Synthesis and biological activity of new metronidazole derivatives

Article abstract of DOI:10.1007/s00706-015-1612-7

The development of new antimicrobial and antiparasitic agents offers the possibility of generating structures of increased potency. To this end, three sulphonate ester derivatives of metronidazole were synthesized. Treatment of the tosylate analogue with NaSPh and NaN₃ gave the thiophenolate and azide derivatives, respectively. Oxidation of phenylthio derivative with mCPBA afforded the sulfonyl analogue. Similarly, cycloaddition of azido-metronidazole with various symmetric acetylene compounds furnished the 1,2,3-triazole analogues. Treatment of dimethyl dicarboxylate metronidazole derivative with guanidine hydrochloride in the presence of base resulted in the formation of the ring-expanded (fat) derivative, triazolo-diazepam derivative of metronidazole. Treatment of chlorometronidazole with silylated quinolones gave the quinolone analogues of metronidazole. The antigardiasis and antifungal activities of the synthesized compounds were investigated. In addition, all synthesized compounds were evaluated for their in vitro anti-HIV activity in MT-4 cells as non-nucleoside reverse transcriptase inhibitors.

Full text of DOI:10.1007/s00706-015-1612-7

Monatsh Chem
DOI 10.1007/s00706-015-1612-7
ORIGINAL PAPER
Synthesis and biological activity of new metronidazole derivatives
Najim A. Al-Masoudi1 Zina A. A. Abbas1
•
Received: 5 August 2015 / Accepted: 19 November 2015
Ó Springer-Verlag Wien 2015
Abstract The development of new antimicrobial and
antiparasitic agents offers the possibility of generating
structures of increased potency. To this end, three sul-
phonate ester derivatives of metronidazole were
synthesized. Treatment of the tosylate analogue with
NaSPh and NaN3 gave the thiophenolate and azide
derivatives, respectively. Oxidation of phenylthio deriva-
tive with mCPBA afforded the sulfonyl analogue.
Similarly, cycloaddition of azido-metronidazole with var-
ious symmetric acetylene compounds furnished the 1,2,3-
triazole analogues. Treatment of dimethyl dicarboxylate
metronidazole derivative with guanidine hydrochloride in
the presence of base resulted in the formation of the ring-
expanded (fat) derivative, triazolo-diazepam derivative of
metronidazole. Treatment of chlorometronidazole with
silylated quinolones gave the quinolone analogues of
metronidazole. The antigardiasis and antifungal activities
of the synthesized compounds were investigated. In addi-
tion, all synthesized compounds were evaluated for their
in vitro anti-HIV activity in MT-4 cells as non-nucleoside
reverse transcriptase inhibitors.
Keywords Anti-HIV activity Á
Antiparasitic/antifungal activity Á Cycloadditions Á
Metronidazole Á Triazoles
Introduction
Nitroimidazoles have important pharmaceutical applica-
tions, particularly as anaerobic antibacterials and
antiprotozoal agents [1]. The antimicrobial activity of these
chemotherapeutic agents inhibits the growth of both
anaerobic bacteria and certain anaerobic protozoa such as
Trichomonas vaginalis, Entamoeba histolytica, and Giar-
dia lamblia [2]. Some nitroimidazoles were reported as
potent radiosensitizers in treatment of cancer [3–5], anti-
tubercular therapy [6, 7], control of fertility [8], and as anti-
Helicobacter pylori agents [9, 10]. H. pylori, a Gram-
negative microaerophilic spiral bacterium, is the major
factor in peptic ulcer diseases; highly effective treatment
for H. pylori infections includes a combination of antise-
cretory and antimicrobial agents such as metronidazole.
Furthermore, some 5-nitroimidazole have been tested in
cell-based assays and in enzyme assays against HIV-1
recombinant reverse transcriptase [11, 12] and as anti-
cancer agents [12]. In addition, others are under
investigation for their use as hypoxic cell cytotoxines [13].
Graphical abstract
Metronidazole,
2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethanol (Flagyl) [14–16] has been regarded as the drug
of choice that is commonly used for the treatment of
infections caused by Giardia lamblia and Trichomonas
vaginalis, and also acts as an amoebicidal in cases of
amoebiasis caused by Entamoeba histolytica; thus several
metronidazole derivatives were reported as potent
antiparasitic agents [17, 18]. In 2009, Mital [19] has
& Najim A. Al-Masoudi
najim.al-masoudi@gmx.de
1
Chemistry Department, College of Science, University of
Basrah, Basrah 1006, Iraq
123

N. A. Al-Masoudi, Z. A. A. Abbas
reviewed a brief account of various biological activities
exhibited by synthetic nitroimidazole derivatives as well as
their structure–mutagenicity relationships. Recently,
Mubarak et al. [20–23] have reported the synthesis of new
metronidazole derivatives with evaluation of their
antibacterial and antifungal activity.
new metronidazole with triazolo-diazepine moiety as a new
potential antiviral candidate. Treatment of 7 with guanidine
hydrochloride in the presence of sodium methoxide at
room temperature afforded 10 in 40 % yield (Scheme 1).
