Synthesis, crystal structure, and enhancement of the efficacy of metronidazole against Entamoeba histolytica by complexation with palladium(II), platinum(II), or copper(II)

Article abstract of DOI:10.1002/1522-2675(200209)85:9<2704::AID-HLCA2704>3.0.CO;2-X

Reaction of trans-[PdCl₂(DMSO)₂], cis-[PtCl₂(DMSO)₂], and [Cu(OAc)₂]·H₂O with metronidazole (mnz) leads to the formation of new complexes, i.e., trans-[PdCl₂(mnz)₂] (1), trans-[PtCl₂(mnz)₂], (2), and trans-[Cu₂(OAc)₄(mnz)₂] (3), respectively. Complexes 1-3 crystallize all in the centrosymmetric monoclinic space group P2₁/c with Z=8. Unit-cell parameters for these complexes are: 1, a=7.1328(14) A, b=20.699(4) A, c= 7.1455(14) A, and β=116.17(3)°; 2, a=6.9169(14) A, b=21.853(4) A, c=6.7218(13) A, and β=110.79(3)°; 3, a=9.1663(18) A, b=19.129(4) A, c=8.9446(18) A, and β=116.44(3)°. The complexes 1 and 2 maintain an ideal square-planar geometry. In complex 3, the H₂O molecules of the starting complex are replaced by metronidazole while maintaining a dimeric structure of [Cu(OAc)₂]. Each Cu ion has an ideal octahedral structure, though distortion occurs in the equatorial position where the acetato ligands are attached. The Cu-Cu separation of 2.6343(8) A indicates considerable metal-metal interaction. The testing of the antiamoebic activity of these complexes against the protozoan parasite Entamoeba histolytica suggests that compound 1-3 might be endowed with important antiamoebic properties since they showed IC₅₀ values in a μM range better than metronidazole (Table 2). Thus, compound 1 displayed more effective amoebicidal activity than metronidazole (IC₅₀ values of 0.103 μM vs. 1.50 μM, resp.).

Full text of DOI:10.1002/1522-2675(200209)85:9<2704::AID-HLCA2704>3.0.CO;2-X

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Helvetica Chimica Acta Vol. 85(2002)
Synthesis, Crystal Structure, and Enhancement of the Efficacy of
Metronidazole Against Entamoeba histolytica by Complexation with
Palladium(II), Platinum(II), or Copper(II)
by Neelam Bhartia), Shailendraa), Simon J. Colesb), Michael B. Hursthouseb), Thomas A. Mayerb),
M. T. Gonzalez Garzac), Delia E. Cruz-Vegac), Benito D. Mata-Cardenasc), Fehmida Naqvia),
Mannar R. Mauryad), and Amir Azama)*
a) Department of Chemistry, Jamia Millia Islamia, New Delhi-110025, India
b) Department of Chemistry, EPSRC National Crystallography Service, University of Southampton, Highfield,
Southampton, U.K.
c) Division de Biologia Celular y Molecular, Centro de Investigacion Biomedica del Noreste, IMSS, Monetary,
NL, Mexico
d) Department of Chemistry, Indian Institute of Technology, Roorkee, Uttranchal, India
Reaction of trans-[PdCl2(DMSO)2], cis-[PtCl2(DMSO)2], and [Cu(OAc)2] ¥ H2O with metronidazole
(mnz) leads to the formation of new complexes, i.e., trans-[PdCl2(mnz)2] (1), trans-[PtCl2(mnz)2] (2), and trans-
[Cu2(OAc)4(mnz)2] (3), respectively. Complexes 1 3 crystallize all in the centrosymmetric monoclinic space
group P21/c with Z ˆ 8. Unit-cell parameters for these complexes are: 1, a ˆ 7.1328(14) ä, b ˆ 20.699(4) ä, c ˆ
7.1455(14) ä, and b ˆ 116.17(3)8; 2, a ˆ 6.9169(14) ä, b ˆ 21.853(4) ä, c ˆ 6.7218(13) ä, and b ˆ 110.79(3)8; 3,
a ˆ 9.1663(18) ä, b ˆ 19.129(4) ä, c ˆ 8.9446(18) ä, and b ˆ 116.44(3)8. The complexes 1 and 2 maintain an
ideal square-planar geometry. In complex 3, the H2O molecules of the starting complex are replaced by
metronidazole while maintaining a dimeric structure of [Cu(OAc)2]. Each Cu ion has an ideal octahedral
structure, though distortion occurs in the equatorial position where the acetato ligands are attached. The
CuÀCu separation of 2.6343(8) ä indicates considerable metal-metal interaction. The testing of the
antiamoebic activity of these complexes against the protozoan parasite Entamoeba histolytica suggests that
compound 1 3 might be endowed with important antiamoebic properties since they showed IC50 values in a mm
range better than metronidazole (Table 2). Thus, compound 1 displayed more effective amoebicidal activity
than metronidazole (IC50 values of 0.103 mm vs. 1.50 mm, resp.).
