Synthesis of some N-substituted nitroimidazole derivatives as potential antioxidant and antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2008.05.016

Some new nitroimidazole derivatives have been synthesized by treating 4,5-dinitro- and 2-methyl-4,5-dinitroimidazoles with epoxypropane, epichlorohydrin or phenacyl bromide in alkylation reactions. The nitro group in N-substituted 4,5-dinitro- and 2-methyl-4,5-dinitroimidazoles has been replaced with primary and secondary amines to afford 4-amino-5-nitroimidazole derivatives. Some of the compounds have been tested for their antioxidant and antifungal properties against fungi species acting on timber. Nearly all of them have shown significant antioxidant activity in comparison with that of tocopherol, which is used as a reference substance. Two compounds from those tested have revealed very strong fungistatic activity against Sclerophoma pityophila.

Full text of DOI:10.1016/j.ejmech.2008.05.016

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 645e652
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some N-substituted nitroimidazole derivatives
as potential antioxidant and antifungal agents
a
Dorota Olender , Justyna Zwawiak , Victor Lukianchuk , Roman Lesyk ,
a
b
c
_
Aleksandra Kropacz d, Andrzej Fojutowski d, Lucjusz Zaprutko a,
*
a
´
Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
b Department of Pharmacology, Lugansk State Medical University, 50 Years Defence Lugansk 1, 91045 Lugansk, Ukraine
c Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University,
Pekarska 69, 79010 Lviv-10, Ukraine
d
´
Department of Environmental Protection and Wood Conservation, Wood Technology Institute, Winiarska 1, 60-654 Poznan, Poland
Received 18 December 2007; received in revised form 17 May 2008; accepted 21 May 2008
Available online 28 May 2008
Abstract
Some new nitroimidazole derivatives have been synthesized by treating 4,5-dinitro- and 2-methyl-4,5-dinitroimidazoles with epoxypropane,
epichlorohydrin or phenacyl bromide in alkylation reactions. The nitro group in N-substituted 4,5-dinitro- and 2-methyl-4,5-dinitroimidazoles
has been replaced with primary and secondary amines to afford 4-amino-5-nitroimidazole derivatives. Some of the compounds have been tested
for their antioxidant and antifungal properties against fungi species acting on timber. Nearly all of them have shown significant antioxidant ac-
tivity in comparison with that of tocopherol, which is used as a reference substance. Two compounds from those tested have revealed very strong
fungistatic activity against Sclerophoma pityophila.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Nitroimidazole; Aminonitroimidazole; Nitro group substitution; Antioxidant activity; Antifungal activity
1. Introduction
structures (CGI-17341, PA-824) might be potential antituber-
cular agents [5e7]. Derivatives of 5-nitroimidazoles have
been tested in cell-based assays and in enzyme assays against
HIV-1 recombinant reverse transcriptase [8,9]. 2-Nitroimida-
zoles play a major role as bioreductive markers for tumour
hypoxia and as radiosensitizers [10e12]. Some of them dem-
onstrate antiprotozoan activity [4].
Our earlier investigations have provided evidence of the anti-
fungal and antibacterial properties of N-phenacyl-4,5-dinitroimi-
dazole derivatives [13]. Some dinitro- and mononitroimidazole
derivatives have been predicted as notable radiosensitizers, anti-
protozoal and antibacterial or antiepileptic agents [14].
Antifungal agents are regarded as efficient drugs with rela-
tively low toxicity. New, effective antifungal substances are
still being sought because of the increasing number of resistant
fungi being isolated.
For a few decades, nitroimidazoles have been the subject of
much interest because of their properties. Depending on the
nature of substituents and the position of the nitro group, the
nitroimidazole derivatives can show various pharmacological
activities [1]. The compounds with nitro group at position 4
are usually less active than the corresponding 5-nitro deriva-
tives. Nitroimidazoles, such as metronidazole, misonidazole,
ornidazole, secnidazole, etanidazole and tinidazole, are com-
monly used as therapeutic agents against a variety of proto-
zoan and bacterial infections of humans and animals [2e4].
It has been suggested by many authors that nitroimidazoles
containing nitroimidazooxazole or nitroimidazooxazine
Antioxidants play a significant role in several important bi-
ological processes such as immunity, protection against tissue
* Corresponding author. Tel.: þ48 61 8546 670; fax: þ48 61 8546 680.
E-mail address: zaprutko@ump.edu.pl (L. Zaprutko).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.016

646
D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
damage, reproduction and growth or development. They pre-
serve adequate function of cells against homeostatic distur-
bances such as those caused by septic shock, aging and, in
general, processes involving oxidative stress. These substances
are classified according to their mode of action. Important an-
tioxidants include the chain-breaking or scavenging sub-
stances (vitamins E, C and A, bilirubin), preventative
(albumin, lactoferrin, haptoblobin) and enzyme antioxidants
(catalase and glutathione peroxidase) [15]. They reduce dam-
age to cells and biochemicals caused by free radicals, which
are normal products of metabolism. Antioxidants can prevent
cardiovascular disease, cancer, cataracts and various other ail-
ments associated with aging [16,17]. The studies suggest that
supplementation with antioxidants may be useful in the pre-
vention and treatment of Parkinson’s disease [18,19]. Oxida-
tive stress is also important in the pathogenesis of
Alzheimer’s disease. The studies suggest that supplementation
with vitamin E might delay the development of Alzheimer’s
disease [20,21].
The broad spectrum of biological activity of nitro com-
pounds was the inspiration to test them for antioxidant and an-
tifungal activities. The results obtained in this study can be
useful for the design and synthesis of new substances with an-
tioxidant and antifungal activities.
