Antimalarial activity of the compounds anisodiamine and erypoegin.

Abstract

Antimalarial activity of anisodamine and erypoegin compounds: Malaria is a parasitic disease that affects a large part of the world's population. In the absence of an effective vaccine, its treatment depends on the use of chemotherapeutic agents, and the need for new treatments is reinforced by the growing resistance identified to current antimalarials. Thus, the present study aimed to evaluate the in vitro antimalarial activity of the compounds against the P. falciparum (W2) strain. To this end, the compounds (S)-3-hydroxy-2-phenyl-propionic acid (1R,3S,5R,6S)-6-hydroxy-8-methyl-8-aza-bic and 7-(2-(4-methoxy-phenyl)-2-oxo-ethoxy)-3,4,8-trimethyl-chromen2-on were tested using the traditional assay. In addition, a cytotoxicity assay was performed using the WI-26VA4 cell line by the MTT assay to verify cell viability.Experimentally, it was verified that the compounds do not exhibit cytotoxicity, and that the compound s)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6s)-6-hydroxy-8-methyl-8-aza-bic has a lower IC50 value. Thus, the results suggest that these compounds can be used as antimalarials. The present invention demonstrates the products (compounds) (s)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6s)-6-hydroxy-8-methyl-8-aza-bic and 7-(2-(4-methoxyphenyl)-2-oxo-ethoxy)-3,4,8-trimethyl-chromen-2-o as antimalarial agents.

Description

[01] The present invention relates to the use as antimalarial agents of the compounds (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-aza-bic, Reference: CDS012493 Packaging / Lot: SPBB3213; and 7-(2-(4METHOXY-PHENYL)-2-OXO-ETHOXY)-3,4,8-TRIMETHYL-CHROMEN-2-ON Reference: R511714-1EA Packaging / Lot: SPBB3207 obtained from Sigma (https: / / www.sigmaaldrich.com / ). STATE OF THE ART

[02] Since the dawn of civilization, the search for treatments for the major diseases that afflict humanity has been a constant concern of the population. In 2015, the Nobel Prize in Physiology or Medicine was awarded to three scientists who made pioneering discoveries in the use of drugs and compounds used in the treatment of neglected tropical diseases (MCKERROW, JH - Recognition of the role of Natural Products as drugs to treat neglected tropical diseases by the 2015 Nobel prize in physiology or medicine. Nat Prod Rep. v. 32(12), p. 1610-1611, 2015). One of these awards was given to the Chinese researcher Youyou Tu, who, guided by ancient Chinese texts, isolated a component of the Artemisia annua plant, called artemisinin, which proved to be highly effective in the treatment of human malaria (TU, YY; LIANG, XT - Studies on the constituents of Artemisia annua. Yaoxue Xuebao. Acta Pharmaceutica Sinica. v. 16, p. 366-369, 1981).Artemisinin has since become the drug of choice for the treatment of human malaria in much of the tropical world. Petition 870190021765, dated 07 / 03 / 2019, page 8 / 33 2 / 16

[03] Malaria remains a deadly disease for children and adults in underdeveloped countries where it is endemic. It is estimated that the disease affects approximately 500 million people in subtropical and tropical areas of the planet, resulting in more than one million deaths each year, the vast majority of whom are children (FRANCISCO, AI. - Ferroquine: the antimalarial of the future. Virtual Chemistry Journal. v. 2, p. 118-129, 2010). Resistance of malaria parasites to the main drugs used in treatment is the main problem in controlling the disease. Resistance to artemisinin has already been described in malaria-endemic areas, which threatens, in the very near future, the continued use of this drug for the treatment of the disease and efforts to eliminate the parasite worldwide (ASHLEY, EA et al. - Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. v. 371(8), p. 786, 2014).