The structures of 2–10 were determined by their 1H, 13C,
and 2D NMR spectra, since metronidazole backbone pro-
tons showed a similar pattern. H-4 of the imidazole ring
appeared as singlet in the region d = 8.05–7.26 ppm,
whereas methyl protons at C-2 of the same ring resonated
as singlets in the region 2.52–2.41 ppm. NCH2 protons
appeared as triplets (J * 5.1 Hz), multiplets, or broad
singlets at 4.65–3.68 ppm, while OCH2 methylene protons
of compounds 2–4 resonated at 3.67 ppm (t, J = 5.0 Hz),
3.46 ppm (br s.), and 4.41 ppm (t, J = 5.1 Hz), respec-
tively. In addition, SCH2 and N3-CH2 protons of 5 and 6
appeared as triplets at 3.79, 3.67 ppm (J = 5.2, 5.1 Hz),
respectively. The methylene protons (Ntriazole-CH2) of 7–10
appeared as triplets or broad singlets at the regions
4.34–3.38 ppm. The other aliphatic and aromatic protons
were fully analysed (cf. ‘‘Experimental’’). The 13C NMR
spectra of 2–10 contained similar resonance signals of the
metronidazole carbon atoms. Carbon atoms 2, 4, and 5 of
the metronidazole scaffold resonated at the regions
152.7–149.6, 133.8–131.4, and 139.7–137.8 ppm, respec-
tively, whereas the carbonyl carbon atom of 3 appeared at
214.0 ppm. Signals at lower field of 164.4 and 161.7 and
167.7 ppm were assigned to C=O carbon atoms of 7 and 8,
whereas the carbonyl carbon atoms at 80 and 60 of 10
appeared at 190.7 and 164.0 ppm, respectively. The
chemical shifts between 48.9 and 41.6 ppm were assigned
to the NimidazoleCH2 carbon atom, except for those of 9,
which appeared at 59.7 ppm. The signals at 69.1, 68.1, and
68.3 ppm were assigned to CH2O carbon atom, while the
chemical shifts in the region 59.2–53.2 ppm were attrib-
uted to NtriazoleCH2 carbon atom of 7–10. The signals of
other carbon atoms were fully analysed (cf.
‘‘Experimental’’).
Based on these pharmacological activities, and in con-
tinuation of our work on nitroimidazoles [24–29], we
report here the synthesis of new mitronidazole derivatives,
with the aim to develop new antifungal, antiparasitic and
anti-HIV agents.
Results and discussion
Chemistry
Prior studies on side chain modifications of metronidazole
did not show remarkable structural themes that could be
exploited to generate compounds with superior activity [30,
31]. However, the nitro group is critical for antimicrobial
activity. We decided here to design new structural diversity
of metronidazole, aiming for development of new
improved antiparasitic agents. Thus, sulfonylation of
metronidazole (1), with 4-bromobenzenesulfonyl, cam-
phorsulfonyl, and 4-methylsulfonyl chlorides in the
presence of dry pyridine afforded the sulfonate ester
derivatives 2–4 in 76, 69, and 82 % yield, respectively.
Displacement of the tosylate group at 4 [32], via SN2
mechanism, by treatment with sodium thiophenolate at
120 °C furnished the phenylthio analogue 5 (71 %).
Analogously, treatment of 4 with sodium azide in DMF
resulted in SN2 displacement of the sulfonate ester group
by the azide residue 6 (89 %) (Scheme 1), which was
identical in the physical properties for those prepared
previously from the mesylate derivative [33]. The azide
alkyne cycloaddition reaction is used widely in synthesis of
various substituted 1,2,3-triazole derivatives [34], since
they are suitable for generating new antimicrobial drugs.
Thus, treatment of the azide 6 with three symmetrical
acetylene compounds: dimethyl acetylenedicarboxylate,
acetylene dicarboxylic acid, and diphenylacetylene in
toluene at 100-120 °C furnished, after purification, the
1,2,3-triazole analogues 7–9 in 43, 37, and 63 % yield
(Scheme 1).
Additionally, some quinolines are a family of synthetic
broad spectrum antibacterial drugs, for example cipro-
floxacin is an antibiotic that can treat a number of bacterial
infections [38]. This has attracted our attention towards the
synthesis of new metronidazole analogues conjugated with
the antibacterial quinolone bases, via silylation method
[39], aiming to evaluate their antimictobial and antiviral
activities. Thus, metronidazole (1) was converted first into
the chloro analogue 12 following Aita method [40].
