Synthesis, antimicrobial and antioxidant activities of imidazotriazoles and new multicomponent reaction toward 5-amino-1-phenyl[1,2,4]triazole derivatives

Article abstract of DOI:10.1007/s00044-015-1322-z

The Groebke-type multicomponent reaction between 5-amino-1,2,4-triazole derivatives, aromatic aldehydes and isocyanides has been studied from the viewpoint of convenient generation of combinatorial arrays of imidazo[2,1-c][1,2,4]triazoles. The reaction is

Full text of DOI:10.1007/s00044-015-1322-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1322-z
ORIGINAL RESEARCH
Synthesis, antimicrobial and antioxidant activities
of imidazotriazoles and new multicomponent reaction
toward 5-amino-1-phenyl[1,2,4]triazole derivatives
•
Monia Aouali Dhekra Mhalla Fatma Allouche
•
•
•
•
•
Laurent El Kaim Slim Tounsi Mohamed Trigui
Fakher Chabchoub
Received: 11 September 2014 / Accepted: 4 January 2015
Ó Springer Science+Business Media New York 2015
Abstract The Groebke-type multicomponent reaction
between 5-amino-1,2,4-triazole derivatives, aromatic
aldehydes and isocyanides has been studied from the
viewpoint of convenient generation of combinatorial arrays
of imidazo[2,1-c][1,2,4]triazoles. The reaction is consid-
ered to proceed via the formation of an iminium species
followed by a [4 ? 1] cyclo addition with the isocyanides
using scandium triflate as a Lewis acid catalyst. The syn-
thesized imidazo[2,1-c][1,2,4]triazole derivatives 4a, 4b,
4f and 4g were screened for antibacterial, antifungal and
antioxidant activities. Among the tested compounds, 4b
followed by 4f showed potent antibacterial and antifungal
activities. All of these compounds were also screened
in vitro for the antioxidant activity using DPPH assay.
Most of them have shown very significant antioxidant
activity.
Introduction
The chemistry of heterocyclic compounds has attracted
much attention in recent times due to its increasing impor-
tance in the field of pharmaceuticals and industrial chemicals
(Al-Tel and Al-Qawasmeh, 2010; Shukla et al., 2012; Bode
et al., 2011). In fact, the development of simple, elegant and
facile methodologies for the synthesis of heterocycles is one
of the most important aspects in organic synthesis. Multi-
component reactions (MCRs), reactions involving at least
three starting materials in a one-pot reaction, have brought
some of the most efficient syntheses of heterocycles (Adib
et al., 2011; Schwerkoske et al., 2005; Rousseau et al., 2007;
Parchinsky et al., 2006). Isocyanide-based MCRs (IMCRs),
such as the Passerini, the Ugi reactions and the Groebke–
´
Blackburn–Bienayme, are very useful for the diversity ori-
ented synthesis of collections of compounds (El Kaim and
Grimaud, 2009; Sadjadi and Heravi, 2011; Khan et al., 2012;
Shaabani et al., 2001; Mandair et al., 2002; Guchhait and
Madaan, 2011; Shukla et al., 2012). They allow a dramatic
increase of structural complexity in just one step, introduc-
ing at the same time three or more diversity inputs with a
high degree of atom economy (Agrebi et al., 2013; Xu et al.,
2012; Shaw et al., 2012; El Kaim et al., 2010).
Keywords 5-Amino-1-phenyl-1,2,4-triazole Á MCR Á
Imidazo[2,1-c][1,2,4]triazole Á Antioxidant Á Antimicrobial
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1322-z) contains supplementary
material, which is available to authorized users.
M. Aouali (&) Á F. Allouche Á F. Chabchoub
´
Laboratoire de Chimie Appliquee: Heterocycles, Corps Gras et
Polymeres, Faculte des Sciences de Sfax, Universite de Sfax,
´ ´
`
´
´
Materials and methods
3018 Sfax, Tunisia
e-mail: fatmaallouch@yahoo.fr
Chemistry
D. Mhalla Á S. Tounsi Á M. Trigui
Biopesticides Team (LPIP), Center of Biotechnology of Sfax,
University of Sfax, P. O. Box «1177», 3018 Sfax, Tunisia
Melting points were measured on an electrothermal appa-
ratus. Reactions were checked with TLC using aluminum
sheets with silica gel 60 F254 from Merck. Spectra IR were
recorded on a Perkin-Elmer PARAGON FT-IR spectrom-
eter covering field 400–4,000 cm-1. The spectra of 1H
L. El Kaim
´ ´
Laboratoire Chimie et Procedes, DCSO, UMR 7652, Ecole
´
Nationale Superieure de Techniques Avancees, 828 Bd des
Marechaux, 91120 Palaiseau, France
´
´
123

Med Chem Res
NMR and 13C NMR were recorded in solution in CDCl3 on
a Bruker spectrometer (1H at 400 MHz, 13C at 100 MHz)
and high-resolution mass spectra (HRMS). The chemical
shifts are expressed in parts per million (ppm) by using
tetramethylsilane (TMS) as internal reference. The multi-
plicities of the signals are indicated by the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quadruplet;
and m, multiplet, and coupling constants are expressed in
Hertz. The chemical reagents used in synthesis were pur-
chased from Fluka, Sigma and Aldrich. The microwave-
assisted reactions were carried out in synthetic microwave:
Monowave 300 with a maximum power of 300 W.