The aim of this study was to determine in vitro the antiox-
idant properties of some N-substituted 4,5-dinitroimidazoles
and their 2-methyl derivatives as well as respective com-
pounds with primary or secondary amino group in C-4 posi-
tion of the nitroimidazole ring. In addition some derivatives
were tested for their antifungal activity against fungi species
attacking timber. Wood can be protected from the attack of
fungi decay by applying suitable chemical preservatives. The
antioxidative activity experiments were carried out in compar-
ison with tocopheryl acetate.
with propylene oxide or with epichlorohydrin without solvent
or in alcoholic solutions (Scheme 1). The reagents were heated
under reflux for about 3 h. The treatment of 2 with an excess of
epoxypropane (1:2) led to new N-(2-hydroxypropyl) derivative
7. While reacting 1 with epoxypropane in ethanol or without
solvent, formation of respective hydroxypropyl derivative of
4,5-dinitroimidazole was not observed. Dinitroimidazoles 1
and 2 alkylated with epichlorohydrin formed compounds 8
and 9, respectively [13]. If a methyl group at C-2 position of
dinitroimidazole ring was present, the solvent was not necessary
but formation of 8 in the reaction without solvent was not
observed. Thus, the presence of a methyl group at C-2 position
of the imidazole ring significantly influenced N-alkylation and
other substitution reactions. Subsequently, derivatives 7e9
were transformed into new N-(2-oxopropyl)-4,5-dinitroimida-
zoles 10e12 (Scheme 1). The process was carried out using
Jones reagent in the oxidation reaction, at room temperature
and by using acetone as a solvent. From this reaction mixture
the isolated and purified derivatives of ketones were readily
1
obtained. H NMR spectra of 10e12 showed only methyl and
methylenic protons assigned to the side chains or proton at C-
2 position of the imidazole ring, but no signals of the hydroxyl
groups. IR spectra of the compounds prepared showed strong
bands in the region 1725e1720 cmꢀ1 attributed to the carbonyl
group.
Derivatives 3e11 were applied to obtain some new prod-
ucts in the reaction with primary and secondary amines such
as o-toluidine, morpholine, pyrrolidine, piperidine and N-
methylpiperazine. These reactions were carried out in THF,
at room temperature [23]. The treatment of 3e6 with the
above-mentioned amines, even at molar ratio 1:5 led to the re-
spective 4-amino-5-nitroimidazole derivatives 13e29 only
(Scheme 2). Formation of aminonitro compounds occurs
very easily and often with high yield. The other solvents (eth-
anol, acetonitrile, methylene chloride) were unsuitable for the
reaction. Under these conditions, the amino products were
formed in longer time and often with poor yields and purity.
With ethanol being used as a solvent, the reaction led to a mix-
ture of both possible isomers: 4-amino-5-nitro- and 5-amino-
4-nitroimidazoles. Moreover, elevated temperatures favour
the formation of by-products. Derivatives 13e17, 20, 22e
24, 27 and 29 are newly synthesized substances but products
18, 19, 21, 25, 26 and 28 have been described earlier [23].
IR spectra of some newly synthesized aminonitroderivatives
2. Results and discussion
2.1. Chemistry
As starting materials 4,5-dinitroimidazole 1 and 2-methyl-
4,5-dinitroimidazole 2 were used [22]. 4,5-Dinitro-1-phenacyli-
midazole derivatives 3e6 of above-mentioned dinitroimidazoles
are known substances [13]. N-Hydroxypropyl derivatives 7e9
were synthesized by the reaction of dinitroimidazoles 1 or 2
O2N
O2N
O2N
N
N
N
ii
i
R1
CH2Y
O2N
R1
O2N
R1
N
N
O2N
N
H
H2C
C
O
H2C CH CH2Y
OH
10-12
7- 9
1, 2
R1= H, 1
R1= CH3
R1= CH3, Y= H 7
R1= H, Y= Cl 8
R1= CH3, Y= Cl 9
R1= CH3, Y= H 10
R1= H, Y= Cl 11
R1= CH3, Y= Cl 12
2
Scheme 1. Synthesis of N-substituted 4,5-dinitroimidazole derivatives. Reagents and conditions: (i) epoxypropane or epichlorohydrin, without solvent or in EtOH,
boiling, 2.5e3.5 h; (ii) Jones reagent, acetone, r.t.

D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
647
R2
O2N
R1
H
H
H
H
R2
X
H
H
H
H
N
N
morpholine
pyrrolidine
o-tolyl.
piperidine
N-methyl-
piperazine
13
14
15
16
O2N
R1
O
O2N
R1
N
N
i
O
H
H
17
CH3 morpholine
CH3 pyrrolidine
H
H
H
H
18
19
20
21
CH3
o-tolyl.
CH3 piperidine
X
X
N-methyl-
piperazine
CH3
H
22
23
3-6
13-29
R1=H, X=H 3
H
H
morpholine Cl
o-tolyl.
Cl 24
R1=CH3, X=H 4
R1=H, X=Cl 5
R1=CH3, X=Cl 6
CH3 morpholine Cl 25
CH3 pyrrolidine Cl
26
27
CH3
o-tolyl.
Cl
CH3 piperidine Cl 28
N-methyl-
piperazine
CH3
Cl
29
R2
O2N
N
N
i
O2N
O2N
N
N
H2C CH CH2Cl
H2C CH CH2Cl
OH
OH
8
30, 31
R2= morpholine 30
R2=N-methylpiperazine 31
R2
O2N
N
N
i
O2N
CH3
CH3
O2N
CH3
N
N
H2C
C
O
H2C
C
O
CH3
32, 33
10
R2=aniline 32
R2=morpholine 33
Scheme 2. Synthesis of N-substituted 4-amino-5-nitroimidazole derivatives. Reagents and conditions: (i) amine, THF, r.t., 4e24 h.