[04] The World Health Organization (WHO), in accordance with the increase in cases of treatment resistance to malaria, recommends that in countries where the Plasmodium falciparum species (which causes the most severe form of the disease) is endemic, treatment policies should be replaced. Those countries that still use monotherapies to treat people infected with P. falciparum, which involve drugs such as chloroquine, amodiaquine and sulfadoxine-pyrimethamine (SP), should replace monotherapy with artemisinin-based combination therapies (ACT) (WHO. - World Malaria Report: WHO Press. Geneva: WHO, 2014). However, cases of resistance of P. falciparum strains to treatment with ACTs have been discovered in Southeast Asia (WHO. - World Malaria Report: WHO Press. Geneva: WHO, 2014; FAIRHURST, RM Understanding artemisinin-resistant malaria: what a difference a year makes. Curr Opin Infect Dis, v. 28, p. 417-425, 2015).In light of this, the resistance of... Petition 870190021765, dated 07 / 03 / 2019, page 9 / 33 The 3 / 16 parasite outbreak extends to many antimalarial drugs, highlighting the urgency of new treatment regimens and the search for new medications aimed at controlling the spread of the disease.

[05] Thus, the use of synthetic compounds can be a strategy for identifying bioactive compounds against malaria.

[06] 7-(2-(4-methoxy-phenyl)-2-oxo-ethoxy)-3,4,8-trimethyl-chromen-2-one is a derivative of the quinazolinone chemical nucleus. Quinazolinones and their derivatives constitute an important class of heterocyclic compounds that have diverse applications in medicinal and pesticide chemistry (AANANDHI, MV et al., - Synthesis, characterization and biological evaluation of 3,4-Dihydro quinazoline2(H)-one derivatives. J. Chem. Pharm. Res, v. 2, p. 676-683, 2011). The antibacterial activity of 4-(4-Oxo-3,4-dihydroquinazoline-2-yl)butanoic acid was identified in in vitro assays against Pseudomonas aeruginosa (PINTO, AF et al., - Identification of inhibitors of Pseudomonas aeruginosa exotoxin-S ADP-ribosyltransferase activity. Journal of biomolecular screening, v. 21, n. 6, p. 590-595, 2016).

[07] (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl ester, a substance known as anisodamine, is a tropane alkaloid, originally isolated from Scopolia tangutica, an herb used in Traditional Chinese Medicine (TCM) and found mainly in the Tibet region (POUPKO, JM et al., - The pharmacological properties of anisodamine, J. Appl. Toxicol. 27 (2007) 116121). Anisodamine was used for its vasoactive activity to treat acute intravascular coagulation in patients with septic shock after its discovery in 1965 (Zhang, Wei-Wei et al., - Differential neuropsychopharmacological influences of naturally occurring tropane alkaloids anisodamine versus scopolamine. Neuroscience letters, v. 443, n. 3, p. 241-245, 2008). In addition, anisodamine is used for its spasmolytic, anesthetic, and ophthalmic effects (Chen, H. et al., - Liquid Petition 870190021765, dated 07 / 03 / 2019, page 10 / 33 4 / 16 chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine. Journal of Chromatography B, v. 824, n. 1-2, p. 21-29, 2005). Thus, the examination of new uses for existing drugs, called drug repositioning or drug repurposing, is a widely used strategy, including for antiparasitic use (Ekins, S. et al., - In silico repositioning of approved drugs for rare and neglected diseases. Drug discovery today, v. 16, n. 7-8, p. 298-310, 2011).

[08] The compound (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6s)-6-hydroxy-8-methyl-8-aza-bic, with the linear formula C17H23NO4, anisodamine (in English) and anisodamina (in Portuguese), has pharmacological uses as an alpha-adrenergic agonist, anticholinergic, anti-ulcer, anti-arrhythmic, non-steroidal anti-inflammatory, parasympathetic nervous system inhibitor, vasodilator and antioxidant, for example [National Center for Biotechnology Information. PubChem Compound Database; CID=6918612, https: / / pubchem.ncbi.nlm.nih.gov / compound / 6918612 (Accessed October 24, 2018)].

