Chagas disease is responsible for a large number of human infections and many are also at risk of infection. There is no effective drug for Chagas disease treatment. The Institute of Pharmaceutical Technology at Fiocruz, Brazil, has designed three nitro analogs of the nitroimidazole-thiadiazole, megazol: two triazole analogs PTAL 05-02 and PAMT 09 and a pyrazole analog PTAL 04-09. A set of
Chagas disease is a protozoan zoonosis caused by
Megazol (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole), CAS number 19622-55-0, synthesized in 1968 by Berkelhammer and Asato [
However, megazol is not used clinically, as mutagenic and carcinogenic activity has been reported and related to its nitro group, which is also responsible for its activity against the protozoan [
Results of structure-mutagenicity relationship and metabolism studies in
Megazol (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole) and its nitro analogs PTAL 05-02 (5-(1-methyl-5-nitro-1H-2-imidazolyl)-1H-1,2,4-triazol-3-amine), PAMT 09 (2-amine-N-(1-methyl-4-nitro-1H-imidazole-5-yl)-5-(trifluoromethyl)-1H-1,2,4-triazole), and PTAL 04-09 (1-(1-methyl-4-nitro-1H-imidazole-5-yl)-1H-pirazole) condensed formulas and IC50 values are shown in Table
Megazol and its nitro analogs condensed formula and IC50.
Compound | Condensed formula | IC50 ( |
Reference |
---|---|---|---|
Megazol | C6H7N6O2S | 9.9 | [ |
PTAL 05-02 | C6H7N7O2 | 552 | [ |
PAMT 09 | C7H7F3N7O2 | 220 | [ |
PTAL 04-09 | C7H7N5O2 | 120 | [ |
Table
|
Reversion event | Genotype | Reference |
---|---|---|---|
TA97 | Frameshifts |
|
[ |
TA98 | Frameshifts |
|
[ |
TA100 | Base-pair substitutions |
|
[ |
TA1535 | Base-pair substitutions |
|
[ |
TA102 | Transitions/transversions |
|
[ |
Megazol and its nitro analogs PTAL 05-02, PAMT 09 and PTAL 04-09, were tested following the protocol proposed by Maron and Ames [
To determine the cytotoxic effects of megazol and its nitro analogs, the pre-incubation assay mixture in the bacterial reverse mutation test was diluted in 0.9% NaCl (w/v). This suspension contained 2.0 × 103 cells/mL. An aliquot (100
An S9 fraction, prepared from livers of Sprague-Dawley rats pretreated with a polychlorinated biphenyl mixture (Aroclor 1254), was purchased from Molecular Toxicology Inc. (Moltox, USA). The S9 metabolic activation mixture (S9 mix) used for the bacterial reverse mutation test and survival test was used as previously described by Maron and Ames [
The RAW 264.7 macrophage strain was used from a confluence culture. 950
For cell treatment, 100
The Eagle’s MEM, 1.8 mM Ca++, was replaced with a cold fixative solution methanol-glacial acetic acid (3 : 1) for 15 min. The fixed cells were rinsed with McIlvaine’s buffer (MI buffer: 21.01 g/L citric acid and 35.60 g/L Na2HPO4, pH 7.5) for 2 min and dried at room temperature. The fixed cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) (0.2
Table
Megazol induction of mutagenicity and cytotoxicity in
Strains | Dose ( |
−S9 | +S9 | ||
---|---|---|---|---|---|
Mean ± S.D. (M.I.a) | Survivalb | Mean ± S.D. (M.I.a) | Survivalb | ||
TA97 | 0 | 49 ± 19 (1.00) | 100 | 123 ± 26 (1.00) | 100 |
0.05 | 90 ± 10 (1.80) | 100 | 176 ± 18 (1.40) | 100 | |
0.1 | 116 ± 7 ( |
100 | 168 ± 7 (1.30) | 100 | |
0.2 | 142 ± 20 ( |
100 | 273 ± 11 ( |
100 | |
0.5 | 165 ± 25 ( |
100 | 408 ± 7 ( |
80 | |
1.0 | 266 ± 31 ( |
100 | 548 ± 111 ( |
80 | |
| |||||
TA98 | 0 | 27 ± 4 (1.00) | 100 | 40 ± 4 (1.00) | 100 |
0.05 | 38 ± 11 (1.30) | 92 | 33 ± 1 (0.80) | 100 | |