Amino-3-benzyl-1-phenyl[1,2,4]triazole (1c)
It was obtained as a yellow solid; yield: 87 %; mp:
1
137–140 °C; IR tmax 3,397, 3,477, 1,620, 1,539 cm-1; H
NMR (CDCl3, 400 MHz): d = 7.54–7.53 (4H, m),
7.45–7.40 (1H, m), 7.35–7.29 (4H, m), 7.21 (1H, t,
J = 7.0 Hz, H-16), 4.88 (2H, s, NH2), 3.90 (2H, s); 13C
NMR (CDCl3, 100 MHz): d = 161.8 (C, C-3), 156.5 (C,
C-5), 139.1 (C, C-13), 138 (C, C-6), 130.7 (CH, C-7,
C-11), 129.8 (CH, C-14, C-18), 129.5 (CH, C-8, C-10),
127.5 (CH, C-16), 129.2 (CH, C-9), 124.9 (CH, C-15,
C-17), 35.2 (CH2), HRMS Calcd. for C15H14N4: 250.1218,
found 250.1213.
General procedure for the synthesis and spectral data
of 5-amino-1-phenyl[1,2,4]triazole derivatives 1a–c
using the one-step method
General procedure for the synthesis and spectral data
of substituted imidazotriazole (4a–i)
General procedure for MCR of 5-amino-1-phenyl-1,2,4-
triazoles, aldehydes and isonitriles: A solution of 5-amino-
1-phenyl-1,2,4-triazole (1 mmol), aldehyde (1 mmol) and
5 % Sc(OTf)3 in 1 ml DMF was heated at 80 °C for
20 min, isonitrile (1.2 mmol) was added, and the mixture
was brought to reflux. After 30 h, the crude product is
isolated by flash chromatography on silica gel [PE/DCM
(8:2)].
To a mixture of derivatives orthoester (1 mmol), phenyl
hydrazine (1.2 mmol), acetic acid (5 mol%) and cyana-
mide (1 mmol) in 3 ml of methanol, it was heated with
microwave radiation (200 W) for 40 min at 80 °C. After
the completion of the reaction, as indicated by TLC
(EtOAc-hexane, 90:10), the precipitated solid was filtered,
washed with ether and was crystallized from a suitable
solvent to obtain pure product.
N-(4-chlorobenzyl)-6-(4-chlorophenyl)-3-methyl-1-phenyl-
1H-imidazo[2,1-c][1,2,4] triazol-5-amine (4a)
Amino-3-methyl-1-phenyl[1,2,4]triazole (1a)
It was obtained as a yellow solid; yield: 72 %; mp:
It was obtained as a yellow solid; yield: 95 %; mp:
131–134 °C; IR tmax: 3,397, 3,477, 1,616, 1,537 cm-1; 1H
NMR (CDCl3, 400 MHz): d = 7.53–7.44 (4H, m, H-7,
H-8, H-10, H-11), 7.35–7.31 (1H, m, H-9), 6.33 (2H, br,
NH2), 2.11 (3H, s, CH3); 13C NMR (CDCl3, 100 MHz):
d = 157.4 (C, C-3), 154.6 (C, C-5), 137.3 (C, C-6), 129.2
(CH, C-8, C-10), 126.4 (CH, C-9), 122.2 (CH, C-7, C-11),
13.8 (CH3); HRMS Calcd. for C9H10N4: 174.0905, found
174.0907.
220–222 °C; IR tmax 3,320, 3,065, 2,835, 1,597, 1,505,
1,489, 1,458, 1,433, 1,406 cm-1
;
1H NMR (CDCl3,
400 MHz): d = 8.16 (2H, d, J = 8.0 Hz, H-10, H-14),
7.94 (2H, d, J = 7.5 Hz, H-17, H-19), 7.52 (2H, t,
J = 7.5 Hz, H-24, H-26), 7.42 (2H, d, J = 7.5 Hz, H-23,
H-27), 7.32 (2H, d, J = 8.8 Hz, H-16, H-20), 7.25–7.22
(3H, m, H-11, H-12, H-13), 4.16 (2H, s, H-21), 3.2 (1H, br,
NH), 2.58 (3H, s, CH3); 13C NMR (CDCl3, 100 MHz):
d = 145.8 (C, C-3), 138.0 (C, C-9), 137.8 (C, C-22), 136.9
(C, C-8), 136.4 (C, C-6), 133.6 (C, C-18), 133.0 (C, C-25),
132.9 (C, C-15), 129.6 (CH, C-11, C-13), 129.3 (CH, C-17,
C-19), 128.9 (CH, C-18, C-20), 128.6 (CH, C-24, C-26),
127.9 (CH, C-23, C-27), 124.4 (CH, C-12), 121.0 (C, C-5),
116.3 (CH, C-10, C-14), 54.1 (C, C-21, CH2), 11.4 (C,
CH3); HRMS Calcd. for C24H19Cl2N5: 447.1018, found
447.1006.