[09] There are some patent applications that demonstrate the use of anisodamine as a component in raw material sets for the treatment of some diseases. These diseases result from functional disorders, which have overlapping genetic and environmental factors, unlike parasitic diseases. Some examples of these patent applications are:

[010] CN106729704, entitled “Western medicine composition for treating rheumatism”, which points to the use of anisodamine in a formulation for the treatment of rheumatism. It has a synergistic effect and low toxic and side effects, low cost, short treatment cycle and satisfactory curative effect, it is a safe and effective medicine. Petition 870190021765, dated 07 / 03 / 2019, page 11 / 33 5 / 16

[011] CN106727496, entitled “Medicine for treating epilepsy, and preparation method and application thereof”, formulation for the treatment of epilepsy. It has rapid action, good long-term results, no toxic or side effects or anaphylactic reaction, a high cure rate, low recurrence, and low price are observed.

[012] CN106420742, entitled “Medicine composition for treating esophagitis”, formulation for the treatment of esophagitis and its adult use is 1.2 mg / kg / day, has activity in reducing inflammation, relieving cough, asthma and pain, as well as a curative effect in the treatment of symptoms caused by esophagitis, such as nausea and vomiting, stomach swelling and hyperacidity. The treatment has a fast result and without side effects or toxic effects.

[013] CN107261093, entitled “Traditional Chinese medicine-western medicine compound preparation for treating alopecia”, formulation for the treatment of alopecia. It has a good treatment effect, low price, fast action; it has no adverse reactions or side effects.

[014] CN106309754, entitled “Chinese herbal medicine for treating gallstones and preparation method of Chinese herbal medicine”, formulation for the treatment of gallstones, helps reduce inflammation and relieves pain and has no side effects.

[015] CN105878664, entitled “Chinese and western medicine combined prescription for treating dental ulcer”, formulation for the treatment of dental ulcers. Allows for ulcer removal, bleeding relief, pain relief; has a high cure rate, as well as a satisfactory healing effect on skin ulcers.

[016] In turn, the compound 7-(2-(4-methoxy-phenyl)-2-oxo-ethoxy)-3,4,8-trimethyl-chromen-2-one, with the linear formula C21H20O5 and the generic name erypoegin, has three variations: A, B, and C. The variations have the same linear formula, however, the chemical structures are different, although all three Petition 870190021765, dated 07 / 03 / 2019, page 12 / 33 6 / 16 receive the same generic name. A) 10-(3-methylbut-2-enyl)-6H[1]benzofuro[3,2-c]chromene-3,9-diol (PUBCHEM code 9973411); B) 4-(3-methylbut-2-enyl)-6H-[1]benzofuro[3,2-c]chromene-3,9-diol (PUBCHEM code 636548) and C) 3-(4-hydroxy-2-methoxyphenyl)-8-(3-methylbut-2-enyl)-2Hchromen-7-ol (PUBCHEM code 5315558).

[017] Patent JP2015081251, entitled “Gyoxalase I inhibitor and use thereof” provides a practical glyoxalase I inhibitor, useful for promoting the accumulation of methylglyoxal (MG) associated with cell death (apoptosis). This provides a glyoxalase I inhibitor comprising an isoflavone compound (also known as Erypoegin K) derived from Leguminosae represented by formula (2) and an antitumor agent. To this end, it presents the use of erypoegin as a glyoxalase inhibitor.

[018] In KR20110093279 “AMPK activators from Erythrina abyssinica, and compositions for the prevention and treatment of metabolic syndromes through the activation of the AMPK enzyme containing the same as an active ingredient”. A compound that activates AMPK derived from Erythrina abyssinica is provided to prevent and treat diabetes, obesity or metabolic syndrome with less cytotoxicity, its pharmaceutical composition also contains a pharmaceutically acceptable vehicle or excipient.

[019] In this way, it points out how a compound derived from erythroegin, called AMPK, can be used for the treatment of metabolic syndromes. Despite the descriptions reported in other patents, the description as an antimalarial has not been previously made.

[020] In the present patent application, we demonstrate how the compounds (S) 3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-aza-bic and 7-(2-(4-methoxy-phenyl)-2-oxo-ethoxy)-3,4,8-trimethyl-chromen-2-on show prospects as antimalarial agents. Petition 870190021765, dated 07 / 03 / 2019, page 13 / 33 7 / 16 STATE-OF-THE-ART PROBLEMS:

[021] The compounds anisodamine and erypoegin are already used as drugs, with anisodamine being used against Alzheimer's and erypoegin being used as an antibiotic. However, this is the first time that antimalarial activities have been demonstrated. DETAILED DESCRIPTION OF THE TECHNOLOGY Solubilization of compounds for biological activity tests.