0.1 | 49 ± 5 (1.80) | 97 | 39 ± 7 (0.90) | 100 | |
0.2 | 81 ± 2 ( |
100 | 41 ± 14 (1.00) | 70 | |
0.5 | 211 ± 22 ( |
93 | 87 ± 2 ( |
75 | |
1.0 | 356 ± 35 ( |
|
116 ± 14 ( |
77 | |
| |||||
TA100 | 0 | 129 ± 12 (1.00) | 100 | 171 ± 1 (1.00) | 100 |
0.05 | 228 ± 13 (1.70) | 72 | 324 ± 47 (1.80) | 100 | |
0.1 | 366 ± 59 ( |
98 | 569 ± 58 ( |
100 | |
0.2 | 739 ± 29 ( |
70 | 691 ± 106 ( |
100 | |
0.5 | 1302 ± 247 ( |
80 | 1203 ± 190 ( |
100 | |
1.0 | 2109 ± 476 ( |
|
2041 ± 319 ( |
|
|
| |||||
TA102 | 0 | 197 ± 88 (1.00) | 100 | 193 ± 77 (1.00) | 100 |
0.05 | 280 ± 4 (1.40) | 100 | 408 ± 24 ( |
100 | |
0.1 | 289 ± 45 (1.40) | 100 | 459 ± 50 ( |
100 | |
0.2 | 185 ± 80 (0.90) | 100 | 594 ± 8.0 ( |
100 | |
0.5 | 347 ± 38 (1.70) | 100 | 680 ± 60 ( |
100 | |
1.0 | 313 ± 37 (1.70) | 100 | 666 ± 27 ( |
100 |
bPercent of cellular survival: <70% was considered toxic dose and it is marked in bold.
*Representation of a significant response (
Negative control was DMSO. Positive controls and their M.I., with and without S9 mix, respectively, were 4NQO (10) and 2AA (6.0) for TA97, 4NQO (15) and 2AA (10) for TA98, AS (16) and 2AA (11) for TA100, and MitC (8.7 and 4.3) for TA102.
Megazol exhibited dose-dependent mutagenic activity for a wide range of concentrations and strain in the absence and presence of S9 mix.
TA97 and TA100 were the most responsive strains in the presence and absence of S9 mix. TA100 was the most responsive strains in terms of mutagenic index.
However, there was no mutagenic activity in the absence of S9 mix for the strain TA102. Unlike the others strains, TA102 has its excision repair mechanism intact and this might explain the absence of mutagenic activity. On the other hand, there was mutagenic activity in the presence of S9 mix, suggesting the generation of metabolites by S9 mix metabolization.
Tables
PTAL 05-02 induction of mutagenicity and cytotoxicity in
Strains | Dose ( |
−S9 | +S9 | ||
---|---|---|---|---|---|
Mean ± S.D. (M.I.a) | Survivalb | Mean ± S.D. (M.I.a) | Survivalb | ||
TA97 | 0 | 87 ± 9.8 (1.00) | 100 | 97 ± 25 (1.00) | 100 |
0.05 | 89 ± 5.0 (1.03) | 90 | 67 ± 3.5 (0.70) | 100 | |
0.1 | 89 ± 12 (1.03) | 100 | 68 ± 4.0 (0.70) | 92 | |
0.5 | 86 ± 8.5 (0.99) | 100 | 74 ± 0.0 (0.76) | 90 | |
1.0 | 83 ± 1.7 (0.95) | 100 | 69 ± 4.5 (0.71) | 80 | |
| |||||
TA98 | 0 | 45 ± 1.5 (1.00) | 100 | 18 ± 6.4 (1.00) | 100 |
0.05 | 30 ± 2.1 (0.67) | 100 | 18 ± 5.5 (0.96) | 95 | |
0.1 | 38 ± 5.0 (0.83) | 98 | 21 ± 2.0 (1.16) | 90 | |
0.5 | 38 ± 11 (0.84) | 100 | 19 ± 1.7 (1.02) | 81 | |
1.0 | 58 ± 11 (1.28) | 94 | 22 ± 5.2 (1.18) | 91 | |
| |||||
TA100 | 0 | 120 ± 23 (1.00) | 100 | 117 ± 19 (1.00) | 100 |
0.05 | 136 ± 15 (1.13) | 100 | 121 ± 25 (1.04) | 79 | |
0.1 | 102 ± 25 (0.85) | 90 | 106 ± 12 (0.91) |
|
|
0.5 | 84 ± 10 (0.70) | 100 | 128 ± 18 (1.09) |
|
|
1.0 | 105 ± 9.8 (0.88) | 100 | 129 ± 13 (1.10) |
|
|
| |||||
TA1535 | 0 | 22 ± 3.2 (1.00) | 100 | 5.3 ± 1.5 (1.00) | 100 |
0.05 | 21 ± 3.2 (0.94) | 100 | 5.3 ± 0.5 (1.00) |
|
|
0.1 | 23 ± 2.0 (1.03) | 100 | 5.3 ± 0.5 (1.00) |
|
|
0.5 | 22 ± 4.0 (1.00) | 97 | 6.6 ± 1.5 (1.25) |
|
|
1.0 | 26 ± 2.0 (1.15) | 93 | 6.3 ± 2.0 (1.19) |
|
|
| |||||
TA102 | 0 | 254 ± 16 (1.00) | 100 | 265 ± 12 (1.00) | 100 |
0.05 | 254 ± 31 (1.00) | 79 | 253 ± 19 (0.95) | 100 | |
0.1 | 276 ± 14 (1.09) | 84 | 225 ± 4.0 (0.85) | 100 | |
0.5 | 253 ± 11 (1.00) | 79 | 238 ± 16 (0.90) | 100 | |
1.0 | 261 ± 14 (1.03) | 78 | 256 ± 8.3 (0.97) | 100 |
bPercent of cellular survival: <70% was considered toxic dose and it is marked in bold.