1
(15, 20, 24, and 27) show characteristic absorption bands at
about 3300e3240 cmꢀ1 and near 1600 cmꢀ1. These bands
correspond to the NeH groups. Besides, the signal near
1700 cmꢀ1 corresponds to a carbonyl group. The absorption
bands of nitro groups were observed at ranges 1560e
1500 cmꢀ1 and 1360e1305 cmꢀ1. The corresponding very
strong molecular ions [Mþ] were detected in the mass spectra
of all synthesized compounds. The fragmentation way ex-
hibited a loss of a nitro group and formation of the ion
[Mþ ꢀ 46] in all aminonitroderivatives. Besides the fragmen-
tation ions, two strong fragments (m/z 100%) were observed,
the first one corresponding to the ion at m/z 105 originating
from the phenacyl group (13e22) and the second one corre-
sponding to its p-chloro substituted derivatives (m/z 139;
23e29). 1H NMR spectra of new compounds are also in agree-
ment with their molecular formula, for instance the spectra of
the derivatives prepared from o-toluidine (15, 20, 24 and 27)
show a singlet in the range 9.27e9.56 ppm, assigned to the
proton attached to the amine nitrogen atom. The compounds
obtained in this work are 4-amino-5-nitroimidazoles. A
comparison of the H NMR spectra of 5-amino-4-nitroimida-
zoles reported in literature [24,25] with the spectra of the com-
pounds studied reveals significant differences allowing the
empirical differentiation of the isomeric compounds. In the
1H NMR spectra of 4-amino-5-nitro derivatives the signals
of the CH2 protons are shifted towards lower values and are
in the range of 5.65e5.80 ppm. This suggests that the amino
groups in compounds 13e29 are present in the C-4 position
of the heterocyclic ring. The signals of other H atoms are sim-
ilar and could not be related with structure, but were observed
in the expected regions. Finally, the structure of 4-amino-5-ni-
troimidazoles was confirmed by X-ray analysis [23]. Substitu-
tion mechanism of only one nitro group among two or three,
e.g. for o-dinitrobenzene derivatives [26] and 2,4,6-trinitro-
benzonitrile [27] was described earlier. The reason for C-4 ni-
tro group substitution in N-phenacyl-4,5-dinitroimidazoles is
non-equivalence of these nitro groups. The considerable re-
duction of C-5eNO2 distance is observed with respect to the
C-4eNO2 bond length. This indicates weak conjugation be-
tween the C-5 nitro group and the imidazole ring. Moreover,

648
D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
the C-4 nitro group is significantly deflected and forms a dihe-
dral angle of about 46ꢁ with the plane of the imidazole ring
[28]. This deflection also favoured C-4 nitro group towards
the nucleophilic substitution with amines.
acid reactive substances (TBARS) assay [29]. The lipid perox-
idation (LPO) inhibitory activity of each compound was calcu-
lated from the appropriate equation under Section 3.2. and the
results were expressed as % of antioxidant activity (AOA) rel-
ative to the control blank probe (Table 2). Tocopheryl acetate
was used as a reference substance.
As shown in Table 2, compounds 5, 6, 8, 11, 13, 14, 21 and
30 show the strongest antioxidant properties at closing time of
the experimental model used. They are more effective than the
reference substances. It is also noted that three compounds:
15, 20 and 24 with o-toluidine substituent showed negative
or very low values of AOA. These compounds can act as
strong oxidant agents under these conditions. The most active
were the derivatives bearing chlorine atom in 2-hydroxypropyl
and 2-oxopropyl chains or phenacyl group at N-1 position of
the imidazole ring.
Under the conditions used for preparing N-phenacyl deriv-
atives of 4-amino-5-nitroimidazoles, some N-(2-hydroxy-
propyl) and N-(2-oxopropyl) derivatives were also subjected
to reactions with amines. Similar products were formed on re-
acting respective 1-(3-chloro-2-hydroxypropyl)-4,5-dinitroi-
midazole (8) with morpholine and N-methylpiperazine
(30,31) or 2-methyl-4,5-dinitro-1-(2-oxopropyl)-imidazole
(10) with aniline and morpholine (32,33). The molar ratio of
dinitroimidazole and amine (1:2.2e1:5) and the type of
amines used had essential influence on the structure and quan-
tity of the product obtained. The reaction of dinitroimidazoles
with amine in a molar ratio of 1:2.2 led to a mixture of prod-
ucts in the form of oils. It is worth noting that the size and na-
ture of the N-1 substituent determined the susceptibility of the
imidazole nitro group in the substitution reactions with
amines. N-(3-Chloro-2-hydroxypropyl) and N-(2-oxopropyl)
derivatives in the reaction with amines gave mixtures of iso-
meric substitution products with prevalence of the respective
4-amino-5-nitro-isomers or sometimes diaminoimidazoles
[13]. These reactions proceeded more readily and unidirec-
tionally when the molar ratio of dinitroimidazole and amine
was 1:5. The reaction gave the desired product in good yield
in each case.
2.3. Antifungal activity
Compounds 5, 6, 13, 21, 26 and 28 were tested for their an-
tifungal activity using the standard nutrient method against
Sclerophoma pityophila. This fungus species is one of the
most destructive basidiomycetes in coniferous forests and it
is the main cause of needle disease in pine thickets. The results
of the antifungal activity are given in Table 3 and are ex-
pressed as the ED50 (substance concentrations retarding the
fungal growth rate by 50% in comparison with plates where
the agent studied was absent), the effective dose ED100 (sub-
stance concentrations retarding the fungal growth rate by
The general synthetic pathways are given in Schemes 1 and 2.
Some experimental data for new compounds are tabulated
in Table 1.
