J=6.9 Hz, 2×CH3), 1.78 (4H, m,
pyrrolidine), 2.78 (1H, hept, J=6.9 Hz, CH), 3.48 (2H,
m, pyrrolidine), 3.90 (2H, m, pyrrolidine), 5.57 (2H, s,
NH2, exch. D2O), 11.16 (1H, brs, NH, exch. D2O). 7e:
D2O), 7.50 (3H, m, ArH), 8.05 (1H, d, ArH), 11.6 (1H,
brs, NH, exch. D2O). 7o: yield 15%; m.p. 149–150 °C
1
(EtOH); H NMR (DMSO-d6): l 1.81 (4H, m, pyrro-
lidine), 3.60 (2H, m, pyrrolidine), 4.02 (2H, m, pyrro-
lidine), 5.84 (2H, s, NH2, exch. D2O), 7.15 (2H, m,
ArH), 7.95 (1H, m, ArH), 11.5 (1H, brs, NH, exch.
D2O). 7p: yield 16%; m.p. 231–233 °C (EtOH); 1H
NMR (DMSO-d6): l 1.80 (4H, m, pyrrolidine), 3.41
(2H, m, pyrrolidine), 4.00 (2H, m, pyrrolidine), 5.93
(2H, s, NH2, exch. D2O), 7.46 (1H, d, ArH), 7.64 (1H,
s, ArH), 7.81 (1H, d, ArH), 11.71 (1H, brs, NH, exch.
D2O).
1
yield 56%; m.p. 128–129 °C (EtOH–Et2O); H NMR
(DMSO-d6): l 0.88 (3H, t, J=7.5 Hz, CH3), 1.29 (2H,
hex, J=7.5 Hz, CH3CH2CH2), 1.56 (2H, p, J=7.4 Hz,
CH3CH2CH2), 1.79 (4H, m, pyrrolidine), 2.45 (2H, t,
J=7.4 Hz, CH3CH2CH2CH2), 3.37 (2H, m, pyrro-
lidine), 3.61 (2H, m, pyrrolidine), 5.63 (2H, brs, NH2,
exch. D2O), 11.2 (1H, brs, NH, exch. D2O). 7f: yield
21%; m.p. 217–218 °C (EtOH–Et2O); 1H NMR
(DMSO-d6): l 1.80 (4H, m, pyrrolidine), 3.46 (2H, m,
pyrrolidine), 4.08 (2H, m, pyrrolidine), 5.92 (2H, brs,
NH2, exch. D2O), 7.31 (1H, t, ArH), 7.41 (2H, t, ArH),
7.84 (2H, d, ArH), 12.10 (1H, s, NH, exch. D2O). 7g:
4.4. General procedure for preparation of 2-substituted
4-diazo-5-(1-pyrrolidinyl)carbonylimidazoles (1a–p)
A stirred solution of sodium nitrite (0.18 g, 2.6
mmol) in water (3 ml) was cooled in an ice bath and
treated dropwise with a solution of the appropriate
amine (1.9 mmol) in dilute hydrochloric acid (4 ml).
The solution was stirred at 0 °C for 30 min, then at r.t.
for 1 h. The aqueous solution was extracted with
dichloromethane (3×20 ml), the organic layer was
washed with saturated aqueous sodium hydrogen car-
bonate (2×10 ml), dried and evaporated to afford an
oily residue which was purified by column chromatog-
raphy eluting with 8:2 AcOEt–petroleum ether and
crystallized.
1
yield 10%; m.p. 200 °C (EtOH); H NMR (DMSO-d6):
l 1.78 (4H, m, pyrrolidine), 3.46 (2H, m, pyrrolidine),
4.06 (2H, m, pyrrolidine), 5.91 (2H, s, NH2, exch.
