- Email: [email protected]
Adducts of nitroimidazole derivatives with rhodium(II) carboxylates: Syntheses, characterization, and evaluation of antichagasic activities
Adducts of Nitroimidazole Derivatives With Rhodium(II) Carboxylates: Syntheses, Characterization, and Evaluation of Antichagasic Activities Michael Simon Nothenberg”, Gentilda Kazuko Funayama Takeda, and Renato Na$ar MSN. Faculty of Pharmaceutical Sciences.-RN. Institute of Chemistry,
Sciences.-GGBFT. Institute of Biomedical University of SGo Paulo, Sdo Paulo, Brazil
ABSTRACT Adducts of several rhodium(I1) carboxylates with two antiparasitic nitroimidazole ligands were prepared and characterized by elemental microanalysis, thermogravimetry, spectrophotometry (IR, UV, and visible), and proton magnetic resonance. Results of elemental and thermogravimetric analyses were consistent with the general formula Rh,(RC00),.2L (R = aliphatic or aromatic carboxylic groups; L = metronidazole or benznidazole). The reddish-brown color of the adducts as well as their visible spectra suggest axial coordination of the nitroimidazole ligands through nitrogen atoms. NMR spectra indicate N, as the coordinating atoms. Screening tests performed on cultures of T. cruzi indicate that aliphatic complexes-particularly propionate and acetate adducts-were more active than their aromatic counterparts, the same being observed with benznidazole adducts in relation to their metronidazole analogues. Evaluated for their usefulness as transfusion prophylactic agents against Chagas’ disease, propionate derivatives failed to sterilize T. cruzi infected blood. An oral toxicity assay in mice showed mild toxic effects with daily doses of 5 mg/kg for 20 days.
INTRODUCTION Chagas’
disease,
a blood
and tissue
parasitosis caused by the flagellate protozoan throughout Latin America. A 1988 report from [l] estimates the number of infected people as being
Trypanosoma cruzi, is still endemic the World
Health
Organization
*This work was abstracted from MSN’s Ph.D. thesis. Address reprint requests to: Michael S. Nothenberg, P. 0. Box 30.786, SBo Paulo, SHo Paul0 01051 Brazil. Journal of Inorganic Biochemistry, 42, 217-229 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
217 0162-0134/91/$3.50
218 M. S. Nothenberg
et al.
FIGURE 1. Spatial arrangement of rhodium carboxylates. R= alkyl or aryl groups; L = metronidazole or benznidazole molecules, bonded through imidazole N, atoms.
R
16 to 18 million out of 90 million at direct risk. The development of effective trypanocidal agents is thus considered a priority, especially in Brazil, whose T. cruzi strain does not respond well to presently available antichagasic drugs, nifurtimox and benznidazole [2]. Recent work has been directed towards the preparation and evaluation of transition metal derivatives as trypanocidal drugs [3, 41. The renewed interest in the chemotherapeutic potential of this class of compounds has two main reasons: the discovery of the antineoplastic activity of cisplatin, today’s leading and most widely used anticancer drug in the United States [5], and the similarities in metabolic aspects between rapidly dividing tumor cells and trypanosomas. Both exhibit inefficient or nonfunctional mitochondrial systems and depend primarily on aerobic enzymes like hexokinase. pyruglycolysis, and its metal-susceptible “pacemaking” vie kinase, and phosphofructokinase, to satisfy their energetic needs 16, 71. Filardi et al. [8] observed a loss of virulence in blood forms of 7’. cruzi previously incubated with cisplatin. Other metal-complex evaluations against try panosomas followed, including those of Wisor [9], who tested the effect of cisplatin administration of mice infected with 7’. rhodesiense. an agent of one form of African sleeping sickness, and the assay of pentamidine. f .2-diamminecyclohexane, metafluorobenzoic platinum(H) complexes and rhodium(III)-thiazole derivatives against T. cruzi by Ruiz-PCrez et al. [lo, 111. Rhodium carboxylates (Fig. I), a peculiar class of dimeric complexes which acquired some notoriety as potential anticancer drugs after the thorough studies of Bear et al. [ 12, 131, were tested against African trypanosomas by Farrell et al. [ 141. Rhodium acetate and an adduct prepared by attaching two diminazene aceturate (Berenil, a veterinary trypanocidal drug) molecules to the axial positions of the cage-like carboxylate structure, assayed among several platinum and ruthenium compounds, showed trypanocidal activity along with marked toxic effects. As shown in this paper, we attempted the synthesis and biological assays of adducts of several carboxylates with two chemotherapeutically active nitroimidazoles, benznidazole (N-benzyl-2-nitroI-imidazoleacetamide) and metronidazole (,l(2-hydroxyethyl)-2-methyl-S-nitroimidazole) (Fig. 2). Benznidazole, as already men4
5
NO2
02N
OH
FIGURE 2. Benznidazole (BE) and rnetronidazole (ME).
N,
2
N,
ADDUCTS OF NITROIMIDAZOLE
DERIVATIVES
219
tioned, is one of the two antichagasic agents presently endorsed by the World Health Organization and metronidazole is a widely used antiprotozoal and antibacterial agent recently evaluated against African trypanosomiasis [ 15, 16, 171, malaria [18], and Chagas’ disease [19].
EXPERIMENTAL Materials Trihydrated rhodium chloride was purchased from Fluka AG (Buchs, FRG). Benznidazole and metronidazole were kindly provided by Produtos Roche, Quimicos e Farmaceuticos S. A. and ICN-Usafarma, Industria Farmaciktica Ltda. (SHo Paulo, Brazil) respectively. Syntheses
Rhodium acetate, Rh,(RCOO),, and hydrocinnamate, Rh,(C,H,CH,CH,COO),, were prepared according to Rempel et al. [20] and Najjar et al. [21], respectively. The remaining carboxylates-propionate, Rh,(CH,CH,COO),; butyrate, Rh,(CH$H,CH,COO),; trifluoroacetate, Rh,(CF,COO),; benzoate, Rh,(C,H,COO),; and phenylacetate, Rh,(C,H,CH,COO),-were synthesized by a simplified variant of Rempel’s acetate method: 5 mm01 RhCl, .3H,O and 25 mm01 of the corresponding sodium carboxylate (prepared, whenever necessary, from the corresponding carboxylic acid by a method described by Childers and Struthers [22]), were dissolved in a small volume (ca. 20 mL) of absolute ethanol and retluxed, under a nitrogen blanket, for about 1 hr. After cooling to room temperature, the mixture was filtered, the solid residue disregarded, and the solution concentrated to about 20% of the original volume over a stream bath. After addition of about 30 mL of water to the mixture, the rhodium carboxylate was extracted with ethyl ether (propionate) or dichloromethane (trifluoroacetate and butyrate) until the extract was colorless rather than bluish-green. The solvent was evaporated over a steam bath and the residue redissolved in acetone and left to crystallize overnight in a refrigerator. Finally, after removal of the supematant, the green crystals were dried in a vacuum desiccator at 78°C for 24 hr. Benzoate and phenylacetate were obtained by simple crystallization during a 24 hr rest period of the solution, resulting from filtration of the refluxed mixture, in a refrigerator. The resulting crystals were washed several times with hot water. Adducts were prepared by direct reaction of warm ethanolic solutions of the rhodium carboxylate and the ligand (1 mmol and 2.2 mmol, respectively). The resulting solution was left to crystallize for 24 hr in a refrigerator. The supematant was removed with a pipette and the residue washed with several portions of an alcohol-water 1:l mixture. The crystals were collected and heated in vacua at 78°C for 3 hr. Physical Methods
Elemental analysis were performed at the Microanalytical Laboratory of the Institute of Chemistry, University of SZo Paulo. Thermogravimetric curves were recorded on a Perkin-Elmer TSl apparatus under nitrogen atmosphere, at a heating rate of lO”C/min. Visible ultraviolet spectra were determined on a Beckman DU-70 spectrophotometer. Aliphatic carboxylates and adducts were dissolved in ethanol whereas aromatic
220
M. S. Nothenberg
et al.
