Synthesis of new compounds derived from metronidazole and amino acids and their esters as antiparasitic agents

Article abstract of DOI:10.1007/s00044-011-9689-y

A number of new compounds derived from metronidazole and amino acids and their esters have been synthesized through a reaction between 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetic acid and a number of amino acid esters in the presence of N,N'carbonyldiimidazole (CDI). Hydrolysis of the esters derivatives with sodium hydroxide (4%) followed by acidification with hydrochloric acid (3 M) afforded the corresponding acids. The newly synthesized compounds were characterized by elemental analysis and by spectroscopic techniques such as ¹H-NMR, ¹³C-NMR, and mass spectrometry. Some of the prepared compounds exhibited lethal activity against pathogenic protozoan parasites. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9689-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1700–1707
DOI 10.1007/s00044-011-9689-y
ORIGINAL RESEARCH
Synthesis of new compounds derived from metronidazole
and amino acids and their esters as antiparasitic agents
•
Ahmed T. Al-Masri Haythem A. Saadeh
•
•
Ibrahim M. Mosleh Mohammad S. Mubarak
Received: 1 February 2011 / Accepted: 28 May 2011 / Published online: 9 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A number of new compounds derived from
metronidazole and amino acids and their esters have been
synthesized through a reaction between 2-(2-methyl-5-
nitro-1H-imidazol-1-yl)acetic acid and a number of amino
acid esters in the presence of N,N0carbonyldiimidazole
(CDI). Hydrolysis of the esters derivatives with sodium
hydroxide (4%) followed by acidification with hydrochlo-
ric acid (3 M) afforded the corresponding acids. The newly
synthesized compounds were characterized by elemental
analysis and by spectroscopic techniques such as 1H-NMR,
13C-NMR, and mass spectrometry. Some of the prepared
compounds exhibited lethal activity against pathogenic
protozoan parasites.
peptic ulcer diseases; highly effective treatment for
H. pylori infections includes a combination of antisecretory
and antimicrobial agents such as metronidazole (Cavalcanti
et al., 2004). The need for additional drugs of anti-H. pylori
is an absolute necessity due to metronidazole-resistant
H. pylori strains (Jenks and Edwards, 2002).
Mertonidazole, 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethanol (1) and its derivatives have a wide range of bio-
logical activity (Bertinaria et al., 2003; Martino et al.,
2005; Hay et al., 2003; Gu¨nay et al., 1999). They are
highly effective against trichomoniasis, various forms of
amoebiasis, and infection with anaerobic bacteria and
protozoa (Goldman and Wuest, 1981); it can kill or inhibit
the majority of anaerobic bacteria when the metronidazole
concentration in serum is in the range from 2 to 8 lg per ml
(Salimi et al., 1997). In view of the wide interest in the
activity and profile of metronidazole, and as part of our
ongoing research in the synthesis of new compounds of
pharmacological interests (Al-Zghoul et al., 2005; Salih
et al., 2006, 2007; Al-Soud et al., 2008; Saadeh et al.,
2009; Abu-Shaireh et al., 2009, Saadeh et al., 2010), we
report, herein, on the synthesis and characterization of a
number of new compounds derived from metronidazole
and amino acids and their esters, which, to the best of our
knowledge, have not previously been described in the lit-
erature. In addition, the antiparasitic activities of the newly
prepared compounds are presented.
Keywords Metronidazole ꢀ Amino acid esters ꢀ
N-N0-carbonyldiimidazole ꢀ Antiamoebic activity
Introduction
Nitroimidazole derivatives are an extremely important
class of compounds; they are extensively used in the
treatment of anaerobic infections and are under continuing
investigation regarding their use as hypoxic cell cytotoxins,
radiation sensitizers, and as anti-Helicobacter pylori agents
(Cavalcanti et al., 2004), H. pylori, a Gram-negative
microaerophilic spiral bacterium, is the major factor in
A. T. Al-Masri ꢀ H. A. Saadeh ꢀ M. S. Mubarak (&)
Department of Chemistry, University of Jordan,
Amman 11942, Jordan
Results and discussion
e-mail: mmubarak@ju.edu.jo
Chemistry
I. M. Mosleh
Department of Biological Sciences, University of Jordan,
Amman 11942, Jordan
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetic acid (2), the
starting material for compounds 4a–h and 5b–g was
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Med Chem Res (2012) 21:1700–1707
1701
synthesized through Jones oxidation of metronidazole (1)
as shown in Scheme 1. Reaction of compound 2 with a
solution of the appropriate amino acid ester (3) and triethyl
amine in DMF, in presence of N,N0-carbonyldiimidazole,
under nitrogen afforded the corresponding substituted
(2-(2-methyl-5-nitro-1H-imidazol-1-yl) acetamido)amino
esters, 4a–h as given in Scheme 1. The structures of the
prepared compounds were confirmed by NMR, mass
spectrometry, and elemental analysis. The 1H and 13C-
NMR spectra of all prepared compounds are in total
agreement with the suggested structures. DEPT experi-
ments were employed to differentiate secondary and qua-
ternary carbons from primary and tertiary carbons.