D2O), 7.25 (2H, t, ArH), 7.85 (2H, t, ArH), 12.09 (1H,
brs, NH, exch. D2O). 7h: yield 20%; m.p. 258–260 °C
1
(MeOH); H NMR (DMSO-d6): l 1.80 (4H, m, pyrro-
lidine), 3.42 (2H, m, pyrrolidine), 4.04 (2H, m, pyrro-
lidine), 5.94 (2H, s, NH2, exch. D2O), 7.45 (2H, d,
J=8.6 Hz, ArH), 7.82 (2H, d, J=8.6 Hz, ArH), 12.14
(1H, brs, NH, exch. D2O). 7i: yield 25%; m.p. 243–
1
245 °C (EtOH); H NMR (DMSO-d6): l 1.81 (4H, m,
1a: yield 22%; m.p. 52–53 °C (EtOH–Et2O); IR:
1
pyrrolidine), 2.30 (3H, s, CH3), 3.42 (2H, m, pyrro-
lidine), 4.05 (2H, m, pyrrolidine), 5.85 (2H, s, NH2,
exch. D2O), 7.19 (2H, d, J=8 Hz, ArH), 7.70 (2H, d,
J=8 Hz, ArH), 11.97 (1H, brs, NH, exch. D2O). 7j:
yield 15%; m.p. 285–286 °C (EtOH); 1H NMR
(DMSO-d6): l 1.80 (4H, m, pyrrolidine), 3.40 (2H, m,
pyrrolidine), 4.09 (2H, m, pyrrolidine), 6.18 (2H, s,
NH2, exch. D2O), 8.02 (2H, d, J=9 Hz, ArH), 8.26
(2H, d, J=9 Hz, ArH), 12.5 (1H, brs, NH, exch. D2O).
7k: yield 15%; m.p. 131–133 °C (EtOH); 1H NMR
(DMSO-d6): l 1.77 (4H, m, pyrrolidine), 3.44 (2H, m,
pyrrolidine), 4.02 (2H, m, pyrrolidine), 5.97 (2H, s,
NH2, exch. D2O), 7.15 (1H, m, ArH), 7.40 (1H, m,
ArH), 7.60 (2H, m, ArH), 12.15 (1H, brs, NH, exch.
D2O). 7l: yield 16%; m.p. 188–190 °C (MeOH); 1H
NMR (DMSO-d6): l 1.85 (4H, m, pyrrolidine), 3.62
(2H, m, pyrrolidine), 4.12 (2H, m, pyrrolidine), 5.90
(2H, s, NH2, exch. D2O), 7.52 (2H, m, ArH), 7.95 (1H,
d, ArH), 8.09 (1H, s, ArH), 12.1 (1H, brs, NH, exch.
D2O). 7m: yield 20%; m.p. 84–85 °C (EtOH); 1H NMR
(DMSO-d6): l 1.85 (4H, m, pyrrolidine), 3.45 (2H, m,
pyrrolidine), 4.03 (2H, m, pyrrolidine), 5.84 (2H, s,
NH2, exch. D2O), 7.30 (3H, m, ArH), 7.95 (1H, t,
ArH), 11.49 (1H, brs, NH, exch. D2O). 7n: yield 42%;
m.p. 120–122 °C (EtOH); 1H NMR (DMSO-d6): l
1.80 (4H, m, pyrrolidine), 3.43 (2H, m, pyrrolidine),
4.00 (2H, m, pyrrolidine), 5.94 (2H, s, NH2, exch.
2122 (CN+2 ), 1610 (CꢁO); H NMR (DMSO-d6): l 1.86
(4H, m, pyrrolidine), 2.36 (3H, s, CH3), 3.49 (2H, t,
pyrrolidine), 3.93 (2H, t, pyrrolidine); 13C NMR
(DMSO-d6): l 18.09, 23.60, 26.40, 47.06, 48.65, 102.72,
158.31, 158.49, 159.22. 1b: yield 35%; m.p. 28–30 °C
1
(EtOH–Et2O); IR: 2125 (CN+2 ), 1610 (CꢁO); H NMR
(DMSO-d6): l 1.22 (3H, t, J=7.5 Hz, CH3), 1.85 (4H,
m, pyrrolidine), 2.70 (2H, q, J=7.5 Hz, CH2), 3.47
(2H, t, pyrrolidine), 3.93 (2H, t, pyrrolidine); 13C NMR
(DMSO-d6): l 12.77, 23.60, 25.20, 26.41, 47.03, 48.63,
102.43, 158.21, 158.53, 164.06. 1c: yield 74%; m.p.