Amino-3-ethyl-1-phenyl[1,2,4]triazole (1b)
It was obtained as a yellow solid; yield: 94 %; mp:
1
120–124 °C; IR tmax 3,396, 3,470, 1,622, 1,537 cm-1; H
NMR (CDCl3, 400 MHz): d = 7.57–7.51 (4H, m, H-7,
H-8, H-10, H-11), 7.46–7.42 (1H, m, H-9), 4.88 (2H, br,
NH2), 2.59 (2H, q, J = 7.7 Hz, CH2), 1.29 (3H, t,
J = 7.7 Hz, CH3); 13C NMR (CDCl3, 100 MHz):
d = 164.1 (C, C-3), 156.3 (C, C-5), 138.1 (C, C-6), 130.7
(CH, C-8, C-10), 129.2 (CH, C-9), 124.9 (CH, C-7, C-11),
22.4 (CH2), 12.8 (CH3); HRMS Calcd. for C10H12N4:
188.1062, found 188.1063.
6-(4-chlorophenyl)-N-Cyclohexyl-3-methyl-1-phenyl-1H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4b)
It was obtained as a yellow solid; yield: 57 %; mp:
220–223 °C; IR tmax 3,294, 3,067, 2,925, 2,853, 1,609,
123

Med Chem Res
1,598, 1,505, 1,488, 1,459, 1,440 cm-1; H NMR (CDCl3,
400 MHz): d = 8.06 (2H, d, J = 8 Hz, H-11, H-13), 7.95
(2H, d, J = 8.8 Hz, H-16, H-20), 7.40 (2H, t, J = 8.0 Hz,
H-10, H-14), 7.31 (2H, d, J = 8.0 Hz, H-17, H-19), 7.11
(1H, t, J = 7.5 Hz, H-12), 2.81 (1H, br, NH), 2.75 (2H, br,
H-21), 2.62 (3H, s, CH3), 1.76–1.35 (5H, m, H-22, H-23,
H-24, H-25, H-26), 1.10–1.08 (5H, m, H-22, H-23, H-24,
H-25, H-26); 13C NMR (CDCl3, 100 MHz): d = 145.9 (C,
C-3), 138.3 (C, C-9), 137.9 (C, C-8), 136.6 (C, C-6), 133.4
(C, C-18), 132.7 (C, C-15), 129.2 (CH, C-11, C-13), 128.4
(CH, C-17, C-19), 128.1 (CH, C-16, C-20), 124.3 (CH,
C-12), 121 (C, C-5), 116.3 (CH, C-10, C-14), 58.6 (CH,
C-21), 33.7 (CH, C-22, C-26), 25.7 (CH, C-24), 24.8 (CH,
C-23, C-25), 11.7 (CH3); HRMS Calcd. for C23H24ClN5:
405.1720, found 405.1713.
25.7 (CH, C-24), 24.6 (CH, C-23, C-25), 11.6 (CH3);
HRMS Calcd. for C23H23Cl2N5: 439.1331, found
439.1351.
1
N-(4-methoxybenzyl)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-imidazo[2,1-c][1,2,4] triazol-5-amine (4e)
It was obtained as a white solid; yield: 72 %; mp:
170–175 °C; IR tmax 3,322, 3,063, 2,999, 2,922, 2,834,
1
1,594, 1,541, 1,504, 1,488, 1,458 cm-1; H NMR (CDCl3,
400 MHz): d = 8.18 (2H, d, J = 7.0 Hz, H-16, H-20),
7.99 (2H, d, J = 8.0 Hz, H-17, H-19), 7.51 (2H, d,
J = 7.5 Hz, H-23, H-26), 7.42 (2H, d, J = 8.4 Hz, H-24,
H-25), 7.25–7.21 (3H, m, H-11, H-12, H-13), 6.89 (2H, d,
J = 7.1 Hz, H-10, H-14), 4.15 (2H, s, H-21), 3.85 (3H, s,
OCH3), 3.16 (1H, br, NH), 2.58 (3H, s, CH3); 13C NMR
(CDCl3, 100 MHz): d = 159.2 (C, C-25), 155.2 (C, C-3),
145.8 (C, C-9), 137.9 (C, C-8), 136.6 (C, C-6), 133.1 (C,
C-22), 132.6 (C, C-18), 130.7 (C, C-15), 129.5 (CH, C-11,
C-13), 129.3 (C, C-17, C-19), 128.6 (C, C-16, C-20), 127.9
(CH, C-23, C-27), 124.3 (CH, C-12), 121.5 (C, C-5), 116.3
(CH, C-10, C-14), 114.1 (CH, C-24, C-26), 55.3 (CH3,
OCH3), 54.4 (CH2), 11.3 (CH3); HRMS Calcd. for
C25H22ClN5O: 443.1513, found 443.1551.