Additional support of the proposed structures comes from
mass spectral data; mass spectra of the prepared com-
pounds showed the correct molecular ions as suggested by
their molecular formulas. The 1H-NMR spectra of 4a–
h showed singlets at d = 2.22–2.47 ppm attributed to the
metronidazole methyl group CH3-C2 and singlets at
d * 7.90 ppm ascribed to the C4-H of the metronidazole
ring. Detailed 1H-NMR signals are described in the
experimental section. The 13C-NMR spectra of the afore-
mentioned compounds reveal clearly the presence of CH3
signal d = 14.0–14.1 ppm for the CH3 carbon and a car-
bonyl amide carbon signal at d = 171.2–173.1 ppm.
Moreover, polarimetric measurements indicated that the
coupling reactions to produce the esters have proceeded
with retention of configuration and racemization has been
excluded. The same applies for the base promoted hydro-
lysis of the esters to the corresponding acids. For example,
[a]DMF for 4b is -49.0o and for 5b -49.0o.
diamino acid ester, lysine methyl ester (6) with compound
2 using the same general procedure employed for the
synthesis of compounds 4b–h as shown in Scheme 2. The
solution was filtered off to yield the desired product in
fairly good yield. The identity of the product is confirmed
1
from H-NMR data—which shows two signals for the two
secondary amino groups—mass spectrometry, and by ele-
mental analysis.
Base-pormoted hyrolysis of compounds 4a–g, and 7
with 4% NaOH solution followd by acidification with HCl
(3 M) yielded the corresponding amino acid derivatives of
metronidazole 5b–g and 8. The identities of these com-
pounds were confirmed by their 1H NMR, 13C NMR, mass
spectra, and elemental analyses. The 1H and 13C-NMR
spectra of all prepared compounds are in total agreement
with their proposed structures.
Biological activity
In vitro antiamoebic and antigiardial activity
and cytotoxicity
The newly synthesized metronidazole-derivatives were
tested for their antiamoebic and antigiardial activity using
in vitro bioassays. The cytotoxicity of these compounds on
the two cell lines, Hep-2 and Vero cells, was also inves-
tigated and compared with that of the standard drug,
metronidazole. The IC50 of the tested compounds against
Entamoeba histolytica, Giardia intestinalis, and the two
cell lines is given in Table 1. As indicated in the table,
compounds 4a, 4c, 4h, and 7 showed more potent activi-
ties against Entamoeba and Giardia than the standard drug
with IC50 values ranging from 2.10 to 3.95 lM.
Ethyl 2,6-bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetamido)hexanoate (7) was synthesized by reacting the
Scheme 1 Synthesis of (2-(2-
methyl-5-nitro-1H-imidazol-1-
yl) acetamido)esters (4a–h) and
the corresponding acids 5b–g
N
N
N
CH3
R
O2N
CDI / TEA
N
O
Con.H2SO4
CH3
OH
O2N
+
H
OCH3
CH3
OH
O2N
N
N
H2N
HCl
N
Na2Cr2O7 /H2O
OCH3
O
O
R
O
1
3
4b-h
2
N
NaOH
then H+
CH3
O2N
N
O
H
N
OH
O
R
5b-g
c
g
b
d
e
h
a
f
R
Ala
Asp
Tyr
Try
Pro
Pha
His
Lys
(aminoacid
residue)
* 4a is the ethyl ester analogue of 4
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Med Chem Res (2012) 21:1700–1707
Scheme 2 Synthesis of Ethyl
2,6-bis(2-(2-methyl-5-nitro-1H-
imidazol-1-yl) acetamido)
hexanoate (7) and 2,6-bis
(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)acet-
NO2
N
CH3
N
N
N
NH2
O
CDI
O2N
N
O
CH3
CH3
OH
OEt
N
+
H2N
HN
O
TEA
O2N
OEt
N
H
amido)hexanoic acid (8)
O
O
7
6
2
NO2
N
CH3
N
N
1. NaOH
2. HCl
N
O
CH3
HN
O
O2N
OH
N
H
O
8
Compound 4a displayed the highest activity against the
two parasites. However, when the cytotoxicity of the
prepared molecules is considered, compound 4c appears to
be better; its cytotoxicity was around four times less than
that of 4a (Table 1). The amino acid part apparently seems
to influence the antiparasitic activity of the compound;
introduction of some amino acid moieties such as alanine,
aspartic, and proline increased the antiparasitic activity of
the prepared compounds. In addition, data in Table 1 also
reveal that the introduction of some groups on metroni-
dazole can decrease its biological activities against the two
tested parasites to variable degrees. Interestingly, the
majority of the tested compounds exhibited almost similar
activities against both G. intestinalis and E. histolytica
(Table 1), indicating that each compound affects both
parasites by a similar mechanism of action. The activities
exhibited by the newly prepared compounds suggest that
these new derivatives may be used as starting points in the
development of new antiparasitic drugs. Moreover, the
importance of such biologically active, noncytotoxic
molecules, lies in their potential contribution to overcome
the problem of resistance of pathogens to the standard
drugs (Elizondo et al., 1996; Hager and Rapp, 1992).