48–50 °C (EtOH–Et2O); IR: 2130 (CN+2 ), 1610 (CꢁO);
1H NMR (DMSO-d6): l 0.91 (3H, t, J=7.4 Hz, CH3),
1.70 (2H, hex, J=7.4 Hz, CH3CH2CH2), 1.85 (4H, m,
pyrrolidine), 2.64 (2H, t, J=7.4 Hz, CH3CH2CH2),
3.46 (2H, t, pyrrolidine), 3.92 (2H, t, pyrrolidine); 13C
NMR (DMSO-d6): l 13.79, 21.24, 23.26, 26.07, 33.43,
46.67, 48.29, 102.33, 157.81, 158.18, 162.68. 1d: yield
33%; oil; IR: 2125 (CN+2 ), 1615 (CꢁO); 1H NMR
(DMSO-d6): l 1.25 (6H, d, J=6.9 Hz, 2×CH3), 1.86
(4H, m, pyrrolidine), 3.00 (1H, hept, J=6.9 Hz, CH),
3.48 (2H, t, pyrrolidine), 3.95 (2H, t, pyrrolidine); 13C
NMR (DMSO-d6): l 21.49, 23.15, 25.95, 30.68, 46.56,
48.16, 101.96, 157.62, 158.17, 167.25. 1e: yield 40%; oil;
1
IR: 2122 (CN+2 ), 1610 (CꢁO); H NMR (DMSO-d6): l
0.88 (3H, t, J=7.3 Hz, CH3), 1.33 (2H, hex, J=7.3
Hz, CH3CH2CH2), 1.66 (2H, p, J=7.3 Hz,

L. Varoli et al. / Il Farmaco 56 (2001) 885–890
889
CH3CH2CH2), 1.85 (4H, m, pyrrolidine), 2.67 (2H, t,
J=7.3 Hz, CH3CH2CH2CH2), 3.47 (2H, t, pyrrolidine),
3.93 (2H, t, pyrrolidine); 13C NMR (DMSO-d6): l
14.07, 22.12, 23.50, 26.31, 30.27, 31.33, 46.93, 48.53,
102.56, 157.92, 158.43, 163.04. 1f: yield 43%; m.p.
137–138 °C (EtOH–Et2O); IR: 2130 (CN+2 ), 1600
1n: yield 25%; m.p. 105–106 °C (EtOH–Et2O); IR:
1
2140 (CN+2 ), 1600 (CꢁO); H NMR (CDCl3): l 1.99
(4H, m, pyrrolidine), 3.68 (2H, t, pyrrolidine), 4.21 (2H,
t, pyrrolidine), 7.41 (2H, d, ArH), 8.13 (2H, d, ArH);
13C NMR (CDCl3): l 23.88, 26.73, 47.24, 48.97, 105.29,
128.67, 129.00, 129.30, 131.99, 135.66, 157.32, 158.77,
159.09. 1o: yield 24%; oil; IR: 2120 (CN+2 ), 1600 (CꢁO);
1H NMR (CDCl3): l 1.99 (4H, m, pyrrolidine), 3.68
(2H, t, pyrrolidine), 4.18 (2H, t, pyrrolidine), 6.94 (2H,
m, ArH), 8.19 (1H, m, ArH); 13C NMR (CDCl3): l
23.78, 26.63, 47.14, 48.86, 105.07, 111.50, 111.75,
118.00, 128.65, 132.22, 132.36, 155.62, 158.44, 158.63.