Introduction.
Entamoeba histolytica (E. histolytica), the cause of amoebic
dysentery and amoebic liver abscesses, remains a significant threat to health in large
parts of the world. More than 50 million people are affected, and only malaria and
schistosomiasis surpass amoebiasis as a parasitic cause of death [1]. It is responsible for
100000 deaths annually [2]. The morbidity and mortality associated with amoebiasis
have persisted despite the availability of effective antiamoebic therapy, suggesting a
need for alternative measures of disease control. Nitroimidazole drugs such as
metronidazole are highly effective against the acute disease but are relatively
ineffective in eradicating parasites from the gut lumen [3]. Moreover, tolerance of
these drugs is often poor and the mutagenic effects of metronidazole in bacteria have
raised fear that the drug may be carcinogenic in man [4], although no evidence of
carcinogenicity was found when exploratory studies were carried out [5][6]. To date,
there have been a few reports of resistance of E. histolytica to metronidazole, but the
possibility of this developing must be borne in mind [7], especially as emetine-resistant
mutants have been isolated in the laboratory [8]. Metal ions are known to accelerate

Helvetica Chimica Acta Vol. 85(2002)
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drug action, and the efficacy of a therapeutic agent may be enhanced upon
coordination with a metal ion [9]. Many neutral palladium(II) and palladium(IV)
complexes were found to exhibit potential antitumor activity [10][11]. Copper(II) is a
biologically active essential ion; its chelating ability and positive redox potential allow
its participation in biological transport [12]. Some metal complexes have been proved
to be active against trypanosoma [13 15], leishmania [14], and malaria [16]; their
potential as antiamoebic agents has so far been little explored [17 19].
In this paper, we present a novel approach towards the development of chemo-
therapy against amoebiasis, consisting of the modification of metronidazole (ˆ2-
methyl-5-nitro-1H-imidazole-1-ethanol; mnz) by coordination with PdII, PtII, and CuII.
These new complexes are active against E. histolytica in vitro and in vivo. This study
establishes the potential for developing new antiamoebic drugs that are more effective
than mnz and which may prove to be useful clinical alternatives to mnz.
Results and Discussion.
Synthesis and Characterization of the Complexes.