Because of their low solubility in DMSO, the rest of the
prepared compounds were not tested against the afore-
mentioned parasites.
Experimental section
Chemicals
The following chemicals and materials were used without
further purification:
Metronidazole, N,N0-carbonyldiimidazole, L-phenylala-
nine methyl ester hydrochloride, L-phenylalanine ethyl
ester hydrochloride, and L-alanine methyl ester hydro-
chloride were obtained from Acros. L-Lysine ethyl ester
dihydrochloride, L-lysine methyl ester dihydrochloride, and
L-proline methyl ester dihydrochloride were purchased
from Fluka. L-aspartic acid dimethyl ester hydrochloride,
L-tyrosine methyl ester, L-tryptophan methyl ester hydro-
chloride, and L-histidine methyl ester dihydrochloride were
obtained from Aldrich. Thin-layer chromatography was
carried out using glass plates, precoated with silica gel 60
GF254, supplied by Fluka.
Instrumentation
Melting points were measured with a Fischer-Johns melt-
1
ing point apparatus and were uncorrected. The H and 13C
NMR spectra were acquired with the aid of a Bruker-DPX
300 MHz spectrometer (Germany) and are reported in ppm
(d) relative to TMS as an internal standard and in DMSO-
d6 as solvent. Coupling constants are in Hz; abbreviations:
s = singlet, d = doublet, t = triplet, m = complex multi-
plets; bs = broad singlet. EIMS spectra were measured
using a Finnegan MAT TSQ-70 spectrometers (Finnegan
MAT, USA); ion source temperature = 200°C. High res-
olution mass spectral data were obtained with a Brucker
APEX (IV) mass spectrometer (Bremen, Germany). Ele-
mental analyses were obtained using a Eurovector Euro
EA3000, C, H, N, and S elemental analyzer and the
obtained results agreed with the calculated percentages to
Conclusions
We have successfully synthesized and characterized a
number of new compounds derived from metronidazole
and amino acids and their esters. The newly synthesized
compounds were screened for their antiparasitic activity
and results showed that these compounds exhibited lethal
activity against pathogenic protozoan parasites.
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Med Chem Res (2012) 21:1700–1707
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Table 1 The biological activity of the derivative compounds and metronidazole against the parasites, Entamoeba histolytica and Giardia
intestinalis, and cancer (Hep-2) and nonmalignant (Vero) cells
Compound
IC50 SD (half the growth in negative control) lM
IC0 SD (growth as in negative control) lM
Entamoeba histolytica
Giardia intestinalis
Hep-2 cells
Vero cells
4a
2.10 0.20
22.13 2.90
2.21 0.20
18.29 2.60
8.29 1.10
8.91 0.4
2.18 0.30
44.43 4.83
2.26 0.34
17.79 1.25
16.57 0.98
8.93 1.08
2.70 0.75
3.84 0.55
22.22 1.05
18.00 0.65
17.21 0.70
5.17 0.75
69.74 2.70
72.17 2.02
4b
4c
304.16 8.58
168.85 5.01
314.00 12.77
203.62 15.32
4d
4e
4g
4h
2.54 0.55
3.95 0.25
23.45 1.55
17.25 1.15
16.17 1.21
4.40 0.60
7
5b
5e
5f
Metronidazole
869.33 25.32
903 16.09
within 0.4%. Compounds were checked for their purity
by TLC using glass plates, precoated with silica gel 60
GF254, supplied by Fluka.
and kept in the freezer for 1 h. The desired products were
collected by filtration. Using the aforementioned general
procedure, the following compounds were synthesized.
Ethyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-
Synthesis of 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetamido)-3-phenylpropanoate (4a)
yl)acetic acid (2)
1
Yield 1.20 g (73%), Mp 69–71°C. H-NMR (DMSO-d6) d
The title compound was synthesized according to the
following general procedure: metronidazole (1) (5 g,
29.0 mmol) was added to a solution of sulfuric acid (5 ml)
and ice-water (20 ml); the solution was stirred in an ice-
bath until all metronidazole was dissolved. A solution of
sodium dichromate (5 g, 20 mmol) in water (5 ml) was
added drop-wise and the mixture was stirred overnight at
room temperature, where the solution color changed to
green. The solution was then extracted several times with
ether; evaporation of the ether under reduced pressure gave
2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetic acid (2) (3 g,
55%, Mp 172–174°C, Lit. Mp 176–178°C) (Khabnadideh
et al., 2007).
(ppm) = 1.26 (t, 3H, J = 7.14 Hz), 2.39 (s, 3H), 2.98–3.15
(m, 2H), 4.18 (q, 2H, J = 7.14 Hz), 4.82–4.98 (m, 3H)
6.50 (d, 1H, J = 7.81 Hz), 7.04–7.06 (m, 2H),7.21–7.28
(m, 3H), 7.91 (s, 1H). 13C-NMR (DMSO-d6):
d
(ppm) = 14.1 (CH3), 14.1 (CH3), 37.7 (CH2), 48.4 (CH2),
53.4 (CH), 62.1 (CH2); 127.4 (CH), 128.7 (CH), 129.3
(CH); 132.9 (CH); 135.4 (C); 138.4 (C); 151.4 (C); 164.7
(C); 171.2 (C). MS (70 eV) (C18H22N4O4), m/z (% rel.