compounds demanded dimethylformamide for complete solubilization at 10 - ‘M (visible) and 10 m41\/1 (ultraviolet) concentrations. Infrared spectra were recorded from KBr pellets on a Perkin-Elmer FTIR-1750 spectrophotometer. Proton magnetic resonance curves were obtained from deuterated acetone solutions on a Varian EM360 60MHz spectrometer, using TMS as internal reference. Biological
Studies In vitro incubation studies were performed with each of 32 compounds including, besides rhodium carboxylates and their adducts, control substances like rhodium(II1) chloride, metronidazole, benznidazole, nifurtimox, and allopurinol, among others. Exactly 1 mg of each compound was suspended-only nonbonded rhodium acetate and hydrophilic control substances could be dissolved-in 0.5 mL of PBS (phosphate buffer saline) in small glass tubes. T. cruzi strains Y (231 and Fr (isolated from a human clinical case under study at the Department of Parasitology, Institute of Biomedical Sciences, University of SZo Paulo), cultured in LIT (liver infusion tryptose) medium, were diluted to 7.8 x 10” and 7.4 x 10’ parasites/mL for strains Y and Fr, respectively. Precisely measured volumes of 400 pl_ of the cultures and 100 PL of the suspensions were transferred into 128 tubes, allowing activity evaluations of the 32 compounds under test after 24 and 72 hr incubation periods, at 28”C, against both strains. The amount of the compounds added to each tube (0.2 mg) corresponds to 5 mg/kg/day administered to 21) g mictt. ‘Three drugless control tubes were incubated w-ith each of the four groups. In order to test the efficacy of some of the adducts as preventive agents against infections through blood transfusions? 1 mL of blood of Y strain infected mice (about 3.4 x 10’ parasites) was transferred into glass tubes and mixed with 30 FL dimethylformamide solutions containing 0.1 mg of rhodium(I1) propionatemetronidazole and benznidazole adducts as well as the isolated carboxylate, imidazole derivatives, and controls. Quantitative and qualitative evaluations of the trypanosomas were performed after incubation periods of 5 and 24 hr at 6°C. Single 0.1 mL doses of the incubated blood samples were suhsequentiy administered by
TABLE 1. Elemental Analysis Data Calculated (76) C
H
N 10.72 10.00 9.38 8.40 (i. 14 7i. 11‘. 7.73 Il.64 il.00 10.43 9.51 9,26 X.84 x.47
Acetate-2ME Propionate-2ME Butyrate-2ME Trifluoroacetate-2ME Benzoate-2ME Phenylacetate-2ME Hydrocinnamate-2ME
30.63
37.51 24.02 46.52 48.54 SO.36
-3.86 4.56 5.17 1.81 3 71 ‘1 26 4.75
Acetate-2BE Propionate-2BE Butyrate-2BE Trifluoroacetate-2BE Benzoate-2BE Phenylacetate-2BE Hydrocinnamate-2BE
39.93 42.45 44.70 32.62 51.58 53.09 54.47
3.77 4.35 4.88 2.05 3.66 4.14 4.51
34.30
Found C
H
(% I N
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
221
TABLE 2. Thermogravimetric Analysis Data Decomp. Temperature:
(“C)
(“C)
Acetate Propionate Butyrate Trifluoroacetate Benzoate Phenylacetate Hydrocinnamate
295.4 289.4 240.0 308.2 315.0 296.6 288.0
296.2 289.4 280.0 340.0 388.0 304.8 372.0
53.39 58.64 62.83 65.12 70.16 72.40 75.43
53.40 59.50 62.58 91.50* 69.61 71.69 73.72
42.53 49.01 54.17 58.20 63.20 67.53 69.70
Acetate-2ME Propionate-2ME Butyrate-2ME Trifluoroacetate-2ME Benzoate-2ME Phenylacetate-2ME Hydrocinnamate-2ME
168.8 244.4 200.0 161.0 287.0 215.8 210.0
304.8 285.0 350.0 377.0 300.0 316.6 380.0
73.74 75.49 77.02 79.40 80.0 78.34 82.0
74.59 75.66 75.48 79.37 78.77 81.08 77.17
67.62 69.78 71.67 74.61 75.40 76.67 77.81
Acetate-2BE Propionate-2BE Butyrate-2BE Trifluoroacetate-2BE
216.6 201.5 200.0 194.0
317.6 303.0 321.0
78.60 79.78 80.83 82.52
79.96 85.71 83.48 83.52
73.61 75.07 76.37 78.44
Beuzoate-2BE
247.0 201.0 192.0
410.0 301.4 400.0
82.99 83.74 84.42
77.26 82.71 78.84
79.02 79.95 80.80
Phenylacetate-2BE Hydrocinnamate-2BE
Residue:
Calc. (W)
Found (W)
Calc. (%)
*Subject to partial sublimation
intraperitoneal injections into batches of ten mice (average weight 20 g). Parasitemia was microscopically checked in samples of tail blood for 30 days after infection. An oral toxicity assay was performed on four batches of 15 mice (average weight 25 g) treated with rhodium propionate, its adducts with benznidazole and metronidazole, and benznidazole alone. Drugs were administered by means of gastric intubation of aqueous dispersions in arabic gum in doses of 5, 50, and 100 mg/kg/day for 20 days.
RESULTS
AND DISCUSSION
Results of elemental analyses (Table 1) performed on all adducts comply with the general formula Rh,(RCOO),.2L, with metronidazole (ME) or benznidazole (BE) as axial ligands (L). Adducts, excluding those formed with trifluroacetate which are magenta, exhibit brownish tints. Aliphatic carboxylate adducts dissolve in hot ethanol and acetone while those formed with aromatic ligands are soluble in dimethylformamide. Thermogravimetry Table 2 compiles data derived from thermogravimetric curve analysis. Curves corresponding to acetate and propionate derivatives (Fig. 3) show reasonably defined steps related to the loss of the two axial ligands. The decomposition of the remaining compounds occurs in poorly defined steps, suggesting that the loss of the two ligand molecules and the breakdown of the carboxylate cage might be simultaneous events, as advanced by Bear et al. [24] in thermal studies of similar complexes.
