DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2018.05

Research Article

Synthesis and evaluation trypanosomicidal activity of new derivatives of megazol

Helena B. Leites1, Flávia S. Damasceno2, Ariel M. Silber2, Ronaldo Z. Mendonça3, Cristina N. Albuquerque1*

1Departament of Biochemical-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, University of São Paulo. Av. Prof. Lineu Prestes, 580, bloco 16, Cidade Universitária, 05508-900, 2Laboratory of Biochemistry of Tryps - LaBTryps, Departament of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Prédio Biomédicas II, Av.Prof. Lineu Prestes 1374, Sala 24, Cidade Universitária, 05508-000, 3Laboratory of Parasitology and Entomology, Butantã Insitute, University of São Paulo. Av. Vital Brasil, 1500, Cidade Universitária, 05503-900, São Paulo, SP, Brazil

*For correspondence

Dr. Cristina N. Albuquerque,

Departament of Biochemical-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, University of São Paulo. Av. Prof. Lineu Prestes, 580, bloco 16, Cidade Universitária, 05508-900, São Paulo, SP, Brazil.

Email: northfle@usp.br

 

 

 

 

 

 

 

Received: 20 February 2018

Revised: 19 March 2018

Accepted: 20 March 2018

ABSTRACT

Objective: This work aims at the synthesis of megazol analogs with antitrypanosomicidal activity. Chagas'disease is caused by Trypanosoma cruzi and is a debilitating disease that has both acute and chronic forms. Many South Americans suffer from the chronic form of Chagas'disease, and there is no treatment currently available.

Methods: In the chemical part, classical techniques of heterocyclic synthesis as well as usual methods of identification were used. In the biological part the cell proliferation test was used in vitro and the IC 50.

Results: We synthesized a series of derivatives of 2-(1-methyl-5-nitro-2-imidazolyl)-5-substituted-1,3,4-thiadiazoles where 1-acetyl, 1-propyl and 1-nonyl were used as the substituent (4,6,7). Derivatives without nitro group were also synthesized (3,12) along with thiosemicarbazones (8,9,10) and a 5-(5-nitro-2-furanyl)-1,3,4-thiadiazol-2-amine (11). These compounds were evaluated using an in vitro test where were measured the cell proliferation. The derivatives that obtained the best results underwent further tests, in which their IC50 was calculated. The data revealed that two compounds (4,6) were effective against the parasite (IC50= 0.354 µM; IC50= 2.13 µM) and besides that, obtained the same results as the positive control, antimycim and rotenone. All proposed structures were obtained in satisfactory yields and purities.

Conclusions: In conclusion, the in vitro trypanocidal activity makes these compounds promising leads in the development of an effective therapeutic agent. However, this study must be completed by additional tests with in vitro amastigote/macrophage models or in vivo mouse models. Analyzing the amide derivatives, compounds (4) and (6) were the ones that presented the best results.

Keywords: 5-nitroimidazole, 1,3,4-Thiadiazole, Megazol, Trypanosoma cruzi, Antichagasic activity

Introduction

Chagas'disease is one of major causes of morbidity and death in Latin America, where it has been estimated that 16–18 million people are chronically infected by the T. cruzi parasite.1 No definitive immunological or chemotherapeutic treatment for this illness is available. This protozoan has a complex life cycle that involves different forms of the parasite in both the mammalian host and the insect vector. During the acute and chronic stages of the infection, the parasite presents numerous antigenic determinants to the mammalian immune system.2-5

The humoral and cellular immune response caused by the vertebrate host is able to keep the parasitemia at low levels and prevent the acute state after a new infection, but it is unable to eradicate the parasite. To better understand the biology of T. cruzi, the identification and characterization of its parasitic antigens important. This will also help in its diagnosis and immunoprophylaxis. Despite the socioeconomic importance of these tropical infections, efforts directed toward the discovery of new drugs and/or vaccines are underdeveloped.6