Treatment of the silylated quinolones 14 and 16, prepared
by refluxing bases 13 and 15 in hexamethyldisilazane
(HMDS), with 12 in the presence of n-Bu4NI as catalyst in
dry acetonitrile as solvent at 25 °C to give 17 (67 %) and
18 (64 %), respectively (Scheme 2).
Recently, Hosmane et al. [35–37] have reported
numerous papers concerning the chemical and biological
activity of ring-expanded (fat) heterocycles, containing the
imidazo[4,5-e][1,3]diazepine ring system with their
potency as inhibitors of NTPases/helicases of flaviviridae
including the West Nile virus (WNV), hepatitis C virus
(HCV), hepatitis B virus (HBV), and Japanese encephalitis
virus (JEV). Accordingly, we have undertaken synthesis of
1
Structures of 17 and 18 were confirmed from their H,
13C NMR, and mass spectra. Compound 17 has been
123

Synthesis and biological activity of new metronidazole derivatives
Scheme 1
Scheme 2
selected for further NMR studies. From the gradient
selected HMBC [41] spectrum of 17, H-2 of the quinolone
3
backbone at dH = 8.85 ppm showed a JC,H heteronuclear
correlation with CH2 carbon atom at N-10 of imidazole ring
at dC = 47.0 ppm. Two similar correlations between CH2
protons at N-1 of quinolone moiety at dH = 3.41 ppm and
C-2 at dC = 143.7 ppm as well as with C-8a of quinolone
ring at dC = 137.7 ppm were observed (Fig. 1).
Antigiardiasis and antifungal activity
Compounds 2, 3, 5, 6, 8–11, 17, and 18 were screened for
their antimicrobial activity by use of in vitro bioassays and
Fig. 1 JC,H correlations in the HMBC NMR spectrum of 17
123

N. A. Al-Masoudi, Z. A. A. Abbas
Table 1 Minimum inhibitory concentrations (MIC) of the tested
substances and reference drug against the tested microorganisms
Table 2 In vitro anti-HIV-1 and HIV-2 activity of some new
metronidazole analogues
Compd.
MIC/lM
Compd.
Virus strain
EC50/lMa
CC50/lMb
SIc
G. intestinals
C. tropicalis
C. albicans
2
IIIB
[19.92
[19.92
[105.00
[105.00
[95.70
[95.70
[18.32
[18.32
2.23
19.92
19.92
\1
\1
X1
X1
\1
\1
X1
X1
11
ROD
IIIB
2
7.90
10.24
3.25
0.89
2.22
2.85
5.23
3.21
8.31
6.81
5.03
432
[500
[500
263
[500
[500
[500
87
3
[105.00
[105.00
95.70
3
ROD
IIIB
5
4
6
ROD
IIIB
95.70
8
[500
[500
[500
182
[500
[500
[500
122
5
[18.32
[18.32
24.75
9
ROD
IIIB
10
6
11
ROD
IIIB
[24.75
[87.34
[87.34
[102.30
[102.30
[97.80
[97.80
[68.9
24.75
\1
X1
X1
X1
X1
X1
X1
\1
\1
4
17
182
122
7
[87.34
[87.34
[102.30
[102.30
[97.80
[97.80
68.9
18
138
79
ROD
IIIB
Metronidazole
128
64
8
ROD
IIIB
9
assessed as minimal inhibitory concentration (MIC) values.
Results are summarised in Table 1, in which the data for
metronidazole [42] was included for comparison. The
fungal strains utilised in the assays were C. tropicalis
ATCC 13803 and C. albicans ATCC 40227. As shown in
Table 1, compound 6 was the most potent candidate
against Giardia, with an IC50 value of 0.89 lM compared
with 5.03 lM for metronidazole. Table 1 revealed also that
all synthesized compounds were inactive or moderately
active against C. tropicalis and C. albicans, with IC50
values ranging from [500 lM to 87 lM compared to
metronidazole.
ROD
IIIB
10
ROD
IIIB
[68.9
68.9
11
[7.28
29.12
ROD
IIIB
[29.12
[32.23
[32.23
[19.22
[19.22
0.05
29.12
\1
\1
\1
\1
\1
[80
\1
17
32.23
ROD
IIIB
23.23
18
19.22
ROD
IIIB
19.22
Nevirapine
[4
ROD
[4
[4
On the contrary, structural modification of the hydroxyl
group of metronidazole into sulphonyl group positively
influenced the antigiardial activity, in addition to moderate
activity in antifungal potency against C. albicans.