[022] For the biological tests, the compounds were solubilized with dimethyl sulfoxide (DMSO) solvent, resulting in an initial concentration of 10000 pg / mL. This solution is kept refrigerated at approximately 4°C. During the cytotoxicity and traditional tests, dilutions were performed using incomplete RPMI medium, resulting in the following concentrations from the stock solution: 1000 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL. From these concentrations, serial dilutions were performed, where the final concentration range of the compounds in the cell plate was 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL. The final volume of DMSO concentration was 0.01%. Biological Assays: Cultivation of human cell lines

[023] The human cell line used for toxicity analysis was WI26VA4 (ATCC CCL-75 lung fibroblast). This cell line is part of the animal cell bank of the Cell Biology Service (SBC) of the Ezequiel Dias Foundation (FUNED) in Belo Horizonte - Minas Gerais, which has a laboratory with ABNT NBR ISO9001 / 2008 certification and stands out in supporting the development of research projects that require access to techniques. Petition 870190021765, dated 07 / 03 / 2019, page 14 / 33 8 / 16 of cell and tissue culture, establishing itself as a reference in animal cell culture.

[024] The cells were cultured from cryopreserved ampoules, which were thawed at 37°C, and their contents transferred to a 15mL tube with 10mL of RPMI 1640 medium. The cells were centrifuged at 1200 rpm for five minutes, the supernatant discarded and the sediment resuspended in RPMI 1640 medium supplemented with 10% bovine serum. Heat-inactivated fetal fat (SBF) (complete medium). Cells were transferred to 10 mL T75 (75 cm²) plastic cell culture flasks treated to promote cell adhesion (Corning Costar Inc., USA) and incubated as monolayers at 37°C in an incubator (Thermo Electron Co., USA) with a 5% CO₂ humid atmosphere. The culture medium was replaced every 48 hours of incubation. Contamination, cell morphology, and monolayer formation were observed under an inverted microscope at 100x magnification (Olympus model, CKX 41). These cells were then frozen in cryopreservation ampoules containing a solution of complete RPMI medium with 5% DMSO and stored in liquid nitrogen in a cryopreservation bank (CryoPlus 7405 / Thermo Scientific, USA). After 80% of the culture in the T75 flask had confluenced, the cells were subcultured or used in cytotoxicity assays (PATRA, N. et al.).- A novel epoxypropoxy flavonoid derivative and topoisomerase II inhibitor, MHY336, induces apoptosis in prostate cancer cells. European Journal of Pharmacology, v. 658, p. 98-107, 2011; PEREIRA, JR et al. Evaluation of cytotoxicity of marine alkaloid analogs of viscosaline and theonelladin C. Biomedicine & Preventive Nutrition, v. 2. p. 145-148, 2012). Petition 870190021765, dated 07 / 03 / 2019, p. 15 / 33 9 / 16 Cytotoxicity Assays

[025] The most widely used chemosensitivity test currently in preclinical practice is the MTT test, referring to the abbreviation of the reagent used in the final evaluation, namely 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyl-2H bromotetrazolate (MATSUZAKI, WS et al. - Use of chemosensitivity test for choosing adjuvant chemotherapy in advanced gastric cancer. Revista do Colégio Brasileiro de Cirurgiões. v.33, n. 4, p. 228-234, 2006). This test consists of the rapid colorimetric evaluation of the growth of cell lines in vitro. The assay is based on the reduction of the tetrazolate salt by the enzyme succinate dehydrogenase present in the mitochondria of viable eukaryotic cells, forming a water-insoluble crystal known as formazan crystal (CARMICHAEL, J. et al. - Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Research, v. 47, p.943-946, 1987; PARK, JG et al.- Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium-based colorimetric assay. Cancer research, v. 47, p. 5875-5879, 1987). When the crystal is dissolved in an organic solvent, it acquires a violet color that is evaluated by spectrophotometry.