222
M. S. Nothenberg et al.
The final residues after heating at 3OO”C-400°C clearly correspond to metallic rhodium for aliphatic adducts. Results for aromatic complexes leave margin for uncertainty as calculated values for possible residues Rh” and Rh,O, are similar due to the higher molecular weights of the parent compounds. Electronic
Spectra Visible spectra of rhodium carboxylate solutions present two low intensity bands, around 580-600 nm (band I) and 440-450 nm (band II) [25]. The position of the lower energy band I is sensitive to the nature of the axial ligand and defines the apparent color of the adducts [26, 271. Axial oxygen coordination, as in water, produces green colored complexes, while nitrogen or sulfur atoms render reddish-brown compounds by promoting hypsochromic shifts of about 30-40 nm on band I. Brown colored metronidazole adducts’ visible spectra exhibit peaks at about 550 nm and 565 nm for aliphatic (in ethanol) and aromatic carboxylates (in dimethylformamide). respectively, suggesting nitrogen axial coordination. On the other hand, benznidazole complexes, although similarly colored in solid form, revert to green in solution. This fact, evidenced by the absence of the hypsochromic shift in their visible spectra, indicates the low stability of these adducts in solution (Table 3 and Fig. 4). For their turn, the so-called bands II of the visible spectra of rhodium carboxylates depend on the equatorial ligands, that is, the nature of the carboxylate itself. This explains the 465.5 nm absorption of the bluish-green trifluoroacetate as opposed to the typical 440 nm peak presented by the remaining carboxylates. Ultraviolet spectra are not useful for the characterization of the adducts as the typical low intensity carboxylate absorption bands (III and IV [28]) are completely overlapped by the strong nitroimidazole-derived peaks.
Infrared
Spectra Figure 5 shows the IR spectra of rhodium propionate and its adducts while Table 4 lists wavenumbers corresponding to absorption maxima assigned to asymmetric (v,,~~,) and symmetric stretching ( Y,~,,,) vibrations, respectively, of the coordinated carboxylate groups for all the prepared compounds. Average A ( va,ym-vSy,) values allow us to preclude the occurrence of monodentate coordination in the complexes (29, 301.
C.
z4: 5:
Y)C
.___%
200 _/ 3OC -
I
406 ~~.
xi: _ ~. 600
‘2 .4 -5
FIGURE 3. Thermogravimetric curves for rhodium propionate (A) and its adducts with metronidazole (B) and benznidazole (C).
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
223
TABLE 3. Visible and Ultraviolet Spectrophotometric Readings Compound Metronidazole
Benmidazole
Acetate
5470 2350 3970 8180 16660
250.0 220.5 200.5 309.0 222.0 200.5 314.0 208.0
5000 16640 14060 17230 19170 19910 15880 30990
250.0 222.0 201.0 311.0 225.5 202.5 315.0 211.0
5000 14810 14960 17790 18750 18530 16720 29590
250.0 222.5 201.0 311.0 226.0 203.0 315.5 211.0
5000 15640 15920 18730 20570 20370 16880 30320
587.0 442.0
244 118
Acetate-2ME
547.0
199
Acetate-2BE
586.0
190
589.0 441.0
232 115
Propionate-2ME
550.0
225
Propionate-2BE
590.0 439.0
161 89
589.0 438.0
242 146
Butyrate-2ME
549.0
239
Butyrate-2BE
591.0 440.0
201 119
575.0 465.5
198 84
256.0 228.0 199.0
5500 14900 8790
Trifluoroacetate-2ME
557.0
169
Trifluoroacetate-2BE
577.0 462.0
185 87
309.0 230.5 202.0 315.0 229.5 209.5
17120 19690 16990 15370 21260 29330
285
271.0
24960
Benmate+2ME
584.0 450.0 565.5
310
Benmate-2BE
584.0
344
322.0 272.0 322.0 272.0
19250 30540 14990 3m60
586.0 443.5 567.0
248 118 250
267.0
6020
586.0 440.0
321
323.0 266.0 232.5 266.0
16500 1084 15230 1152
Propionate
Butyrate
Trifluoroacetate
Benmate
Phenylacetate Phenylacetate-2ME Phenylacetate-2BE
EtOH
311.0 224.0 200.0 314.0 203.0
EtOH
EtOH
EtOH
DMF
DMF
224
M. S. Nothenberg et al.
TABLE 3. Continued. Compound Hydrocinnamate Hydrocinnamate-2ME Hydrocinnamate-2BE
Solvent DMF
588.0 444.0 567.0
301 136 176
587.0 435.0
280 150
267 0
9010
322 0 266.0 323.5 266.5
18500 13080 17720 136!90
Proton Magnetic Resonance As the 0.9 ppm (triplet) methylene and 2.1 ppm (quadruplet) methyl proton chemical shifts seem unaffected by the nature of the axial substituents (Table S), the primary concern in the evaluation of PMR spectra of rhodium propionate and its adducts are ligand protons. The 7.99 ppm singlet associated with the sole carbon-bonded proton (H-C,) in free metronidazole [31, 321 is down-shifted to 8.4 ppm in the adduct, suggesting decreased electronic shielding. On the other hand, the three methyl protons (H-C,) seem inversely effected by the carboxylate linkage, exhibiting up-shifts. from 3.70 to 3.05 ppm. Both phenomena are related with the higher electron density, i.e., basicity, of the coordinating nitrogen, N,, in relation to N, 1331. The benznidazole adduct NMR spectrum reinforces previous remarks on the weakness of the compound’s Rh-N linkage; chemical shifts for carboxylate and FIGURE 4.
Ultraviolet and visible spectra of rhodium propionate with metronidazole (C and D) and benznidazole (E and F!.
(A and B) and its adducts
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
225
10 8 6 4 2 0 4.0
3.5
3.0
2.5
2.0
1.5
1.0
3.5
3.0
2.5
2.0
1.5
1.0
3.5
3.0
2.5
2.0
1.5
1.0
8 6 4 2 0 4.0
10 8 6 4 2 0 4.0
.5.,-'
(x7000,
FIGURE 5. Infrared spectra for rhodium propionate (B) and benznidazole (C).
TABLE 4. Infrared Spectrometry: Stretching Frequencies
Asymmetric
Water Adducts Bands (cm- ‘) Acetate Propionate Butyrate Trifluoroacetate Benzoate Phenylacetate Hydrocinnamate
(A) and its adducts with metronidazole
and Symmetric Carboxylate
Metronidazole Adducts
Benznidazole Adducts
Yasym
Vsym
A”
Vasym
Y,yrn
A”
v,,yrn
%ym
A”
1588 1570 1575 1661 15524 1577 1569
1438 1425 1422 1459 1399 1408 1414
150 145 145 202 153 169 155
1593 1586 1585 1670 1602 1595 1591
1429 1422 1415 1478 1398 1399 1416
164 164 170 192 166 196 175
1598 1588 1588 1660 1602 1596 1591
1435 1420 1417 1456 1398 1402 1415
163 168 171 207 204 194 176
TABLE 5. Proton Magnetic Resonance Data for Rhodium Propionate and Adducts
Qw) Propionate Propionate-2ME Propionate-2BE
o,o(t, 12H,CH,); 2,l(q,
8H, CH,); 3,3(s,4H, H,O) 0,9(t, 12H,CH,); 2,l(q,8H,CH,); 3,05(s,6H,CH,-im); 4,2(r,4H,CH,-0); 4,8(t,4H,N-CH,); 8,4(s,2H,H,-im) 0,9(r, 12H, CH,); 2,l(q, 8H, CH,); 2,9(s,4H, N-CH,-CO) 4,45(d, 4H, CH,-Ar); 5,3(s, 2H, CONH); 7,12(s., 2H, H,-im) 7,28(s, 5H, ArH); 7,45(s, 2H, H,-im)
226
M. S. Nothenberg
et al.