Table 2
Antioxidant activity of nitroimidazole derivatives
2.2. Antioxidant activity
Compound
AOA (%)
18.9 ꢂ 1.8
All compounds were tested for their in vitro antioxidant ac-
tivity using the method of non-enzymatic initiation of lipid
peroxidation by Fe2þ ions and determination of thiobarbituric
Tocopheryl acetate
3
17.8 ꢂ 1.8
39.8 ꢂ 1.5
35.3 ꢂ 1.5
60.0 ꢂ 1.6
57.6 ꢂ 1.8
79.9 ꢂ 1.0
25.8 ꢂ 0.7
76.2 ꢂ 0.7
67.7 ꢂ 1.5
44.2 ꢂ 1.3
7.4 ꢂ 1.5
18.9 ꢂ 1.8
34.5 ꢂ 1.9
33.8 ꢂ 2.3
67.8 ꢂ 0.9
71.7 ꢂ 1.2
78.6 ꢂ 1.1
25.4 ꢂ 1.2
83.1 ꢂ 0.9
63.8 ꢂ 2.0
80.1 ꢂ 1.1
1.2 ꢂ 2.8
40.6 ꢂ 1.3
26.4 ꢂ 1.4
64.1 ꢂ 0.8
60.2 ꢂ 0.8
56.5 ꢂ 1.6
13.1 ꢂ 1.2
63.7 ꢂ 1.6
56.3 ꢂ 1.6
61.8 ꢂ 2.9
2.9 ꢂ 2.6
4
5
6
8
Table 1
Yields and basic physical properties of nitroimidazole derivatives
10
11
13
14
15
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Compound
Yield (%)
m.p. (ꢁC)
Rf
Crystals
7
71
51
74
82
42
71
67
45
77
27
88
35
80
82
78
54
65
44
153e155
90e93
0.60
0.60
0.30
0.51
0.60
0.77
0.65
0.26
0.83
0.30
0.53
0.77
0.80
0.30
0.75
0.21
0.57
0.46
Light yellow needles
Light yellow needles
Yellow crystals
Light yellow needles
Yellow needles
Yellow needles
Yellow needles
Yellow needles
Orange needles
Light yellow needles
Yellow needles
Yellow needles
Orange plates
10
11
13
14
15
16
17
20
22
23
24
27
29
30
31
32
33
158e160
192e194
166e167
204e207
135e138
126e129
165e168
155e157
192e194
242e244
215e218
178e180
150e151
161e163
193e195
124e126
45.6 ꢂ 2.9
45.2 ꢂ 0.7
ꢀ354.8 ꢂ 11.9
48.6 ꢂ 2.8
13.1 ꢂ 1.2
37.3 ꢂ 2.3
ꢀ1.5 ꢂ 6.6
35.9 ꢂ 2.1
20.8 ꢂ 2.5
12.2 ꢂ 1.9
39.9 ꢂ 2.3
37.1 ꢂ 1.8
51.6 ꢂ 1.8
39.1 ꢂ 3.9
17.5 ꢂ 2.7
39.9 ꢂ 4.8
39.9 ꢂ 1.8
54.1 ꢂ 1.1
ꢀ14.8 ꢂ 2.9
65.9 ꢂ 1.3
17.7 ꢂ 1.9
21.0 ꢂ 2.1
ꢀ31.9 ꢂ 4.0
43.0 ꢂ 0.8
39.0 ꢂ 2.1
11.8 ꢂ 1.9
36.9 ꢂ 0.8
47.0 ꢂ 0.9
68.8 ꢂ 1.0
62.7 ꢂ 1.3
15.1 ꢂ 1.2
23.2 ꢂ 1.5
50.9 ꢂ 1.4
61.9 ꢂ 2.0
7.3 ꢂ 3.2
66.5 ꢂ 1.5
13.0 ꢂ 2.3
25.3 ꢂ 1.4
ꢀ35.3 ꢂ 3.7
30.1 ꢂ 2.1
37.1 ꢂ 2.3
13.0 ꢂ 1.3
41.6 ꢂ 1.4
53.2 ꢂ 1.5
69.7 ꢂ 2.7
60.9 ꢂ 1.8
27.4 ꢂ 2.9
23.1 ꢂ 1.3
Yellow needles
Yellow needles
Yellow needles
Yellow crystals
Yellow needles

D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
649
Table 3
Antifungal activity of nitroimidazole derivatives
stirred. The precipitate was filtered off, air-dried and crystal-
lized from 40% ethanol, yielding 1.63 g of 7 (71%).
IR: 3305 (n OH); 1525, 1340 (n CeNO2); 1115 (n CeOH).
MS: m/z (rel. int.%) ¼ 230 (3.5) Mþ, 215 (2.3), 183 (100.0),
166 (3.1), 156 (40.6), 111 (39.3), 82 (33.6). 1H NMR
(DMSO-d6): d ¼ 5.25 (s, 1H, OH), 4.30 and 4.05 (two m,
2H, CH2), 3.90 (m, 1H, CH), 2.49 (s, 3H, CH3eIm), 1.13
(d, J ¼ 6.0, 3H, CH3-chain).
Compound
ED50
ED100
LD
5
1000e2500
500e750
<25
>5000
>5000
<25
>5000
>5000
>5000
>5000
>5000
>5000
6
13
21
26
28
>5000
>5000
<25
>5000
>5000
<25
3.1.2. General procedure for the preparation
of 1-(2-oxopropyl)-derivatives of 4,5-dinitroimidazole
100% in comparison with plates where the agent studied was
absent), and LD values (concentrations causing death of inoc-
ulum) of the compounds examined.
To a solution of respective N-hydroxypropyl derivative 7, 8
or 9 (10 mmol) in acetone (100 mL), oxidising Jones reagent
(10 mL) was dropped at room temperature. After 24 h isopro-
panol (10 mL) was added. The dark green precipitate was fil-
tered and washed with a small volume of acetone. The
combined filtrates were poured into water (250 mL) and the
solution was held for 2e3 days to afford the crystalline solid.