Anti-HIV-1 activity measured on strain IIIB
Anti-HIV-2 activity measured using strain ROD
a
Compound concentration required to achieve 50 % protection of
MT-4 cells from HIV-1 and HIV-2 induced cytopathogenic effects
b
Compound concentration that reduces the viability of mock-in-
fected MT-4 cells by 50 %
In vitro anti-HIV activity
c
Selectivity index (CC50/EC50
)
Compounds 2–11, 17, and 18 were tested for their anti-
HIV-1 activity in vitro, using IIIB strain in human MT-4
cells, based on a Microculture Tetrazolium (MTT) assay
[43, 44]. Cytotoxicity was also measured on MT-4 cells,
where results are summarized in Table 2, nevirapine (BOE/
BIRG587) [45] was used as a reference drug. All com-
pounds were found to be inactive except compounds 6 and
11, which showed remarkable anti-HIV-1 activity with
EC50 values of [2.23 and [7.28 lM, and CC50 values of
24.75 and 29.12 lM, resulting in SI values of 11 and 4,
respectively.
other functional groups. Based on the above data, com-
pounds 6 and 11 might be considered new promising anti-
HIV-1 agents to act as NNRTIs for further structural
modifications and pharmacological evaluation.
Conclusion
In conclusion, we have synthesized new metronidazole
analogues, including the derivatization of the hydroxyl
group with various potential groups. Some of these ana-
logues exhibited activity against Giardia and HIV-1 which
are considered as promising antiparasitic and anti-HIV
With respect to SAR, derivatization of the hydroxyl
group of metronidazole with sulphonyl group (e.g. 6) or
triazolo-diazepam (e.g. 11) moieties has considerably
increased the anti-HIV activity compared for those with
123

Synthesis and biological activity of new metronidazole derivatives
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (7,7-dimethyl-
2-oxobicylo[2.2.1]heptan-1-yl)methanesulfonate
(3, C16H23N3O5S)
candidates for further structural development and phar-
macological study.
From 501 mg camphorsulphonyl chloride. Yield: 530 mg
(69 %) as colouirless crystals; m.p.: 135–137 °C;
Rf = 0.62; H NMR (400 MHz, DMSO-d6): d = 7.94 (s,
Experimental
1
1H, H-4), 4.65 (br s, 4H, NCH2?OCH2), 3.46 (d, 2H,
J = 6.5 Hz, CH2SO2), 2.93 (m, 2H, CH2-200), 2.52 (s, 3H,
C2-Me), 1.91 (m, 1H, H-40), 1.45 (m, CH2-50?CH2-60),
Melting points were measured on a Bu¨chi melting point
¨
apparatus B-545 (BUCHI Labortechnik AG, Switzerland).
Elemental analyses (C, H, N, S) were conducted using a
Vario Elementar apparatus (Shimadzu, Japan), their results
were found to be in good agreement ( 0.3 %) with the
calculated values. NMR spectra were recorded on 400 and
600 MHz (1H) and at 100 and 150.91 MHz (13C) spec-
trometers (Bruker, Germany) with TMS as internal
standard and on d scale in ppm. Heteronuclear assignments
were verified by 1H-13C HMBC experiment. Metronidazole
tosylate (4) (m.p.: 152 °C; Ref. [32] 153 °C) and azide
analogue 7 (m.p.: 55–58 °C; Ref. [33] 56–58 °C) were
synthesized and purified according to published
procedures.
0.81 (s, 6H, C7 -Me2) ppm; 13C NMR (150.91 MHz,
00
DMSO-d6): d = 214.5 (C=O), 151.4 (C-2), 138.2 (C-5),
133.0 (C-4), 68.5 (OCH2), 57.8 (C-40), 48.2 (CH2SO2),
45.7 (CH2-20), 42.6 (C-70), 42.4 (NCH2), 35.2 (C-60), 31.9
(C-50), 29.7 (C-7), 19.4 (C7-Me2), 14.1 (C2-Me) ppm; MS
(FAB): m/z = 408 ([M?Na]?).