1p: yield 50%; m.p. 90–92 °C (EtOH); IR: 2139 (CN2+
), 1622 (CꢁO); 1H NMR (CDCl3): l 1.91 (4H, m,
pyrrolidine), 3.62 (2H, t, pyrrolidine), 4.11 (2H, t,
pyrrolidine), 7.26 (1H, d, ArH), 7.45 (1H, s, ArH), 7.95
(1H, d, ArH); 13C NMR (CDCl3): l 23.64, 26.49, 46.98,
48.77, 104.08, 127.00, 130.76, 131.79, 132.61, 133.50,
135.26, 156.69, 157.76, 158.47.
1
(CꢁO); H NMR (CDCl3): l 1.90 (4H, m, pyrrolidine),
3.54 (2H, t, pyrrolidine), 4.08 (2H, t, pyrrolidine), 7.38
(3H, m, ArH), 8.12 (2H, d, ArH); 13C NMR (CDCl3):
l 23.16, 26.01, 46.55, 48.19, 103.87, 126.67, 128.21,
129.02, 132.92, 157.92, 158.28, 158.44. 1g: yield 30%;
m.p. 128–130 °C (EtOH–Et2O); IR: 2125 (CN+2 ), 1600
1
(CꢁO); H NMR (CDCl3): l 1.91 (4H, m, pyrrolidine),
3.55 (2H, t, pyrrolidine), 4.09 (2H, t, pyrrolidine), 7.06
(2H, t, ArH), 8.10 (2H, t, ArH); 13C NMR (CDCl3): l
23.07, 25.92, 46.43, 48.10, 103.87, 114.82, 115.10,
128.53, 128.64, 129.20, 129.23, 157.49, 157.83, 158.57.
1h: yield 50%; m.p. 135–137 °C (EtOH–Et2O); IR:
1
2130 (CN+2 ), 1600 (CꢁO); H NMR (CDCl3): l 1.91
(4H, m, pyrrolidine), 3.54 (2H, t, pyrrolidine), 4.09 (2H,
t, pyrrolidine), 7.41 (2H, d, J=8.6 Hz, ArH), 8.08 (2H,
d, J=8.6 Hz, ArH); 13C NMR (CDCl3): l 23.07, 25.92,
46.47, 48.05, 104.16, 128.04, 128.35, 131.70, 134.12,
156.89, 157.76, 158.19. 1i: yield 45%; m.p. 153–154 °C
(MeOH); IR: 2120 (CN+2 ), 1608 (CꢁO); 1H NMR
(CDCl3): l 1.90 (4H, m, pyrrolidine), 2.33 (3H, s, CH3),
3.52 (2H, t, pyrrolidine), 4.06 (2H, t, pyrrolidine), 7.17
(2H, d, J=7.9 Hz, ArH), 7.97 (2H, d, J=7.9 Hz,
ArH); 13C NMR (CDCl3): l 20.94, 23.08, 25.95, 46.45,
48.08, 103.56, 126.57, 128.81, 130.24, 138.72, 157.83,
158.28, 158.48. 1j: yield 32%; m.p. 158–160 °C (dec.)
4.5. Antimicrobial assays
The minimal inhibitory concentrations (MICs) were
determined by the two-fold agar dilution method [6].
Mueller–Hunton Agar (BBL) was used to test the
antibacterial activity against: S. aureus ATCC 29213, E.
faecalis ATCC 8043, E. coli ATCC 11105, P. mirabilis
MB 81. Sabouraud Dextrose Agar (Difco) was used to
evaluate the antimycotic activity against: C. albicans
ATCC 10231, C. lypolitica CBS 6124, C. neoformans L
29, A. niger L 32. MB and L strains were obtained from
our collection. All experiments were performed in tripli-
cate, using inocula of 106 cfu (colony forming units)/ml
of yeasts and bacteria, and 105 conidia/ml of mould.
