BR102018067503A2 - USE OF SYNTHETIC HYDROXYLATED COMPOUNDS DERIVED FROM INDAN-1,3-DIONA AS ANTI-VIRALS AND PHARMACEUTICAL COMPOSITIONS - Google Patents

Abstract

use of hydroxylated synthetic compounds derived from indan-1,3-dione as antivirals and pharmaceutical compositions the present invention relates to the use of two synthetic hydroxylated derivatives of indan-1,3-dione and pharmaceutical compositions containing such compounds for the treatment of infections caused by the West Nile (WNV), Dengue (Denv) and Zika (ZikV) viruses and covers the field of medicinal chemistry.

Description

Descriptive report USE OF SYNTHETIC COMPOUNDS DERIVED FROM INDAN-1,3-DIONA AS ANTI-VIRALS AND COMPOSITIONS

PHARMACEUTICAL

FIELD OF THE INVENTION 01. The present invention covers the field of medicinal chemistry and relates to the use of two synthetic hydroxylated compounds derived from indan-1,3-dione and pharmaceutical compositions containing such compounds for the treatment of infections caused by West viruses Nile (WNV), Dengue (DENV) and Zika (ZIKV).

STATE OF THE TECHNIQUE 02. Dengue is an arbovirus caused by a virus belonging to the Flavirity family, of the genus Flavivirus. There are four serotypes of the dengue virus, DENV-1, DENV-2, DENV-3 and DENV-4, in addition to a fifth emerging serotype, all of which are transmitted by Aedes aegypti and more rarely by the Aedes albopictus mosquito (Schuller, A. , et al., Tripeptide inhibitors of dengue and West nile virus NS2B-NS3 protease. Antiviral Res, 2011. 92: p. 96-101; Behnam, MAM, et al., Discovery of nanomolar dengue and west nile virus protease inhibitors containing a 4-benzyloxyphenyl glycine residue (J Med Chem, 2015. 58: p. 9354-9370). 03. The West Nile virus (WNV, English), since its initial isolation in Uganda in 1937, has become an important cause of human and animal diseases worldwide. WNV, also of the genus Flavivirus, has increased its geographic distribution, reaching Africa, the Middle East, southern Europe, western Russia, southern western Asia, Australia and North America. This virus can cause severe neurological disease resulting in long-term sequelae or death (Chancey, C., et al., The global ecology and epidemiology of West Nile virus. Biomed Res Int, 2015. 2015: p. 376230). Recently, the first human case of WNV was reported in Brazil, with the development of encephalitis, in the state of Piauí. It is possible that sporadic cases or small groups of WNV disease have already occurred in different regions of the country and have remained undiagnosed (Vieira, MA, et al., West Nile Virus Encephalitis: The First Human Case Recorded in Brazil. Am J Trop Med Hyg , 2015. 93: p. 377-9). However, the circulation of the virus had already been confirmed in Brazil in an investigation that studied horses and birds in the period 2008-2010 (Ometto, T., et al., West Nile virus surveillance, Brazil, 2008-2010. Trans R Soc Trop Med Hyg, 2013. 107: p. 723-30). WNV is now the most common cause of neurological disease caused by an arbovirus in the world (Gould, L.H. and E. Fikrig, West Nile virus: a growing concern? J Clin Invest, 2004. 113: p. 1102-7). 04. Since the emergence of the Zika virus in Brazil in May 2015, it has generated global concern due to its association with a significant increase in the number of children born with microcephaly and neurological disorders, such as Guillain-Barré syndrome (Abushouk, AI , A. Negida, and H. Ahmed, An updated review of Zika virus. J Clin Virol, 2016. 84: p. 53-58). More than 440,000 to 1.3 million individuals were estimated to be infected with ZIKV in 2015, and their global distribution has already exceeded 50 countries (Musso, D., Zika Virus Transmission from French Polynesia to Brazil. Emerg Infect Dis, 2015. 21 (10): p. 1887). 05. Although some flaviviruses already have available vaccines, DENV, WNV and ZIKV do not yet have an efficient and global vaccination. In view of the challenges for the development of a vaccine, research for antiviral therapies has become relevant, especially for severe cases of the three diseases. The development of antiflaviviral therapies remains a high priority for the World Health Organization (Noble, C.G., et al., Strategies for development of Dengue virus inhibitors. Antiviral Res, 2010. 85: p. 450-62). 06. In the present application for patent protection, two hydroxylated compounds derived from indan-1,3-dione (compounds (2) and (4), Figures 2 and 3) that have relevant antiviral activity against DENV, WNV and ZIKV. 07. Indan-1,3-dione (Figure 1) is an important member of the class of 1,3-dicarbonylated compounds that have a wide range of molecules of chemical and pharmaceutical interest. The diverse chemistry of indan-1,3-dione attracts constant interest, since it is a valuable synthetic precursor and its derivatives have been used as dyes; as semiconductors; in forensic chemistry, for fingerprint detection; and also used in the synthesis of drugs and agrochemicals. 08. Considering the antiviral activity, several studies indicate the action of indan-1,3-dione derivatives against human papillomavirus (HPV). Goudreau and colleagues synthesized a derivative of indan-1,3-dione capable of inhibiting the interaction of HPV11 E1 and E2 proteins with an IC50 of 11 pmol L-1 (Goudreau, N., et al., Optimization and determination of the absolute configuration of a series of potent inhibitors of human papilloma virus type-11 E1-E2 protein-protein interaction: a combined medicinal chemistry, NMR and computational chemistry approach. Bioorg Med Chem, 2007. 15: p. 2690-700). In another investigation, HPV11 inhibition was observed by the same mechanism of action; however, the IC50 was 610 nmol L-1 (Davidson, W., et al., Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry. Anal Chem, 2004. 76: p. 2095-102). Yokim and collaborators also obtained a similar result, with the same mechanism of action, and an IC50 value of 11 pmol L-1 (Yoakim, C., et al., Discovery of the first series of inhibitors of human papiloma virus type 11: inhibition of the assembly of the E1-E2-Origin DNA complex (Bioorg Med Chem Lett, 2003. 13: p. 2539-41). 09. The viral enzymatic inhibition activity for indan-1,3-dione derivatives stands out, as demonstrated in the study by Artico et al., In which derivatives inhibited the HIV 1 virus integrase enzyme, with an IC50 of 3 ± 0 , 5 umol L-1 (Artico, M., et al., Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling. J Med Chem, 1998. 41: p. 3948 -60). In the research conducted by Liu and colleagues, derivatives of indan-1,3-dione were evaluated against the hepatitis C virus (HCV), which also belongs to the family Flaviridae such as WNV, DENV and ZIKV. Four compounds were found capable of inhibiting HCV virus protease with IC 50 values of 12; 3.6; 3.1 and 1.7 umol L-1 (Liu, Y., et al., Investigating the origin of the slow-binding inhibition of HCV NS3 serine protease by a novel substrate based inhibitor. Biochemistry, 2003. 42: p. 8862-9). 10. In a search carried out in the national and international patent banks, some documents were found that present technologies involving indan-1,3-diones for biological use, but different from the one presented in the current patent application. As an example and differentiation, some of them are presented below. 11. The patent CN20141505723 describes the synthesis of indan-1,3-dione derivatives with pharmacological properties, showing activity in the treatment of hepatitis B and also with anti-inflammatory activity. 12. The patent CN20151813066 describes the synthesis of indan-1,3-dione derivatives with pharmacological properties, showing antitumor activity. 13. Patents US19780936396, US19550517095 describe the synthesis of indan-1,3-dione derivatives as a method of eliminating animal parasites. 14. The patents US19750576347, US19530336440, US19720260512, HU1977RE00608, WO2004GB05369, GBD1350900, GB19680053666, GB19680053650, GB19760046858, GB19650043477, GB196505020033, 2008, 2009, 2006 use as rodenticide. 15. JP19930031963 describes the synthesis of indan-1,3-dione derivatives with herbicidal properties. 16. Patents GB19580010410, CAD614511, DE19702045466 describe the synthesis of indan-1,3-dione derivatives with anticoagulant property. 17. Although several compounds belonging to the class of indan-1,3-diones have been reported, no records related to the antiviral application of compounds (2) and (4) or antiviral activity of these compounds against viruses have been found in the patent database. WNV, DENV and ZIKV similar to those described in the present invention, object of patent application. Specifically with respect to the application of compounds (2) and (4) in the field of medicinal chemistry, it was found only that compound (2) showed, in vitro, moderate antibacterial activity against Proteus vulgaris. For the evaluation of bacterial activity, the assay using the agar diffusion disc test was used. Compound (2) showed an inhibition halo of 16 mm, while the amikacin compound (positive control) showed an inhibition halo of 28 mm. This same compound (2) showed, in vitro, considerable antifungal activity against Candida albicans (inhibition halo equal to 25 mm); the positive control griseofulvin showed a 25 mm inhibition zone (MEENA, S., et al., Synthesis of 2- (aryl methylene) - (1H) -indane-1,3- (2H) -diones as potential fungicidal and bactericidal agents, Indian J Chem, 2006. 45B: p. 1572-1575). 18. Thus, the object of this patent application is the use of hydroxylated synthetic compounds derived from indan-1,3-dione, as well as compositions containing such compounds, such as antiviral drugs against West Nile, Dengue-1 viruses, Dengue-2, Dengue-3, Dengue-4 and Zika.