In addition, the drugs currently in use are expensive and require long-term treatment. The development of new, effective, cheap, and safe drugs for the treatment of Chagas' disease is an urgent task.7,8 A pharmaceutical interest in the imidazole ring is well established. Drugs containing the nitroimidazole ring are widely found in the therapy of amoebiasis, trichomonal, giardial and anaerobic bacteria, or as hypoxic cell radiosensitizers.9 Metronidazole and N-substituted imidazoles (ketoconazole, fluconazole and itraconazole) are well-tolerated drugs that are potentially active against different parasites.10 However, the antiparasitic property of 1,3,4-thiadiazoles is also well documented. Depending upon the type of substituent and position on ring, the attachment of 1,3,4-thiadiazoles to other heterocycles often ameliorates or diminishes the bioresponses.11

Derivative CL 64855 (5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole-2-amine), which is commonly called megazol (5) (Figure 1), is a nitroimidazole effective against T. cruzi. The compound is particularly interesting because it is active against all strains of the parasite. Due to the low efficiency and severe side effects of nifurtimox and benznidazole, megazol (5) presents a promising alternative.12 In previews papers, megazol (5) has exhibited high activity against T. brucei in conjunction with suramin or melarsoprol.13

Figure 1: Megazol structure.

In the 1980s, Brazilian researchers at the Oswaldo Cruz Institute and the René Rachou Center discovered a great number of chemical substances that are active against T. cruzi, which commonly causes Chagas'disease in Brazil. One of these substances, megazol (5), was of great interest because it was highly effective during in vivo rat studies, even when the rats received only one dose. As a result of this discovery, more extensive studies of analogous molecules have been deemed essential for the determination of the mechanism of action for megazol (5) and its potential toxic effects.14

Megazol (5) was initially synthesized in 1968 by Asato and Berkelhammer using 5-nitroimidazole as their starting material. Other synthetic routes were described by Albuquerque in 1995.15 Megazol (5) was a prototypical 5-nitroimidazole-1,3,4-thiadiazole derivative that showed promising antichagasic properties. The biological importance of nitroimidazoles and the previous papers in the field led us to propose the preparation of amide derivatives of 2-(1-methyl-5-nitro-2-imidazolyl)-5-substituted-1,3,4-thiadiazoles'as potential new antichagasic agents.16-25 Analogues containing 5-nitrofuran, thiosemicarbazone and without nitro group were also synthesized, to evaluate the importance of these structures to antichagasic activity.

Materials and Methods

Materials

1-methyl-2-imidazole-carboxyaldehyde (Sigma-Aldrich), 2-thiophene-carboxaldehyde

(Sigma-Aldrich), 5-nitro-1-methyl-2imidazole-carboxyaldehyde (Sigme-Aldrich), 5-

nitro-2-furaldehyde (Sigma-Aldrich), Acetic Anhydride (Synth), Acetone (Synth),

Ammonium hydroxide (Synth), Anhydrous sodium sulphate (Synth), Deuterated

dimethylsulfoxide (Sigma-Aldrich), Dimethylsulfoxide (Synth), Ethanol (Synth), Ethyl

Acetate (Synth), Hydrochloric acid (Synth), Nitric acid (Synth), Nonanoyl chloride

(Sigma-Aldrich), Propanoyl chloride (Sigma-Aldrich), Tetrahydrofuran (Synth),

Trichlorethylene (Synth), Thiosemicarbazide (Sigma-Aldrich).

Chemistry

The melting points were determined on a Kofler hot-stage apparatus and are uncorrected. The IR spectra were obtained on a Perkin-Elmer 1610 spectrophotometer using potassium bromide disks.

The 1H-NMR spectra were recorded on a Bruker spectrometer (Advance DPX-300, 300 MHz), and the chemical shifts are reported in parts per million () relative to tetramethylsilane (TMS) as an internal standard. The 75MHz 13C-NMR spectra were recorded on a Bruker DPX-75 spectrometer.

The elemental analyses were performed on a CHNO rapid elemental analyzer (GmbH Germany) for C, H and N, and the results are within ± 0.4% of the theoretical values.

Merck silica gel 60 F254 plates were used for analytical TLC and column chromatography was performed on Merck silica gel (70 – 230 mesh).