2-Methyl-5-nitro-1-[2-(phenylthio)ethyl]-1H-imidazole
(5, C12H13N3O2S)
To a solution of 650 mg 4 (2.00 mmol) in 10 cm3 DMF
was added 330 mg sodium thiophenolate (2.00 mmol) and
the mixture was stirred at 120 °C for 6 h. After cooling, the
mixture was evaporated to dryness and the residue was
partitioned between CHCl3 (3 9 15 cm3) and 15 cm3
water. The combined organic extracts was dried (Na2SO4),
filtered, and evaporated to dryness. The crude product was
purified on a short SiO2 column (5 g), by elution in
gradient with MeOH (0–5 %) and chloroform as eluent to
give 5 (400 mg, 71 %) as colourless crystals. M.p.:
General procedure for the synthesis of the imidazole
arylsulfonate esters 2–4
A solution of arylsulphonyl chloride (2.00 mmol) in
10 cm3 dichloromethane was added dropwise onto a stir-
red, cooled (*10 °C) solution of 342 mg metronidazole 1
(2.00 mmol) in 10 cm3 dry pyridine. The mixture was
stirred at room temperature for 6–10 h and the progress of
reaction was followed by TLC. After completion of the
reaction, few drops of water were added and the mixture
was stirred for 1 h. Chloroform (20 cm3) was added and
the solution was portioned successively with 10 % sul-
phuric acid (3 9 20 cm3), 20 cm3 10 % NaHCO3, and
finally with water. The combined organic extracts was
dried (Na2SO4), filtered, and evaporated to dryness. The
crude product was recrystallized from EtOH to give the
desired sulfonate ester derivative.
1
154–156 °C; Rf = 0.80; H NMR (400 MHz, DMSO-d6):
d = 8.00 (s, 1H, H-4), 7.63–7.11 (m, 5H, Harom), 4.34 (t,
2H, J = 5.2 Hz, NCH2), 3.40 (t, 2H, J = 5.2 Hz, OCH2),
2.45 (s, 3H, C2-Me), 7.92 (s, 1H, H-4), 7.60 (d, 2H,
J2 ,3 = 8.3 Hz, Harom.-30?Harom.-50), 7.40 (d, 2H,
0
0
J5 ,6 = 8.3 Hz, Harom.-20?Harom.-60), 4.57 (t, 2H,
J = 5.0 Hz, NCH2), 4.41 (t, 2H, J = 5.0 Hz, OCH2),
2.41 (s, 3H, C2-Me), 2.40 (s, 3H, Me-Ar) ppm; 13C NMR
(150.91 MHz, DMSO-d6): d = 151.9 (C-2), 137.8 (C-
5?Carom.-10), 132.9 (C-4), 132.8 (Carom.-20?Carom.-60),
128.4 (Carom.-3?Carom.-60), 125.5 (Carom.-40), 59.7
(NCH2), 34.0 (SCH2), 14.1 (C2-Me) ppm; MS (FAB):
m/z = 264 ([M?H]?).
0
0
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl
4-bromobenzenesulfonate (2, C12H12BrN3O5S)
From 511 mg 4-bromobenzenesulfonyl chloride. Yield:
2-Methyl-5-nitro-1-[2-(phenylsulfonyl)ethyl]-1H-imidazole
(6, C12H13N3O4S)
580 mg (76 %) as colourless crystals; m.p.: 144–145 °C;
1
Rf = 0.48; H NMR (400 MHz, DMSO-d6): d = 7.94 (s,
To a stirred cold solution of 74 mg 5 (0.28 mmol) in
10 cm3 CH2Cl2 was added 100 mg m-chloroperbenzoic
acid (mCPBA, 0.58 mmol). After stirring for 6 h at 25 °C,
the solution was evaporated to dryness and the residue was
partitioned between CHCl3 (2 9 10 cm3) and 10 cm3
water. The combined organic extracts were dried (Na2-
SO4), filtered, and evaporated to dryness. The residue was
poured onto a SiO2 column (10 g), with MeOH, in gradient
(0–10 %), and CHCl3 as eluent to give 6 (60 mg, 73 %), as
white crystals. M.p.: 149–151 °C; Rf = 0.8; 1H NMR
1H, H-4), 7.51 (d, 2H, J2 ,3 = 8.2 Hz, Harom.-30?Harom.
-
0
0
50), 7.21 (d, 2H, J5 ,6 = 8.2 Hz, Harom.-20?Harom.-60), 4.35
(t, 2H, J = 5.0 Hz, NCH2), 3.67 (t, 2H, J = 5.0 Hz,
OCH2), 2.50 (s, 3H, C2-Me) ppm; 13C NMR (150.91 MHz,
DMSO-d6): d = 151.6 (C-2), 145.2 (Carom.-10), 138.2 (C-
5), 132.9 (C-4), 132.9 (Carom.-30 ? Carom.-50), 129.4
(Carom.-20 ? Carom.-60), 129.2 (C–Br), 69.1 (OCH2), 44.9
(NCH2), 13.9 (C2-Me) ppm; MS (FAB): m/z = 312/314
([M?Na]?).