DESCRIPTION OF THE FIGURES 19. Figure 1: Basic structure of indan-1,3-dione. 20. Figure 2: Synthesis of the compound (2). 21. Figure 3: Synthesis of the compound (4). 22. Figure 4: Antiviral screening for WNV protease. Compounds (2) and (4) of indan-1,3-dione were analyzed at a unique concentration of 16.6 μmol L-1 against WNV protease. One-way ANOVA multiple comparisons statistical test was used, with a p-value <0.05. 23. Figure 5: Enzymatic inhibition of WNV protease in the presence of varying concentrations of compounds (2) and (4). The enzymatic inhibition was analyzed in the presence of seven concentrations (final concentration of 33 μmol L-1 to 0.5 μmol L-1) of compounds (2) and (4), in A and B, respectively. The regression calculation was based on Hill's coefficient. The IC50 values are 11.27 μmol L-1 and 3.19 μmol L-1 for compounds (2) and (4), respectively. [] corresponds to the concentration of the compounds in μmol L-1. 24. Figure 6: Enzymatic kinetics of the WNV protease in the presence of compounds (2) and (4). Michaelis-Menten plots for compounds (2) and (4) are represented in C and E, respectively. In D and F are the Lineweaver-Burk plots for compounds (2) and (4), respectively. 25. Figure 7: Assay for cytotoxicity assessment of compounds (2) and (4) against Vero cells. In G is cell viability for compound (2) and in H is cell viability for compound (4). The regression calculation was based on Hill's coefficient. The CC50 values for compounds (2) and (4) are, respectively, 89.72 pmol L-1 and 267.60 pmol L-1, respectively. 26. Figure 8: Pre-treatment antiviral assay for compounds (2) and (4) at a concentration of 100 pmol L-1 in Vero cells for the DENV-2 and ZIKV viruses. 27. Figure 9: Post-treatment antiviral assay for compounds (2) and (4) at a concentration of 100 pmol L-1 in Vero cells for the DENV-2 and ZIKV viruses.