*Representation of a significant response (
Negative control was DMSO. Positive controls and their M.I., with and without S9 mix, respectively, were 4NQO (7.19) and 2AA (9.55) for TA97, 4NQO (10) and 2AA (3.95) for TA98; AS (13) and 2AA (2.10) for TA100, AS (3.31) and 2AA (6.25) for TA1535, and MitC (9.28 and 6.86) for TA102.
PAMT 09 induction of mutagenicity and cytotoxicity in
Strains | Dose ( |
−S9 | +S9 | ||
---|---|---|---|---|---|
Mean ± S.D. (M.I.a) | Survivalb | Mean ± S.D. (M.I.a) | Survivalb | ||
TA97 | 0 | 87 ± 9.8 (1.00) | 100 | 97 ± 25 (1.00) | 100 |
0.05 | 89 ± 4.5 (1.02) | 100 | 74 ± 5.5 (0.76) | 100 | |
0.1 | 87 ± 3.6 (1.02) | 95 | 84 ± 7.3 (0.87) | 96 | |
0.5 | 92 ± 4.9 (1.07) | 85 | 87 ± 2.0 (0.90) | 95 | |
1.0 | 81 ± 1.4 (0.93) | 83 | 80 ± 8.7 (0.82) | 100 | |
| |||||
TA98 | 0 | 45 ± 1.5 (1.00) | 100 | 18 ± 6.4 (1.00) | 100 |
0.05 | 58 ± 9.6 (1.28) | 100 | 17 ± 1.0 (0.91) | 100 | |
0.1 | 66 ± 3.0 (1.46) | 93 | 17 ± 0.5 (0.93) | 100 | |
0.5 | 50 ± 5.8 (1.10) | 97 | 21 ± 4.0 (1.14) | 100 | |
1.0 | 48 ± 5.8 (1.07) | 78 | 32 ± 9.0 (1.73) | 74 | |
| |||||
TA100 | 0 | 120 ± 23 (1.00) | 100 | 117 ± 19 (1.00) | 100 |
0.05 | 100 ± 18 (0.83) | 100 | 104 ± 11 (0.89) | 73 | |
0.1 | 96 ± 9.5 (0.80) | 100 | 126 ± 12 (1.08) | 73 | |
0.5 | 95 ± 9.1 (0.79) | 100 | 107 ± 2.6 (0.91) | 72 | |
1.0 | 100 ± 14 (0.83) | 100 | 107 ± 10 (0.92) | 71 | |
| |||||
TA1535 | 0 | 22 ± 3.2 (1.00) | 100 | 5.3 ± 1.5 (1.00) | 100 |
0.05 | 19 ± 1.1 (0.94) | 100 | 7.5 ± 0.7 (1.41) |
|
|
0.1 | 19 ± 2.3 (0.92) | 100 | 9.5 ± 2.1 (1.78) |
|
|
0.5 | 32 ± 5.5 (1.56) | 100 | 8.6 ± 1.5 (1.63) |
|
|
1.0 | 25 ± 0.5 (1.21) | 85 | 7.0 ± 1.0 (1.31) |
|
|
| |||||
TA102 | 0 | 254 ± 16 (1.00) | 100 | 265 ± 12 (1.00) | 100 |
0.05 | 285 ± 30 (1.12) | 79 | 275 ± 17 (1.04) | 100 | |
0.1 | 289 ± 9.0 (1.14) | 76 | 242 ± 11 (0.91) | 100 | |
0.5 | 282 ± 28 (1.11) | 73 | 256 ± 10 (0.97) | 100 | |
1.0 | 307 ± 24 (1.21) |
|
240 ± 27 (0.91) | 100 |
bPercent of cellular survival: <70% was considered toxic dose and it is marked in bold.