[026] After achieving 80% confluence, the cells were trypsinized with 1.5 mL of trypsin enzyme (1:250 Sigma), incubated at 37°C for five minutes, resuspended in 10 mL of complete medium, and centrifuged at 1200 rpm for five minutes. The supernatant was discarded, and the sediment was resuspended with complete medium. The cells were distributed in 96-well microplates at a density of 4 x 10⁵ cells / µL per well and incubated in a CO₂ incubator at 37°C for 24 h to adhere to the plate. After this adhesion period, the medium was removed, and 180 µL of RPMI medium supplemented with [missing information] was added. Petition 870190021765, dated 07 / 03 / 2019, page 16 / 33 10 / 16 1% of FBS was added, and 20 μl of the diluted compound was added in RPMI medium containing different concentrations of the tested compounds from the initial dilutions. The compounds were added in triplicate. The plates were incubated for a further 48 hours. At the end of the treatment period, 100 μl / well of a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazol (MTT) bromide solution (Sigma), the tetrazolium salt, at a concentration of 0.5 mg / mL in RPMI 1640 without phenol was added (DENIZOT, F., LANG, R. - Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal Immunol Methods. V. 89, n. 2, p. 271-277, 1986). After three hours of incubation in a CO2 incubator at 37°C in the dark with the MTT assay, the supernatant was aspirated and the formazan crystals were dissolved in 50 µl / well of DMSO and kept at 37°C for 10 minutes.Well absorbance was measured at a wavelength of 550 nm using Gen5 (Data Analysis Software - Bio-Tek). The data were analyzed from independent experiments. The minimum lethal dose that inhibits cell growth by 50% (IC50) in the presence of the test compounds was determined in comparison with cells cultured without the presence of compounds (considered 100% growth). The calculations were performed from sigmoidal dose-response concentration curves using OriginPro software version 8.0 (OriginLab Corporation, Northampton, MA, USA) (PATRA, N. et al. A novel epoxypropoxy flavonoid derivative and topoisomerase II inhibitor, MHY336, induces apoptosis in prostate cancer cells. European Journal of Pharmacology, v. 658, p. 98-107, 2011; PEREIRA, JR et al. Cytotoxicity evaluation of marine alkaloid analogues of viscosaline and theonelladin C. Biomedicine & Preventive Nutrition, v. 2. p. 145-148, 2012). Petition 870190021765, dated 07 / 03 / 2019, p. 17 / 33 11 / 16 In vitro cultivation Plasmodium sp.

[027] Currently, the only species capable of being continuously cultured in vitro is Plasmodium falciparum, due to its ability to invade young and mature erythrocytes (TRAGER, W., JENSEN, JB. - Human Malaria in Continuous Culture. Journal of Parasitology. p. 673-675, 1976). P. vivax invades only reticulocytes (young erythrocytes), which is one of the factors that makes continuous culture unsatisfactory (MORENO-PÉREZ, DA, RUÍZ, JA, PATARROYO, MA - Reticulocytes: Plasmodium vivax target cells. Biol. Cell. v. 105, p. 251-60, 2013). Therefore, the most commonly used model for screening new compounds with antimalarial activity focuses on P. falciparum in in vitro tests (AGUIAR, ACC et al. - Antimalarial activity and mechanisms of action of two novel 4-aminoquinolines against chloroquine-resistant parasites. PLoS One. V. 7, 2012). In vitro culture of intraerythrocytic phases of Plasmodium falciparum

[028] Parasites of the W2 strain (chloroquine-resistant) were cultured in human red blood cells in vitro under conditions established by Trager and Jensen (TRAGER, W., JENSEN, JB - Human Malaria in Continuous Culture. Journal of Parasitology p. 673-675, 1976). The parasites were cultured in Petri dishes with a 5% hematocrit using complete culture medium (RPMI 1640 supplemented with 25mM Hepes, 21mM sodium bicarbonate, 300μM hypoxanthine, 11mM glucose, 40μg / ml gentamicin and 10% (v / v) heat-inactivated human plasma). The plates were maintained at 37°C and the appropriate oxygen concentration was obtained by burning a candle, with daily medium changes being performed. Parasitemia was monitored daily in smears stained with Giemsa, under an optical microscope (1000x). Petition 870190021765, dated 07 / 03 / 2019, page 18 / 33 12 / 16 Determination of parasitemia