0
0
123

N. A. Al-Masoudi, Z. A. A. Abbas
(400 MHz, DMSO-d6): d = 8.11 (m, 2H, Harom), 7.91 (s,
1H, H-4), 7.65–7.48 (m, 3H, Harom), 4.32 (t, 2H,
J = 5.1 Hz, NCH2), 3.48 (t, 2H, J = 5.1 Hz, OCH2),
2.42 (s, 3H, C2-Me) ppm; 13C NMR (150.91 MHz, DMSO-
d6): d 151.3 (C-2), 138.0 (C-5?Carom.-10), 132.3 (C-
(400 MHz, DMSO-d6): d = 8.01 (s, 1H, H-4), 7.48-7.10
(m, 10H, Harom), 4.35 (t, 2H, J = 5.0 Hz, NimidazoleCH2),
3.67 (t, 2H, J = 5.0 Hz, NtriazoleCH2), 2.45 (s, 3H, C2-Me)
ppm; 13C NMR (150.91 MHz, DMSO-d6): d = 151.9 (C-
2), 142.4 (Ctriazole-4), 139.7 (Ctriazole-5), 137.0 (Cimidazole
-
4?Carom.-4), 131.3 (Carom.-20?Carom.-60), 128.6 (Carom.
-
5), 133.8 (Ctriazole-4), 132.8, 127.9, 127.4, 125.4 (Carom.),
59.6 (Ntriazole-CH2), 48.1 (Nimidazole-CH2), 14.2 (C2-Me)
ppm; MS (FAB): m/z = 375 ([M?H]?).
3?Carom.-60), 125.5 (Carom.-40), 59.4 (SCH2), 35.1 (NCH2),
14.0 (C2-Me) ppm; MS (FAB): m/z = 296 ([M?H]?).
6-Amino-8-hydroxy-3-[2(2-methyl-5-nitro-1H-imidazol-1-
yl)ethyl]-[1,2,3]triazolo[4,5-e][1,3]diazepine-4-one
(11, C11H11N9O4)
General procedure of cycloaddition reaction
of metronidazole azide 7 with substituted acetylene
compounds
To a cooled stirred solution of sodium methoxide (from
750 mg sodium and 15 cm3 MeOH) was added 380 mg
guanidine hydrochloride (4.00 mmol) and the solution was
stirred at ice-bath for 30 min then filtered from sodium
chloride. The filtrate was added to a stirred cooled solution
of 332 mg 8 (1.00 mmol) in 20 cm3 MeOH and the stirring
was continued at room temperature for 48 h. The mixture
was filtered and the filtrate was neutralised with 2 M
hydrochloric acid and the precipitate was filtered and dried,
followed by suspension with 20 cm3 methanol. The
suspension was heated under reflux for 30 min and filtered
as a hot mixture. The filtrate was evaporated to dryness to
give 11 (133 mg, 40 %) as a yellow crystals. M.p.:
To a stirred solution of 392 mg 7 (2.00 mmol) in 10 cm3
dry toluene was added acetylene derivative (2.10 mmol)
and the mixture was heated, with stirring, at 100–120 °C
for 5–6 h. After cooling, the mixture was evaporated to
dryness and the residue was partitioned between CHCl3
(3x15 cm3) and 15 cm3 water. The combined organic
extracts was dried (Na2SO4), filtered, and evaporated to
dryness. The residue was purified on a short column of
silica gel (5 g) and elution, in gradient, with MeOH
(0–20 %) and CHCl3 as eluent to afford the desired
1,2,3-triazole analogue.
1
Dimethyl 1-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]-
1,2,3-triazole-4,5-dicarboxylate (8, C12H14N6O6)
195–198 °C; Rf = 0.31; H NMR (600 MHz, DMSO-d6):
d = 8.95 (br s, 2H, NH2), 7.26 (s, 1H, H-4), 5.50 (br s, 1H,
OH), 3.68 (s, 2H, NimidazoleCH2), 3.38 (s, 2H, NtriazoleCH2),
2.43 (s, 3H, C2-Me) ppm; 13C NMR (150.91 MHz,0DMSO-
From 296 mg dimethyl acetylenedicarboxylate. Yield:
291 mg (43 %) as yellow powder. M.p.: 260–263 °C
(dec.); Rf = 0.89; 1H NMR (600 MHz, DMSO-d6):
d = 8.01 (s, 1H, H-4), 4.35 (m, 4H, NimidazoleCH2?Ntria-
zoleCH2), 3.67, 3.36 (2 s, 6H, 2xCO2Me), 2.45 (C2-Me)
ppm; 13C NMR (150.91 MHz, DMSO-d6): d = 164.4,
161.7 (CO2Me), 152.7 (C-2), 139.5 (C-5), 133.2 (C-4),
121.5 (Ctriazole-4), 117.7 (Ctriazole-5), 53.9 (CO2Me), 53.2
(Ntriazole-CH2), 41.6 (Nimidazole-CH2), 11.4 (C2-Me) ppm;
MS (FAB): m/z = 339 ([M?H]?).