Reaction of mnz with trans-[PdCl2(DMSO)2] [20], cis-[PtCl2(DMSO)2] [20], and
[Cu(OAc)2] ¥ H2O in refluxing MeOH resulted in the formation of trans-[PdCl2(mnz)2]
(1), trans-[PtCl2(mnz)2] (2), and trans-[Cu2(OAc)4(mnz)2] (3), respectively. Whether
the starting material was cis- or trans-configured, the resulting complexes were trans
only (vide infra) under the reaction conditions. These complexes were purified by
recrystallization. All complexes 1 3 are stable in air, soluble in coordinating solvents
such as MeOH, EtOH, DMSO, and DMF, and insoluble in noncoordinating solvents
like C6H6, CHCl3, and CH2Cl2. Besides elemental analysis, all the new complexes were
well-characterized by UV/VIS, IR, and 1H-NMR spectroscopy and by crystal-structure
analysis. Thermogravimetric analysis of the complexes showed them to be very stable
and to decompose above 2008 in two or three steps. These decomposition steps
overlapped, and it was impossible to judge exactly which group was lost. The IR spectra
of the complexes exhibit a sharp band at 1558 1575 cmÀ1 due to nÄ(CˆN) (ring); a
positive shift DnÄ ˆ 21 38 cmÀ1 with respect to the corresponding absorption of the free
ligand indicates the coordination of the ring N-atom to the metal ion. The alcoholic
group remains free and does not participate in bond-formation as nÄ(CÀO) (alcohol)
appears at a nearly constant position of 1187 cmÀ1 in both mnz and its complexes. To
determine the coordination geometry of the Pd and Pt complexes, far-IR studies were
undertaken; thus, both complexes 1 and 2 exhibit a single MÀCl stretch at 374 and
361 cmÀ1, respectively. The appearance of a single IR-active band indicates that the two
ClÀ ligands of 1 and 2 are trans to each other. In addition to this, all complexes exhibit a
broad band due to nÄ (OH) at ca. 3400 cmÀ1. This is further supported by the 1H-NMR
data of 1 3 (t at d ca. 6.0 due to free OH; moreover d 4.36 4.49 (t) and 3.56 3.71 (q)
for CH2Cl2). The structures of complexes 1 3 were confirmed by X-ray-diffraction
studies.
Crystal Structures of Complexes 1 3. The Pd and Pt complexes 1 and 2 crystallized
i
from PrOH/MeOH and the Cu complex 3 from dimethylsulfoxide (DMSO)/MeOH.
The molecular structures and atomic numbering for 1 3 are shown in Figs. 1 3.
Selected bond distances and angles for all complexes are summarized in Table 1.
Complexes 1 and 2 are isostructural, exhibiting similar unit-cell volumes and
crystallizing in the same space group. Moreover, an overlap plot of the two molecules

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Helvetica Chimica Acta Vol. 85(2002)
Fig. 1. Molecular structure and atomic numbering (arbitrary) for complex 1. Ellipsoids at 30% probability.
Fig. 2. Molecular structure and atomic numbering (arbitrary) for complex 2. Ellipsoids at 30% probability.
shows all ligand substituents to be in the same orientation. The molecules are
centrosymmetric and, hence, the metal ions have trans geometries. The central atoms
Pd and Pt both have an ideal square-planar coordination geometry with MÀN and
MÀCl distances in accordance with reported ones [21]. The mnz ligands exhibit the
expected geometries. The imidazole rings form dihedral angles of 85.98(5) and
73.73(2)8 with the ClÀMÀCl plane for 1 and 2, respectively, indicating that the rotation
of the mnz ligand about the MÀN bond is more restricted in the Pd complex 1. Both

Helvetica Chimica Acta Vol. 85(2002)
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Fig. 3. Molecular structure and atomic numbering (arbitrary) for complex 3. Ellipsoids at 30% probability.