*Representation of a significant response (
Negative control was DMSO. Positive controls and their M.I., with and without S9 mix, respectively, were: 4NQO (7.19) and 2AA (9.55) for TA97, 4NQO (10) and 2AA (3.95) for TA98; AS (13) and 2AA (2.10) for TA100, AS (18) and 2AA (6.25) for TA1535, and MitC (9.28 and 6.86) for TA102.
PTAL 04-09 induction of mutagenicity and cytotoxicity in
Strains | Dose ( |
−S9 | +S9 | ||
---|---|---|---|---|---|
Mean ± S.D. (M.I.a) | Survivalb | Mean ± S.D. (M.I.a) | Survivalb | ||
TA97 | 0 | 87 ± 9.8 (1.00) | 100 | 97 ± 25 (1.00) | 100 |
1.0 | 85 ± 95 (0.98) | 97 | 74 ± 13 (0.76) | 77 | |
5.0 | 100 ± 5.7 (1.15) | 100 | 80 ± 13 (0.82) | 73 | |
10 | 115 ± 16 (1.33) | 100 | 78 ± 16 (0.80) | 77 | |
50 | 143 ± 11 (1.65) | 100 | 82 ± 9.6 (0.85) | 77 | |
| |||||
TA98 | 0 | 45 ± 1.5 (1.00) | 100 | 18 ± 6.4 (1.00) | 100 |
1.0 | 66 ± 11 (1.46) | 93 | 18 ± 8.5 (0.98) | 100 | |
5.0 | 64 ± 3.0 (1.40) | 100 | 17 ± 2.8 (0.91) | 100 | |
10 | 65 ± 5.1 (1.43) | 100 | 15 ± 1.5 (0.82) | 71 | |
50 | 66 ± 7.0 (1.45) | 100 | 16 ± 2.0 (0.88) |
|
|
| |||||
TA100 | 0 | 120 ± 23 (1.00) | 100 | 117 ± 19 (1.00) | 100 |
1.0 | 113 ± 12 (0.94) | 100 | 129 ± 18 (1.11) |
|
|
5.0 | 165 ± 12 (1.37) | 100 | 144 ± 14 (1.23) |
|
|
10 | 192 ± 9.4 (1.59) | 100 | 169 ± 9.4 (1.45) |
|
|
50 | 439 ± 39 ( |
100 | 297 ± 12 ( |
|
|
| |||||
TA1535 | 0 | 22 ± 3.2 (1.00) | 100 | 5.3 ± 1.5 (1.00) | 100 |
1.0 | 21 ± 1.5 (0.94) | 97 | 7.3 ± 1.1 (1.38) |
|
|
5.0 | 24 ± 2.0 (1.09) | 95 | 8.3 ± 2.3 (1.56) |
|
|
10 | 25 ± 4.5 (1.10) | 100 | 8.6 ± 1.1 (1.63) |
|
|
50 | 26 ± 1.5 (1.18) | 93 | 5.6 ± 1.1 (1.06) |
|
|
| |||||
TA102 | 0 | 254 ± 16 (1.00) | 100 | 265 ± 12 (1.00) | 100 |
1.0 | 258 ± 19 (1.02) | 100 | 266 ± 24 (1.01) | 100 | |
5.0 | 262 ± 24 (1.03) | 100 | 248 ± 9.1 (0.94) | 100 | |
10 | 262 ± 31 (1.03) | 100 | 260 ± 30 (0.98) | 100 | |
50 | 274 ± 16 (1.08) | 100 | 265 ± 7.6 (1.00) | 100 |
bPercent of cellular survival: <70% was considered toxic dose and it is marked in bold.
*Representation of a significant response (
Negative control was DMSO. Positive controls and their M.I., with and without S9 mix, respectively, were: 4NQO (7.19) and 2AA (9.55) for TA97, 4NQO (10) and 2AA (3.95) for TA98; AS (13) and 2AA (2.10) for TA100, AS (18) and 2AA (6.25) for TA1535, and MitC (9.28 and 6.86) for TA102.