FIGURE 6. Screening of rhodium carboxylates and adducts (upper graph) and control substances (lower graph) on T. cruzi strain Fr cultures. The shaded bars record relative (%) culture development under the influence of the screened compounds 24 and 72 hr (initial count I= 100%).
after incubation periods of
ligand remain unchanged, acetone used as solvent.
molecules
suggesting
them to be nonbonded
in deuterated
In vitro screening Figures 6 and 7 illustrate the relative sensibilities of T. cruzi strains Fr and Y, respectively, to the screened compounds. Initial countings were normalized to 100%. Shaded bars indicate the quantitative development of the cultures after 24 and 72 hr incubation period. Both strains reacted similarly to the assayed drugs and controls but strain Y showed more sensibility as benznidazole, metronidazole, nifurtimox. and most benznidazole derivatives were able to promote the apparent disappearance of all parasites in 24 hr. Aliphatic oarboxylates showed higher than their aromatic counterparts while benznidazole adducts were more active than their metronidazole counterparts or water adducts. Blood Incubation
Assay
Rhodium propionate exhibited maximum activity among its group, followed by the metronidazole and benznidazole adducts, in that order, against T. cru~i strain Y.
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
227
FIGURE 7. Screening of rhodium carboxylates and adducts (upper graph) and control substances (lower graph) on T. cruzi strain Y cultures. The shaded bars record relative (%) culture development under the influence of the screened compounds after incubation periods of 24 and 72 hr (initial count = 100%).
blood forms. Unfortunately, most tubes showed some degree of hemolysis, its intensity being proportional to the activity of the complexes. Morphological alterations, most vacuolization and loss of motility, were noted on microscopic examinations of the parasite cells submitted to the complexes and, to a lesser degree, to free nitroimidazoles and dimethylformamide. Metronidazole induced conversions from trypomastigote to amastigote Trypanosoma forms. Intraperitoneal injections of the infected/treated blood in mice indicated no significant differences in infectivity between blood incubated for 5 and 24 hr. Benznidazole and metronidazole adducts were less active than rhodium propionate, the parasitemia being detected after 15 days from injection of the blood treated with the latter. Subacute
Toxicity
Assay
Rhodium propionate, whose systemic toxicity was previously established by Bear et al. [34] by means of LD,, and LD,, determinations of 2.7 and 4.5 mg/kg, respectively, was assayed along with its adducts in a 20-day oral toxicity test. Results showed good tolerance for doses of 5 mg/kg and the manifestation of toxic nervous effects (agitation) as well as moderate mortality with heavier regimes.
228
M. S. Nothenberg et al.
CONCLUSIONS Analytical procedures confirm the syntheses of the 14 new carboxglate adducts with metronidazole and benznidazole, the latter being unstable upon solubilization. Screenings performed on cultures of T. cruzi indicated that aliphatic complexes, particularly propionate and acetate adducts, were more active than their aromatic counterparts, the same being observed with benznidazole adducts in relation to their metronidazole analogues. Evaluated for their usefulness as a preventive agent against Chagas’ failed
disease to sterilize
dissemination the T
cruzi
through infected
blood
transfusions.
blood at the employed
pro&mate
derivatives
doses
The authors thank the Funda@io de Amparo ci Pesquisa de E:stado de Siio Paulo (FAPESP) and the Conseiho National de De.cenvolvimento Cientjfiw e Tecr~oicigir~o (CNPq) that supported, in part, this work.
REFERENCES Chagas’ Disease in Tropical Disease Research, a Gobal Partnership. 8th Programme Report. World Health Organization. Geneva.. 1987, pp. 87-98. 2. World 2. Essential Drugs WHO Model List, 5th Revision, WHO Drug hformation 1.
Health Organization,
Geneva,
198X.
3. J. Williamson, N. P. Farrell, and D. M. McLaren, Parrzssito/ogy 85, 13 (1982). 4. A. E. Balber. S. L. Gonias, and S. V. Pizza. Exp. Parasitol. 59. 74 (1985). A. T. \xn Oo~reram. and 6’. wn de Putte. 5. J. Reedijk, A. M. J. Fichtinger-Schepman. Strucf. Bond 67, 53 (19811. 6. P. Borst, Trans. R. Sot. Trop. Med. Hyg. 71, 3 (1977). Agent.~ Chemother. 15, 7. K. E. Kinnamon, E, A Stcck, and D. S. Raw. Antimicroh. 157 (1979). 8. L. S. Filardi, W. Leon, F. S. Cruz. and J. J. Frausto. Rev. In.yr. Med Trop. S. PalrIo 20. 248 (1978). 9. M. S. Wisor, Science 217, 454 (1982). 10. L. M. Ruiz-PCrez, A. Osuna, S. Castanys. F. Gamarro, D, Craciunescu. and A. Doadrio, Arzneim-Forsch. 36. 13 (1986). 11 L. M. Ruiz-PCrez. A. Osuna, M. C. Lopez. F. Gamarro, S. Castanys. D Craciunescu, and C. Alonso. Arzneim.-Forsch. 38, 312 (1988). 12. J. L. Bear, R. G. Hughes. and A. P. Kimball. Proc. Anl. .&~oc. Cancer Res. 13. 170 (1972). l?. J. L. Bear, H. B. Gray Jr., L. Rainen, 1. M. Chang, R. Howard. G. Scrio. and A. P. Kimball, Cancer Chemother. Rep. (Part I) 59. 61 IO (197.5) 14, N. P. Farrell, J. Williamson. and D. M. McLaren, Rioch~m. Pkarmacol. 33. 9(11
(1984). 1s. 16. 17. is. 19. 20. 21.
J. Arroz, and M. Djedje, Trans. R. Sot. Trop. Med. HJJ~. 82. 421 ( 1988). 8. H. Raseroka and W. E. Ormerod. Trans. R. Sot. Trap. Med. f-l?;g, 80. 634 (1986). 1. N. Otigbuo and P. ‘I‘. K. Woo, .I. Parasit. 74. 201 i i 98Xi. R. F. James, Lancer 2, 498 (1985). W. Raether aud H. Seidenath, ilnn. Trop. Med. Parasitoi. 77, iS (1983). G. A. Rempel, P. Legzdins. H. Smith, and G. Wilkinson, Inorg. Syn. 13, I@4 (1972). R. Najjar, W. de Olweira. J. B. Carducci, and M. Watanabe. Po@hedron 8, 1 157
11989). 22. E. Childers and C. W. Struthers, 23. L. H. Silva and V. Nussenzweig,
Analyt. Chem. 21, 737 i 1955). F&a Clin. Bioi. 20. I91 (1953).
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
229
24. J. L. Bear, R. A. Howard, A. M. Wytme, and W. W. Wendlandt, J. Inorg. Nucl. Chem. 38, 1015 (1976). 25. L. Dubicky and R. L. Martin, Znorg. Chem. 9, 673 (1970). 26. S. A. Johnson, H. R. Hunt, and H. M. Neumann, Inorg. Chem. 2, 960 (1963). 27. G. Pneumatikakis and P. Psaroulis, J. Chem. Sot. Dalton 4, 596 (1980). 28. V. M. Miskowski, W. P. Schaeffer, B. Sadeghi, and B. D. Santarsiero, Znorg. Chem. 23, 1154 (1984). 29. G. B. Deacon and R. J. Philips, Coord. Chem. Rev. 33, 227 (1980) 30. G. B. Deacon, F. Huber, and R. J. Philips, Znorg. Chim. Acta 104, 41 (1985). 31. G. B. Barlin and T. J. Batterham, J. Chem. Sot. B, 5 16 (1967). 32. J. E. Stambaugh and R. W. Manthei, Can. Spectrosc. 13, 134 (1968). 33. R. J. Sundberg and R. B. Martin, Chem. Rev. 74, 471 (1974). 34. J. L. Bear, A. Erck, L. Rainen, J. Whileyman, I. M. Chang, and A. P. Kimball, Proc. Sot. Exp. Biol. Med. 145, 1278 (1974).