0
0
d6): d = 190.7 (C8 =O), 164.0 (C6 =O), 158.2 (C-4 ), 149.6
(C-2), 139.5 (Cimidazole-5?C-8a0), 139.3 (C-3a0), 131.4
(Cimidazole-4), 59.2 (Ntriazole-CH2), 48.9 (Nimidazole-CH2),
11.3 (C2-Me) ppm; MS (FAB): m/z = 356 ([M?Na]?).
1-(2-Chloroethyl)-5-methyl-2-nitro-1H-imidazole (12)
This compound was prepared according to the procedure of
Atia [40] from 300 mg metronidazole (1, 1.75 mmol) and
240 mg thionyl chloride (2.00 mmol). Yield: 272 mg
(82 %); m.p.: 86–88 °C (Ref. [39] 85–87 °C).
1-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl]-1,2,3-tria-
zole-4,5-dicarboxylic acid (9, C10H10N6O6)
From 240 mg acetylene dicarboxylic acid. Yield: 230 mg
6,7-Dihalo-1-[2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethyl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acids
General procedure. A suspension of the quinolone bases 13
and 15 (0.76 mmol) in 20 cm3 hexamethyldisilazane and a
few crystals of (NH4)2SO4 were heated under reflux for
10 h. After cooling, the solution was evaporated to dryness
and the residue was dissolved in 20 cm3 dry acetonitrile to
give the silylated quinolones 14 and 16. Chloride 12
(100 mg, 0.53 mmol) in 10 cm3 dry acetonitrile was added
dropwise followed by addition of 150 mg n-Bu4NI. After
stirring at 25 °C for 5 h, the solution was evaporated to
dryness and the residue was partitioned between CHCl3
(3 9 15 cm3) and 15 cm3 water. The combined organic
extracts were dried (MgSO4) and evaporated to dryness.
(37 %) as yellow powder. M.p.: 290–294 °C (dec.);
Rf = 0.39; H NMR (400 MHz, DMSO-d6): d = 8.02 (s,
1
1H, H-4), 4.35 (m, 4H, NimidazoleCH2?NtriazoleCH2), 2.46
(C2-Me) ppm; 13C NMR (150.91 MHz, DMSO-d6):
d = 167.7 (CO2H), 151.7 (C-2), 139.7 (Ctriazole-5), 138.2
(C-5), 132.8 (C-4?Ctriazole-4), 59.6 (Ntriazole-CH2), 48.2
(Nimidazole-CH2), 14.1 (C2-Me) ppm; MS (FAB): m/
z = 333 ([M?Na]?).
1-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl]-4,5-
diphenyl-1H-1,2,3-triazole (10, C20H18N6O2)
From 374 mg diphenylacetylene. Yield: 472 mg (63 %) as
1
yellow powder. M.p.: 153–155 °C; Rf = 0.64; H NMR
123

Synthesis and biological activity of new metronidazole derivatives
Crystallization from EtOH/hexane afforded the pure
desired product.
MIC value of the test compound and was expressed in lg/
cm3. Each compound was assayed in duplicate in each of
three independent experiments. The antigiardial activity of
the prepared compounds and metronidazole as the standard
drug were tested as described elsewhere [47]. Briefly, the
tested compounds and metronidazole were dissolved in
DMSO then in medium and filter-sterilized. In a 15-cm3
glass tube, two fold dilutions starting at 15 mg/cm3 were
prepared in a final volume of 15 cm3 to exclude air from
the tube. Each tube was inoculated with 20,000 cells of the
parasite being tested (Giardia). Each compound was
assayed in duplicate in each of three independent experi-
ments. In each assay, the appropriate controls were
performed, including one without any compound and
another with metronidazole as the positive control. The
biological activity of the compounds was evaluated by
counting the parasites in each tube by use of a standard
haemocytometer. In each count, trypan blue was used to
distinguish live from dead parasite.
6,7-Dichloro-1-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethyl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(17, C16H12Cl2N4O5)
From 196 mg 13. Yield: 209 mg (67 %); m.p.: 188–200 °C
1
(dec.); Rf = 0.59; H (600 MHz, DMSO-d6) d = 8.85 (s,
1H, H-2), 8.58 (s, 1H, H-5), 8.01 (s, 1H, H-8), 7.24 (s, 1H,
H-40), 3.89 (s, 2H, NimidazoleCH2), 3.41 (s, 2H, Nquinol.CH2),
2.41 (s, 3H, C2-Me) ppm; 13C NMR (150.91 MHz, DMSO-
d6): d = 177.2 (C-9), 165.0 (C-4), 151.9 (Cimidazol-20),
143.7 (C-2), 139.0 (C-7), 137.7 (C-8a), 137.0 (Cimidazole
-
50), 131.9 (C-6), 128.5 (C-5), 125.6 (C-4a), 118.0 (C-8),
110.1 (C-3), 56.9 (Nquinol.-CH2), 47.0 (Nimidazole-CH2), 14.1
(C2-Me) ppm; MS (FAB): m/z = 411/413 ([M?H]?).