Table 1. Selected Bond Lengths [ä] and Angles [8] for Complexes 1
Complex 2
3
Complex 1
Pd(1)ÀN(1)
Pd(1)ÀCl(1)
2.0054(17) Pt(1)ÀN(1)
2.006(4)
2.292(12)
89.71(11)
2.2980(6)
Pt(1)ÀCl(1)
N(1)ÀPd(1)ÀCl(1) 90.11(5)
Complex 3
N(1)ÀPt(1)ÀCl(1)
Cu(1)ÀO(4)
Cu(1)ÀO(5)
Cu(1)ÀO(6)
Cu(1)ÀO(7)
Cu(1)ÀN(1)
Cu(1)ÀCu(1')
1.971(2)
1.971(2)
1.963(3)
1.971(2)
2.152(3)
2.6343(8)
O(4)ÀCu(1)ÀO(5) 168.32(10)
O(5)ÀCu(1)ÀN(1)
95.62(10)
O(4)ÀCu(1)ÀO(6)
O(4)ÀCu(1)ÀO(7)
O(4)ÀCu(1)ÀN(1)
O(5)ÀCu(1)ÀO(6)
O(5)ÀCu(1)ÀO(7)
89.87(11)
91.66(10)
96.05(10)
89.27(11)
86.75(10)
O(6)ÀCu(1)ÀO(7) 167.66(9)
O(6)ÀCu(1)ÀN(1)
O(7)ÀCu(1)ÀN(1)
96.52(10)
95.50(10)
structures adopt the same three-dimensional supramolecular assembly, forming classic
OÀH ¥¥¥ Cl and CÀH ¥¥¥ O H-bonds (O(1)ÀH(1) ¥¥¥ Cl(1) and C(6)ÀH(6) ¥¥¥ O(1)
D ¥¥¥ A distance ˆ 3.166(2) and 3.189(3) ä for Pd and 3.172(4) and 3.271(6) ä for Pt,
resp.).
The copper complex 3, with the same ligand mnz in the presence of acetate forms a
dimer through a CuÀCu interaction and four bridging m2-acetato ligands. The structure
is centrosymmetric with the Cu-centers forming a distorted octahedral coordination
geometry. This distortion occurs only in the equatorial positions, which are occupied by
the bridging acetato ligands whose donor groups are not sufficiently separated to span
the CuÀCu bond and form ideal octahedral geometry. The mnz ligands occupying the
apical coordination sites exhibit the expected geometry, and the orientation of the
imidazole ring with respect to the Cu-coordination sphere is determined by an

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intramolecular interaction between the Me group at C(1) (arbitrary numbering) and an
O-donor of one of the acetato bridges (D ¥¥¥ A for C(2)ÀH(2C) ¥¥¥ O(6) ˆ 3.185(3) ä).
A three-dimensional network in the crystal structure is formed by a number of
intermolecular interactions (O(1)ÀH(1) ¥¥¥ O(5) ˆ 2.829(4) ä, C(3)ÀH(3A) ¥¥¥
O(7) ˆ 3.103(6) ä, C(3)ÀH(3B) ¥¥¥ O(4) ˆ 3.428(4) ä, and C(8)ÀH(8a) ¥¥¥ O(1) ˆ
3.421(3) ä).
Biological Activity. In vitro antiamoebic activity of complexes 1 3 was determined
with the HM1:1MSS strain of E. histolytica. The biological tests were carried out with
DMSO as the solvent, in which the complexes are stable. The NMR spectra of 1 3 in
(D6)DMSO remained unchanged at room temperature for several days, showing no
evidence of displacement of the mnz ligand by the solvent. The maximum
concentration of DMSO in the test did not exceed 0.1%, at which level no inhibition
of amoebal growth occurred [22]. Two-fold serial dilutions were made in either the free
ligand mnz or the corresponding complexes 1 3 in 96-well microtiter plates and were
incubated for 72 h. Cell viability was assessed, and the concentration of drug for 50%
inhibition (IC50) was determined by the microdilution method [23]. The effect of
assayed compounds on E. histolytica growth is summarized in Table 2. Experiments
were repeated thrice and the IC50 value was estimated to be 1.36 1.64 mm for mnz,
whereas that of complex 1 was in the range of 0.085 0.148 mm. The ratio of IC50 of
complexes 1 3 to mnz were 14-, 7-, and 13-fold, respectively, which indicates that
complexes 1 3 are manyfold more active than mnz. The IC50 values for the Pd-, Pt-,
and Cu-complex precursors were also determined establishing that these metal-
complex precursors have no activity against E. histolytica (Table 3).
Table 2. IC50 Values Obtained for Compounds 1 3 and Free Ligand mnz against E. histolytica (HM1 : 1MSS)
IC50 [mm]a)
Ratio
14.6
Complex 1
Complex 2
Complex 3
mnz
0.10 Æ 0.03
0.20 Æ 0.057.6
0.11 Æ 0.04
1.5 Æ 0.2
13.3
a) Mean Æ 2 s.d.