DETAILED DESCRIPTION OF THE INVENTION 28. The present invention relates to the use of compounds 2- (4-hydroxybenzylidene) -1H-indene-1,3 (2H) -dione (compound 2) and 2- (3,4-dihydroxybenzylidene) -1H-indene-1,3 (2H) -dione (compound 4) (Figures 2 and 3, respectively), their pharmaceutically acceptable salts, solvates, hydrates or prodrugs and compositions containing these compounds, such as antivirals. 29. Compounds (2) and (4) have activity against WNV protease and can be applied as antivirals to treat infections caused by flaviviruses WNV, Zika and Dengue. 30. Compounds 2- (4-hydroxybenzylidene) -1H-indene-1,3 (2H) -dione (compound 2) and 2- (3,4-dihydroxybenzylidene) -1H-indene-1,3 (2H) - dione (compound 4) may also be used in the production of pharmaceutical compositions. Such compositions comprise carrier agents, diluents and excipients, such as tablets, lactose, magnesium, stearate, gelling agents, among others.

SUMMARY OF COMPOUNDS 31. The compound of structure (2), shown in Figure 2, was prepared by Knoevenagel condensation, using indan-1,3-dione (1) and 4-hydroxybenzaldehyde as starting material. , using the conditions described in Example 1, below. 32. The synthesis of compound (4) was performed, as shown in Figure 3, by means of demethylation 2- (3,4-dimethoxybenzylidene) -1H-indene-1,3 (2H) -dione (3) using standard procedure described in the literature, that is, demethylation with boron tribromide (BBr3), and also described in Example 2, below. 33. The compounds were characterized by infrared (IR) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The procedures used in the synthesis of compounds (2) and (4) and the data related to their structural characterization are presented below. 34. Example 1: Synthesis of 2- (4-hydroxybenzylidene) -7H-indene-1,3 (2H) -dione (compound 2). 35. Indan-1,3-dione (1) (1.00 mmol), 4-hydroxybenzaldehyde (1.00 mmol), zirconyl chloride octahydrate ZrOCb8H2O (0.04 mmol) and distilled water were added to a flask round bottom (10 mL). The reaction mixture was heated to temperatures ranging from 25 to 90 ° C and kept under magnetic stirring for a few minutes. The reaction was monitored by thin layer chromatography (CCD). After the end of the reaction, the mixture was vacuum filtered and the residue was washed with ice-cold ethanol. Compound (2) was obtained in 76% yield (0.759 mmol) after recrystallization from acetone-dichloromethane (1: 1 v / v). 36. Characteristic: yellow solid. 37. CCD: Rf = 0.38 (hexane-ethyl acetate 2: 1 v / v). 38. Tf = 235.8-236.0 ° C. 39. IV (ATR) v max: 3361-3043 (broadband), 3059, 1715, 1655, 1547, 1505, 1204, 737. 40. 1H NMR (300 MHz, DMSO-cfe) δ: 6.92 ( d, 2H, J = 8.7 Hz); 7.72 (s, 1H); 7.86-7.92 (m, 4H); 8.50 (d, 2H, J = 8.7 Hz); 10.86 (s, 1H, OH). 41. 13C NMR (75 MHz, DMSO-cfe) δ: 116.4; 123.1; 123.2; 125.0; 125.6; 135.8; 135.9; 138.0; 139.6; 142.0; 146.7; 163.7; 189.4; 190.4. 42. Example 2: 2- (3,4-dihydroxybenzylidene) -1H-indene-1,3 (2H) -dione (compound 4). 43. 2- (3,4-dimethoxybenzylidene) -1H-indene-1,3 (2H) -dione (3) (0.679) was added to a bitubulated flask (150 mL) under a nitrogen atmosphere (N2). mmol) together with 5.0 mL of anhydrous dichloromethane. The resulting mixture was kept under magnetic stirring and cooled in an ice bath for a few minutes. Then, 2.7 ml of 1.00 mol L-1 solution of BBr3 in dichloromethane was added dropwise. After the addition, the reaction mixture was kept under stirring for 24 hours at room temperature. Subsequently, 10.0 mL of distilled water was added and the formation of a brown precipitate was observed. The resulting mixture was transferred to a separatory funnel and the aqueous phase was extracted with ethyl acetate (3 x 30.0 ml). The organic extracts were combined, and the resulting organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Compound (4) was obtained as a solid, after washing with dichloromethane and acetone, in 58% yield (0.394 mmol). 44. Characteristic: yellow solid. 45. CCD: Rf = 0.05 (hexane-ethyl acetate 2: 1 v / v). 46. Tf = 238.0-239.4 ° C. 47. IV (ATR) v max: 3492-3109, 3093, 3039, 1720, 1666, 1555, 1384, 1182, 730. 48. 1H NMR (300 MHz, DMSO-cfe) δ: 6.89 (d, 1H, J = 8.1 Hz); 7.64 (s, 1H); 7.83 (dd, 1H, Ji = 8.1 Hz, J2 = 1.8 Hz); 7.86-7.94 (m, 4H); 8.32 (d, 1H, J = 1.8 Hz). 49. 13C NMR (75 MHz, DMSO-cfe) δ: 116.2; 121.0; 123.1; 125.2; 125.5; 130.7; 135.7; 135.9; 139.6; 142.1; 145.7; 147.3; 153.0; 189.4; 190.5.