Fungal and bacteria plates were incubated at 28 °C for
48 h and at 37 °C for 24 h, respectively. Stock solu-
tions of the tested compounds were prepared in N,N-
dimethylformamide (DMF): as this solvent is inhibitory
for some microrganisms, it is essential to reduce the
final concentration in test media to a non-inhibitory
level; a final concentration of less than 1% DMF was
found to be satisfactory. In the assay conditions, all the
tested compounds proved to be stable.
1
(EtOH–Et2O); IR: 2130 (CN+2 ), 1605 (CꢁO); H NMR
(CDCl3): l 1.74 (4H, m, pyrrolidine), 3.39 (2H, t,
pyrrolidine), 3.92 (2H, t, pyrrolidine), 8.02 (4H, m,
ArH); 13C NMR (CDCl3): l 22.96, 25.81, 46.39, 48.06,
102.62, 123.16, 127.04, 138.39, 147.28, 156.26, 157.59,
158.09. 1k: yield 28%; m.p. 121–123 °C (EtOH–Et2O);
IR: 2120 (CN+2 ), 1620 (CꢁO); 1H NMR (CDCl3): l 1.99
(4H, m, pyrrolidine), 3.68 (2H, t, pyrrolidine), 4.21 (2H,
t, pyrrolidine), 7.09 (1H, t, ArH), 7.40 (1H, q, ArH),
7.93 (2H, m ArH); 13C NMR (CDCl3): l 23.63, 26.47,
46.98, 48.72, 103.72, 114.26, 116.46, 122.71, 129.92,
130.14, 135.50, 157.92, 158.54, 158.89. 1l: yield 18%;
m.p. 132–134 °C (EtOH–Et2O); IR: 2110 (CN+2 ), 1605
1
(CꢁO); H NMR (CDCl3): l 1.93 (4H, m, pyrrolidine),
3.59 (2H, t, pyrrolidine), 4.13 (2H, t, pyrrolidine), 7.32
(2H, m, ArH), 7.72 (1H, d, ArH), 7.85 (1H, s, ArH);
13C NMR (CDCl3): l 24.14, 26.99, 47.53, 49.25, 104.83,
125.73, 127.60, 129.81, 130.21, 131.99, 134.96, 158.10,
158.98, 159.48. 1m: yield 37%; m.p. 94–95 °C (EtOH–
Et2O); IR: 2140 (CN+2 ), 1605 (CꢁO); 1H NMR
(CDCl3): l 1.90 (4H, m, pyrrolidine), 3.60 (2H, t,
pyrrolidine), 4.13 (2H, t, pyrrolidine), 7.09–7.29 (3H,
m, ArH), 8.12 (1H, t, ArH); 13C NMR (CDCl3): l
23.65, 26.49, 47.01, 48.77, 104.29, 116.48, 116.92,
121.30, 124.08, 130.79, 131.08, 155.64, 158.22, 158.58.
5. Results and discussion
The results reported in Table 1 show that the intro-
duction in position 2 of alkyl substituents does not
affect activity against C. neoformans, with the exception
of 1d (MIC=100 mg/ml), while this substitution is
detrimental in regard to the activity against C. albicans.
The presence of a methyl group (1a) increases the
activity against C. lypolitica and A. niger, while more
lipophilic substituents do not influence activity against

890
L. Varoli et al. / Il Farmaco 56 (2001) 885–890
study will be devoted to the improvement of the biolog-
ical profile.
C. lypolitica and cause loss of activity against A. niger
(1c–1e). The introduction of a phenyl ring in position 2
of the imidazole moiety affected in a different way the
activity: the unsubstituted phenyl group decreased the
activity against A. niger (MIC\200 mg/ml), a p-
halogenophenyl group led to the most active deriva-
tives: compound 1g showed the best activity against C.
lypolitica and C. neoformans (MIC=10 mg/ml), com-
pound 1h exhibited the broadest antifungal spectrum
(MIC=25 mg/ml) against yeasts and moulds. Particu-
larly, the efficacy of these products against C. lypolitica,
C. neoformans and A. niger can be compared with that
showed by amphotericin B and fluconazole, used as
reference compounds for inhibitory activity against
fungi.