Int.): 358 (8) [M]?; 343 (45); 315 (32); 288 (10); 270 (6);
242 (20); 176 (96); 150 (87); 132 (90); 92 (100); 53 (96).
Anal. Calcd. for C18H22N4O4: C, 60.32; H, 6.19; N, 15.63.
Found: C, 60.11; H, 6.08; N, 15.81.
Compounds 4a–h and 7 were synthesized and purified
according to the following general procedure: A solution of
N,N0-carbonyldiimidazole (CDI) (0.9 g, 5.50 mmol) in
DMF (10 cm3) was added to a solution of (2-methyl-5-
nitro-1H-imidazol-1-yl)acetic acid (2) (1.0 g, 5.40 mmol)
in DMF (3 ml) under N2. After stirring for 15 min, a
solution of the appropriate amino acid ester hydrochloride
(3) (5.40 mmol) and triethylamine (1.0 ml) in DMF (4 ml)
was added. The solution was then stirred overnight under
N2 and monitored by TLC. After consumption of the
starting materials, most of the DMF was evaporated under
reduced pressure and the residue was treated with water
Methyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetamido) propanoate (4b)
1
Yield 0.75 g (80%), Mp 142–144°C. H-NMR (DMSO-
d6): d (ppm) = 1.40 (d, 3H, J = 7.18 Hz), 2.46 (s, 3H),
3.73 (s, 3H), 4.95 (d, 2H, J = 2.52 Hz), 4.52–4.62 (m, 1H),
6.72 (d, 1H, J = 6.85 Hz),7.93 (s, 1H). 13C-NMR (DMSO-
d6): d (ppm) = 14.1 (CH3), 18.2 (CH3), 48.4 (CH2), 48.5
(CH3), 52.8 (CH), 132.7 (CH), 138.5 (C), 151.5 (C), 164.7
(C), 173.1 (C). MS (70 eV) C10H14N4O5, m/z (% rel. Int.):
270 (18) [M]?; 253 (27); 165 (86); 140 (59); 136 (45); 111
123

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Med Chem Res (2012) 21:1700–1707
Methyl 3-(1H-indol-3-yl)-2-(2-(2-methyl-5-nitro-1H-
(88); 102 (67); 80 (92); 74 (82); 53 (100). HRMS (EIMS)
m/z: calcd. for C10H14N4O5 [M]? 270.09642 found
270.09622. Anal. Calcd. for C10H14N4O5: C, 44.44; H,
5.22; N, 20.73. Found: C, 44.51; H, 5.16; N, 20.42.
imidazol-1-yl)acetamido)propanoate (4f)
Yield 1.38 g (69%); Mp 179–181°C. 1H-NMR (DMSO-d6):
d = 2.22 (s, 3H), 3.06–3.14 (m, 2H), 3.55 (s, 3H), 4.56–4.64
(m, 1H), 4.97 (d, 2H, J = 10.8 Hz), 6.96–7.04 (m, 2H), 7.14
(s, 1H), 7.31 (d, 1H, J = 7.9 Hz), 7.45 (d, 1H, J = 7.71 Hz);
7.99 (s, 1H), 8.87 (d, 1H, J = 7.52 Hz), 10.90 (s, 1H,
NH-indole). 13C-NMR (DMSO-d6): = 14.1 (CH3), 27.6
(CH2), 48.2 (CH2), 52.4 (CH), 53.9 (CH3), 109.6 (C), 111.9
(CH), 118.4 (CH); 118.9 (CH), 121.5 (CH), 124.3 (C), 127.6
(C), 132.8 (CH), 136.6 (C), 139.0 (C), 152.2 (C), 166.2 (C),
172.3 (C). MS (70 eV) (C18H19N5O5), m/z (% rel. Int.): 385
(36) [M]?; 326 (5); 280 (1); 201 (93); 197 (21); 170 (23);
131 (100); 103 (43); 80 (48); 53 (98); 28 (46). Anal. Calcd.
for C18H19N5O5: C, 56.10; H, 4.97; N, 20.76. Found: C,
56.24; H, 4.86; N, 20.64.
Methyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetamido)-3-phenylpropanoate (4c)
1
Yield 1.20 g (75%), Mp 88–90°C. H-NMR (DMSO-d6):
d = 2.35 (s, 3H), 2.86–3.06 (m, 2H), 3.70 (s, 3H),
4.50–4.97 (m, 1H), 4.82–4.97 (m, 2H), 7.04–7.06 (m, 2H),
7.19–7.24 (m,3H); 7.88 (s, 1H); 8.89 (d, 1H, J = 7.76 Hz).