Synthesis of megazol

1-methyl-2-imidazolyl-thiosemicarbazone (2)

Thiosemicarbazide (0.350 g, 3.9 mmol) was added with 1-methyl-2-imidazolyl-carboxaldehyde (1) (3 mmol, 0,330 g) in dimethylsulfoxide (DMSO) (3 ml). The mixture was stirred at room temperature for 15 hours. After this time, the solvent was evaporated and a white solid was obtained. Recrystallization: ethanol. Yield: 0.44 g (81%), Mp: 160-162,2ºC. 1H-NMR (DMSO-d6-300MHz): 4,15 (s, 3H, N-CH3), 7,75 (sl, 2H, NH2), 8,11 (s, 1H, HC=N), 8,2 (s, 1H, H4), 8,29 (s, 1H, H5), 11,8 (s, 1H, NH); 13C-NMR (DMSO-d6-75MHz): 35,07 (N-CH3), 124,8 (C4), 131,4 (C5), 133,3 (HC=N), 141,7 (C2), 178,4 (C=S). Elemental Analysis: Found: C, 31,28; H, 3,47; N, 37,08. Calculated for C6H9N5S: C, 31,57; H, 3,53; N, 36,22.

5-(1-methyl-1H-imidazolyl)-1,3,4-thiadiazol-2-amine (3)

Compound (2) (0.3 g, 1.65 mmol) was added in hidrochloric acid (5 ml) and the reaction was maintained at room temperature for 1 hour. Then, the mixture was cooled and neutralized with ammonium hydroxide. After extraction with trichloroethylene (3 x 50 ml), the solvent was evaporated under reduced pressure and a white solid was obtained. Recrystallization: etanol. Yield: 0.21 g (71%). Mp: 240,1-241ºC. 1H-NMR (DMSO-d6-300MHz): 3,96 (s, 3H, N-CH3), 6,0 (s, 1H, H4), 7,3 (s, 1H, H5), 7,72 (sl, 2H, NH2); 13C-NMR (DMSO-d6-75MHz): 34,7 (N-CH3), 124,7 (C4), 128,4 (C5), 137,7 (C2), 149,8 (C*2), 168,2 (C*5). MS (EI) m/z 182 (M+H, 20%). Elemental Analysis: Found: C, 39,46; H, 3,79; N, 37,96; Calculated for C6H7N5S: C, 39,77; H, 3,89; N, 38,65.

5-(1-methyl-5-nitro-1H-imidazolyl)-1,3,4-thiadiazol-2-acetamide (4)

Compound (3) (0.2 g, 1.1 mmol) was added in acetic anhydride (5 ml) and the mixture was heated at 50ºC for 1 hour. After this time, nitric acid 70% (2 ml) was carefully added, and the reaction was heated at 60ºC for 2 hours. Then, the mixture was cooled and neutralized with ammonium hydroxide, obtaining a weak yellow solid. Recrystalization: acetone. Yield: 0.22 g (74,3%). Mp: 235,3-326,3ºC. 1H-NMR (DMSO-d6-300MHz): 2,56 (s, 3H, CH3), 4,41 (s, 3H, CH3), 8,28 (s, 1H, H4); 13C-NMR (DMSO-d6-75MHz): 22,4 (CH3), 35,23 (N-CH3), 133,09 (C4), 141,09 (C2), 147,9 (C*2), 159,7 (C*5), 169,2 (C=O). MS (EI) m/z 269 (M+ + 1, 10%). Elemental Analysis: Found: C, 35,92; H, 3,09; N, 30,35; Calculated for C8H8N6O3S: C, 35,82; H, 2,98; N, 31,34.