Table 3. IC50 Values obtained for Metal-Complex Precursors and Free Ligand mnz against E. histolytica
(HM1 : 1MSS)
IC50 [mm]a)
Ratio
[PdCl2[(DMSO)2]
[PtCl2(DMSO)2]
[Cu(OAc)2] ¥ H2O
mnz
8.0 Æ 1.2
9.3 Æ 1.7
7.7 Æ 1.4
1.6 Æ 0.3
0.20
0.18
0.21
a) Mean Æ 2 s.d.
In a further series of experiments, the in vivo antiamoebic activity of complexes 1 3
was assayed in male golden hamsters infected by amoeba to produce a systematic
experimental amoebic hepatic abscess (EAHA). To increase the parent-strain
virulence, the amoebic cultures were subjected to four liver passages and in vitro

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culture cycles. All compounds were administered orally. Effectiveness as an anti-
amoebic agent was determined as the proportion of score reduction relative to
untreated controls as shown in Fig. 4. Treatment with 0 30 mg kgÀ1 of compounds 1 3
and mnz for 5days administration did not show a clear dose-dependent effect.
Treatment with 0 30mg kgÀ1 of each compound for 10 days produced a reduction of
71 92% (1), 49 71% (2), 58 86% (3), and 40 58% (mnz) in the EAHA score (P ˆ
0.001 compared with controls). All compounds at 10 mg kgÀ1 produced an effect
quantitatively comparable with that produced by 30 mg kgÀ1 of mnz. Compound 1 at
5mg kg À1 produced a reduction in EAHA that was slightly higher (4%) than the effect
of 30 mg kgÀ1 of mnz.
Fig. 4. Effect of mnz and complexes 1 3 on E. histolytica. The average scores of nontreated control and
samples treated with 30 mg of compound/kg of body weight of golden hamster are shown. Scores were
determined after 10 days of treatment; 1, control; 2, mnz; 3, complex 1; 4, complex 2; 5, complex 3.
These results clearly demonstrate that the (metronidazole)metal complexes 1 3
are potent inhibitors of the development of E. histolytica both in vitro and in vivo and
that they are more active than the standard parent compound mnz.
Conclusions. The metronidazole complexes of transition metals Pd, Pt, and Cu
were synthesized by direct reaction of the free mnz with appropriate metal-complex
precursors. In vitro and in vivo tests against E. histolytica showed that the incorporation
of a metal ion generally produced an enhancement of the efficacy of mnz.
Experimental Part
1. General. All solvents were purified prior to use by standard methods. [Cu(OAc)2] ¥ H2O (E. Merck),
PdCl2, K2PtCl4, and metronidazole (ˆ mnz; Aldrich) were used. trans-[PdCl2(DMSO)2] [20] and cis-
[PtCl2(DMSO)2] [20] were prepared according to the literature, and their purity was checked by UV/VIS,

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IR, and 1H-NMR spectra. UV/VIS Spectra: ShimadzuUV-1601-PC -UV-VIS spectrophotometer; lmax in nm. IR
Spectra: Perkin-Elmer 1600-FT-IR (KBr pellet in the 4000 400 cmÀ1 range) and Perkin-Elmer 983
1
spectrometer with DS system (CsI pellet in the 600 150 cmÀ1 range); nmax in cmÀ1. H-NMR Spectra: Bruker
DPX-300 spectrometer; (D6)DMSO solns.; d in ppm rel. to SiMe4 as standard reference. Thermogravimetric
analyses of the complexes were performed under N2 with a TG-51 thermogravimetric analyser, with the heating
rate of 108/min. Elemental analysis was performed by RSIC, Central Drug Research Institute, Lucknow, India.