13C-NMR (DMSO-d6): d = 14.0 (CH3), 37.6 (CH2), 48.2
(CH2), 52.6 (CH3), 53.4 (CH), 127.3 (CH), 128.7 (CH),
129.2 (CH), 132.7 (CH), 135.5 (C), 138.6 (C), 151.5 (C),
165.0 (C); 171.8 (C). MS (70 eV) (C16H18N4O5), m/z (%
rel. Int.): 346 (2) [M]?; 328 (56); 300 (24); 241 (6); 202
(3); 178 (4); 162 (96); 150 (60); 131 (71); 80 (84); 53
(100); 28 (73). Anal. Calcd. for C16H18N4O5: C, 55.49; H,
5.24; N, 16.18. Found: C, 55.57; H, 5.29; N, 16.13.
Methyl 3-(1H-imidazol-4(5)-yl)-2-(2-(2-methyl-5-
nitro-1H-imidazol-1-yl)acetamido)propanoate (4g)
1
Yield 1.31 g, (63%), Mp 220–222°C. H-NMR (DMSO-
Dimethyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-
d6): d = 2.29 (s, 3H), 2.82–2.97 (m, 2H), 3.57 (s, 3H),
4.47–4.54 (m, 1H), 5.00 (s, 2H), 6.8 (s, 1H), 7.49 (s, 1H),
7.99 (s, 1H), 8.79 (d, 2H, J = 6.98 Hz), 11.84 (bs, 1H).
13C-NMR (DMSO-d6): d = 14.1 (CH3), 29.7 (CH2), 48.2
(CH2), 52.4 (CH3), 53.1 (CH), 132.8 (CH), 132.8 (CH),
135.4 (CH), 135.5 (C), 139.0 (C), 152.2 (C), 166.2 (C),
172.2 (C). MS (70 eV) (C13H16N6O5), m/z (% rel. Int.):
336 (4) [M]?; 290 (37); 277 (29); 240 (8); 185 (27); 152
(31); 136 (30); 110 (26); 82 (66); 53 (75); 28 (100). Anal.
Calcd. for C13H16N6O5: C, 46.43; H, 4.80; N, 24.99.
Found: C, 46.52; H, 4.87; N, 25.07.
yl)acetamido)succinate (4d)
1
Yield 1.07 g (64%); Mp 109–110°C. H-NMR (DMSO-
d6): d = 2.47 (s, 3H), 2.81–3.07 (m, 2H), 3.67 (s, 3H), 3.74
(s, 3H), 4.80–4.86 (m, 1H), 4.98 (d, 2H, J = 3.5 Hz), 7.06
(d, 1H, J = 7.85), 7.94 (s, 1H), 7.06 (d, 1H, J = 7.85 Hz).
13C-NMR (DMSO-d6): d = 14.1 (CH3), 35.7 (CH2), 48.4
(CH2), 48.8 (CH), 52.8 (CH3), 53.1 (CH3), 132.7 (CH),
138.5 (C), 151.4 (C), 165.1 (C), 170.7 (C), 171.6 (C). MS
(70 eV) C12H16N4O7, m/z (% rel. Int.): 328 (8) [M]?; 311
(12); 283 (100); 269 (23); 222 (30); 180 (6); 168 (21); 140
(22); 111 (49); 80 (6); 28 (84); 15 (86). Anal. Calcd. for
C12H16N4O7: C, 43.90; H, 4.91; N, 17.07. Found: C, 44.01;
H, 4.84; N, 17.15.
1-[2-(Methyl-5-notroimidazol-1-yl)acetyl]pyrolidine-2-
carboxylic acid methyl ester (4h)
1
Yield 1.60 g (68%); Mp 115–118°C). H-NMR (DMSO-
Methyl 3-(4-hydroxyphenyl)-2-(2-(2-methyl-5-nitro-
d6): d = 1.83–1.90 (m, 2H), 2.12–2.24 (m, 2H), 2.33 (s,
3H), 3.56 (s, 3H), 3.63–3.72 (m, 2H), 4.28–4.32 (m, 1H),
5.25 (s, 2H), 8.01 (s, 1H). 13C-NMR (DMSO-d6): d = 14.0
(CH3), 24.9 (CH2), 29.1 (CH2), 46.4 (CH2), 48.1 (CH2),
52.3 (CH3), 59.2 (CH), 132.7 (CH); 139.1 (C), 152.1
(C),164.8 (C), 172.4 (C). HRMS (ESI) m/z: calcd for
C12H17N4O5 [M ? H]? 297.111990, found 297.12008.
Anal. Calcd. for C12H16N4O5: C, 48.65; H, 5.44; N, 18.91.
Found: C, 48.53; H, 5.39; N, 18.74.
1H-imidazol-1-yl)acetamido)propanoate (4e)
1
Yield 1.05 g (78%); Mp 106–108°C. H-NMR (DMSO-
d6): d = 2.24 (s, 3H), 2.74–2.93 (m, 2H), 3.56 (s, 3H),
4.37–4.44 (m, 1H), 4.96 (d, 2H, J = 8.07 Hz), 6.63 (d, 2H,
J = 8.40 Hz), 6.96 (d, 2H, J = 8.42 Hz), 7.99 (s, 1H),
8.83 (d, 1H, J = 7.82 Hz), 9.26 (s, 1H, OH). 13C-NMR
(DMSO-d6): d = 14.1 (CH3), 36.6 (CH2), 48.1 (CH2), 52.4
(CH), 54.5 (CH3), 115.5 (CH), 127.3 (C), 130.5 (CH),
132.8 (CH), 138.1 (C), 152.2 (C), 156.6 (C), 166.1 (C),
172.1 (C). MS (70 eV) C16H18N4O6, m/z (% rel. Int.): 362
(1) [M]?; 303 (10); 256 (2); 227 (9); 197 (98); 150 (41);
147 (100); 107 (60); 77 (48); 68 (47). Anal. Calcd. for
C16H18N4O6: C, 53.04; H, 5.01; N, 15.46. Found: C, 52.91;
H, 5.12; N, 15.59.