The crude product was filtered, washed with water, air-dried
and crystallized from methanol to yield 10, 11 or 12.
As shown in Table 3, all compounds are weakly active
against the fungus used. On the other hand, derivatives 13
and 28 showed high fungistatic activity (EDs < 25) against
S. pityophila. High effectiveness was induced by the displace-
ment of nitro group at 4-position on the imidazole ring to mor-
pholine or piperidine and by the presence of a chlorine atom at
4-position on the phenyl ring. The results tabulated in Table 3
show that all compounds caused the death of the fungi studied
with a lethal dose (LD) up to 5000 ppm.
3.1.2.1. 2-Methyl-4,5-dinitro-1-(2-oxopropyl)-imidazole (10). IR:
1725 (n C]O); 1525, 1340 (n CeNO2). MS: m/z (rel.
int.%) ¼ 228 (1.1) Mþ, 182 (2.0), 156 (9.2), 93 (2.8), 43
3. Experimental
1
(100.0). H NMR (DMSO-d6): d ¼ 5.39 (s, 2H, NeCH2), 2.41
(s, 3H, CH3eIm), 2.30 (s, 3H, COCH3).
3.1. Chemistry
3.1.2.2. 1-(3-Chloro-2-oxopropyl)-4,5-dinitroimidazole (11). IR:
1720 (n C]O); 1500, 1305 (n CeNO2). MS: m/z (rel.
int.%) ¼ 250 (1.8, Mþ þ 2), 248 (4.1) Mþ, 202 (5.9), 172
Melting points were determined on a Boetius apparatus and
were uncorrected. 1H NMR spectra were recorded on a Varian
Gemini 300 VT spectrometer (300 MHz). Chemical shifts (d)
are expressed in parts per million (ppm) relative to tetrame-
thylsilane (TMS) as an internal standard, using CDCl3,
DMSO-d6 and CD3OD as solvents. Coupling constants (J
values) are expressed in Hertz (Hz). Splitting patterns are des-
ignated as follows: s, singlet; d, doublet; t, triplet; m, multi-
plet. MS spectra were recorded on a 402 AMD INTECTRA
apparatus by the electron impact technique, operating at
75 eV. Infrared (IR) spectra were recorded in KBr tablets using
a Specord 75-IR spectrophotometer and were expressed in
cmꢀ1 scale. The progress of reactions and purity of products
were controlled by thin-layer chromatography (TLC) method
on silica gel plates (60 F254 from Merck) in a CHCl3/MeOH
(9:1, v/v) solvent system at room temperature. The spots on
the plates were visualized using UV lamp (l ¼ 254 nm). Solid
products were purified by the crystallization process. Analyti-
cal data of C, H, N assays for new compounds were within less
than ꢂ 0.3% of the theoretical values and are in good agree-
ment with the proposed structures. From among substances,
which were used as substrates, the epichlorohydrin, the epox-
ypropane and the amines were commercial products.
1
(20.6), 142 (43.1), 77 (100.0). H NMR (DMSO-d6): d ¼ 8.10
(s, 1H, 2-Im), 5.56 (s, 2H, NeCH2), 4.78 (s, 2H, CH2Cl).
3.1.2.3. 1-(3-Chloro-2-oxopropyl)-2-methyl-4,5-dinitroimida-
zole (12). IR: 1720 (n C]O); 1500, 1320 (n CeNO2). MS:
m/z (rel. int.%) ¼ 264 (15.2, Mþ þ 2), 262 (40.9) Mþ, 227
(8.3), 217 (27.5), 186 (18.3), 156 (89.5), 93 (50.3), 77
(100.0). 1H NMR (DMSO-d6): d ¼ 5.49 (s, 2H, NeCH2),
4.78 (s, 2H, CH2Cl), 2.43 (s, 3H, CH3eIm).
3.1.3. General procedure for the preparation
of aminonitroderivatives (13e33)
To a solution of appropriate 4,5-dinitroimidazole or 2-
methyl-4,5-dinitroimidazole derivatives 3e6,
8
or 10
(1 mmol) in THF (5 mL), a respective amine: secondary (mor-
pholine, pyrrolidine, piperidine, or N-methylpiperazine) or pri-
mary (aniline, o-toluidine) (5 mmol) was added slowly and
dropwise at room temperature. After left to rest for 4e24 h,
the precipitate was filtered off, washed with a small quantity
of cold THF and air-dried. The crude solid was crystallized
from a mixture of methanol:water (9:1) to yield 13e33.
3.1.1. 1-(2-Hydroxypropyl)-2-methyl-4,5-
dinitroimidazole (7)
A mixture of 2 (1.72 g, 10 mmol) and epoxypropane
(1.40 mL, 1.16 g, 20 mmol) was heated under reflux for about
3 h. The mixture was poured into ice water (60 mL) and
3.1.3.1. 4-(Morpholin-4-yl)-5-nitro-1-phenacylimidazole
(13). IR: 1680 (n C]O); 1540, 1320 (n CeNO2). MS: m/z
(rel. int.%) ¼ 316 (30.2) Mþ, 299 (34.1), 271 (62.0), 253
1
(44.3), 168 (7.1), 105 (100.0), 77 (52.5). H NMR (CDCl3):
d ¼ 7.98 (m, 2H, 2,6-Ph), 7.67 (m, 1H, 4-Ph), 7.53 (m, 2H,

650
D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
3,5-Ph), 7.37 (s, 1H, 2-Im), 5.70 (s, 2H, NeCH2), 3.85 (m, 4H,
2 ꢃ CH2, 3,5-morpholine), 3.62 (m, 4H, 2 ꢃ CH2, 2,6-
morpholine).