Received July IO, 1990; accepted November 12, 1990
Sciences.-GGBFT. Institute of Biomedical University of SGo Paulo, Sdo Paulo, Brazil
ABSTRACT Adducts of several rhodium(I1) carboxylates with two antiparasitic nitroimidazole ligands were prepared and characterized by elemental microanalysis, thermogravimetry, spectrophotometry (IR, UV, and visible), and proton magnetic resonance. Results of elemental and thermogravimetric analyses were consistent with the general formula Rh,(RC00),.2L (R = aliphatic or aromatic carboxylic groups; L = metronidazole or benznidazole). The reddish-brown color of the adducts as well as their visible spectra suggest axial coordination of the nitroimidazole ligands through nitrogen atoms. NMR spectra indicate N, as the coordinating atoms. Screening tests performed on cultures of T. cruzi indicate that aliphatic complexes-particularly propionate and acetate adducts-were more active than their aromatic counterparts, the same being observed with benznidazole adducts in relation to their metronidazole analogues. Evaluated for their usefulness as transfusion prophylactic agents against Chagas’ disease, propionate derivatives failed to sterilize T. cruzi infected blood. An oral toxicity assay in mice showed mild toxic effects with daily doses of 5 mg/kg for 20 days.
INTRODUCTION Chagas’
disease,
a blood
and tissue
parasitosis caused by the flagellate protozoan throughout Latin America. A 1988 report from [l] estimates the number of infected people as being
Trypanosoma cruzi, is still endemic the World
Health
Organization
*This work was abstracted from MSN’s Ph.D. thesis. Address reprint requests to: Michael S. Nothenberg, P. 0. Box 30.786, SBo Paulo, SHo Paul0 01051 Brazil. Journal of Inorganic Biochemistry, 42, 217-229 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
217 0162-0134/91/$3.50
218 M. S. Nothenberg
et al.
FIGURE 1. Spatial arrangement of rhodium carboxylates. R= alkyl or aryl groups; L = metronidazole or benznidazole molecules, bonded through imidazole N, atoms.
R
16 to 18 million out of 90 million at direct risk. The development of effective trypanocidal agents is thus considered a priority, especially in Brazil, whose T. cruzi strain does not respond well to presently available antichagasic drugs, nifurtimox and benznidazole [2]. Recent work has been directed towards the preparation and evaluation of transition metal derivatives as trypanocidal drugs [3, 41. The renewed interest in the chemotherapeutic potential of this class of compounds has two main reasons: the discovery of the antineoplastic activity of cisplatin, today’s leading and most widely used anticancer drug in the United States [5], and the similarities in metabolic aspects between rapidly dividing tumor cells and trypanosomas. Both exhibit inefficient or nonfunctional mitochondrial systems and depend primarily on aerobic enzymes like hexokinase. pyruglycolysis, and its metal-susceptible “pacemaking” vie kinase, and phosphofructokinase, to satisfy their energetic needs 16, 71. Filardi et al. [8] observed a loss of virulence in blood forms of 7’. cruzi previously incubated with cisplatin. Other metal-complex evaluations against try panosomas followed, including those of Wisor [9], who tested the effect of cisplatin administration of mice infected with 7’. rhodesiense. an agent of one form of African sleeping sickness, and the assay of pentamidine. f .2-diamminecyclohexane, metafluorobenzoic platinum(H) complexes and rhodium(III)-thiazole derivatives against T. cruzi by Ruiz-PCrez et al. [lo, 111. Rhodium carboxylates (Fig. I), a peculiar class of dimeric complexes which acquired some notoriety as potential anticancer drugs after the thorough studies of Bear et al. [ 12, 131, were tested against African trypanosomas by Farrell et al. [ 141. Rhodium acetate and an adduct prepared by attaching two diminazene aceturate (Berenil, a veterinary trypanocidal drug) molecules to the axial positions of the cage-like carboxylate structure, assayed among several platinum and ruthenium compounds, showed trypanocidal activity along with marked toxic effects. As shown in this paper, we attempted the synthesis and biological assays of adducts of several carboxylates with two chemotherapeutically active nitroimidazoles, benznidazole (N-benzyl-2-nitroI-imidazoleacetamide) and metronidazole (,l(2-hydroxyethyl)-2-methyl-S-nitroimidazole) (Fig. 2). Benznidazole, as already men4
5
NO2
02N
OH
FIGURE 2. Benznidazole (BE) and rnetronidazole (ME).
N,
2
N,
ADDUCTS OF NITROIMIDAZOLE
DERIVATIVES
219
tioned, is one of the two antichagasic agents presently endorsed by the World Health Organization and metronidazole is a widely used antiprotozoal and antibacterial agent recently evaluated against African trypanosomiasis [ 15, 16, 171, malaria [18], and Chagas’ disease [19].
EXPERIMENTAL Materials Trihydrated rhodium chloride was purchased from Fluka AG (Buchs, FRG). Benznidazole and metronidazole were kindly provided by Produtos Roche, Quimicos e Farmaceuticos S. A. and ICN-Usafarma, Industria Farmaciktica Ltda. (SHo Paulo, Brazil) respectively. Syntheses
Rhodium acetate, Rh,(RCOO),, and hydrocinnamate, Rh,(C,H,CH,CH,COO),, were prepared according to Rempel et al. [20] and Najjar et al. [21], respectively. The remaining carboxylates-propionate, Rh,(CH,CH,COO),; butyrate, Rh,(CH$H,CH,COO),; trifluoroacetate, Rh,(CF,COO),; benzoate, Rh,(C,H,COO),; and phenylacetate, Rh,(C,H,CH,COO),-were synthesized by a simplified variant of Rempel’s acetate method: 5 mm01 RhCl, .3H,O and 25 mm01 of the corresponding sodium carboxylate (prepared, whenever necessary, from the corresponding carboxylic acid by a method described by Childers and Struthers [22]), were dissolved in a small volume (ca. 20 mL) of absolute ethanol and retluxed, under a nitrogen blanket, for about 1 hr. After cooling to room temperature, the mixture was filtered, the solid residue disregarded, and the solution concentrated to about 20% of the original volume over a stream bath. After addition of about 30 mL of water to the mixture, the rhodium carboxylate was extracted with ethyl ether (propionate) or dichloromethane (trifluoroacetate and butyrate) until the extract was colorless rather than bluish-green. The solvent was evaporated over a steam bath and the residue redissolved in acetone and left to crystallize overnight in a refrigerator. Finally, after removal of the supematant, the green crystals were dried in a vacuum desiccator at 78°C for 24 hr. Benzoate and phenylacetate were obtained by simple crystallization during a 24 hr rest period of the solution, resulting from filtration of the refluxed mixture, in a refrigerator. The resulting crystals were washed several times with hot water. Adducts were prepared by direct reaction of warm ethanolic solutions of the rhodium carboxylate and the ligand (1 mmol and 2.2 mmol, respectively). The resulting solution was left to crystallize for 24 hr in a refrigerator. The supematant was removed with a pipette and the residue washed with several portions of an alcohol-water 1:l mixture. The crystals were collected and heated in vacua at 78°C for 3 hr. Physical Methods
Elemental analysis were performed at the Microanalytical Laboratory of the Institute of Chemistry, University of SZo Paulo. Thermogravimetric curves were recorded on a Perkin-Elmer TSl apparatus under nitrogen atmosphere, at a heating rate of lO”C/min. Visible ultraviolet spectra were determined on a Beckman DU-70 spectrophotometer. Aliphatic carboxylates and adducts were dissolved in ethanol whereas aromatic
220
M. S. Nothenberg
et al.