5-(1-Methyl-5-nitro-1H-2-imidazolyl)-1,3,4-thiadiazol-2-amine (5) – megazol

Compound (4) (0.2 g, 0.75 mmol) was added in hidrochloric acid (5 ml) and solution was stirred at room temperature for 1 hour. After this time, mixture was cooled and neutralized with ammonium hidroxide, obtaining a strong yellow solid. Recrystallization: acetone. Yield: 0,14g (79,7%). Mp: 269,1-270ºC. 1H NMR (DMSO-d6-300MHz): 4,33 (s, 3H, CH3), 7,83 (sl, 2H, NH2), 8,20 (s, 1H, H4); 13C-NMR (DMSO-d6-75MHz): 35,02 (N-CH3), 133,11 (C4), 141,47 (C2), 148,28 (C*2), 170,01 (C*5). MS (ESI=ion) m/z=227. Elemental Analysis: Found: C, 31,61; H, 2,60; N, 36,44; Calculated for: C6H6N6O2S: C, 31,83; H, 2,67; N, 37,15.

General procedure for the synthesis of compounds 6 and 7

Acid chloride (10 mmol) was added with a syringe through a septum cap on (5) (0.8 g, 3.5 mmol) in THF (20 ml) under argon at room temperature. Drops of pyridine were also added. After 3 hours, the precipitate formed was filtered and crystallized (ethanol/acetone, 2/1).

5-(1-methyl-5-nitro-1H-2-imidazolyl)-1,3,4-thiadiazol-2-propanamide (6)

Yield: 0.6 g (60%). Mp: 235-237,2ºC. 1H-NMR (DMSO-d6-300MHz): 1,11 (t, 3H, CH3), 2,50 (q, 2H, CH2), 4,32 (s, 3H, CH3), 8,37 (s, 1H, H4); 13C-NMR (DMSO-d6-75MHz): 19,8 (CH3), 27,35 (CH2), 35,02 (N-CH3), 133,1 (C4), 140,21 (C5), 141,48 (C2), 148,3 (C*2), 170,02 (C=O). Elemental Analysis: Found: C, 38,00; H, 3,14; N, 29,32; Calculated for C8H6O3S: C, 38,29; H, 3,57; N, 29,77.

5-(1-methyl-5-nitro-1H-2-imidazolyl)-1,3,4-thiadiazol-2-nonanoylamide (7)

Yield 0.8 g (62%). Mp.: 246,2-247,1°C. 1H-NMR (DMSO-d6-300MHz) δ 0.85 (s,3H,CH3), 1.25 (m,14H,(CH2)7), 4.39 (s,3H,NCH3), 8.26(s,1H,H4). 13C-NMR (DMSO-d6-75MHz) δ 13.6(CH3), 21.9-34.8((CH2)7), 35.2 (NCH3), 133.1(C4), 140.5(C2), 153.8(CONH), 159.9(C'2), 172.2(C'5). MS (DCI/NH3): m/z 367(M++1, 100%) 384(M++18, 15%). Elemental Analysis: Found: C, 48.89; H, 5.98; N, 21.80. Calculated for C15H22N6O3S: C, 49.17; H, 6.05; N, 22.93.

General procedure for synthesis of compounds 8, 9 and 10

Aldehyde (1 mmol), thiosemicarbazide (0.118 g, 1.3 mmol) and DMSO (3 ml) were mixtured and stirred for 12 hours at room temperature. Then, the solvent was evaporated to give the desired solid. Recristallyzation: ethanol.

2-formyl-5-nitrofuran-thiosemicarbazone (8)

Yield: 0.17 g (80%). Mp: 250,9-251,3ºC. 1H-NMR (DMSO-d6-300MHz): δ 7,42 (d, 1H, H3), 7,84 (d,1H, H4), 8,04 (s, 1H, CH=N), 8,56 (sl, 2H, NH2), 11,88 (s, 1H, NH). 13C-NMR (DMSO-d6-75MHz): δ 113,1 (C3), 115,1 (C4), 129,77 (CH=N), 151,5 (C2), 152,56 (C5), 178,42 (C=S). MS (EI) m/z 214 (M+, 3%). Elemental analysis: Found: C, 33,30; H, 2,60; N, 25,92; Calculated for C6H6N4O3S: C, 33,64; H, 2,82; N, 26,16.