DEMONSTRATION EXPERIMENTS

Evaluation of the antiviral activity of compounds (2) and (4) Experimental conditions for evaluation of the activity of compounds (2) and (4) against the NS2B-NS3 protease of the WNV 50 virus. The purified and activated WNV virus protease provided was used by R&D Systems (Minneapolis, MN, USA), Recombinant Viral WNV NS3 Protease (catalog number 2907-SE). As a fluorescent substrate, pERTKR-AMC (R & D Systems, Minneapolis, MN, USA, catalog number ES013) was used. In order to investigate whether compounds (2) and (4) (Figures 2 and 3) are capable of inhibiting the NS2B-NS3 protease of the WNV virus, a total of 50 pL of the purified protease (final concentration of 1ng juL-1) diluted in buffer solution (50 mmol L-1 Tris, 30% (v / v) Glycerol, pH 9.5) was incubated with 50 pL of the compound (final concentration of 16.6 pmol L-1) in a black 96 wells for 30 min at 21-22 ° C before the start of the reaction by adding 50 pL of the substrate at a concentration of 40 μmol L-1. A positive control without the compound was tested on the same plate, with 50 pL of buffer, in addition to a negative control with 1% DMSO. The blank contained 50 pL of buffer and 100 pL of substrate. The fluorescence intensity was recorded continuously at an excitation wavelength of 360 nm and an emission wavelength of 460 nm using the SpectraMax® M5 (Molecular Devices) fluorescence reader. The assays for calculating the IC50 were performed with a total of 50 pL of purified NS2B-NS3-protease (final concentration of 1ng pL-1) incubated with 50 pL of the compound being evaluated; eight different concentrations (66, 33, 16, 8, 4, 2, 1 and 0.5 pmol L-1) were used. For the kinetics assay, 50 pL of purified NS2B-NS3 protease (final concentration of 1ng pL-1) was incubated with 50 pL of the compound being evaluated, at the final concentration of 2, 4 and 8 pmol L-1, in a plate 96-well black for 30 min at a temperature of 21-22 ° C, before starting the reaction by adding 50 pL of the substrate in different concentrations (8, 16, 24, 32 and 40 pmol L-1). The analysis of the results was conducted using the GraphpadPrism 6 program using the one-way multiple comparison test ANOVA.

Experimental conditions for evaluating antiviral activity in cell 51. Vero cells were used to carry out the experiments, which are continuous cultures of African green monkey kidney (Cercopithecus aethips). The medium used for the growth and maintenance of the cells was the DMEM medium. This medium was supplemented with fetal bovine serum (SFB) in the proportion of 10% for growth promotion and 2% for maintenance of the cell line. To prevent contamination of cell cultures by bacteria and fungi, 1% PSA (10,000 U of Penicillin, 10,000 pg of Streptomycin and 25 pg of Amphotericin B) was added to the medium. To obtain cell subcultures, maintain cells and perform experiments with Vero cells, trypsin (trypsin from pig pancreas prepared in EDTA solution 1: 250) was used as a dissociating agent, which is a proteolytic enzyme that catalyzes reactions of polypeptide chain break in certain amino acid sequences. 52. The cytotoxicity assessment of compounds (2) and (4) was performed against Vero cells. The MTT colorimetric assay was used. A suspension of Vero cells, containing approximately 1x104 cells / mL, obtained by trypsinization of a cell culture flask, was distributed in a 96 well plate per well (100 µl / well). The plate was incubated for 24 h at 37 ° C. After 24 h, with the confluent cell monolayer, the DMEM medium was replaced by 100 pL of the various concentrations of the compounds diluted in incomplete DMEM medium (400, 200, 100, 50, 25, 12 and 6 pmol L-1). The plate was incubated for 72 h at 37 ° C. The medium was then removed and 50 µl MTT (1 mg mL-1) was added, and the plate was incubated again for 4 h. The MTT medium was removed and replaced with 100 µL of DMSO / well; the plate was shaken for 10 minutes and a spectrophotometer reading at 540 nm was performed. The absorbance obtained was directly proportional to cell viability, which was calculated using the absorbances of monolayers treated with test material, compared to cell control. The CC50 values, that is, the concentration of each evaluated compound that reduced cell viability by 50%, was obtained by regression analysis of the percentages referring to the different concentrations of the compounds. The results were treated in GraphpadPrism 6 and the CC50 values represented the average of three independent experiments. 53. The evaluation of the virucidal activity of compounds (2) and (4) was performed on Vero cells, which were cultured in 24-well plates for a total of 8x105 cells per plate with 10% medium for 24 h. A volume of 100 pL of viral suspension (50-100 UFP) was incubated with 100 pL of varying concentrations of compounds (2) and (4) under evaluation (3, 6, 12, 25, 50 and 100 pmol L-1) and incubated at 37 ° C for 1 h. The medium was removed by aspirating the plates and 100 µl of the virus mixture (DENV-1,2,3,4 or ZIKV) and the compound under evaluation were added to the cell monolayer. The plates were incubated for 1 hour, with shaking for better viral distribution. After that time, the viral suspension mixed with the different concentrations of the compound under evaluation was aspirated and 1500 pL of a 3% CMC solution in DMEM medium (twice concentrated) plus 2% SFB and 1% PSA were added to each well. . The plates were incubated for 5-6 days. After this period, the medium was removed and the cells fixed and stained. In this assay it was possible to calculate the EC50 value by regressing the inhibition percentages for the different concentrations of the test compounds with GraphpadPrism 6. 54. For the pretreatment assay, Vero cells were cultured in 96-well plates (2 , 0 x 105 cells / mL, 100 μl / well) until confluence (24 h) using MEM medium supplemented with 5% SFB and incubated at 37 ° C with 5% CO2. After 24 h, the medium was removed by aspiration and 100 pL of the different concentrations of compounds (2) and (4) (100, 50, 25, 12, 6 and 3 pmol L-1) were added, which were in contact with the cellular mat for 3 h at 37 ° C with 5% CO2. After this period, the compounds were carefully aspirated and 200 µl of the viral suspension (MOI = 2) was added. Cell control (100 pL medium / well) and viral control (200 pL viral suspension / well, MOI = 2) were performed on the same plate. The plates were incubated for 4 h in the same conditions mentioned above for viral adsorption. Subsequently, the supernatant was removed and 200 µl of incomplete medium was added. The plates were incubated for 96 h in the same conditions mentioned above. After this period, the medium was aspirated and 50 pL of MTT (1 mg / mL) were added, and the plate was incubated for another 4 hours. The MTT medium was removed and replaced with 100 pL of 0.1 mol L-1 DMSO / well, the plate was shaken for 10 minutes and a spectrophotometer reading at 540 nm was performed. 55. The absorbance values measured for each concentration of each test material were transformed into a percentage (x%) in relation to the cellular and viral controls, using the following equation: 56. For the post-treatment test, Vero cells were cultured in 96 well plates (2.0 x 105 cells / mL, 100 μl / well) until confluence (24 h) using MEM medium supplemented with 5% SFB and incubated at 37 ° C with 5% CO2. After 24 h, the medium was removed by aspiration and 100 pL of the viral suspension was added (MOI = 2). Cell control (100 pL medium / well) and viral control (200 pL viral suspension / well, MOI = 2) were performed on the same plate. The plates were incubated for 4 hours in the same conditions mentioned above for viral adsorption. Subsequently, the supernatant was removed and 200 pL of the different concentrations of compounds (2) and (4) (100, 50, 25, 12, 6 and 3 pmol L-1) were added. The plates were incubated for 96 h in the same conditions mentioned above. After this period, the compounds were aspirated and 50 pL of MTT (1 mg mL-1) was added and the plates were incubated again for 4 h. The MTT medium was removed and replaced with 100 pL of 0.1 mol L-1 DMSO / well, the plates were shaken for 10 minutes and a spectrophotometer reading at 540 nm was performed. The calculations were performed according to what has been described for the pre-treatment.