N-Cyclohexyl-6-(4-fluorophenyl)–3-methyl-1-phenyl-1H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4c)
It was obtained as a yellow solid; yield: 60 %; mp:
182–185 °C; IR tmax 3,219, 3,082, 2,933, 2,918, 2,851,
1
1,648, 1,593, 1,583, 1,501, 1,440 cm-1; H NMR (CDCl3,
400 MHz): d = 8.61 (2H, d, J = 7.8 Hz, H-10, H-14),
8.10 (2H, t, J = 7.8 Hz, H-16, H-20), 7.52 (2H, d,
J = 7.62 Hz, H-11, H-13), 7.26–7.22 (3H, m, H-17, H-19,
H-12), 3.30 (1H, br, NH), 2.82 (3H, s, CH3), 2.05–1.32
(11H, m, H-21, H-22, H-23, H-24, H-25, H-26); 13C NMR
(CDCl3, 100 MHz): d = 144.2 (C, C-3), 137.7 (C, C-9),
137.6 (C, C-8), 136.2 (C, C-6), 133.2 (C, C-18), 133.1 (C,
C-15), 129.7 (CH, C-11, C-13), 129.3 (CH, C-17, C-19),
124.4 (CH, C-16, C-20), 116.1 (CH, C-12), 115.1 (C, C-5),
114.9 (CH, C-10, C-14), 58.4 (CH, C-21), 33.8 (CH, C-22,
C-26), 25.4 (CH, C-24), 24.8 (CH, C-23, C-25), 13.3
(CH3); HRMS Calcd. for C23H24FN5: 389.2016, found
389.2018.
6-(4-chlorophenyl)-N-cyclohexyl-3-phenyl-1-phenyl-1H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4f)
It was obtained as a white solid; yield:60 %; mp: 194–196 °C;
IR tmax 3,299, 3,073, 2,974, 2,933, 2,852, 1,600, 1,587, 1,506,
1,460, 1,440, 1,409 cm-1 1H NMR (CDCl3, 400 MHz):
;
d = 8.20 (2H, d, J = 7.3 Hz, H-11, H-13), 8.05 (2H, d,
J = 8.5 Hz, H-16, H-20), 7.53 (2H, t, J = 7.7 Hz, H-10,
H-14), 7.42 (2H, d, J = 8.5 Hz, H-17, H-19), 7.28 (1H, t,
J = 7.5 Hz, H-12), 3.10 (2H, q, J = 7.1 Hz, CH2), 2.89 (1H,
br, NH), 1.87 (1H, br, H-21), 1.73–1.64 (5H, m, H-22, H-23,
H-24, H-25, H-26), 1.55 (3H, t, J = 7.4 Hz, CH3), 1.2 (5H, m,
H-22, H-23, H-24, H-25, H-26); 13C NMR (CDCl3,
100 MHz): d = 146.0(C,C-3),141.5(C, C-9),138.3(C, C-8),
138.0 (C, C-6), 133.5 (C, C-18), 132.5 (C, C-15), 129.2 (CH,
C-11, C-13), 128.4 (CH, C-17, C-19), 128.2 (CH, C-16, C-20),
124.2 (CH, C-12), 120.6 (C, C-5), 116.4 (CH, C-10, C-14),
58.6 (CH, C-21), 33.8(CH, C-22, C-26), 25.7(CH, C-24), 24.9
(CH, C-23, C-25), 19.6 (CH2), 11.4 (CH3); HRMS Calcd. for
C24H26ClN5: 419.1876, found 419.1877.
N-Cyclohexyl-6-(2,6-dichlorophenyl)-3-methyl-1-phenyl-
1H-imidazo[2,1-c][1,2,4]triazol-5-amine (4d)
It was obtained as a yellow solid; yield: 54 %; mp:
174–178 °C; IR tmax 3,348, 3,065, 2,923, 2,852, 1,585,
1,504, 1,425 cm-1; 1H NMR (CDCl3, 400 MHz): d = 7.99
(2H, d, J = 8.5 Hz, H-17, H-19), 7.37–7.32 (4H, m, H-10,
H-11, H-13, H-14), 7.18 (1H, t, J = 8.5 Hz, H-18), 7.06
(1H, t, J = 7.3 Hz, H-12), 2.64 (3H, s, CH3), 2.59 (2H, br,
NH, H-21), 1.72 (2H, d, J = 8.5 Hz, H-22, H-26),
1.52–1.41 (3H, m, H-23, H-25, H-24), 1.07–0.84 (5H, m,
H-22, H-23, H-24, H-25, H-26); 13C NMR (CDCl3,
100 MHz): d = 145.6 (C, C-3), 138.0 (C, C-9), 136.6 (C,
C-8), 134.5 (C, C-16, C-20), 134.2 (C, C-6), 132.4 (C,
C-15), 130.0 (CH, C-18), 129.1(CH, C-11, C-13), 128.8
(CH, C-10, C-14), 124.1 (CH, C-12), 123.0 (C, C-5), 116.5
(CH, C-17, C-19), 58.3 (CH, C-21), 33.5 (CH, C-22, C-26),
N-cyclohexyl-6-(2,6-dichlorophenyl)-3-ethyl-1H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4g)
It was obtained as a white solid; yield: 70 %; mp:
138–141 °C; IR tmax 3,345, 3,058, 2,933, 2,853, 1,671,
1
1,598, 1,579, 1,504, 1,455, 1,425 cm-1; H NMR (CDCl3,
400 MHz): d = 8.09 (2H, d, J = 8.8 Hz, H-17, H-19),
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Med Chem Res
7.35–7.29 (4H, m, H-10, H-11, H-13, H-14), 7.14 (t,
J = 7.7 Hz, 1H, H-18), 7.03 (t, J = 7.7 Hz, 1H, H-12),
2.99 (q, J = 14.5 Hz, 2H, CH2), 2.58 (br, 2H, NH, H-21),
1.69 (d, J = 12 Hz, 2H, H-22, H-26), 1.49-1.41 (m, 6H,
CH3, H-23, H-25, H-24), 1.07–0.84 (m, 5H, H-22, H-23,
H-24, H-25, H-26); 13C NMR (CDCl3, 100 MHz):
d = 145.7 (C, C-3), 141.6 (C, C-9), 138.1 (C, C-8), 136.6
(C, C-16, C-20), 134.4 (C, C-6), 132.6 (C, C-15), 130.0
(CH, C-18), 129.2 (CH, C-11, C-13), 128.2 (CH, C-10,
C-14), 124.1 (CH, C-12), 123.0 (C, C-5), 116.5 (CH, C-17,
C-19), 58.3 (CH, C-21), 33.5 (CH, C-22, C-26), 25.7 (CH,
C-24), 24.6 (CH, C-23, C-25), 19.65 (CH2), 11.5 (CH3);
HRMS Calcd. for C25H25Cl2N5: 543.1487, found
543.1487.