Ethyl 2,6-bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetamido)hexanoate (7)
1
Yield 0.95 g (69%), Mp 116–118°C. H-NMR (DMSO-
d6): d = 1.13 (t, 3H, J = 7.0 Hz), 1.24–1.39 (m, 4H),
123

Med Chem Res (2012) 21:1700–1707
1705
1.53–1.76 (m, 2H), 2.33 (s, 6H), 3.01–3.08 (m, 2H), 4.04
(q, 2H, J = 7.0 Hz), 4.14–4.22 (m, 1H); 4.90 (s, 2H), 5.01
(s, 2H),7.99 (s, 2H), 8.31 (t, 1H), 8.78 (d, 1H,
J = 7.78 Hz). 13C-NMR (DMSO-d6): d = 14.1 (CH3),
14.2 (CH3), 14.4 (CH3), 22.9 (CH2), 28.9 (CH2), 31.0
(CH2), 38.9 (CH2), 48.2 (CH2), 48.5 (CH2), 52.6 (CH);
61.1 (CH2), 132.8 (CH), 139.0 (C), 152.3 (C), 165.9 (C),
166.3 (C), 172.1 (C). MS (70 eV) (C20H28N8O8), m/z (%
rel. Int.): 508 (31) [M]?; 462 (24); 446 (26); 249 (20); 205
(100); 138 (20); 111 (37). Anal. Calcd. for C20H28N8O8: C,
47.24; H, 5.55; N, 22.04. Found: C, 47.33; H, 5.42; N,
21.89.
1H), 4.94 (d, 2H, J = 11.9 Hz), 7.17–7.27 (m, 5H, aro-
matic hydrogens), 7.98 (s, 1H), 8.73 (d, 1H, J = 8.08 Hz,
NH), 12.87 (bs, 1H). 13C-NMR (DMSO-d6): d = 14.1
(CH3), 37.3 (CH2), 48.1 (CH2), 54.1 (CH), 127.0 (CH),
128.7 (CH); 129.6 (CH); 132.8 (CH), 137.7 (C), 139.0 (C),
152.2 (C), 165.9 (C), 172.9 (C). HRMS (EIMS) m/z: calcd.
for C15H15N4O6 [M-H]? 331.10425, found 331.10414.
Anal. Calcd. for C15H16N4O5: C, 54.21; H, 4.85; N, 16.86.
Found: C, 54.32; H, 4.76; N, 16.81.
2-(2-(2-Methyl-5-nitro-1H-imidazol-1-
yl)acetamido)succinic acid (5d)
1
Syntehsis 2- or 3-substituted 2-(2-(2-methyl-5-nitro-
1H-imidazol-1-yl)acetamido)substituted propanoic acid
(5b–g)
Yield 1.80 g (35%); Mp 215–217°C. H-NMR (DMSO-
d6): d = 2.32 (s, 3H), 3.19 (d, 2H, J = 6.9 Hz), 4.51 (t,
1H), 5.01 (s, 2H), 7.98 (s, 1H), 8.69 (d, 1H, J = 7.7 Hz),
12.68 (bs, 2H). 13C-NMR (DMSO-d6): d = 14.2 (CH3),
36.6 (CH2), 48.3 (CH2), 48.8 (CH), 132.8 (CH), 138.0
(CH), 153.1 (CH), 166.5 (CH), 172.5 (CH), 172.1 (CH).
HRMS (EIMS) m/z: calcd. for C10H11N4O6 [M-H]?
283.06786, found 283.06821. Anal. calcd. for C10H12N4O6:
C, 42.26; H, 4.26; N, 19.71. Found: C, 42.19; H, 4.19, N,
19.63.
These compounds were synthesized via base-pormoted
hyrolysis of their corresponding esters followd by acidifi-
cation with HCl (6 M). The following general procedure
was employed in the synthesis: each of compounds 4b–
g (5.50 mmol) was dissolved in 4% NaOH (40 ml) and
stirred overnight. After the completion of the reaction, the
solution was acidified with HCl (3 M) (4.5 \ pH \ 6.5).
The precipitate was collected by filtration, washed with
distilled water, dried in vacuum and purified using silica
gel plates (CHCl3:MeOH 95: 5) to obtain yellowish crys-
tals. The structures of compounds were confirmed by
NMR, mass spectrometry, and elemental analysis. The
following compounds were synthesized using the afore-
mentioned general procedure.