Evaluation of Antiviral Activity 57. Compounds (2) and (4) were biologically evaluated against WNV viral protease and against DENV-1,2, 3, 4 and ZIKV viruses. 58. To assess the antiviral action of the two compounds, an assay was initially carried out to assess their effect on the WNV protease. The protease was incubated for 30 minutes with the compounds under evaluation. Subsequently, the reaction was initiated with the addition of the fluorescent substrate pERTKRAMC and the reading was performed in Relative Fluorescence Units (RFU) in a microplate reader. The compounds (2) and (4) showed an inhibitory behavior, with a reduction, respectively, of approximately 41% and 100% of the enzymatic action, in the single concentration of 16.6 pmol L-1 of the compounds under investigation (Figure 4). 59. The enzymatic inhibition of WNV protease was evaluated at concentrations of 66, 33, 16, 8, 4, 2, 1 and 0.5 pmol L-1 of compounds (2) and (4). The two compounds showed dose-response inhibition with IC 50 values of 11.27 and 3.19 μmol L-1, respectively (Figure 5). 60. The enzymatic kinetics assay was conducted using varying concentrations of substrate. The Michaelis-Menten equation was used to find the maximum speed and KM values. Subsequently, the Lineweaver-Burk chart was constructed. The compounds (2) and (4) promoted a significant decrease in the maximum enzymatic speed without changes in the KM values, as can be seen in Figure 6. The Lineweaver-Burk graph showed lines that intersect on the x axis. The Ki values were, respectively, 12.31 and 1.25 μmol L-1 for compounds (2) and (4). This inhibition profile suggests a non-competitive inhibition, where the inhibitor and the substrate can simultaneously bind to an enzyme molecule. A non-competitive inhibitor works by decreasing the number of renewals, instead of decreasing the proportion of enzyme molecules that are bound to the substrate. Non-competitive inhibition cannot be canceled out by increasing the concentration of the substrate. The substrate can still bind to the enzyme-inhibitor complex. However, the enzyme-inhibitor-substrate complex does not proceed to form a product; therefore the value of Vmax is decreased to a new value, while the value of KM is not affected (Berg JM, T., JL, Stryer, L, Biochemistry, ed. 6. 2008, Rio de Janeiro: Guanabara Koogan). 61. The cytotoxicity assay was conducted using seven different concentrations of compounds (2) and (4). The evaluation of the cytotoxicity of these compounds was performed on Vero cells, using MTT colorimetric assay. The values of the concentrations of the compounds that reduced 50% of the cell viability (CC50) were determined by non-linear regression and corresponded to 89.72 pmol L-1 and 267.60 μmol L-1, respectively, for (2) and ( 4) (Figure 7). 62. The virucidal assay was conducted at different concentrations (3, 6, 12, 25, 50 and 100 μmol L-1) against the DENV-1-4 and ZIKV viruses, using the lysis plate assay on Vero cells and the results are shown in Table 1. The concentration value of the evaluated compounds that inhibited 50% of the viral infection (EC50) was obtained by non-linear regression, leading to the calculation of the Selectivity Index (IS). 63. Cytotoxicity assessment sensations indicated CC50 values of 89.72 and 267.60 μmol L-1 for (2) and (4), respectively. These values when associated with those of EC50 obtained for DENV and ZIKV, led to high IS values for the compound (4), which was not observed for (2). This result indicates the inhibitory potential of (4) not only for the WNV protease, but also for the DENV and ZIKV viruses, for which good IS values were obtained, with emphasis on the DENV-3 virus, with IS of 147 (Table 1). 64. Table 1 - CCso, ECso and IS values of compounds (2) and (4) in the presence of the DENV1-4 and ZIKV viruses. 65. The protection found in the virucidal assay suggests two mechanisms of action: a) the compound acts directly on the viral particle; or b) the compound acts on cell receptors preventing the virus from entering the cell. To elucidate which of the two mechanisms, a pre-treatment test was performed, where the cells are previously treated with the compound. A protective result in this test indicates that the compound acts directly on the cell surface and not on the viral particle. 66. The pretreatment assay was conducted with different concentrations of compounds (2) and (4) (3, 6, 12, 25, 50 and 100 μmol L-1) against the DENV-2 and ZIKV viruses, via assay colorimetric analysis of MTT on Vero cells. The results did not indicate a significant difference in cellular viral infection in the presence or absence of compounds (2) and (4) as can be seen in Figure 8. 67. Antiviral compounds can act in the phases of viral replication. In this case, the viral particle is adsorbed and internalized, but when its replicative cycle is started, the inhibitor prevents it from occurring, usually by acting on capsid proteins or viral enzymes. To check whether the compound acts in the replication phases, the post-treatment test is performed. In this test the compound is added only after the viral particle has adsorbed and internalized in the cells. A protective result in this test indicates that the compound acts in the replication phases. 68. The post-treatment test was conducted with different concentrations of compounds (2) and (4) (3, 6, 12, 25, 50 and 100 μmol L-1) against the DENV-2 and ZIKV viruses, using colorimetric assay of MTT on Vero cells and is shown in Figure 9. The results did not indicate significant difference in viral replication in the presence or absence of compounds (2) and (4). 69. The protection identified in the virucidal assay plus the lack of protection in the pre- and post-treatment trials with DENV and ZIKV indicated that compounds (2) and (4) are likely to act directly on the virus. When the viral particle is mature and therefore capable of infecting the cell, its surface is coated with protein dimers that are associated with the virus entering the cell and fusing it to the endosome membrane. Therefore, a molecule that acts directly on the viral particle, is probably acting on the protein's dimers, which ends up preventing viral adsorption or the fusion of the viral and host membranes.