(C, C-9), 138.0 (C, C-28), 136.6 (C, C-16, C-20), 135.5 (C,
C-8), 143.7 (C, C-6), 132.6 (C, C-15), 130.0 (CH, C-18),
129.3 (CH, C-11, C-13), 128.8 (CH, C-29, C-33), 128.7
(CH, C-30, C-32), 128.2 (CH, C-10, C-14), 127.2 (CH,
C-31), 124.4 (CH, C-12), 123.0 (C, C-5), 116.7 (CH, C-17,
C-19), 55.7 (CH, C-21), 33.4 (CH, C-22, C-26), 31.7
(CH2), 25.7 (CH, C-24), 24.6 (CH, C-23, C-25); HRMS
Calcd. for C29H27Cl2N5: 515.1644, found 515.1649(-).
Antimicrobial activity
Authentic pure cultures of bacteria were obtained from
international culture collections (ATCC) and the local
culture collection of the Centre of Biotechnology of Sfax,
Tunisia. They included Gram-positive bacteria: B. cereus
ATCC 14579, S. aureus ATCC 25923, and Gram-negative
bacteria: S. enteritidis (food isolate), E. coli ATCC 25922
and P. aeruginosa ATCC 9027. The fungi tested in the
antifungal test were Aspergillus niger CTM 10099,
Fusarium oxysporum CTM10402, Fusarium culmorum
ISPAVE 21w and Rhizopus Nigerians CTM 10150. The
bacterial strains were grown on Mueller–Hinton broth
(Bio-Rad, France) at 37 °C for 12–14 h and on potato
dextrose agar (PDA) (1.5 % agar) at 28 °C for 4 days for
fungus. Inocula were prepared from an overnight broth
culture by their dilution in saline solution to approximately
107 colony-forming units CFU/ml for bacteria and 106
spores/ml for fungus. The compounds were dissolved in
DMSO.
6-(4-benzonitrile)-N-cyclohexyl-3-ethyl-1-phenyl-1-H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4h)
It was obtained as a white solid; yield: 57 %; mp:
170–173 °C; IR tmax 3,466, 3,062, 2,928, 2,853, 2,226,
1
1,644, 1,601, 1,537, 1,505, 1,450, 1,404 cm-1; H NMR
(CDCl3, 400 MHz): d = 8.11 (2H, d, J = 7.0 Hz, H-17,
H-19), 8.03 (2H, d, J = 7.0 Hz, H-11, H-13), 7.54 (2H, d,
J = 7.0 Hz, H-16, H-20), 7.37 (2H, t, J = 7.5 Hz, H-10,
H-14), 7.09 (1H, t, J = 7.3 Hz, H-12), 2.89 (2H, q,
J = 7.8 Hz, CH2), 2.70 (2H, br, NH, H-21), 1.70 (2H, s,
H-22, H-26), 1.59–1.51 (3H, m, H-23, H-24, H-25), 1.39
(3H, t, J = 7.5 Hz, CH3), 1.08–1.04 (5H, m, H-22, H-23,
H-24, H-25, H-26); 13C NMR (CDCl3, 100 MHz):
d = 146.1 (C, C-3), 141.3 (C, C-9), 139.6 (C, C-8), 137.9
(C, C-15), 137.4 (C, C-6), 132.0 (CH, C-17, C-19), 129.2
(CH, C-11, C-13), 127.0 (CH, C-16, C-20), 124.4 (CH,
C-2), 121.9 (C, C-5), 119.4 (C, CN), 116.6 (CH, C-10,
C-14), 109.6 (C, C-18), 58.7 (CH, C-21), 33.7 (CH, C-22,
C-26), 25.7 (CH, C-24), 24.8 (CH, C-23, C-25), 19.6
(CH2), 11.1 (CH3); HRMS Calcd. for C25H26N6: 410.2219,
found 410.2211.
Antibacterial and antifungal tests were performed by
agar well diffusion method as described by Tagg and
McGiven (1971) and broth microdilution assay using
sterile Mueller–Hinton media (Bio-Rad, France) for bac-
terial strains and potato dextrose agar (Bio-Rad, France) for
antifungal tests. A freshly cell suspension (100 ll) adjusted
to 107 CFU/ml for bacteria and 105 spores/ml for fungus
were inoculated onto the surface of agar plates. Thereafter,
wells with 6 mm in diameter were punched in the inocu-
lated agar medium with sterile Pasteur pipettes and the
extracts were added to each well. Negative controls con-
sisted of DMSO, used to dissolve the synthesized com-
pounds. Gentamicin (15 lg/well) and amphotericin B
(20 lg/well) were used as positive control to determine the
sensitivity of each bacterial and fungal strain, respectively.