7-Chloro-6-fluoro-1-[2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethyl]-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (18, C16H12ClFN4O5)
From 196 mg 15. Yield: 209 mg (67 %); m.p.: 188–200 °C
(dec.); Rf = 0.53; H (600 MHz, DMSO-d6) d = 8.83 (s,
1
HIV-1 reverse transcriptase inhibition assay
1H, H-2), 8.25 (d, 1H, JH-5,F = 8.5 Hz, H-5), 8.03 (d, 1H,
JH-8,F = 5.8 Hz, H-8), 7.23 (s, 1H, H-40), 3.87 (s, 2H,
Evaluation of the antiviral activity of compounds 2–11, 17,
and 18 against the HIV-1 strain (IIIB) and the HIV-2 strain
(ROD) in MT-4 cells was performed using an MTT assay
as described previously [43, 44]. In brief, stock solutions
(10 times final concentration) of test compounds were
added in 25-mm3 volumes to two series of triplicate wells
to allow simultaneous evaluation of their effects on mock
and HIV-infected cells at the beginning of each experi-
ment. Serial 5-fold dilutions of test compounds were made
directly in flat-bottomed 96-well microtiter trays using a
Biomek 3000 robot (Beckman instruments). Untreated
control, HIV and mock-infected cell samples, were inclu-
ded for each sample. HIV-1 (IIIB) [48] or HIV-2 (ROD)
[49] stock (50 mm3) at 100–300 CCID50 (50 % cell culture
infectious dose) or culture medium was added to either of
the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test
compound on uninfected cells to assess the cytotoxicity of
the test compounds. Exponentially growing MT-4 cells
[50] were centrifuged for 5 min at 1000 rpm, and the
supernatant was discarded. The MT-4 cells were resus-
pended at 6 9 105 cells per cm3, and 50-mm3 volumes
were transferred to the microtiter tray wells. Five days after
infection, the viability of the mock- and HIV-infected cells
was examined spectrophotometrically by the MTT assay.
N
imidazoleCH2), 3.40 (s, 2H, Nquinol.CH2), 2.43 (s, 3H, C2-
Me) ppm; 13C NMR (150.91 MHz, DMSO-d6): d = 177.8
(CO2H), 166.3 (C-4), 156.7 (d, JC-6,F = 248 Hz, C-6),
151.7 (Cimidazol-20), 148.3 (C-2), 137.2 (Cimidazole-50), 136.6
(C-8a), 128.0 (d, JC-4a,F = 5.6 Hz, C-4a), 126.1 (d, JC-
= 21 Hz, C-7), 120.2 (C-8), 119.5 (C-3), 113.2 (d, JC-
7,F
= 23 Hz, C-5), 56.6 (Nquinol.-CH2), 47.5 (Nimidazole
-
5,F
CH2), 14.0 (C2-Me) ppm; MS (FAB): m/z = 394/396
([M?H]?).
Antifungal and antigiadriasis activity
The antifungal activity of the synthesized compounds and
metronidazole, determined as MIC, was assessed using
microdilution method recommended by NCCLS [46]
against C. tropicalis ATCC 13803 and C. albicans ATCC
40227. Test compounds were dissolved in 10 % DMSO, to
produce a 2000 lg/cm3 stock solution. These test tubes
were serially diluted to give a concentration of 100, 50, 25,
12.5, 6.25, 3.12, 1.56, and 0.78 lg/cm3. SDB (Sabouraud
Dextrose Broth) was used for fungus. The cell density of
each inoculum was adjusted in sterile water of a 0.5
McFarland standard. A final concentration of 106 CFU/
cm3 was obtained for fungus, Microbial inocula were
added to the two fold diluted samples. The test tubes were
incubated 18–24 h at 37 1 °C for 2–5 days at 25 1 °C
for fungus. Metronidazole was used as standard drug. The
highest dilution of the test compound that completely
inhibited the growth of test organism was considered as the
Acknowledgements We thank Prof. C. Pannecouque of Rega
Institute for Medical Research, Katholieke Universiteit, Leuven,
Belgium, for the anti-HIV screening. Miss A. Friemel of the Chem-
istry Department, University of Konstanz, Germany, is highly
acknowledged for the NMR experiments.
123

N. A. Al-Masoudi, Z. A. A. Abbas
28. Al-Soud YA, Al-Masoudi NA, Hassan HG, De Clercq E, Pan-
necouque E (2007) Acta Pharm 57:379
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