Structure-activity relationship and in silico calculations of the physical-chemical properties of the compounds (2) and (4) 70. Considering the therapeutic potential of the compounds, a computational study was carried out to predict the physical-chemical properties of the compounds (2) and (4). These parameters influence pharmacokinetic properties, such as absorption, distribution, metabolism and excretion (ADME). Screening was carried out to assess whether the compounds may have the characteristics of drug candidates based on Lipinski's rule of five (Lipinski, CA, et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Review, 1997. 23: p.3-25) and other related criteria added by Veber et al. (Veber, DF et al., Molecular properties that influence the oral bioavailability of drug candidates. Journal of Medicinal Chemistry, 2002. 45: p. 2615-2623). These characteristics, theoretically, determine whether a compound has good absorption in the membrane and permeability through it, properties that probably make it an active drug orally in humans. 71. The molecular parameters analyzed were the octanol / water partition coefficient (LogP <5), the amount of hydrogen bonding donors (HBD <5), the amount of hydrogen bonding receptors (HBA <10), the molecular mass of drugs (MW <500), number of connections capable of rotating (nRotb <10) and polar surface area (PSA <140 A2). The values in parentheses represent the ideal values according to Lipinski (Lipinski, C.A., et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.

Advanced Drug Delivery Review, 1997. 23: p.3-25) and Veber (Veber, DF et al., Molecular properties that influence the oral bioavailability of drug candidates. Journal of Medicinal Chemistry, 2002. 45: p. 2615-2623 ). The physical-chemical parameters were calculated using the software Osiris Property Explorer (Tetko, IV, Computing chemistry on the web. Drug Discovery Today, 2005. 10: p. 1497-1500) and Molinspiration (http://www.molinspiration.com / cgi-bin / properties), computational tools of free use that help to predict the pharmacokinetic properties of drug candidates used by several research groups (Abrigachi, F., et al., Synthesis, biological screening, POM, and 3D- QSAR analyzes of some novel pyrazolic compounds.Medicinal Chemistry Research, 2017. 26: p. 1784-1795; Amin, SA, et al., Pharmacoinformatics study of Piperolactam A from Piper betle root as new lead for non steroidal anti fertility drug development. Computational Biology and Chemistry, 2017. 67: p.213-224; Al-Maqtari, HM, et al., Synthesis, characterization, POM analysis and antifungal activity of novel heterocyclic chalcone derivatives containing acylatedpyrazole.Research on Chemical Intermediates, 2017. 43: p.1893-1907; Klen, E. E., et al., 3-Substituted Thietane-1,1-Dioxides: Synthesis, Antidepressant Activity, and in Silico Prediction of Their Pharmacokinetic and Toxicological Properties, Pharmaceutical Chemistry Journal, 2017. 50: p.642-648; Gabr, M. T., Isatin-b-thiocarbohydrazones: Microwave-assisted synthesis, antitumor activity and structure-activity relationship, European Journal of Medicinal Chemistry, 2017. 128: p.36-44). 72. The chemical and physical-chemical parameters that influence the pharmacokinetic properties of a drug were evaluated using the OsirisProperty Explorer software. The software allows the calculation of lipophilicity, inferred from the LogP value, solubility in water, expressed as LogS, molecular mass, drug-likeness and drug-scores. Low hydrophilicity and, therefore, high LogP values, can cause poor absorption or penetration. In addition, Osiris software calculates various parameters of the compounds, such as toxicity risks (mutagenic, irritant, carcinogenic and reproductive effects), drug-likeness and drug-scores (Lipinski, CA, Lead- and drug-like compounds: the rule -of-five Revolution, Drug Discovery Today Technologies, 2004. 1: p.337-341; Proudfoot, R., Drugs, leads, and drug-likeness: an analysis of some recently launched drugs. Bioorganicand Medicinal Chemistry, 2012.12: p 1647-1650). LogP and molecular mass calculations, as well as the total polar surface area (TPSA), number of rotating bonds (nRotB), number of hydrogen bond donors (HBD), number of hydrogen bond acceptor bonds ( HBA) and bioactivity scores (Jarrahpour, A., et al., Petra, Osirisandmolinspiration (POM) together as a successful support in drug design: antibacterial activity and biopharmaceutical characterization of some azo schiff bases. Medicinal Chemistry Research, 2012. 21: 1984-1990) were determined by the Molinspiration software. Tables 2 and 3 show the results of the calculations based on these computational packages. 73. Table 2 - Predicted drug-likeness properties and toxicity risks for compounds (2) and (4) calculated by the Osiris software.