The plate was allowed to stand for 2 h at 4 °C to permit the
diffusion of the extracts followed by incubation at 37 °C
for 24 h for bacterial strains and 72 h for fungi at 28 °C.
The antibacterial activity was evaluated by measuring the
zones of inhibition (clear zone around the well) against the
test microorganisms. All tests were repeated three times.
Minimum inhibitory concentrations of the synthesized
compounds were determined according to Eloff (1998) in
sterile 96-well microplates with a final volume in each
3-Benzyl-6-(4-chlorophenyl)-N-cyclohexyl-1-phenyl-1H-
imidazo[2,1-c][1,2,4]triazol-5-amine (4i)
It was obtained as a white solid; yield: 62 %; mp:
179–182 °C; IR tmax 3,294, 3,067, 2,926, 2,853, 1,662,
1,609, 1,597, 1,539, 1,505, 1,488, 1,458, 1,440,
1
1,405 cm-1; H NMR (CDCl3, 400 MHz): d = 8.03 (2H,
d, J = 8.0 Hz, H-17, H-19), 7.35–7.10 (10H, m, H-10,
H-11, H-12, H-13, H-14, H-29, H-30, H-31, H-32, H-33),
7.05 (1H, t, J = 7.5 Hz, H-12), 4.35 (2H, s, H-27), 2.40
(1H, br, NH), 2.35 (1H, br, H-21), 1.59 (2H, d,
J = 12.0 Hz, H-22, H-26), 1.41–1.34 (3H, m, H-23, H-24,
H-25), 0.87–0.69 (5H, m, H-22, H-23, H-24, H-25, H-26);
13C NMR (CDCl3, 100 MHz): d = 145.7 (C, C-3), 138.7
123

Med Chem Res
microplate well of 200 ll. A stock solution of the extract
(50 mg/ml) was prepared in 20 % DMSO. Thereafter, a
twofold serial dilution of the extract was prepared in the
microplate wells over the range 0.039–10 mg/ml. To each
test, well was added 10 ll of cell suspension to a final
inoculum concentrations of 106 CFU/ml for bacteria and
105 spores/ml for fungus. Positive growth control wells
consisted of bacteria or fungi only in their adequate med-
ium. DMSO was used as negative control. The plates were
then covered with the sterile plate covers and incubated at
37 °C for 24 h for bacterial strains and 72 h for fungi at
28 °C. The MIC was defined as the lowest concentration of
the extract at which the microorganism does not demon-
strate visible growth after incubation. As an indicator of
microorganism growth, 25 ll of p-iodonitrotetrazolium
violet (INT) (0.5 mg/ml) dissolved in sterile water were
added to the wells and incubated at 37 °C for 30 min.
Following the determination of the MIC, the MBC was
determined by transferring a 10 ll aliquot from each of the
wells at the concentration corresponding to the MIC and
those concentrations above into agar plate and were incu-
bated under the same conditions. The MBC was considered
to be the lowest concentration of the compound at which no
growth occurred. All experiments were performed in triplicate.
Results and discussion
Chemistry
The synthesis of 5-amino-1-phenyl-1,2,4-triazole
derivatives 1a–c in one-step method
Due to the convenience and high degree of atom economy,
MCRs have become one of the most efficient tools for
rapid scaffold construction and introduction of molecular
diversity (Chen et al., 2014). In our search for operationally
simple, resource and cost-effective processes, we have
been investigating 5-amino-1-phenyl-1,2,4-triazole-based
MCRs.
The advent of single-mode microwave reactors, which
enable precise control of reaction conditions, has opened
the way for the exploration of microwave-assisted syn-
thetic methods in combinatorial and parallel synthesis. As
part of an ongoing program of research, we decided to
investigate the possibility of using microwaves to accel-
erate the synthesis of a range of fused 5-aminotriazoles via
a three-component reaction. The synthesis of this latter was
obtained after using both classical and microwave heating.
Their classical synthesis in two steps was well described in
the literature (Chihaoui et al., 1981).
Determination of DPPH radical-scavenging capacity
The one-pot method in the multicomponent reaction of
orthoester, phenylhydrazine and cyanamide under micro-
wave irradiation led to the synthesis of aminotriazole with
high yield (40 min, 200 W, 80 °C) but in classical condi-
tions need 48 h (Table 1). In this case, we obtained
essentially a convenient and rapid method that gives better
yields and higher purity of the products than the conven-
tional method for an appropriate time (Scheme 1).
The radical-scavenging activity of each synthesized com-
pounds was evaluated on the basis of its activity in scav-
enging the stable DPPH radical, according to the method
described by Kirby and Schmidt (1997). In this procedure,
1 ml of a 4 % solution of DPPH radical in methanol (w/v)
was mixed with 500 ll of sample solutions at different
concentrations. The scavenging capacity was determined
spectrophotometrically after 20 min of incubation by
monitoring the decrease of the absorbance at 517 nm.