2-thiophenyl-thiosemicarbazone (9)

Yield: 0.15 g (84%). Mp: 164,1-165ºC. 1H-NMR (DMSO-d6 300MHz): δ 7,12 (d, 1H, H3), 7,43 (dd, 1H, H4), 7,53 (d, 1H, H5), 8,00 (sl, 2H, NH2), 8,36 (s, 1H, CH=N), 10,39 (s, 1H, NH). 13C-NMR (DMSO-d6-75MHz): 127,8 (C4), 129,66 (C3), 130,5 (CH=N), 137,6 (C5), 139,1 (C2), 179,4 (C=S). Elemental analysis: Found: C, 38,70; H, 3,54; N, 22,62. Calculated for C6H6N4O2: C, 38,91; H, 3,78; N, 22,70.

1-methyl-5-nitro-2-imidazolyl-thiosemi-carbazone (10)

Yield: 0,15% (66%). Mp: 239-240ºC. 1H-NMR (DMSO-d6 300MHz): δ 4,15 (s, 3H, CH3), 8,11 (s, 1H, CH=N), 7,75-8,54 (sl, 2H, NH2); 11,8 (s, 1H, NH). 13C-NMR (DMSO-d6-75MHz): 35,07 (CH3N), 133,3 (CH=N), 133,4 (C4), 140,2 (C5), 144,6 (C2), 178,4 (C=S). MS (EI) m/z 229 (M + H, 80%). Elemental analysis: Found: C, 31, 28; H, 3,47; N, 37,08. Calculated for C6H8N6O2S: C, 31,57; H, 3,53; N, 36,82.

General procedure for synthesis of compounds 11 and 12

Compound (8-9) (0.1 g) was added in hidrochloric acid (2 ml) and reaction was mantained at room temperature for 1 hour. Then, the mixture was cooled and neutralized with ammonium hydroxide. After extraction with trichloroethylene (3 x 50 ml), the solvent was evaporated under reduced pressure and a solid was obtained. Recrystallization: acetone.

5-(5-nitro-2-furanyl)-1,3,4-thiadiazol-2-amine (11)

Yield: 0.059 g (60%) Mp: 280,3-281ºC. 1H-NMR (DMSO- d6-300MHz): 7,31 (d, 1H, H4), 7,83 (d, 1H, H3), 7,87 (sl, 2H, NH2). 13C-NMR (DMSO-d6-75MHz): 111,15 (C4), 115,10 (C3), 144,47 (C5), 147,8 (C2), 151,30 (C*2), 168,05 (C*5). MS (EI) m/z 212 (M+, 2%). Elemental analysis: Found: C, 33,80; H, 1,76; N, 25,85; Calculated for C6H6N4O3S: C, 33,96; H, 1,90; N, 26,40.

5-(2-thiophenyl)-1,3,4-thiadiazol-2-amine (12)

Yield: 0.072 g (73%). Mp:205-206,3ºC. 1H-NMR (DMSO-d6-300MHz): 7,13 (dd, 1H, H4), 7,41 (d,1H, H3), 7,43 (sl, 2H, NH2), 7,62 (d, 1H, H5). 13C-NMR (DMSO-d6-75MHz): 127,46 (C4), 127,49 (C3), 127,90 (C5), 133,25 (C2), 150,61(C*2), 168,05 (C*5). Elemental analysis: Found: C, 39,14; H, 2,52; N, 22,80; Calculated for C6H7N3S2:. C, 39,34; H, 2,73; N, 22,95.

Biological study

Culture of epimastigote forms

The assays were performed with the epimastigote form of Trypanosoma cruzi, which was cultured in Liver Infusion Triptose (LIT) medium and maintained in its exponential phase of growth. The LIT medium contains liver infusion (5.0 g/l), Tryptose (5.0 g/l), NaCl (4.0 g/l), KCl (0.4 g/l), Na2HPO4 (8.0 g/l), glucose (2.0 g/l), hemin (10.0 g/l) supplemented with 10% fetal bovine serum (FBS) at pH=7.2. The medium was maintained at 28°C.26

Evaluation of cell proliferation

The parasites were treated with the compounds at the final concentration of 5 μM. As a negative control, DMSO was used. For positive control (inhibition), a combination of Rotenone (60 μM) and Antimycin (0.5 μM) was used. The parasites (2.5 x 106) were tranferred to 96-well culture plates (200μl/well) and incubed at 28°C. Its proliferation was estimated by measuring the absorbance of the wells for 8 days, using a wavelength at 620 nm. After this process, the value obtained was converted into the number of parasites, The absorbance was transformed into cell density (cell / ml) values using a linear regression equation that was previously obtained under the same conditions.27 The GraphPadPrism5 program was used to construct the graphs.