compounds demanded dimethylformamide for complete solubilization at 10 - ‘M (visible) and 10 m41\/1 (ultraviolet) concentrations. Infrared spectra were recorded from KBr pellets on a Perkin-Elmer FTIR-1750 spectrophotometer. Proton magnetic resonance curves were obtained from deuterated acetone solutions on a Varian EM360 60MHz spectrometer, using TMS as internal reference. Biological
Studies In vitro incubation studies were performed with each of 32 compounds including, besides rhodium carboxylates and their adducts, control substances like rhodium(II1) chloride, metronidazole, benznidazole, nifurtimox, and allopurinol, among others. Exactly 1 mg of each compound was suspended-only nonbonded rhodium acetate and hydrophilic control substances could be dissolved-in 0.5 mL of PBS (phosphate buffer saline) in small glass tubes. T. cruzi strains Y (231 and Fr (isolated from a human clinical case under study at the Department of Parasitology, Institute of Biomedical Sciences, University of SZo Paulo), cultured in LIT (liver infusion tryptose) medium, were diluted to 7.8 x 10” and 7.4 x 10’ parasites/mL for strains Y and Fr, respectively. Precisely measured volumes of 400 pl_ of the cultures and 100 PL of the suspensions were transferred into 128 tubes, allowing activity evaluations of the 32 compounds under test after 24 and 72 hr incubation periods, at 28”C, against both strains. The amount of the compounds added to each tube (0.2 mg) corresponds to 5 mg/kg/day administered to 21) g mictt. ‘Three drugless control tubes were incubated w-ith each of the four groups. In order to test the efficacy of some of the adducts as preventive agents against infections through blood transfusions? 1 mL of blood of Y strain infected mice (about 3.4 x 10’ parasites) was transferred into glass tubes and mixed with 30 FL dimethylformamide solutions containing 0.1 mg of rhodium(I1) propionatemetronidazole and benznidazole adducts as well as the isolated carboxylate, imidazole derivatives, and controls. Quantitative and qualitative evaluations of the trypanosomas were performed after incubation periods of 5 and 24 hr at 6°C. Single 0.1 mL doses of the incubated blood samples were suhsequentiy administered by
TABLE 1. Elemental Analysis Data Calculated (76) C
H
N 10.72 10.00 9.38 8.40 (i. 14 7i. 11‘. 7.73 Il.64 il.00 10.43 9.51 9,26 X.84 x.47
Acetate-2ME Propionate-2ME Butyrate-2ME Trifluoroacetate-2ME Benzoate-2ME Phenylacetate-2ME Hydrocinnamate-2ME
30.63
37.51 24.02 46.52 48.54 SO.36
-3.86 4.56 5.17 1.81 3 71 ‘1 26 4.75
Acetate-2BE Propionate-2BE Butyrate-2BE Trifluoroacetate-2BE Benzoate-2BE Phenylacetate-2BE Hydrocinnamate-2BE
39.93 42.45 44.70 32.62 51.58 53.09 54.47
3.77 4.35 4.88 2.05 3.66 4.14 4.51
34.30
Found C
H
(% I N
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
221
TABLE 2. Thermogravimetric Analysis Data Decomp. Temperature:
(“C)
(“C)
Acetate Propionate Butyrate Trifluoroacetate Benzoate Phenylacetate Hydrocinnamate
295.4 289.4 240.0 308.2 315.0 296.6 288.0
296.2 289.4 280.0 340.0 388.0 304.8 372.0
53.39 58.64 62.83 65.12 70.16 72.40 75.43
53.40 59.50 62.58 91.50* 69.61 71.69 73.72
42.53 49.01 54.17 58.20 63.20 67.53 69.70
Acetate-2ME Propionate-2ME Butyrate-2ME Trifluoroacetate-2ME Benzoate-2ME Phenylacetate-2ME Hydrocinnamate-2ME
168.8 244.4 200.0 161.0 287.0 215.8 210.0
304.8 285.0 350.0 377.0 300.0 316.6 380.0
73.74 75.49 77.02 79.40 80.0 78.34 82.0
74.59 75.66 75.48 79.37 78.77 81.08 77.17
67.62 69.78 71.67 74.61 75.40 76.67 77.81
Acetate-2BE Propionate-2BE Butyrate-2BE Trifluoroacetate-2BE
216.6 201.5 200.0 194.0
317.6 303.0 321.0
78.60 79.78 80.83 82.52
79.96 85.71 83.48 83.52
73.61 75.07 76.37 78.44
Beuzoate-2BE
247.0 201.0 192.0
410.0 301.4 400.0
82.99 83.74 84.42
77.26 82.71 78.84
79.02 79.95 80.80
Phenylacetate-2BE Hydrocinnamate-2BE
Residue:
Calc. (W)
Found (W)
Calc. (%)
*Subject to partial sublimation
intraperitoneal injections into batches of ten mice (average weight 20 g). Parasitemia was microscopically checked in samples of tail blood for 30 days after infection. An oral toxicity assay was performed on four batches of 15 mice (average weight 25 g) treated with rhodium propionate, its adducts with benznidazole and metronidazole, and benznidazole alone. Drugs were administered by means of gastric intubation of aqueous dispersions in arabic gum in doses of 5, 50, and 100 mg/kg/day for 20 days.
RESULTS
AND DISCUSSION
Results of elemental analyses (Table 1) performed on all adducts comply with the general formula Rh,(RCOO),.2L, with metronidazole (ME) or benznidazole (BE) as axial ligands (L). Adducts, excluding those formed with trifluroacetate which are magenta, exhibit brownish tints. Aliphatic carboxylate adducts dissolve in hot ethanol and acetone while those formed with aromatic ligands are soluble in dimethylformamide. Thermogravimetry Table 2 compiles data derived from thermogravimetric curve analysis. Curves corresponding to acetate and propionate derivatives (Fig. 3) show reasonably defined steps related to the loss of the two axial ligands. The decomposition of the remaining compounds occurs in poorly defined steps, suggesting that the loss of the two ligand molecules and the breakdown of the carboxylate cage might be simultaneous events, as advanced by Bear et al. [24] in thermal studies of similar complexes.
222
M. S. Nothenberg et al.
The final residues after heating at 3OO”C-400°C clearly correspond to metallic rhodium for aliphatic adducts. Results for aromatic complexes leave margin for uncertainty as calculated values for possible residues Rh” and Rh,O, are similar due to the higher molecular weights of the parent compounds. Electronic
Spectra Visible spectra of rhodium carboxylate solutions present two low intensity bands, around 580-600 nm (band I) and 440-450 nm (band II) [25]. The position of the lower energy band I is sensitive to the nature of the axial ligand and defines the apparent color of the adducts [26, 271. Axial oxygen coordination, as in water, produces green colored complexes, while nitrogen or sulfur atoms render reddish-brown compounds by promoting hypsochromic shifts of about 30-40 nm on band I. Brown colored metronidazole adducts’ visible spectra exhibit peaks at about 550 nm and 565 nm for aliphatic (in ethanol) and aromatic carboxylates (in dimethylformamide). respectively, suggesting nitrogen axial coordination. On the other hand, benznidazole complexes, although similarly colored in solid form, revert to green in solution. This fact, evidenced by the absence of the hypsochromic shift in their visible spectra, indicates the low stability of these adducts in solution (Table 3 and Fig. 4). For their turn, the so-called bands II of the visible spectra of rhodium carboxylates depend on the equatorial ligands, that is, the nature of the carboxylate itself. This explains the 465.5 nm absorption of the bluish-green trifluoroacetate as opposed to the typical 440 nm peak presented by the remaining carboxylates. Ultraviolet spectra are not useful for the characterization of the adducts as the typical low intensity carboxylate absorption bands (III and IV [28]) are completely overlapped by the strong nitroimidazole-derived peaks.