Lower absorbance of the reaction mixture indicates higher
free radical-scavenging activity. Ascorbic acid was used as
standard. The percent DPPH scavenging effect was calcu-
lated using the following equation: DPPH scavenging
effect (%) = (Acontrol - Asample/Acontrol) 9 100. A
control is the absorbance of the control reaction where the
sample is replaced by 500 ll of methanol. Tests were
carried out in triplicates.
The synthesis of imidazo[2,1-c][1,2,4]triazole derivatives
(4a–i)
The reaction of 5-amino-1,2,4-triazoles 1a–c with alde-
hydes aromatic and isocyanides using scandium triflate as
catalyst was carried out in DMF as solvent and has been
reported to produce the expected imidazo[2,1-c][1,2,4]tri-
azoles 4a–i into a reliable process that would enable the
production of compound arrays in a combinatorial fashion.
Herein, we present the results of these studies (Scheme 2).
Table 1 Microwave-assisted synthesis of compounds 1a–c
Compounds
R1
Classical condition
Yields (%)
Microwave
Time (h)
Yields (%)
Time (min)
1a
1b
1c
CH3
85
88
65
48
48
48
95
94
87
40
40
40
C2H5
Ph-CH2
123

Med Chem Res
Scheme1 Synthesis of
5-amino-1-phenyl-1,2,4-
triazoles derivatives 1a–c
Ph
OEt
OEt
OEt
N
Ph
N
H2N
NC
HN
AcOH
MeOH
R1
R1
NH2
N
NH2
1 a-c
(in mm) around wells, minimum inhibitory concentration
(MIC), as determined by the dilution method and minimum
bactericidal concentration (MBC) for bacteria and mini-
mum fungicidal concentration (MFC) for fungi. The results
are summarized in (Table 3). The antibacterial activity was
observed with 4a, 4b and 4f against only Gram-positive
bacteria B. cereus and S. aureus one of the most common
Gram-positive bacteria causing food poisoning. However,
these compounds are inactive against Gram-negative bac-
teria. Particularly, para-chloro substituted compound 4b
exhibited excellent inhibition of the Gram-positive bacteria
with inhibition zone diameter ranged from 29 to 20 mm
and MIC values of 78 and 312 lg/ml against B. cereus and
S. aureus, respectively. Unlike compound 4g was inactive
in our bacterial growth-inhibiting assay due to the cyano
substituents. As with the antibacterial activity, we evalu-
ated antifungal activity by means of inhibition zone, MIC
and MFC observations, and the results are summarized in
(Table 3). Antifungal activity was seen in three of the
derivatives 4a, 4b and 4f. The compound 4b demonstrated
an excellent in vitro activity and a wide spectrum of anti-
fungal activity with inhibition zones diameters varied from
9 to 30 mm and MIC range from 0.156 to 0.625 lg/ml. The
MBC determination showed a bactericidal effect of the
compound 4b with MBC/MIC ratio less than or equal to 4
with A. niger, F. culmorum, and R. nigricans (Fig. 1).
N
N
R1
N
N
N
NH2
Sc(OTf)3
DMF
N
R1
N
NC
R3
O
H
HN
R3
R2
R2
4a-i
Scheme 2 Synthesis of imidazo[2,1-c][1,2,4]triazoles derivatives
4a–i
A number of isocyanides were combined in dimethyl-
formamide with 5-amino-1,2,4-triazoles and a variety of
heterocyclic aldehydes (Table 2) and heated at reflux for
30 h in the presence of scandium triflate Sc(OTf)3.
A mechanistic rationalization for this scandium triflate
´
triggered Groebke–Bienayme–Blackburn reaction is pro-
vided in (Scheme 3).
In these cases, the condensation of amine group with
aromatic aldehyde, gives an alternative imine. The trapping
of the more electrophilic iminium derivatives allows a far
more efficient coupling with a wide range of aldehydes
aromatics, which is then trapped by the isocyanide to give
imidazo[2,1-c][1,2,4] triazoles in modest yields. The crude
products were purified by column chromatography on silica
gel using appropriate gradients of petroleum ether in
dichloromethane (8:2 PE/DCM) as eluent to provide
moderate to excellent yields of 4a–i (Table 2).
Antioxidant activity
The synthesized compounds 4a, 4b, 4g and 4f were tested
for their antioxidant properties by using the free stable
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). This assay
was used to test the capacity of the antioxidative com-
pounds as proton radical scavengers or hydrogen donors.
The DPPH radical reacts with suitable reducing agents;
then, electrons become paired off, and the solution loses
color, observed by the decrease in absorbance at 517 nm,
stoichiometrically with the number of electrons taken up.
The antioxidant efficiency can be also evaluated by the
determination of the EC50 value corresponding to the
amount of the compound required to scavenge 50 % of
DPPH radicals present in the reaction mixture. High IC50
values indicated low antioxidant activity. As shown in
Fig. 2, the DPPH radical scavenger capacities of com-
pounds were in the following order: 4g [ 4a [ 4b [ 4f.
The structures of the products 4a–i were deduced from
1
their IR, H NMR, 13C NMR spectra and HRMS.