The nitro analog PTAL 05-02 did not present a positive response for the
The nitro analog PAMT 09 did not present a mutagenic response for the
The mutagenicity assays with PTAL 04-09 presented a positive response for the
Table
Micronucleus test with RAW 264.7 macrophages using megazol analogs.
Compound | Dose ( |
‰ Mi.I. |
% Apoptosis | % Necrosis | % Survival | % Micronucleus ± S.D. |
---|---|---|---|---|---|---|
DMSO | 6.0 ± 4.2 | 0.06 | 0.12 | 99.8 | 0.7 ± 0.4 | |
PTAL 05-02 | 1.0 | 0.8 ± 0.0* | 5.90* | 0.16 | 93.9* |
|
10 | 3.0 ± 2.2 | 7.14* | 0.02 | 92.9* |
| |
100 | 1.2 ± 0.8* | 11.4* | 0.06 | 88.5* |
| |
PAMT 09 | 1.0 | 2.8 ± 1.6 | 2.68 | 0.00 | 97.3 |
|
10 | 1.2 ± 0.7* | 7.32 | 0.06 | 92.6 |
| |
100 | 1.2 ± 1.7* | 24.7* | 0.22 | 75.0* | 0.4 ± 0.2 | |
PTAL 04-09 | 1.0 | 3.2 ± 2.4 | 1.50* | 0.00 | 98.5* | 1.2 ± 0.8 |
10 | 1.2 ± 1.4* | 2.60* | 0.00 | 97.4* |
| |
100 | 0.4 ± 1.4* | 10* | 0.00 | 90.0* |
| |
MNNG | 5.2 ± 3.7 | 0.30 | 0.00 | 99.7* |
|
Percentage (%) and per mil (‰) mean and standard deviation (S.D.) of the ratio (samples/negative control) values of at least five experiments.
Negative control was DMSO; positive control was MNNG 0.5
All of the nitro analogs were capable of inducing the formation of micronuclei. However, this induction decreases as the concentration increases (PTAL 05-02 and PAMT 09). Moreover, apoptosis (all analogs) and necrosis (PAMT 09) increase as the concentration increases suggesting there is a cytotoxic activity which inhibits the formation of micronuclei.
The mutagenic and genotoxic assays with the new compounds were designed with recommended
In this work, the
Megazol was able to induce DNA lesions in different sites, since a positive response was found for almost all the tested concentrations and
Considering the number of highly DNA-reactive products that are formed during megazol metabolism, Farmanguinhos has designed three new synthetic megazol nitro analogs with efficacy against
Our results show that the nitro analogs did not present mutagenic activity for most of the
However, TA100 and TA1535 exhibited cytotoxic activity for most nitro analogs, only in the presence of S9 mix, suggesting that generated metabolites in this condition are more likely to induce base-pair substitutions mutations that lead to cell death.
Nevertheless, the mutagenic response of the nitro analogs was detected at higher concentration than the lowest megazol concentration to yield mutagenic activity showing the advances made in the development of new substances.
The structural modifications made on megazol to diminish mutagenic activity and improve antiparasitic potential are reported. The replacement of the megazol’s thiadiazole moiety for the triazole moiety on PTAL 05-02 and PAMT 09 and for the pyrazole moiety on PTAL 04-09 is known to improve the trypanocidal activity and reduce nonspecific toxicity [
In the genotoxic evaluation (micronucleus test), the analogs were capable of inducing the formation of micronuclei in macrophage cells. In addition, it was important to take into account the mitotic index, since mitosis is a prerequisite for micronucleus formation, and a low mitotic rate might explain the decreased rates of micronucleated cells at the highest concentrations. Furthermore, apoptosis, necrosis, and survival rates suggest cytotoxic activity, which might also be implicated in the low mitotic index and micronucleated cell formation.
It is reported the cytotoxic activity of megazol by genotoxic assays in cultures without endogenous activation by cytochrome P450 enzymes, while there was not the same activity when present [
PTAL 04-09 showed no genotoxic activity at the lower concentration and its cytotoxic activity was less prominent compared to other nitro analogs. Its structural modifications might be better suitable for the design of promising new drugs candidate for Chagas’ disease treatment. Furthermore, assays with the combination of
The demonstration that our compounds exhibited a lower mutagenic, cytotoxic, and genotoxic activity compared to megazol thanks to molecular structure modifications shows that the advances in the rational design of new molecules with lower toxicity and similar pharmacological profile may be of significance in safety evaluations for new drug development.
The authors declare that there is no conflict of interests.