Concentration inhibitory 50% (IC50)

The compounds that showed good activity against Trypanosoma cruzi underwent further tests, in which their IC50 was calculated. The parasite (2.5 x 106 ml-1) were also incubated in 96-well plates (200 μl / well) at 28°C for eight days. The IC50 was determined during exponential growth phase (fifth day). The compounds were used at concentrations 0.5 μM - 1.0 μM - 2.0 μM - 3.0 μM - 4.0 μM - 5.0 μM, resulting in a dose - response curve. The IC50 was determined by adjusting the data of this curve, using the OriginPro program 8.27

Results and Discussion

Chemistry

The synthesis of the 2-substituted-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazoles (6–7) began with the following versatile and efficient synthetic route (Figure 2) using 1-methyl-2-imidazolyl-carboxaldehyde (1) to obtain megazol (5).

Figure 2: Synthetic route to obtain megazol (5).

The reaction of 1-methyl-2-imidazolyl-carboxaldehyde (1) with thiosemicarbazide in DMSO gaves the thiosemicarbazone (2) correspondent. The nitrogen in position 1, a stronger nucleophile, reacts with carbonyl, which characterizes a substituition nucleophillic of carbonilic compounds. The yield obtained (81%) agrees with the literature (80-90%), which proves that the methodology was efficient. Then, compound (2) obtained reacted with hydrochloric acid, in a cyclization reaction, originated 5-(1-methyl-1H-imidazolyl)-1,3,4-thiadiazol-2-amine (3).28,29 To form 5-(1-methyl-5-nitro-1H-imidazolyl)-1,3,4-thiadiazol-2-acetamide (4) a nitration reaction was made using a mixture of nitric acid 70% and acetic anhydride. The anhydride is necessary for the protection of the amine group present in the 1,3,4-thiadiazolic ring because the temperature used in the reaction (60ºC) causes it to be protonated, being able to be attacked by the nitronium ion, impairing the yield. Then, a deprotection reaction was made using hydrochloric acid to give megazol (5).

To obtain derivatives (6) and (7), Megazol (5) was treated with an acid chloride in refluxing THF (Figure 3 A).

The compounds containing thiosemicarbazones (8-10) were obtained by reaction of respectives aldehydes with thiosemicarbazide in DMSO (Figure 3 B).

Finally, (8) and (9) were cyclizated by action of hydrochloric acid, giving the compounds (11) and (12) (Figure 3 C).

These reactions are standard in organic chemistry and are currently in scale-up production; therefore, we started an optimization study of the operational conditions.

Biology

For the assays, the epimastigote form of the parasite was used, in order to obtain data for studies of structure-activity-relationship (SAR) in this morphological type.

Cell proliferation assay

The parasites were plated on culture plates, and the compounds were added at the concentration of 5 μM. Thereafter, the absorbance of the wells was measured for 8 days. The values obtained were converted into number of parasites / ml.27 The substances that showed the best results underwent further tests, in which they had their IC50 calculated.

Inhibitory concentration assay 50% (IC 50)

Compounds (4) and (6) were selected for the IC50 assay after showing interesting antichagasic activity. Megazol (5), as might be expected, also obtained excellent action against Trypanosoma cruzi, and also had its IC50 calculated for comparison. The assay is performed using six known concentrations of the compounds, so that each will inhibit a given amount of protozoan. After the absorbance value is converted into a number of parasites, the program calculates the IC50.27,28 This parameter is calculated on the fifth day.

Figure 3: Synthetic pathway to compounds A=(6,7); B=(8,9,10); C=(11,12).