Biological activities
Antimicrobial activity
The new synthesized compounds were screened for their
antibacterial activity against two Gram-positive bacteria
Bacillus cereus and Staphylococcus aureus species and
three Gram-negative bacteria Escherichia coli, Pseudo-
monas aeruginosa and Salmonella enteritidis. This activity
was evaluated by means of the diameter of inhibition zones
123

Med Chem Res
Table 2 Synthesis of imidazo[2,1-c][1,2,4]triazoles derivatives 4a–i
The compounds 4a–i were obtained by a mixture of triazole (1 eq), aldehyde (1 eq), isonitrile (1,2 eq) and 5 % Sc(OTf)3 in DMF of 80 °C in
30 h
These compounds exhibited significantly high DPPH rad-
ical-scavenging activity when compared to the standard
BHA and a-tocopherol. The synthesized 5-amino-3-
methyl-1-phenyl-1,2,4-triazole and the 5-amino-3-ethyl-1-
benzyl-1,2,4-triazole used in the preparation of 4a, 4b, 4d,
4f and 4g showed a low inhibition capacities of 5 and 7 %,
respectively, at 1.2 mg/ml and therefore a low antioxidant
activity.
123

Med Chem Res
Ph
Ph
N
N
N
H
O
R2
N
N
NH2
N
C
R3
N
N
R2
R1
R1
HN
R3
Sc (OTf)3
- Sc(OTf)3
Ph
Ph
Ph
N
Sc(OTf)3
N
N
N
Sc(OTf)3
N
N
N
Sc(OTf)3
R2
N
H
R1
N
N
N
R1
N
R1
C
N
R2
N+
N
R3
R3
R3
Scheme 3 Mechanism for the synthesis of imidazo[2,1-c][1,2,4]triazoles derivatives 4a–i
Table 3 Antimicrobial activities of 4a, 4b, 4f and 4g against fungi, foodborne and spoiling bacteria and the determination of the MIC, MBC and
MFC
Synthesized compounds
4a
4b
4f
4g
Genta
I Z
IZ
MIC (MBC)
IZ
MIC (MBC)
IZ
MIC (MBC)
IZ
MIC
MIC
Bacterial strains
Gram positive
B. cereus ATCC 14579
S. aureus ATCC 25923
Gram negative
10
0
0.312 (0.972)
–
29
20
0.078 (0.087)
0.312 (0.624)
18
09
0.624 (1.248)
0
0
–
–
20
25
0.001
0.002
1.248 ([5)
E. coli ATCC 25922
P. aeruginosa ATCC 27853
S. enteritidis (food isolate)
0
0
0
–
–
–
0
0
0
–
–
–
0
0
0
–
–
–
0
0
0
–
–
–
21
18
16
0.004
0.004
0.007
IZ
MIC (MFC)
IZ
MIC (MFC)
IZ
MIC (MFC)
IZ
Ampho B
IZ
MIC
Fungal strains
Aspergillus niger CTM 10099
Fusarium oxysporum CTM10402
Fusarium culmorum ISPAVE 21w
Rhizopus nigricans CTM 10150
0
09
12
30
18
0.625 (1.25)
0.312 (2.5)
0.156 (0.156)
0.625 (2.5)
0
0
0
0
0
15
0.002
0.008
0.004
0.016
0
0
12.5
14
0
13
14
5 ([5)
2.5 (5)
10
2.5 ([5)
12
Diameter of inhibition zones of various extracts including the diameter of the disk (6 mm)
MIC minimal inhibition concentration, MBC minimum bactericidal concentration, Genta Gentamicin was used as a standard antibiotic at 15 lg/
ml, Amph: Amphotericine B was used as antifungal standard at 20 lg/ml, MFC minimum fungicidal concentration
–: activity not detected
Conclusion
fused 5-amino-1-phenyl-1,2,4-triazoles in good to excellent
yields via MCRs. This methodology should prove useful for
the synthesis of libraries of such derivatives with improved
efficiency using high-throughput synthesis methods. The
In conclusion, We have developed a rapid and efficient
microwave-assisted method for the synthesis of a range of
123

Med Chem Res
Fig. 1 Plate showing antifungal
activity of 4a and 4b and 4f
against R. nigricans and F.
culmorum
Fig. 2 Scavenger effect of 4a,
4b, 4g and 4f fractions at
different concentrations on the
stable 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH).
Results are expressed as
percentage decrement of
absorbance at 517 nm with
respect to control. BHA and a-
tocopherol were used as a
standard. Each value represents
the mean SD of three
experiments
140
120
100
80
114
57.53
55
60
33.5
40
28.18
14.92
20
0
Compounds
imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrimidine derivatives
as potential antimicrobial agents. Eur J Med Chem 45:5848–5855
Bode ML, Gravestock D, Moleele SS, Van Der Westhuyzen CW,
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diversity offered by the process have been further enhanced
by the use of these compounds in a Groebke three-compo-
nent forming fused imidazo[2,1-c][1,2,4]triazoles 4a–i in
good yields. Derivatives were tested for antifungal, anti-
bacterial and antioxidant activity. The compound 4b showed
very good antifungal and antioxidant activity, which sug-
gests a possible clinical significance.
´
Acknowledgments The authors thank the Ecole Nationale Superi-
eure de Techniques Avancees (ENSTA) through a financial support.
´
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conventional interactions between isocyanides and iminium
species. Tetrahedron 65:2153–2171
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