3-(4-Hydroxyphenyl)-2-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl) acetamido)propanoic acid (5e)
Yield 2.00 g (46%); Mp 254–256°C. [a]DMF = ?25.2o. 1H-
NMR (DMSO-d6): d = 2.23 (s, 3H), 2.70–2.95 (m, 2H),
4.31–4.37 (m, 1H,), 4.95 (d, 2H, J = 9.68 Hz), 6.63 (d, 2H,
J = 8.24 Hz), 6.97 (d, 2H, J = 8.22 Hz), 7.98 (s, 1H),
8.66 (d, 1H, J = 7.96 Hz); 9.20 (s, 1H), 12.81 (bs, 1H,
OH). 13C-NMR (DMSO-d6): d = 14.0 (CH3), 36.6 (CH2),
48.1 (CH2), 54.4 (CH), 115.4 (CH), 127.7 (C), 130.6 (CH),
132.8 (CH), 139.2 (C), 152.2 (C), 156.5 (C), 165.9 (C),
173.0 (C). MS (70 eV) (C15H16N4O6), m/z (% rel. Int.):
348 (6) [M]?; 330 (2); 242 (11); 185 (75); 164 (77); 128
(35); 108 (49); 77 (73); 53 (100); 28 (88). HRMS (EIMS)
m/z: calcd. for C15H15N4O6 [M-H]? 347.09971 found
347.09971. Anal. Calcd. for C15H16N4O6: C, 51.72; H,
4.63; N, 16.09. Found: C, 51.83; H, 4.68; N, 16.21.
2-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetamido)
propanoic acid (5b)
1
Yield 1.50 g (90%); Mp 183–185°C. H-NMR (DMSO-
d6): = 1.25 (d, 3H, J = 6.85 Hz), 2.47 (s, 3H), 4.15–4.20
(m, 1H), 4.97 (d, 2H, J = 4.80 Hz), 7.97 (s, 1H); 8.70 (d,
1H, J = 6.60 Hz). 13C-NMR (DMSO-d6): d = 14.0 (CH3),
17.7 (CH3), 48.2 (CH2), 48.4 (CH), 132.6 (CH), 139.0 (C),
152.3 (C), 166.0 (C); 174.2 (C). MS (70 eV) C9H12N4O5,
m/z (% rel. Int.): 239 (24); 224 (2); 210 (100); 164 (29);
136 (35); 111 (65); 94 (28); 80 (74); 51 (70); 28 (85); 18
(60). HRMS (EIMS) m/z: calcd. for C9H11N4O5 [M-H]?
255.07295 found 255.07349. Anal. Calcd. for C9H12N4O5:
C, 42.19; H, 4.72; N, 21.87. Found: C, 42.05; H, 4.86; N,
21.99.
3-(1H-indol-3-yl)-2-(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)acetamido)propanoic acid (5f)
Yield 2.10 g (94%); Mp 174–176°C. 1H-NMR (DMSO-d6)
d (ppm): 2.19 (s, 3H), 3.03–3.13 (m, 2H), 4.51–4.57 (m,
1H), 4.98 (d, 2H, J = 10.81 Hz), 6.92–7.05 (m, 2H), 7.13
(s, 1H), 7.29 (d, 1H, J = 7.87 Hz); 7.49 (d, 1H,
J = 7.43 Hz), 7.98 (s, 1H), 8.68 (s, 1H), 10.91 (s, 1H).
13C-NMR (DMSO-d6): d = 14.0 (CH3), 27.9 (CH2), 48.3
(CH2), 54.0 (CH), 110.2 (C), 111.8 (CH), 118.7 (CH),
118.9 (CH), 121.4 (CH), 124.1 (CH), 127.8 (C), 132.7
2-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetamido)-
3-phenylpropanoic acid (5c)
1
Yield 1.90 g (81%); Mp 230–232°C. H-NMR (DMSO-
d6): d = 2.21 (s, 3H), 2.86–3.06 (m, 2H), 4.43–4.47 (m,
123

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Med Chem Res (2012) 21:1700–1707
(CH), 136.5 (C), 139.0 (C), 152.2 (C), 165.8 (C), 172.3 (C).
HRMS (EIMS) m/z: calcd. for C17H16N5O5 [M-H]?
370.11515, found 370.11553. Anal. Calcd. for C17H17
N5O5: C, 54.98; H, 4.61; N, 18.86. Found: C, 54.85; H,
4.66; N, 18.76.
were incubated on a 158 horizontal slant at 36–37°C. Culture
maintenance and sub-culturing was performed as described
in a previous publication (Saadeh et al., 2009). Entamoeba
and Giardia were harvested from confluent cultures by
chilling of the tubes on ice for 5–10 min to detach cells,
followed by centrifugation at 8009g for 5 min.
3-(1H-imidazol-4-yl)-2-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)acetamido)propanoic acid (5g)
Antiamoebic and antigiardial activity assay
1
Yield 1.85 g (32%); Mp 250–253°C). H-NMR (DMSO-
The antiamoebic and antigiardial activities of the prepared
compounds and metronidazole (the reference drug) were
tested as previously described (Saadeh et al., 2009).