J ¼ 4.9, 4H, 2 ꢃ CH2, 2,6-piperazine), 2.58 (t, J ¼ 4.9, 4H,
2 ꢃ CH2, 3,5-piperazine), 2.35 (s, 3H, pNeCH3), 2.32 (s,
3H, CH3eIm).
3.1.3.2. 5-Nitro-1-phenacyl-4-(pyrrolidin-1-yl)-imidazole (14). IR:
1680 (n C]O); 1550, 1340 (n CeNO2). MS: m/z (rel.
int.%) ¼ 300 (24.8) Mþ, 283 (68.5), 255 (19.0), 224 (12.4),
197 (10.9), 146 (12.9), 105 (100.0). 1H NMR (CDCl3):
d ¼ 7.98 (m, 2H, 2,6-Ph), 7.64 (m, 1H, 4-Ph), 7.51 (m, 2H,
3,5-Ph), 7.32 (s, 1H, 2-Im), 5.67 (s, 2H, NeCH2), 3.68 (t,
J ¼ 6.6, 4H, 2 ꢃ CH2, 2,5-pyrrolidine), 1.98 (m, 2 ꢃ CH2,
3,4-pyrrolidine).
3.1.3.8. 1-(4-Chlorophenacyl)-4-(morpholin-4-yl)-5-nitroimi-
dazole (23). IR: 1670 (n C]O); 1520, 1320 (n CeNO2).
MS: m/z (rel. int.%) ¼ 352 (12.1, Mþ þ 2), 350 (28.5) Mþ,
333 (25.6), 305 (51.1), 287 (34.1), 168 (11.2), 139 (100.0).
1H NMR (CDCl3): d ¼ 7.95 (m, 2H, 2,6-Ph), 7.52 (m, 2H,
3,5-Ph), 7.36 (s, 1H, 2-Im), 5.65 (s, 2H, NeCH2), 3.86 (m,
4H, 2 ꢃ CH2, 3,5-morpholine), 3.63 (m, 4H, 2 ꢃ CH2, 2,6-
morpholine).
3.1.3.3. 5-Nitro-1-phenacyl-4-(o-tolylamino)-imidazole (15). IR:
3275 (n NeH); 1685 (n C]O); 1600 (s NeH); 1520, 1340
(n CeNO2). MS: m/z (rel. int.%) ¼ 336 (63.7) Mþ, 305 (8.9),
3.1.3.9. 1-(4-Chlorophenacyl)-5-nitro-4-(o-tolylamino)-imidaz-
ole (24). IR: 3240 (n NeH); 1670 (n C]O); 1600 (s NeH);
1500, 1340 (n CeNO2). MS: m/z (rel. int.%) ¼ 372 (38.4,
Mþ þ 2), 370 (91.4) Mþ, 324 (28.5), 296 (5.5), 185 (15.2),
1
290 (17.1), 283 (68.5), 170 (8.9), 157 (12.3), 105 (100.0). H
1
NMR (CDCl3): d ¼ 9.28 (s, 1H, NH), 8.27 (d, J ¼ 7.9, 1H, 6-
tolyl), 8.01 (m, 2H, 2,6-Ph), 7.68 (m, 1H, 4-Ph), 7.55 (m,
2H, 3,5-Ph), 7.47 (s, 1H, 2-Im), 7.28 (m, 2H, 4,5-tolyl),
7.09 (m, 1H, 3-tolyl), 5.77 (s, 2H, NeCH2), 2.40 (s, 3H,
CH3etolyl).
158 (19.0), 139 (100.0). H NMR (CDCl3): d ¼ 9.27 (s, 1H,
NH), 8.27 (d, J ¼ 7.7, 1H, 6-tolyl), 7.96 (m, 2H, 2,6-Ph),
7.53 (m, 2H, 3,5-Ph), 7.47 (s, 1H, 2-Im), 7.28 (m, 2H, 4,5-
tolyl), 7.07 (m, 1H, 3-tolyl), 5.72 (s, 2H, NeCH2), 2.40 (s,
3H, CH3etolyl).
3.1.3.4. 5-Nitro-1-phenacyl-4-(piperidin-1-yl)-imidazole (16). IR:
1680 (n C]O); 1550, 1320 (n CeNO2). MS: m/z (rel.
int.%) ¼ 314 (20.6) Mþ, 297 (53.7), 269 (24.1), 238 (10.1),
183 (9.1), 161 (40.0), 105 (100.0). 1H NMR (CDCl3):
d ¼ 8.01 (m, 2H, 2,6-Ph), 7.64 (m, 1H, 4-Ph), 7.53 (m, 2H,
3,5-Ph), 7.35 (s, 1H, 2-Im), 5.69 (s, 2H, NeCH2), 3.56 (m,
4H, 2 ꢃ CH2, 2,6-piperidine), 1.73 (m, 6H, 3 ꢃ CH2, 3,4,5-
piperidine).
3.1.3.10. 1-(4-Chlorophenacyl)-2-methyl-5-nitro-4-(o-tolyla-
mino)-imidazole (27). IR: 3280 (n NeH); 1690 (n C]O);
1620 (s NeH); 1520, 1340 (n CeNO2). MS: m/z (rel.
int.%) ¼ 386 (38.0, Mþ þ 2), 384 (83.2) Mþ, 338 (34.4),
297 (13.6), 269 (8.2), 199 (13.2), 158 (20.2), 139 (100.0).