[029] Blood smears were prepared for culture, air-dried, fixed with methanol, and stained with freshly diluted Giemsa solution at a ratio of three drops per 1 mL of buffered saline solution pH 6.8. After 10 minutes, the slides were washed in running water, air-dried, and examined under an optical microscope with an immersion objective (1000x). Parasitemia was determined by counting the number of infected red blood cells. For parasitemia greater than 5%, 1000 red blood cells were counted. In the case of very low infections, less than 5%, 6000 red blood cells were counted. In this case, the evaluation was done by estimating the total number of red blood cells per microscopic field in a total of 50 to 100 fields, estimating the number of infected red blood cells. Parasitemia was expressed as a percentage of parasitized red blood cells. Synchronization of Plasmodium falciparum cultivation

[030] The cultured parasites were synchronized using the sorbitol method (LAMBROS, C. and VANDERBERG JP. - Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65 418-420, 1979).

[031] Cultures with a predominance of juvenile forms (rings), obtained immediately after synchronization, were used in chemotherapeutic assays. After the addition of 10 ml of sorbitol, the parasites were kept under the same environmental conditions as the culture (37 °C and 5% CO2) for 10 min. After the sorbitol action time, the contents were centrifuged in a Falcon tube at 2500 RPM for 5 min and the sediment volume was used to determine the hematocrit. The supernatant liquid was removed and the red blood cells were resuspended with RPMI culture medium with the hematocrit adjusted to 2%. Petition 870190021765, dated 07 / 03 / 2019, page 19 / 33 13 / 16 In vitro schizonticidal tests with Plasmodium falciparum using a traditional test.

[032] Initially, a preliminary assay was performed to evaluate whether the compounds showed activity against the W2 strain. To assess parasitemia reduction, synchronized cultures with 2% parasitemia in the ring stage and 2% hematocrit were distributed in 96-well microplates, with 180 μL per well. Subsequently, 20 μL of RPMI medium containing different concentrations of the compounds (50 - 0.024 μg / mL) was added to each well in triplicate. After 24 h of incubation of the parasites at 37°C, the culture medium with or without the addition of the compounds was changed in each well. Blood smears from all samples were prepared after 48 h, stained with Giemsa, and examined under an optical microscope to determine parasitemia. The average parasitemia calculated in the wells without the addition of compounds, considered as 100% parasite growth, was compared with the parasitemias in the wells that received the test compounds.The activity of the compounds was expressed as the percentage reduction in parasitemia compared to controls without the compounds. All experiments were performed in triplicate. Selectivity Index

[033] An important criterion in research on active compounds with therapeutic potential is determining the absence of toxic effects on host cells, through the selectivity index (SI), which measures how active the compound is against the parasite without causing damage to the viability of mammalian cells. Thus, the higher the proportion, the greater the selectivity of the compound for parasite cells. The selectivity index is calculated according to the formula below. Petition 870190021765, dated 07 / 03 / 2019, page 20 / 33 14 / 16 IS = IC50 of the compound in mammalian cell lines IC50 of the compound in parasitic cell lines IC0

[034] The antimalarial and cytotoxic effect values ​​corresponding to half the maximum inhibitory concentration (IC50) were determined for the compounds (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-aza-bic, Reference: CDS012493 Packaging / Lot: SPBB3213; 7-(2-(4METHOXY-PHENYL)-2-OXO-ETHOXY)-3,4,8-TRIMETHYL-CHROMEN-2-ON Reference: R511714-1EA Packaging / Lot: SPBB3207 obtained from Sigma (https: / / www.sigmaaldrich.com / ). These were determined from the cytotoxicity test, using a human fibroblast cell line (WI-26VA4 # ATCC) and the traditional method against the W2 strain of P. falciparum (chloroquine-resistant). In addition to the aforementioned controls, determinations were made for standard antimalarials (chloroquine and artemether). The experiments were performed in triplicate.