2. [SP-4-1]-Dichlorobis(2-methyl-5-nitro-1H-imidazole-1-ethanol-kN3)palladium (trans-[PdCl2(mnz)2]; 1)
and [SP-4-1]Dichlorobis(2-methyl-5-nitro-1H-imidazole-1-ethanol-kN3)platinum) (trans-[PtCl2(mnz)2]; 2). A
soln. of mnz (684 mg, 4 mmol) in MeOH (20 ml) was added to a soln. of [PdCl2(DMSO)2] (665mg, 2 mmol) or
[PtCl2(DMSO)2] (844 mg, 2 mmol) in MeOH (20 ml) with stirring, and the mixture was refluxed at 808 for 4 h.
After concentration to ca. 10 ml, the mixture was kept overnight in the refrigerator at 108. The crystalline solid
i
obtained was filtered off, washed with cold MeOH (2 Â 5ml), dried in vacuo, and recrystallized from Pr-OH/
MeOH 4 :6.
Data of 1: Yield 63%. Greenish-orange crystals. UV/VIS: 318. IR: 1559 (CˆN), 1190 (CÀO), 374
(PdÀCl). 1H-NMR (CD3)2SO): 3.06 (s, 2 Me); 3.71 (q, 2 CH2); 4.44 (t, 2 CH2); 5.13 (t, 2 OH); 8.29
(s, 2 arom. H). Anal. calc. for C12H18Cl2N6O6 Pd: C 27.74, H 3.46, N 16.18; found: C 27.96, H 3.63, N 16.04.
Data of 2: Yield 57%. Yellow-orange crystals. UV/VIS: 310. IR: 1558 (CˆN), 1187 (CÀO), 361 (PtÀCl).
1H-NMR (CD3)2SO): 3.0 (s, 2 Me); 3.71 (q, 2 CH2); 4.49 (t, 2 CH2); 5.16 (t, 2 OH); 8.24 (s, 2 arom. H). Anal.
calc. for C12H18Cl2N6O6 Pt: C 23.68, H 2.96, N 13.71; found: C 23.80, H 3.15, N 13.47.
3. Tetrakis[m-(acetato-kO : kO')bis(2-methyl-5-nitro-1H-imidazole-1-ethanol-kN3)dicopper ([Cu2(m-
OAc)4(mnz)2]; 3). A soln. of [Cu(OAc)2] ¥ H2O (400 mg, 2 mmol) in MeOH (50 ml) was added with stirring
to mnz (342 mg, 2 mmol) in MeOH (15ml). The mixture was stirred at r.t. for 48 h. On concentration of the
mixture, greenish-blue crystals precipitated, which were filtered off, washed with cold MeOH, dried in vacuo,
and recrystallized from MeOH/DMSO 9 :1: 3 (63%). Greenish-blue crystals. UV/VIS: 317. IR: 1548 (CˆN),
1189 (CÀO). Anal. calc. for C20H32Cu2N6O14: C 34.09, H 4.26, N 11.93; found: C 34.26, H 4.46, N 11.90.
4. Crystal Structures of Complexes 1 3. Crystals of 1 3 were mounted on glass fibers by means of the oil-
drop technique, and intensity data were collected at 150 K. A summary of crystal data, data collection strategy,
and mode-refinement parameters are given in Table 4. Data were measured on a Nonius Kappa CCD area-
detector diffractrometer with a rotating anode molybdenum (l(Mo-Ka) 0.71073 ä) target, controlled by
DENZO and COLLECT [24] software. The structures were solved via direct methods and then subjected to
full-matrix least-squares refinement on Fo2 with the SHELX-97 [25] suite of programs.
For all structures, non-H-atoms were made anisotropic, with H-atoms in calculated positions (CÀH ˆ
0.96 ä, with Uiso tied to Ueq of the parent atoms) for structures 2 and 3, whilst H-atom parameters were freely
refined for structure 1. Absorption corrections were performed by means of comparison of symmetry-related
and equivalent reflections with the method employed in SORTAV [26]; however, due to the large size of the
crystal of 3, some large residual-density peaks remained around the Cu center. Moreover, the alcohol group at
C(4) (arbitrary numbering) of complex 3 was disordered over two sites (in an 80 :20 ratio) with a fractionally
occupied H-atom completing the site requirements. Full crystallographic data for 1 3 have been submitted to
CCDC (deposition Nos. 155790 155792).