Bioavailability and Drug-Score Toxicity Risks3 Compound _____________________________________________________________________ _________________________________________________ cLogP LogS MM Drug-likeness Drug-Score MTIR 2 2.77 -3.90 250.0 -1.32 030 - - - + 4 2.43 -3.60 266 , 0 -0.37 0.36 - - - + cLogP, calculated lipophilicity; LogS, logarithm of aqueous solubility measured in mol L-1; MM, molecular weight; M, mutagenic effect; T, teratogenic effect; I, irritating effect; R, reproductive effect, aClassified according to: (-), no negative effect; (±), average negative effect; (+), negative effect 74. A parameter used to determine a good absorption of the compounds is the cLogP value. As an indication of good absorption, the LogP value should not be greater than 5.0. The cLogP values of indan-1,3-diones (2) and (4) are in the range of 2.43-2.77. Normally, drugs that interact with enzyme inside the human body have LogP values between 2 and 5 (Tambunan, USF, et al., In silico modification of suberoyl anilide hydroxamicacid (SAHA) as potential inhibitor for class II histone of acetylase (HDAC ) BMC Bioinformatics, 2011.12: Suppl13, S23). In this context, the compounds described in this invention have values within the desirable range (Table 2). 75. The two compounds tested had molecular mass less than 500. Drugs with low molecular mass (<500) are transported, diffused and absorbed without difficulty when compared to molecules with high molecular masses, being an important aspect in the action of drugs. 76. Toxicity risk alerts are an indication that the structure of a compound can be harmful. Theoretical analysis of toxicity risks for compounds (2) and (4), using the Osiris software, revealed that the compounds examined were non-mutagenic, non-carcinogenic and non-irritating. The evaluated compounds had a negative effect on the mammal reproduction criterion (Table 2). However, this classification does not preclude the therapeutic potential of the compounds. Compounds (2) and (4) were evaluated as potential drugs through drug-likeness calculations, with negative values equal to -1.32 (compound 2) and -0.37 (compound 4) (Table 2), which indicates that they do not contain fragments that are frequently present in commercial drugs (Lipinski, CA, Lead- and drug-like compounds: the rule-of-five Revolution, Drug Discovery Today Technologies, 2004. 1: p.337-341; Proudfoot , R., Drugs, leads, and drug-likeness: an analysis of some recently launched drugs. Bioorganicand Medicinal Chemistry, 2012.12: p. 16471650). Π. The water solubility of a compound significantly affects absorption and distribution characteristics. In general, it is desirable to obtain compounds that are soluble in the biological environment. One parameter to assess solubility is LogS. More than 80% of drugs on the market have estimated LogS values greater than -4 (Alafeefy, A M., et al., Quinazoline-etyrphostin as a new class of antitumoral gents, molecular properties prediction, synthesis and biological testing. European Journal of Medicinal Chemistry, 2012. 53: p. 133-140). The LogS values of indan-1,3-diones (2) and (4) are respectively equal to -3.90 and -3.60, which corresponds to the desirable range (Table 2). 78. Drug-score values can be used to assess the overall potential of the compound that qualifies it as a potential drug. The values are the combination of drug-likeness, the risk of toxicity, and some physical-chemical parameters, such as cLogP, solubility and molecular mass (Lipinski, CA, Lead- and drug-like compounds: the rule-of -five Revolution, Drug Discovery Today Technologies, 2004. 1: p.337-341; Proudfoot, R., Drugs, leads, and drug-likeness: an analysis of some recently launched drugs. Bioorganicand Medicinal Chemistry, 2012. 12: p 1647-1650). These values are used to assess the potential of a drug candidate. As can be seen in Table 2, the drug-score values for compounds (2) and (4) are equal, respectively, to 0.30 and 0.36. 79. Table 3 - Drug-likeness calculations for compounds (2) and (4) using the Molinspiration software. Molinspiration Calculations Bioactivity Score Calculations3 Compound ________________________________________________________________ _________________________________________________ cLogP TPSAb HBDC HBA Volume nRotB Violations GPCRL ICM Kl NRL PI El 2 Ζ4Ϊ 54.37 1 3 218.01 Ϊ Õ -0.49 -0.67 -0.05 -0.23 -0.65 -0.12 4 1.92 1 0 -0.44 -0.67 -0.04 -0.21 -0.60 -0.11 aGPCRL: GPCR ligand; ICM: Tonic modulator channel; Kl: Kinase Inhibitor; NRL: Nuclear Receptor Ligand; PI: Protease inhibitor; El: Enzyme Inhibitor, b TPSA: Total Polar Surface Area. CHBD: number of hydrogen bonding donors; dHBA: number of hydrogen bonding receptors. 80. In addition to LogP, the total polar surface area (TPSA) is an important descriptor for predicting drug transport properties, including intestinal absorption, permeability of the Caco-2 monolayer, and penetration into the blood-brain barrier (Clark , DE, Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena, 1. Prediction of Intestinal Absorption.Journal of Pharmacy Science, 1999. 88: p. 807-814; Ertl, P., et al. , Fast Calculation of Molecular Polar Surface Area as a Sum of Fragment-Based Contributions and Its Application to the Prediction of Drug Transport Properties. Journal of Medicinal Chemistry, 2000. 43: p. 37143717). This parameter was calculated using the Molinspiration software based on the sum of the surfaces belonging to polar atoms (usually oxygen, nitrogen and hydrogen) (Jarrahpour, A., et al., Petra, Osiris and molinspiration (POM) together as a successful support in drug design: antibacterial activity and biopharmaceutical characterization of some azo schiff bases (Medicinal Chemistry Research, 2012. 22: p. 1984-1990). Compounds with TPSA> 140 A2 tend to have low oral bioavailability and substances with TPSA <61 A2 tend to have adequate bioavailability (Clark, DE, Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena, 1. Prediction of Intestinal Absorption, Journal of Pharmacy Science, 1999. 88: pp. 807-814). Considering the TPSA values, it is expected that compounds (2) and (4) (Table 3) present appropriate bioavailability, based on the acceptable range (TPSA <140 A2). 81. The number of hydrogen bond acceptors (HBA) was determined by considering the number of nitrogen and oxygen atoms in chemical structures. The number of hydrogen bonding donors (HBD) corresponds to the sum of the hydrogen atoms attached to the oxygen and nitrogen atoms (Lipinski, CA, Lead- and drug-like compounds: the rule-of-five revolution. Drug Discovery Today Technologies , 2004. 1: p. 337-341). The values of these parameters for compounds (2) and (4) are within the limit established by Lipinski's rules. This rule suggests that two or more violations for a compound may present problems with availability bioavailability (Lipinski, CA, et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Review, 2001. 46: p. 3-26). All compounds in Table 3 show no violation of the Lipinski rule. 82. The number of bonds capable of rotating is a simple topological parameter of molecular flexibility and has been shown to be a good descriptive indicator of oral bioavailability of drugs (Veber, DF et al., Molecular properties that influence the oral bioavailability of drug candidates, Journal of Medicinal Chemistry, 2002. 45: p. 2615-2623). Compounds 2 and 4 in Table 3 do not violate this criterion. 83. The bioactivity of the compounds was analyzed under different receptors in the human body, with six drug activity criteria being observed: GPCR ligand activity, ion channel modulation, inhibition of kinase activity, nuclear receptor ligand activity, inhibition protease activity, and the enzyme inhibiting activity. The higher the value of the numerical assignment, the better the probability of a molecule being active. The results of these parameters are presented (Table 3). The investigated compounds showed negative numerical values for human body receptors. 84. Quantum-chemical calculations were performed using the molecular modeling software Spartan 10 (Spartan'10, Wavefunction) and Gaussian 09 (Frisch, MJ, et al., Gaussian '09, Revision A.1, 2009, Wallingford CT). The molecular geometry of the compounds was optimized with semi-empirical calculations with the Spartan 10 tool package using the PM6 method. The outputs obtained with the Spartan 10 software were used as inputs to the Gaussian 09 software. The lower energy conformers were optimized in the gas phase using the theory level B3LYP / 6- 311 ++ G (2d, p) (Becker , AD Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys., 1993, 98: p. 5648-5653. The energies of the boundary orbitals and the dipole moment are important properties in several chemical processes and pharmacological (Arantes, FFP, et al., A quantum chemical and chemometric study of sesquiterpene lactones with cytotoxicity against tumor cells, Journal of Chemometrics. 2011. 25: p. 401407). The highest-occupied molecular orbital , Highest Occupied Molecular Orbital - HOMO), and the lowest unoccupied molecular orbital (of English, Lowest Unoccupied Molecular Orbital - LUMO) determine how a molecule interacts with other species. The difference between the energies of the HOMO-LU orbitals MO is called GAP and helps to characterize the chemical reactivity as well as the kinetic stability of the compound (Kumar, A., et al., Combined experimental (FT-IR, UV-visiblespectra, NMR) andtheoreticalstudiesonthe molecular structure, vibrationalspectra, HOMO, LUMO, MESP surfaces, reactivity descriptor and molecular docking of Phomarin. Journalof Molecular Structure, 2015. 1096: p. 94-101). The dipole moment is an important molecular descriptor that provides a measure of the polarity of the bond throughout the molecule. The calculated values are listed in Table 4. 85. Table 4 - Quantum-chemical descriptors for compounds (2) and (4).