The introduction in para position of the phenyl ring
of substituents with different electronic and steric prop-
erties caused a decrease of activity against A. niger (1i)
with regard to compound 1h or a reduced susceptibility
against both yeasts and moulds (1j). meta- (1k, 1l) and
ortho-substitution (1m, 1n) or disubstitution (1o, 1p)
gave less active compounds.
The obtained results suggest that the introduction of
a substituent in position 2 of the imidazole moiety with
increasing lipophilicity does not significantly influence
antifungal activity with the exception of a para-substi-
tuted phenyl ring which is important for improved
activity and spectrum. The activity seems independent
of the electron nature of the substituent (1h compared
with 1i or 1j), p-chloro- and p-fluoro-phenyl groups
impart the highest activity and the broadest spectrum.
The Cl group on the phenyl ring results in a more
suitable substituent, it retains some activity also in
ortho- or ortho,para-positions.
Acknowledgements
This work was supported by Ministero della Ricerca
Scientifica e Tecnologica (MURST, grant 60%).
References
[1] M.F.G. Stevens, The Medicinal Chemistry of 1,2,3-Triazines, in:
G.P. Ellis, G.B. West (Eds.), Progress in Medicinal Chemistry,
vol. 13, North-Holland, Amsterdam, 1976, pp. 205–269.
[2] Y.F. Shealy, Syntheses and biological activity of 5-aminoimida-
zoles and 5-triazenoimidazoles, J. Pharm. Sci. 59 (1970) 1533–
1558.
[3] L. Varoli, S. Burnelli, L. Garuti, A. Guarnieri, M. Rossi, Synthe-
sis and antimicrobial activity of N,N-dialkyl-2-substituted-5-dia-
zoimidazole-4-carboxamides, Pharmazie 52 (1997) 578–581.
[4] C.J. Rose, A.T. Ainsworth, Antiinflammatory 5-acetamido-4-
carbamoylimidazoles, West German Patent, 2 358 509; Chem.
Abstr. 81 (1974) 63630y.
[5] A.H. Cook, I. Heilbron, A.L. Levy, Studies in the azole series.
Part I. A novel route to 5-aminothiazoles, J. Chem. Soc. (1947)
1594–1598.
[6] W.B. Hugo, A.D. Russel (Eds.), Pharmaceutical Microbiology,
3rd ed., Blackwell Scientific, Oxford, 1983.
8
[7] J. Houben, R. Zivadinovitsch, Uber Imino-kohlensa¨ure-ester,
Imino-thiol-, Oximino-thiol- und Dithio-ameisensa¨ure-ester,
Berichte B 69 (1936) 2352–2360.
[8] E. Lunt, C.G. Newton, C. Smith, G.P. Stevens, M.F.G. Stevens,
C.G. Straw, R.J.A. Walsh, P.J. Warren, C. Fizames, F. Lavelle,
S.P. Langdon, L.M. Vickers, Antitumor imidazotetrazines. 14.
Synthesis and antitumor activity of 6- and 8-substituted imi-
dazo[5,1-d]-1,2,3,5-tetrazinones and 8-substituted pyrazolo[5,1-
d]-1,2,3,5-tetrazinones, J. Med. Chem. 30 (1987) 357–366.
[9] H. Bader, J.D. Downer, P. Driver, New 5-aminoglyoxalines as
potential adrenergic agents, J. Chem. Soc. (1950) 2775–2784.
[10] J. Heyes, N. Ward, Antiinflammatory 5-amino-2-phenylimida-
zoles, Ger. Offen. 2 142 831; Chem. Abstr. 76 (1972) 153744q.
Considering the antifungal activity of compounds 1g
and 1h, particularly against the mould A. niger, further