5. Organism Culture and in vitro Testing against E. histolytica. Preliminary experiments were carried out to
determine the antiamoebic activities of the in vitro culture against the HM1 :1MSS strain of E. histolytica as
previously described [23]. The E. histolytica strain HM1 :1MSS was cultured in tubes (15ml) with Diamond
TYIS-33 medium [27]. All the compounds were dissolved in DMSO (40 ml) [22][28], followed by adding enough
culture medium to obtain a concentration of 1 mg/ml. Stock solutions of the compounds were prepared freshly
before use at a concentration of 0.1 mg/ml. Two-fold serial dilutions were made in the wells of 96-well microtiter
plate (Costar). Each test included mnz as a standard amoebicidal drug, with control wells (culture medium plus
amoebae) and a blank (culture medium only). The amoebae suspension was prepared from a confluent culture
by pouring off the medium at 378 and adding 5ml of fresh medium, chilling the culture tube on ice to detach the
organisms from the side of the flask. The number of amoeba/ml was estimated with a heamocytometer, by
means of trypan-blue exclusion to confirm the viability. The suspension was diluted to 105 organism/ml by adding
fresh medium, and 170 ml of this suspension was added to the test and control wells in the plate so that the wells
were completely filled (total volume, 340 ml). An inoculum of 1.7 ¥ 104 organisms/well was chosen so that
confluent, but not excessive growth, took place in control wells. Plates were sealed and gassed for 10 min with N2
before incubation at 378 for 72 h. After incubation, the growth of amoebae in the plate was checked with a low-
power microscope. By inverting the plate, the culture medium was removed with gentle shaking and then
immediately washed with 0.9% NaCl soln. at 378. This procedure was completed quickly, and the plate was not

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Table 4. Crystal Data and Structure-Refinement Details for Compounds 1
3
3
1
2
Empirical formula
Mr
C12H18Cl2N6O6Pd
519.62
C12H18Cl2N6O6Pt
608.31
C20H30CuN6O14
352.79
Crystal size [mm]
Crystal system
Space group
0.22 Â 0.2 Â 0.050.2
Monoclinic
P21/c
 0.2  0.2
Monoclinic
P21/c
0.33 Â 0.3 Â 0.25
Monoclinic
P21/c
a [ä]
b [ä]
c [ä]
7.1328(14)
20.699(4)
7.1455(14)
116.17(3)
946.8(3)
6.9169(14)
21.853(4)
6.7218(13)
110.79(3)
949.9(3)
2
9.1663(18)
19.129(4)
8.9446(18)
116.44(3)
1404.3(5)
2
b [8]
Volume [ä3]
Z
2
Density (calc.) [Mg/m3]
Absorption coefficient [mmÀ1
F(000)
1.823
1.3057.710 92
520
2.127
1.669
]
1.5
584
724
qmax
8
27.51
10091
21652123
0.0487
27.49
4908
3137
0.0439
27.45
10714
Reflections collected
Independent reflections
R (int)
0.0748
Transmission factors
Final R indices
F2 > 2sF2
0.9376, 0.7578
R1 ˆ 0.0229
wR2 ˆ 0.0538
(1872 reflections)
0.786, À0.716
0.3077, 0.3077
R1 ˆ 0.0433
wR2 ˆ 0.1192
(1832 reflections)
3.764, À3.210
0.6916, 0.6256
R1 ˆ 0.0555
wR2 ˆ 0.1507
(2718 reflections)
2.229, À0.856
D1 max, min [eäÀ3
]
allowed to cool to prevent the detachment of amoebae. It was allowed to dry at r.t. After drying, amoebae were
fixed with chilled MeOH by keeping it in an ice bath for 15min, dried, and stained with 0.5% aq. eosin soln. for
15min. The stained plate was washed once with tap water, and then twice with distilled water and allowed to dry.