Infrared
Spectra Figure 5 shows the IR spectra of rhodium propionate and its adducts while Table 4 lists wavenumbers corresponding to absorption maxima assigned to asymmetric (v,,~~,) and symmetric stretching ( Y,~,,,) vibrations, respectively, of the coordinated carboxylate groups for all the prepared compounds. Average A ( va,ym-vSy,) values allow us to preclude the occurrence of monodentate coordination in the complexes (29, 301.
C.
z4: 5:
Y)C
.___%
200 _/ 3OC -
I
406 ~~.
xi: _ ~. 600
‘2 .4 -5
FIGURE 3. Thermogravimetric curves for rhodium propionate (A) and its adducts with metronidazole (B) and benznidazole (C).
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
223
TABLE 3. Visible and Ultraviolet Spectrophotometric Readings Compound Metronidazole
Benmidazole
Acetate
5470 2350 3970 8180 16660
250.0 220.5 200.5 309.0 222.0 200.5 314.0 208.0
5000 16640 14060 17230 19170 19910 15880 30990
250.0 222.0 201.0 311.0 225.5 202.5 315.0 211.0
5000 14810 14960 17790 18750 18530 16720 29590
250.0 222.5 201.0 311.0 226.0 203.0 315.5 211.0
5000 15640 15920 18730 20570 20370 16880 30320
587.0 442.0
244 118
Acetate-2ME
547.0
199
Acetate-2BE
586.0
190
589.0 441.0
232 115
Propionate-2ME
550.0
225
Propionate-2BE
590.0 439.0
161 89
589.0 438.0
242 146
Butyrate-2ME
549.0
239
Butyrate-2BE
591.0 440.0
201 119
575.0 465.5
198 84
256.0 228.0 199.0
5500 14900 8790
Trifluoroacetate-2ME
557.0
169
Trifluoroacetate-2BE
577.0 462.0
185 87
309.0 230.5 202.0 315.0 229.5 209.5
17120 19690 16990 15370 21260 29330
285
271.0
24960
Benmate+2ME
584.0 450.0 565.5
310
Benmate-2BE
584.0
344
322.0 272.0 322.0 272.0
19250 30540 14990 3m60
586.0 443.5 567.0
248 118 250
267.0
6020
586.0 440.0
321
323.0 266.0 232.5 266.0
16500 1084 15230 1152
Propionate
Butyrate
Trifluoroacetate
Benmate
Phenylacetate Phenylacetate-2ME Phenylacetate-2BE
EtOH
311.0 224.0 200.0 314.0 203.0
EtOH
EtOH
EtOH
DMF
DMF
224
M. S. Nothenberg et al.
TABLE 3. Continued. Compound Hydrocinnamate Hydrocinnamate-2ME Hydrocinnamate-2BE
Solvent DMF
588.0 444.0 567.0
301 136 176
587.0 435.0
280 150
267 0
9010
322 0 266.0 323.5 266.5
18500 13080 17720 136!90
Proton Magnetic Resonance As the 0.9 ppm (triplet) methylene and 2.1 ppm (quadruplet) methyl proton chemical shifts seem unaffected by the nature of the axial substituents (Table S), the primary concern in the evaluation of PMR spectra of rhodium propionate and its adducts are ligand protons. The 7.99 ppm singlet associated with the sole carbon-bonded proton (H-C,) in free metronidazole [31, 321 is down-shifted to 8.4 ppm in the adduct, suggesting decreased electronic shielding. On the other hand, the three methyl protons (H-C,) seem inversely effected by the carboxylate linkage, exhibiting up-shifts. from 3.70 to 3.05 ppm. Both phenomena are related with the higher electron density, i.e., basicity, of the coordinating nitrogen, N,, in relation to N, 1331. The benznidazole adduct NMR spectrum reinforces previous remarks on the weakness of the compound’s Rh-N linkage; chemical shifts for carboxylate and FIGURE 4.
Ultraviolet and visible spectra of rhodium propionate with metronidazole (C and D) and benznidazole (E and F!.
(A and B) and its adducts
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
225
10 8 6 4 2 0 4.0
3.5
3.0
2.5
2.0
1.5
1.0
3.5
3.0
2.5
2.0
1.5
1.0
3.5
3.0
2.5
2.0
1.5
1.0
8 6 4 2 0 4.0
10 8 6 4 2 0 4.0
.5.,-'
(x7000,
FIGURE 5. Infrared spectra for rhodium propionate (B) and benznidazole (C).
TABLE 4. Infrared Spectrometry: Stretching Frequencies
Asymmetric
Water Adducts Bands (cm- ‘) Acetate Propionate Butyrate Trifluoroacetate Benzoate Phenylacetate Hydrocinnamate
(A) and its adducts with metronidazole
and Symmetric Carboxylate
Metronidazole Adducts
Benznidazole Adducts
Yasym
Vsym
A”
Vasym
Y,yrn
A”
v,,yrn
%ym
A”
1588 1570 1575 1661 15524 1577 1569
1438 1425 1422 1459 1399 1408 1414
150 145 145 202 153 169 155
1593 1586 1585 1670 1602 1595 1591
1429 1422 1415 1478 1398 1399 1416
164 164 170 192 166 196 175
1598 1588 1588 1660 1602 1596 1591
1435 1420 1417 1456 1398 1402 1415
163 168 171 207 204 194 176
TABLE 5. Proton Magnetic Resonance Data for Rhodium Propionate and Adducts
Qw) Propionate Propionate-2ME Propionate-2BE
o,o(t, 12H,CH,); 2,l(q,
8H, CH,); 3,3(s,4H, H,O) 0,9(t, 12H,CH,); 2,l(q,8H,CH,); 3,05(s,6H,CH,-im); 4,2(r,4H,CH,-0); 4,8(t,4H,N-CH,); 8,4(s,2H,H,-im) 0,9(r, 12H, CH,); 2,l(q, 8H, CH,); 2,9(s,4H, N-CH,-CO) 4,45(d, 4H, CH,-Ar); 5,3(s, 2H, CONH); 7,12(s., 2H, H,-im) 7,28(s, 5H, ArH); 7,45(s, 2H, H,-im)
226
M. S. Nothenberg
et al.
FIGURE 6. Screening of rhodium carboxylates and adducts (upper graph) and control substances (lower graph) on T. cruzi strain Fr cultures. The shaded bars record relative (%) culture development under the influence of the screened compounds 24 and 72 hr (initial count I= 100%).
after incubation periods of
ligand remain unchanged, acetone used as solvent.
molecules
suggesting
them to be nonbonded
in deuterated
In vitro screening Figures 6 and 7 illustrate the relative sensibilities of T. cruzi strains Fr and Y, respectively, to the screened compounds. Initial countings were normalized to 100%. Shaded bars indicate the quantitative development of the cultures after 24 and 72 hr incubation period. Both strains reacted similarly to the assayed drugs and controls but strain Y showed more sensibility as benznidazole, metronidazole, nifurtimox. and most benznidazole derivatives were able to promote the apparent disappearance of all parasites in 24 hr. Aliphatic oarboxylates showed higher than their aromatic counterparts while benznidazole adducts were more active than their metronidazole counterparts or water adducts. Blood Incubation
Assay
Rhodium propionate exhibited maximum activity among its group, followed by the metronidazole and benznidazole adducts, in that order, against T. cru~i strain Y.