Compound Moment HOMO LUMO GAP (HOMO-LUMO) dipole 2 -6.46 -2.80 3.66 2.2983 4 -6.32 -2.86 3.46 2.67371 86. The energy difference between the HOMO and LUMO (GAP) orbitals were observed for compounds (2) (3.66 eV) and (4) (3.46 eV), respectively. There is a variation between the dipole moment and boundary orbital values for the compounds under investigation, it was not possible to establish a clear correlation between the energy values of HOMO, LUMO, GAP and dipole moment with the biological activity data.

CONCLUSION 87. Using Knoevenagel condensation reactions and demethylation, two synthetic hydroxylated compounds derived from indan-1,3-dione were synthesized. These compounds were evaluated with respect to their antiviral activities for the WNV, DENV and ZIKV viruses. The compounds (2) and (4) evaluated showed important antiviral activity with reduced IC 50 and Ki values for the WNV protease. The inhibition mechanism identified was non-competitive. The tests conducted directly on the DENV-1-4 and ZIKV viruses demonstrated that the compounds also showed virucidal activity under them. Considering the significant antiviral activity presented by the compounds, as well as the favorable values of the Selectivity Index, it can be said that they can be used as chemotherapeutics to treat infections caused by the dengue and Zika viruses. This aspect is relevant considering that the antiviral treatment for the viruses evaluated here is still based on the patient's symptoms. The fact that the presence of a hydroxyl group in the aromatic ring of the arylidene portion of indan-1,3-diones seems to enhance biological activity is highlighted here.

Claims

Claims (3)

1. Pharmaceutical compositions of synthetic hydroxylated compounds derived from indan-1,3-dione characterized by comprising 2- (4-hydroxybenzylidene) -1 H-indene-1,3 (2H) -dione and / or 2- (3,4 -dihydroxybenzylidene) -1H-indene-1,3 (2H) -dione, and / or its salts, solvates, hydrates and / or prodrugs, associated with pharmaceutically acceptable ingredients. 2. Use of synthetic hydroxylated compounds 2- (4-hydroxybenzylidene) -1 H-indene-1,3 (2H) -dione, 2- (3,4-dihydroxybenzylidene) -1H-indene-1,3 (2H) - dione, its pharmaceutically acceptable salts, solvates, hydrates or prodrugs, characterized by being for the preparation of antiviral pharmaceutical compositions. 3. Use of the hydroxylated synthetic compounds 2- (4-hydroxybenzylidene) -1 H-indene-1,3 (2H) -dione and 2- (3,4-dihydroxybenzylidene) -1H-indene-1,3 (2H) - dione, its pharmaceutically acceptable salts, solvates, hydrates or prodrugs, according to claim 3, characterized for being for the preparation of agents for the treatment of diseases caused by viruses of the genus flavivirus, such as West Nile, Dengue and Zika viruses .