A 200-ml portion of 0.1n NaOH was added to each well to dissolve the protein and release the dye. The optical
density of the resulting soln. in each well was determined at 490 nm with a microplate reader. The % inhibition
of amoebal growth was calculated from the optical densities of the control and test wells and plotted against the
logarithm of the dose of the drug tested. Linear-regression analysis was used to determine the best-fitting
straight line from which the IC50 value was found.
6. In vivo Antiamoebic Activity. Animals. Male golden hamsters (Mesocricetus auratus), aged one month
and weighing 50 70 g were used. During this study, the animals were maintained in polypropylene cages, with
food and water ad libitum. Amoebae. E. histolytica, strain HM1 :1MSS, trophozoites were used in all the
experiments. Amoeba cultures were maintained under axenic conditions in TYIS-33 medium with a subculti-
vation frequency of 4 days. These amoebae were used to produce EAHA in hamsters. To increase the parent-
strain virulence, the amoebic cultures were subjected to four liver passages and in vitro culture cycles [29].
Experimental Amoebic Hepatic Abscess (EAHA) Production. The animals were anesthetized with 6.3 mg
i.p./100 g of body weight of sodium pentobarbital (Anestesal¾, from Smith Kline & French, Mexico D.F.). A
laparatomy was practiced on each animal under aseptic conditions. The left hepatic lobe was exposed and
inoculated with 5¥ 10 5 trophozoites suspended in 0.1 ml of fresh TYI-33 medium (without serum). The
inoculation was done with a disposable tuberculin syringe, which was armed with a 25-gauge hypodermic needle.
The incision was closed with a continuous suture. The animals were maintained with food and water ad libitum in
their cages during the following seven days. Afterwards, another laparatomy was practiced to verify the EAHA
development in each animal. The treatment was started 24 h after exploratory laparatomy.
Drugs. Compounds 1 3 or mnz were dissolved in DMSO. The concentration of each of the above drugs was
properly adjusted with buffer, to be administered at the required doses, in 0.5-ml volumes. These were
administered orally with an intragastric canula.
Treatment Schemes. The animals were divided into ten groups (I X) for each 1 3 and mnz. Groups I and X
were untreated controls, groups II, III, IV, and V were treated with 5, 10, 20, and 30 mg kgÀ1 of each compound

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Helvetica Chimica Acta Vol. 85(2002)
for five days, and groups VI, VII, VIII, and IX were treated with 5, 10, 20, and 30 mg kgÀ1 of each compound for
ten days. Twenty-four hours after the last dose of drug, the animals were killed with an i.p. injection of sodium
pentobarbitone, and the EAHA in each animal was scored according to the following criteria: 0, liver showing
the inoculation wound only or EAHA < 1 mm in diameter; 1, 1 5mm diameter lesions; 2, 5 10 mm diameter
lesions; 3, 10 15mm diameter lesions, with minimal increase in lobe volume; 4, 15 20 mm diameter lesions
with multiple satellite lesions of 1 5mm diameter; 5, 20 30 mm diameter lesions with EAHA equivalent to
75% or more of the lobe volume, showing multiple satellite lesions and noticeable increase in the lobe volume.
Effectiveness as an antiamoebic agent was determined as the proportion of score reduction by the drug relative
to untreated controls. The significance of the above comparisons was estimated by applying the Kruskal-Wallis×s
one-way ANOVA nonparametric test, with the help of the SPSS statistical package program installed on a
personal computer (SPSS, Inc. Ver 5.0).
N. Bharti acknowledges the Council of Scientific and Industrial Research, New Delhi, India, for a Senior
Research Fellowship. The authors are thankful to Prof. Alok Bhattacharya and Dr. Sudha Bhattacharya, School
of Life Sciences and School of Environmental Sciences, respectively, Jawaharlal Nehru University, New Delhi,
for providing laboratory facilities for the determination of biological activities.
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Received March 8, 2002