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
227
FIGURE 7. Screening of rhodium carboxylates and adducts (upper graph) and control substances (lower graph) on T. cruzi strain Y cultures. The shaded bars record relative (%) culture development under the influence of the screened compounds after incubation periods of 24 and 72 hr (initial count = 100%).
blood forms. Unfortunately, most tubes showed some degree of hemolysis, its intensity being proportional to the activity of the complexes. Morphological alterations, most vacuolization and loss of motility, were noted on microscopic examinations of the parasite cells submitted to the complexes and, to a lesser degree, to free nitroimidazoles and dimethylformamide. Metronidazole induced conversions from trypomastigote to amastigote Trypanosoma forms. Intraperitoneal injections of the infected/treated blood in mice indicated no significant differences in infectivity between blood incubated for 5 and 24 hr. Benznidazole and metronidazole adducts were less active than rhodium propionate, the parasitemia being detected after 15 days from injection of the blood treated with the latter. Subacute
Toxicity
Assay
Rhodium propionate, whose systemic toxicity was previously established by Bear et al. [34] by means of LD,, and LD,, determinations of 2.7 and 4.5 mg/kg, respectively, was assayed along with its adducts in a 20-day oral toxicity test. Results showed good tolerance for doses of 5 mg/kg and the manifestation of toxic nervous effects (agitation) as well as moderate mortality with heavier regimes.
228
M. S. Nothenberg et al.
CONCLUSIONS Analytical procedures confirm the syntheses of the 14 new carboxglate adducts with metronidazole and benznidazole, the latter being unstable upon solubilization. Screenings performed on cultures of T. cruzi indicated that aliphatic complexes, particularly propionate and acetate adducts, were more active than their aromatic counterparts, the same being observed with benznidazole adducts in relation to their metronidazole analogues. Evaluated for their usefulness as a preventive agent against Chagas’ failed
disease to sterilize
dissemination the T
cruzi
through infected
blood
transfusions.
blood at the employed
pro&mate
derivatives
doses
The authors thank the Funda@io de Amparo ci Pesquisa de E:stado de Siio Paulo (FAPESP) and the Conseiho National de De.cenvolvimento Cientjfiw e Tecr~oicigir~o (CNPq) that supported, in part, this work.
REFERENCES Chagas’ Disease in Tropical Disease Research, a Gobal Partnership. 8th Programme Report. World Health Organization. Geneva.. 1987, pp. 87-98. 2. World 2. Essential Drugs WHO Model List, 5th Revision, WHO Drug hformation 1.
Health Organization,
Geneva,
198X.
3. J. Williamson, N. P. Farrell, and D. M. McLaren, Parrzssito/ogy 85, 13 (1982). 4. A. E. Balber. S. L. Gonias, and S. V. Pizza. Exp. Parasitol. 59. 74 (1985). A. T. \xn Oo~reram. and 6’. wn de Putte. 5. J. Reedijk, A. M. J. Fichtinger-Schepman. Strucf. Bond 67, 53 (19811. 6. P. Borst, Trans. R. Sot. Trop. Med. Hyg. 71, 3 (1977). Agent.~ Chemother. 15, 7. K. E. Kinnamon, E, A Stcck, and D. S. Raw. Antimicroh. 157 (1979). 8. L. S. Filardi, W. Leon, F. S. Cruz. and J. J. Frausto. Rev. In.yr. Med Trop. S. PalrIo 20. 248 (1978). 9. M. S. Wisor, Science 217, 454 (1982). 10. L. M. Ruiz-PCrez, A. Osuna, S. Castanys. F. Gamarro, D, Craciunescu. and A. Doadrio, Arzneim-Forsch. 36. 13 (1986). 11 L. M. Ruiz-PCrez. A. Osuna, M. C. Lopez. F. Gamarro, S. Castanys. D Craciunescu, and C. Alonso. Arzneim.-Forsch. 38, 312 (1988). 12. J. L. Bear, R. G. Hughes. and A. P. Kimball. Proc. Anl. .&~oc. Cancer Res. 13. 170 (1972). l?. J. L. Bear, H. B. Gray Jr., L. Rainen, 1. M. Chang, R. Howard. G. Scrio. and A. P. Kimball, Cancer Chemother. Rep. (Part I) 59. 61 IO (197.5) 14, N. P. Farrell, J. Williamson. and D. M. McLaren, Rioch~m. Pkarmacol. 33. 9(11
(1984). 1s. 16. 17. is. 19. 20. 21.
J. Arroz, and M. Djedje, Trans. R. Sot. Trop. Med. HJJ~. 82. 421 ( 1988). 8. H. Raseroka and W. E. Ormerod. Trans. R. Sot. Trap. Med. f-l?;g, 80. 634 (1986). 1. N. Otigbuo and P. ‘I‘. K. Woo, .I. Parasit. 74. 201 i i 98Xi. R. F. James, Lancer 2, 498 (1985). W. Raether aud H. Seidenath, ilnn. Trop. Med. Parasitoi. 77, iS (1983). G. A. Rempel, P. Legzdins. H. Smith, and G. Wilkinson, Inorg. Syn. 13, I@4 (1972). R. Najjar, W. de Olweira. J. B. Carducci, and M. Watanabe. Po@hedron 8, 1 157
11989). 22. E. Childers and C. W. Struthers, 23. L. H. Silva and V. Nussenzweig,
Analyt. Chem. 21, 737 i 1955). F&a Clin. Bioi. 20. I91 (1953).
ADDUCTS
OF NITROIMIDAZOLE
DERIVATIVES
229
24. J. L. Bear, R. A. Howard, A. M. Wytme, and W. W. Wendlandt, J. Inorg. Nucl. Chem. 38, 1015 (1976). 25. L. Dubicky and R. L. Martin, Znorg. Chem. 9, 673 (1970). 26. S. A. Johnson, H. R. Hunt, and H. M. Neumann, Inorg. Chem. 2, 960 (1963). 27. G. Pneumatikakis and P. Psaroulis, J. Chem. Sot. Dalton 4, 596 (1980). 28. V. M. Miskowski, W. P. Schaeffer, B. Sadeghi, and B. D. Santarsiero, Znorg. Chem. 23, 1154 (1984). 29. G. B. Deacon and R. J. Philips, Coord. Chem. Rev. 33, 227 (1980) 30. G. B. Deacon, F. Huber, and R. J. Philips, Znorg. Chim. Acta 104, 41 (1985). 31. G. B. Barlin and T. J. Batterham, J. Chem. Sot. B, 5 16 (1967). 32. J. E. Stambaugh and R. W. Manthei, Can. Spectrosc. 13, 134 (1968). 33. R. J. Sundberg and R. B. Martin, Chem. Rev. 74, 471 (1974). 34. J. L. Bear, A. Erck, L. Rainen, J. Whileyman, I. M. Chang, and A. P. Kimball, Proc. Sot. Exp. Biol. Med. 145, 1278 (1974).
Received July IO, 1990; accepted November 12, 1990