1H NMR (CDCl3): d ¼ 9.55 (s, 1H, NH), 8.36 (d, J ¼ 7.4,
1H, 6-tolyl), 7.97 (m, 2H, 2,6-Ph), 7.53 (m, 2H, 3,5-Ph),
7.27 (m, 2H, 4,5-tolyl), 7.06 (m, 1H, 3-tolyl), 5.75 (s, 2H,
NeCH2), 2.41 (s, 3H, CH3etolyl), 2.39 (s, 3H, CH3eIm).
3.1.3.5. 4-(4-Methylpiperazin-1-yl)-5-nitro-1-phenacylimidazole
(17). IR: 1690 (n C]O); 1520, 1320 (n CeNO2). MS: m/z
(rel. int.%) ¼ 329 (15.9) Mþ, 312 (36.6), 284 (22.8), 269
(7.4), 180 (11.7), 162 (29.5), 105 (100.0). 1H NMR
(CDCl3): d ¼ 7.98 (m, 2H, 2,6-Ph), 7.66 (m, 1H, 4-Ph), 7.51
(m, 2H, 3,5-Ph), 7.37 (s, 1H, 2-Im), 5.70 (s, 2H, NeCH2),
3.67 (t, J ¼ 4.9, 4H, 2 ꢃ CH2, 2,6-piperazine), 2.63 (t,
J ¼ 4.9, 4H, 2 ꢃ CH2, 3,5-piperazine), 2.38 (s, 3H, pNeCH3).
3.1.3.11. 1-(4-Chlorophenacyl)-2-methyl-4-(4-methylpipera-
zin-1-yl)-5-nitroimidazole (29). IR: 1680 (n C]O); 1520,
1320 (n CeNO2). MS: m/z (rel. int.%) ¼ 379 (7.9, Mþ þ 2),
377 (18.6) Mþ, 360 (78.4), 332 (47.8), 307 (15.8), 289
1
(15.3), 194 (40.2), 139 (100.0). H NMR (CDCl3): d ¼ 7.94
(m, 2H, 2,6-Ph), 7.49 (m, 2H, 3,5-Ph), 5.65 (s, 2H, Ne
CH2), 3.66 (t, J ¼ 4.9, 4H, 2 ꢃ CH2, 2,6-piperazine), 2.57 (t,
J ¼ 4.9, 4H, 2 ꢃ CH2, 3,5-piperazine), 2.35 (s, 3H, pNe
CH3), 2.32 (s, 3H, CH3eIm).
3.1.3.6. 2-Methyl-5-nitro-1-phenacyl-4-(o-tolylamino)-imidaz-
ole (20). IR: 3300 (n NeH); 1700 (n C]O); 1620 (s Ne
H); 1540, 1360 (n CeNO2). MS: m/z (rel. int.%) ¼ 350
(100.0) Mþ, 333 (4.4), 319 (11.4), 304 (35.3), 263 (13.1),
3.1.3.12. 1-(3-Chloro-2-hydroxypropyl)-4-(morpholin-4-yl)-5-
nitroimidazole (30). IR: 3400e3500 (n OeH); 1520, 1340 (n
CeNO2). MS: m/z (rel. int.%) ¼ 292 (36.3, Mþ þ 2), 290
(87.4) Mþ, 273 (80.9), 245 (82.7), 227 (100.0), 208 (24.4),
1
157 (22.0), 105 (78.4). H NMR (CDCl3): d ¼ 9.56 (s, 1H,
NH), 8.37 (d, J ¼ 7.4, 1H, 6-tolyl), 8.02 (m, 2H, 2,6-Ph),
7.66 (m, 1H, 4-Ph), 7.54 (m, 2H, 3,5-Ph), 7.27 (m, 2H, 4,5-
tolyl), 7.05 (m, 1H, 3-tolyl), 5.80 (s, 2H, NeCH2), 2.40 (s,
3H, CH3etolyl), 2.39 (s, 3H, CH3eIm).
1
154 (24.9), 113 (49.2). H NMR (CDCl3): d ¼ 7.83 (s, 1H,
2-Im), 5.63 (m, 1H, OH), 4.58 (m, 1H, CH), 4.03 (m, 2H,
CH2Cl), 3.54 (m, 10H, 4 ꢃ CH2 morpholine and NeCH2).
3.1.3.7. 2-Methyl-4-(4-methylpiperazin-1-yl)-5-nitro-1-phena-
cylimidazole (22). IR: 1700 (n C]O); 1520, 1340 (n Ce
NO2). MS: m/z (rel. int.%) ¼ 343 (12.8) Mþ, 326 (52.5), 298
3.1.3.13. 1-(3-Chloro-2-hydroxypropyl)-4-(4-methylpiperazin-
1-yl)-5-nitroimidazole (31). IR: 3400e3500 (n OeH); 1520,
1340 (n CeNO2). MS: m/z (rel. int.%) ¼ 305 (10.1,
Mþ þ 2), 303 (24.1) Mþ, 286 (49.8), 258 (34.4), 221 (28.7),
180 (11.5), 163 (21.2), 137 (27.7), 70 (100.0). 1H NMR
(CDCl3): d ¼ 7.79 (s, 1H, 2-Im), 5.65 (m, 1H, OH), 4.58 (m,
1
(26.8), 273 (12.6), 228 (10.4), 160 (28.4), 105 (100.0). H
NMR (CDCl3): d ¼ 8.01 (m, 2H, 2,6-Ph), 7.66 (m, 1H, 4-
Ph), 7.53 (m, 2H, 3,5-Ph), 5.73 (s, 2H, NeCH2), 3.67 (t,

D. Olender et al. / European Journal of Medicinal Chemistry 44 (2009) 645e652
651
1H, CH), 4.02 (m, 2H, CH2Cl), 3.62 (m, 2H, NeCH2), 3.47 (t,
J ¼ 5.6, 4H, 2 ꢃ CH2, 2,6-piperazine), 2.42 (t, J ¼ 4.8, 4H,
2 ꢃ CH2, 3,5-piperazine), 2.21 (s, 3H, NeCH3).