Initially, the tests would be performed at a minimum concentration of 0.5 μM and a maximum of 5 μM. However, when megazol (5) and (4) were tested, the minimum value already showed inhibition greater than 50%, having to be reduced. For these compounds was used the minimum concentration of 0.05 μM and maximum of 1 μM.

Chemistry

Proton NMR was used to confirm the structures of compounds. The spectra lacked a peak corresponding to a substituted amide group at position 5 of thiadiazole ring and contained a peak corresponding to the –NCH3 moiety at the same position. A singlet peak corresponding to the H4 of the nitroimidazole ring was also indicative of the desired products. In the 13C-NMR spectra, we confirmed the five carbons of the nitroimidazole ring and the CH peaks. The IR spectrum of the compounds contained a strong absorption at 3395-3135 cm-1 for –NH and at 1670-1690 cm-1 for C=O. Taken together with the LCMS and elemental analysis, these data confirmed the structure.

Figure 4: Growth curve of compounds synthesized.

In the 1H NMR spectra for structures (3), (10), (11) and (12) we observed the absence of the amide peak. In the spectra of (10), the peak of acid hydrogen (NH) of thiosemicarbazone was confirmed. In relation to (11), the peaks of H3 and H4 were confirmed, obtaining values of chemical shifts very close to those of furan. Finally, peaks of H3, H4 and H5 were confirmed for (12), with chemical shifts very close to those of thiophene. Elemental analysis also confirms the structure of such synthesized compounds.

Furthermore, the percent yields of these reactions make them suitable for adaptation into scale-up studies.

Biology

The growth curve of Trypanosoma cruzi is of the sigmoid type (Figure 4). The parasites begin to multiply slowly, and reach the exponential phase on the fifth day.

In relation to compound (11), it is known that 5-nitrofurans have wide antiparasitic activity, as can be seen in nifurtimox and nitrofurazone.28,29 However, by evaluating the growth curve, it can be seen that the compound didn't present such activity. Initially, (11) showed interesting antichagasic activity due to Trypanosoma cruzi still being in the stationary phase of growth. But after the fifth day, when the protozoan reached the exponential phase, it was observed that there was a great increase in the cellular proliferation of the parasites. So, in this case, the exchange of the imidazole by furan worsened the interaction with the target, not contributing to the antichagasic activity.

Compound (10) was the only derivative of the series containing a thiosemicarbazone which was tested. Several studies have already proven the ability of this group to inhibit cruzain, an essential enzyme for the survival of the parasite in the host. The mechanism of inhibition, as already proven by molecular modeling studies, occurs through the nucleophilic attack of Cys-25 present in the cruzaine to the thiocarbonyl carbon of thiosemicarbazone. Then, the His-159 residue donates a proton to the carbon-bound sulfur, forming a tetrahedral intermediate.30-32 However, when tested, it was found that (10) only showed interesting activity until the stationary phase of Trypanosoma cruzi. After the fifth day, when the protozoan reached the exponential phase, the compound couldn't contain its growth, not presenting the expected activity.

This can be explained by the absence of benzene rings as substituents. Studies have shown that such groups covalently bind to the s2 substitium of cruzaine, favoring more the inhibition of this enzyme.31,33 As compound (10) doesn't have this point of interaction, it can't bind to subsite s2, not so favoring activity.

Analyzing the amide derivatives, compounds (4) and (6) were the ones that presented the best results.

It is known that the parasites have enzymes called peptidases, which hydrolyze the amide bond to the amine. Thus, these results may be related to the protection of the molecule by the transformation of the amine functionality, which could be linked to the drug removal and breakdown processes by theses enzymes. Therefore, these derivatives that contain the megazol (5) moiety may serve as prodrugs. In the case of compound (7), which didn't show good results, it is possible that the peptidases failed to hydrolyze the lable bond due to long carbon chain. To confirm this hypothesis, further studies are necessary.

When analyzing (4) (Figure 5), it's noted from the low IC50 (IC50=0.354 µM) that the peptidases succeeded in hydrolyzing the short carbon chain, releasing the active compound (5). In addition, when the compound was used in the amount of 1 μm, almost 100% inhibition was obtained (Table 1). This derivative obtained results very close to those of the positive control (Antimicyn and Rotenone), which makes it a promising compound.