Briefly, the prepared compounds and metronidazole were
separately dissolved in dimethyl sulfoxide (DMSO) then in
medium and filter sterilized. Two-fold dilutions ranging
from 0.1 to 60 lg/ml were prepared in a final volume of
15 ml to exclude air from the tube. Each tube was inocu-
lated with 20,000 cells of the parasite under investigation.
Each compound was assayed in duplicates in each of three
independent experiments. In each assay, the appropriate
controls were performed, including the one without any of
the prepared compounds or metronidazole and another with
metronidazole as the positive control. The biological
activity of the compounds was evaluated by counting the
parasites in each tube using the standard hemacytometer. In
each count, trypan blue was employed to distinguish live
from dead parasites (Aley et al., 1994). The 50% inhibitory
concentration (IC50) was employed as a parameter for the
antiamoebic and antigiardial activity. The IC50 is the
concentration of tested compound or metronidazole which
cuts the number of parasites to half that in the negative
control (growth medium ? DMSO ? parasites).
d6): d = 2.25 (s, 3H), 2.78–2.99 (m, 2H), 4.18–4.22 (m,
1H), 5.00 (d, 2H, J = 7.98 Hz), 6.72 (s, 1H), 7.47 (s, 1H),
7.96 (s, 1H), 8.41 (m, 1H), 11.84 (bs, 1H). 13C-NMR
(DMSO-d6): d = 14.1 (CH3), 29.7 (CH2); 48.3 (CH2); 54.6
(CH), 119.2 (CH), 132.7 (CH), 134.8 (CH), 139.1 (C),
152.2 (C), 165.2 (C), 174.3 (C), 182.3 (C). HRMS (EIMS)
m/z: calcd. for C12H13N6O5 [M-H]? 322.09474, found
322.09451. Anal. Calcd. for (C12H14N6O5): C, 44.72; H,
4.38; N, 26.82. Found: C, 44.67; H, 4.34; N, 26.73.
Synthesis of 2,6-bis(2-(2-methyl-5-nitro-1H-imidazol-
1-yl)acetamido)hexanoic acid (8)
The title compound was prepared, as given in Scheme 2,
by dissolving compound 7 (2.80 g, 5.50 mmol) in 4%
NaOH (40 ml) and the solution was stirred overnight at
room temperature. The solution was then acidified with
HCl (3 M) at pH 4.5, and the precipitate was collected by
filtration, washed with distilled water, dried in vacuum, and
purified using silica gel plates (CHCl3:MeOH 95:5) to
obtain yellowish crystals. (1.40 g, 54%, m.p. 220–221°C).
Yield 1.40 g, (54%); Mp 220–221°C. 1H-NMR (DMSO-
d6): d = 1.23–1.46 (m, 4H), 1.51–1.82 (m, 2H), 2.33 (s, 6H),
2.93–3.14 (m, 2H), 4.14-4.19 (m, 1H), 4.91 (s, 2H), 5.01 (d,
2H, J = 7.95 Hz), 7.99 (s, 2H), 8.36 (t, 1H), 8.70 (d, 1H,
J = 7.78 Hz). 13C-NMR (DMSO-d6): d = 14.2 (CH3), 14.2
(CH3), 23.0 (CH2), 28.9 (CH2), 31.1 (CH2), 39.0 (CH2), 48.2
(CH2), 48.2 (CH2), 52.4 (CH), 132.7 (CH), 139.0 (C),
152.3 (C), 165.8 (C); 166.2 (C), 173.7 (C). HRMS (EIMS)
m/z: calcd. for C18H23N8O8 [M-H]? 479.16389, found
479.16353. Anal. Calcd. for C18H24N8O8: C, 45.00; H, 5.04;
N, 23.32. Found: C, 45.18; H, 4.95; N, 23.43.
Cytotoxicity assay
The cytotoxicity of the prepared compounds and metroni-
dazole was investigated on Hep-2 and Vero cells using the
standard cytotoxicity assay and the trypan blue exclusion
method as described before (Saadeh et al., 2009). Briefly,
100 ll portions of each cell suspension containing 105
cells/ml RPMI medium-10% heat inactivated fetal calf
serum (FCS) were added to the wells of 96-well plates,
incubated for 24 h, and the RPMI-FCS medium in each
well was then replaced with 150 ll fresh medium. Solu-
tions of the compounds and metronidazole were dissolved
in DMSO, prepared in the RPMI-FCS medium, and filter
sterilized. Then, 150 ll two-fold serial dilutions of each of
the compounds and the reference drug starting at a con-
centration of 500 lg/ml in RPMI-FCS medium were pre-
pared in the plates. After 48 h incubation, the number of
cells in each well was determined using a hemacytometer.
Each tested compound or metronidazole was assayed in
duplicate in each of the three independent experiments. In
Biological activity
Test organisms
Entamoeba histolytica HK-9 strain (ATCC number 30015)
was cultured in LYI-S-2 medium supplemented with anti-
biotics. Giardia intestinalis WB strain (ATCC number
30957) was grown in a modified YI-S medium with antibi-
otics. Both parasites were cultivated in 15 ml screw-capped
borosilicate glass tubes containing 13 ml medium. The tubes
123

Med Chem Res (2012) 21:1700–1707
1707
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