organic phase was measured at 532 nm. The antioxidant activ-
ities (AOA) (in %) were determined using the formula:
CK ꢀ CC
AOA ¼
ꢃ 100
3.1.3.14. 2-Methyl-5-nitro-4-1-(2-oxopropyl)-phenylaminoimi-
dazole (32). IR: 3275 (n NeH); 1720 (n C]O); 1615 (s
NeH); 1520, 1325 (n CeNO2). MS: m/z (rel. int.%) ¼ 274
(100.0) Mþ, 242 (2.0), 228 (7.2), 187 (34.1), 145 (43.2), 104
CK
where:
1
(50.9), 77 (73.9). H NMR (CDCl3): d ¼ 9.43 (s, 1H, NH),
CK e concentration of thiobarbituric acid reactants
(TBARS) in control assay, nmol/mL;
CC e concentration of thiobarbituric acid reactants
(TBARS) in assay with investigated compound, nmol/mL.
7.71 (m, 2H, 2,6-Ph), 7.37 (m, 3H, 3,4,5-Ph), 5.12 (s, 2H,
NeCH2), 2.36 (s, 3H, CH3eIm), 2.30 (s, 3H, COCH3).
3.1.3.15. 2-Methyl-4-(morpholin-4-yl)-5-nitro-1-(2-oxopropyl)-
imidazole (33). IR: 1720 (n C]O); 1560, 1340 (n CeNO2).
MS: m/z (rel. int.%) ¼ 268 (84.2) Mþ, 251 (89.8), 223
(100.0), 205 (86.9), 193 (24.0), 182 (18.1), 152 (19.5), 123
Each determination was repeated three times; the results
obtained were subjected to statistical analysis by the stu-
dent’s-t test.
1
(25.1), 98 (91.1). H NMR (CD3OD): d ¼ 5.20 (s, 2H, Ne
CH2), 3.81 (m, 4H, 2 ꢃ CH2, 3,5-morpholine), 3.54 (m, 4H,
2 ꢃ CH2, 2,6-morpholine), 2.30 (s, 3H, CH3eIm), 2.27 (s,
3H, COCH3).
3.3. Antifungal assay
In the study performed, the fungal species S. pityophila,
which is highly aggressive to timber, was used. The strain S.
pityophila (Corda) v. Hohn, S 231 originated from the collec-
¨
tion of Wood Technology Institute, Poznan, Poland.
3.2. In vitro antioxidant assay
´
The fungicidal effectiveness of some of the compounds ob-
Antioxidant activity (AOA) of nitroimidazole derivatives
was studied by in vitro assays. AOA in modelling system
was determined under the conditions of non-enzymatic initia-
tion of lipid peroxidation by Fe2þ ions [29]. The suspension of
egg lipoproteins (SEL) was used as a substrate and fat-soluble
antioxidant e tocopheryl acetate was used as a reference
substance.
_
tained was determined using the method proposed by Wazny
and Thornton [30] and later used in other tests with biocides
designed for wood protection [31,32]. The fungal growth rates
were measured in 90 mm diameter Petri dishes using the agar
dilution test. Nine concentrations of the compounds were stud-
ied in a geometric progression from 25 to 5000 ppm. Stock
mixtures of each concentration were produced in sterile malt
agar (1.5% agar and 4% malt-extract), 20 mL of which was
added to each Petri dish. Three replicate plates of each con-
centration of each chemicals were centrally inoculated with
a 5-mm diameter disc taken from the submargin of 10-day-
old cultures of the desired test fungus grown on malt agar.
The plates were incubated at 22 ꢂ 1 ꢁC in darkness. The dura-
tion of the test was determined by waiting for complete plate
coverage by the fungus mycelium that is until 12 days for the
researched species of S. pityophila. If the growth did not begin
on the chemical-containing agar after 12 days, the inoculum
was removed and transferred to a fresh malt agar plate for de-
termination of the fungal viability. The results obtained were
used to calculate ED50, the effective dose ED100, and LD for
all compounds examined.
Free-radical reaction was induced by Fe2þ ions and the in-
tensity of the lipid peroxidation processes in model system
was estimated by the spectrophotometric determination of thi-
obarbituric acid (TBA) and their reactants (TBARS) concen-
tration, by taking into account the coefficient of molar
extinction for malonodialdehyde (MDA), which is the main
lipooxidation product. SEL was prepared by homogenisation
of an egg yolk with full volume of the phosphate buffer pH
7.4. The substances investigated (as 10ꢀ3 mol/mL solution)
and 1 mL of 0.7% solution of FeSO4$7H2O were added to
1 mL of the suspensions. Mixtures were incubated at 37 ꢁC.
For each substance three samples were subjected to measure-
ments: the experimental one (containing the investigated com-
pound and iron(II) sulphate), the control (containing iron(II)
sulphate only) and the blind test (without Fe2þ ions and inves-
tigated compounds). Antioxidant activity of nitroimidazole de-
rivatives was investigated after 15, 30 and 60 min since the
moment of initiation of the free-radical oxidation. After the in-
tervals mentioned, the reactions were terminated by the addi-
tion of 1 mL of 25% trichloroacetic acid solution (containing
2.5 mg Trilone B in 100 mL of solution for linkage of Fe2þ).
Subsequently, the samples were centrifuged and the superna-
tants were incubated in an acidic medium with the same vol-
ume of thiobarbituric acid solution at 95 ꢁC for 1 h. After
cooling, the reaction product was extracted by n-butanolepyr-
idine mixture (15:1 by volume) and the optical density of the
Acknowledgements
This study was partially supported by Polish State Commit-
tee for Scientific Research (grant no. 2 PO5F 03927).
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