Table 1: Concentration of compounds 4 and 5 (log/µm).

Concentration
(µm) Log (µm)
0,05 -1,3
0,1 -1,0
0,2 -0,69
0,3 -0,52
0,5 -0,3
1,0 0,0

Compound (6) was shown to be less potent than (4), although it also presented interesting activity, obtaining IC50 of 2.13 µM (Figure 7). This molecule is more lipophilic than (4) and (5) due to higher cLogP (Table 4). In this way, it should be more easily internalized by biological membranes, which would contribute to biological activity. However, in this case, the increase of lipophilicity is not related to the increase of the absorption, and therefore, to the increase of the antiparasitic activity. Nevertheless, further studies are needed to confirm this hypothesis.

It is known that most megazol is internalized into the cell by passive diffusion, therefore, the value of cLogP has great influence. But another part binds to the p2-amino-purine transporter, which also carries melarsoprol, adenins, and adenosines. The carrier binds the four substances by means of the C=N-NH2 moiety, so the amine group must be free.14,34,35 The insertion of carbon chains may have somewhat disrupted the absorption of this compound by means of this carrier.

Figure 5: Growth curve in the presence of different concentrations of compound 4.

Figure 6: Growth curve in the presence of different concentrations of compound 5.

Table 2: Concentration of compound 6 (log/µm).

Concentration
(µm) Log (µm)
0,5 -0,3
1 -0,0
2 -0,3
3 -0,47
4 -0,6
5 0,69

Finally, it can be observed that compounds (3) and (12) (Figure 4) could not contain cell proliferation of the parasites, even in the stationary growth phase, obtaining the same results as the negative control, which confirms a bad activity. None of the compounds contain the nitro group in its structure, confirming its importance for biological activity.

Figure 7: Growth curve in the presence of different concentrations of compound 6.

Table 3: Value of cLogP for compounds (4), (5) and (6).

Compound cLogP
Megazol- 5 0,14
5-(1-methyl-5-nitro-1H-imidazolyl)-1,3,4-thiadiazol-2-acetamide - 4 0,21
5-(1-methyl-5-nitro-1H-2-imidazolyl)-1,3,4-thiadiazol-2-propanamide – 6 0,91

Thus, the in vitro trypanocidal activity of nitroimidazolyl-1,3,4-thiadiazole may be due to the potential of reducing the transfer of a single electron ArNO2/ArNO2-.

Nitroheterocylic drugs (e.g., nifurtimox, metronidazole, benznidazole and the own megazol (5)) are generally believed to exert their cytotoxic effects only after their activation by a single-electron reduction of the corresponding nitro anion radicals. Under anaerobic conditions, the radical anion can be transformed into the corresponding nitroso derivative. This nitroso form has been put forth as an efficient scavenger of essential thiols in the cell. Under aerobic conditions, the nitro radical anion reacts with oxygen to form a superoxide anion and a hydroxyl radical. The resulting oxygen-derived free radicals would damage the enzyme, its DNA or other important structures in the surrounding cell, and result in a cytotoxic action.

Thus, it follows that the trypanocidal activity of these compounds, which are structurally related to megazol (5) and other nitroimidazoles, is associated with oxygen metabolism interference and their role as thiol scavengers.

In conclusion, the in vitro trypanocidal activity makes these compounds promising leads in the development of an effective therapeutic agent. However, this study must be completed by additional tests with in vitro amastigote/ macrophage models or in vivo mouse models.

From the structure activity relationship studies, we observed that the position and number of carbons in the alkyl chain moiety is important in reasoning the activity of the 1,3,4-thiazole derivatives (4,6,7). Other derivatives of this class are being analyzed and will assist in a better understanding of the activity and effectiveness of these antichagasic drugs.

Acknowledgements

The authors would like to thank the management of FAPESP and CNPq for supporting this work. The authors also appreciate the cooperative collaboration with their analytical research department.

Funding: No funding sources

Conflict of interest: None declared

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