[035] The cytotoxicity of the compounds was evaluated against a human fibroblast cell line (WI-26-VA4 # ATCC). The test used consists of a colorimetric method (MTT - [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which aims to evaluate cell viability. In the tests performed, cytotoxicity was recorded as the percentage reduction in absorbance. The compounds did not show cytotoxic activity, with IC50 values ​​> 10.6 μM (Table 1).

[036] In the determination of the IC50 by traditional test, the value was similar to the control antimalarials; suggesting potential drugs. In addition, the selectivity index (SI) was obtained, which corresponds to the relationship between the cytotoxic and antiparasitic activities of each compound (Table 1). Petition 870190021765, dated 07 / 03 / 2019, page 21 / 33 15 / 16

[037] The main verification and validation criteria for antimalarials, defined by the committee coordinated by the Global Health Innovative Technology (GHIT) foundation, were guided by the specific requirements for the disease, considering the target product and candidate profiles. The criteria were divided into: validation and activity / mechanism of action (KATSUNO, K., JN BURROWS, et al.. - Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat Rev Drug Discov 14(11): 751-758, 2015). For validation of compounds effective against Plasmodium spp., the IC50 values ​​must be <1 μM. The selectivity index must be greater than 10 times (IC50 for cell line relative to the IC50 of the parasite) for Plasmodium spp (KATSUNO, K., JN BURROWS, et al.. - Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat Rev Drug Discov 14(11): 751-758, 2015).These values ​​can be observed for the compounds tested (Table 1). The compounds were satisfactory according to this criterion, since the cytotoxicity shown against the WI26VA-4 cell line was comparable to that of chloroquine and artemether. Thus, the IC50 values ​​and the selectivity index values ​​for the compounds studied in this work were comparable to the antimalarial drugs used as controls.

[038] As shown in Table 1, the compounds do not exhibit cytotoxic activity. And although toxicologists prefer analyses of the effects of substances on primary culture cells, cytotoxic activity was evaluated in vitro in a cell line, as this presents a faster and less expensive alternative, since it has a higher growth rate (SCHOONEN, WGEJ, et al.. - Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I Mechanical assays on ROS, glutathione depletion and calcein uptake. Petition 870190021765, dated 07 / 03 / 2019, p. 22 / 33 16 / 16

[039] For activity / mechanism of action, compounds should be tested according to the study of the Plasmodium life cycle correlating malaria manifestations. And the main assays according to the target should be performed in order to ensure the characterization of the potential target candidate (Katsuno, Burrows et al. 2015). In vitro culture of P. falciparum strains has been a good assay model for screening new compounds and for monitoring resistance to antimalarials. In this methodology, the compounds were active with IC50 values ​​from 0.098 μM determined for P. falciparum (Table 1). The compound (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-aza-bic has the lowest IC50 value. Table 1: In vitro results for the antimalarial activity (IC50) of the compounds against the W2 strain of P. falciparum, IC50 in the human cell line WI-26-VA-4 and selectivity index. Compounds IC50 (pM) P. falciparum ± DP Antimalarial Activity IC50 (pM) WI-26-VA4 ± DP IS (S)-3-hydroxy-2-phenyl-propionic acid (1r,3s,5r,6S)-6-hydroxy-8-methyl-8-azabic / SIGMA Reference: CDS012493 Packaging / Lot: SPBB3213 0.098 ± 0.021 Active 97 ± 0.45 989.80 7-(2-(4-METHOXY-PHENYL)-2-OXOETHOXY)-3,4,8-TRIMETHYL-CHROMEN2-ON / SIGMA Reference: R511714-1EA Packaging / Lot: SPBB3207 0.116 ± 0.018 Active 1.23 ± 0.68 10.6 Chloroquine 0.57 ± 0.014 Active >100 175 Artemether 0.099 ± 0.09 Active >100 >1000

Claims

1. Use of the compounds erypoegin and anisodamine characterized by being in the preparation of a pharmaceutical composition for the treatment of malaria.