Pharmaceutical formulations, dibenzoylmethane derivative and uses for leishmanicidal and parasiticidal activity.

Description

Pharmaceutical formulations, dibenzoylmethane derivative and uses for leishmanicidal and parasiticidal activity. Field of invention

[001] The present invention relates to a novel compound, derived from dibenzoylmethane, to pharmaceutical formulations comprising the compounds 1-(2(benzyloxy)phenyl)-3-phenylpropane-1,3-dione (DBMObz), 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP), 1-(2-hydroxyphenyl)-3-phenylpropane-1,3-dione (DBMOH6) and 1,3-diphenyl-2-propargyl-1,3-propanedione (DBP) and to their use for the prevention (parasiticide) and treatment of Leishmaniasis. The compounds exhibit significant leishmanicidal activity, without significant cytotoxic effects on mammalian cells. The compounds proved to be stable at room temperature, easy to handle and low cost to produce. Situated within the field of medicinal chemistry, the invention could be applied by the pharmaceutical industry in the manufacture of medicines intended for the prevention and treatment of cutaneous leishmaniasis. Fundamentals of the invention

[002] Leishmaniasis is a group of diseases caused by parasitic protozoa belonging to more than 20 species of Leishmania. These parasites are transmitted to humans by the bite of infected female sandflies. Most people infected with the parasite do not experience symptoms throughout their lives. Therefore, the term "leishmaniasis" refers to the condition of becoming ill as a result of infection with Leishmania, and not simply to the presence of the parasite in the body.

[003] The disease can manifest in different forms. Cutaneous Leishmaniasis (CL), also known as Tegumentary Leishmaniasis (TL), mainly affects the skin and mucous membranes, causing lesions that can develop into ulcers. Visceral Leishmaniasis (VL), on the other hand, is a more severe form of the disease that affects internal organs such as the liver, spleen, and bone marrow, and can be fatal if left untreated. Furthermore, canine leishmaniasis is a significant veterinary concern, as dogs are reservoirs of the parasite and can transmit the disease to humans through the bite of phlebotomine sandflies. Treatment for each type of leishmaniasis may vary, but it is essential to control the spread and complications associated with the disease (Ferreira, Cassio Porto. Description of clinical and laboratory aspects of patients with Petition 870250014195, dated 21 / 02 / 2025, page 15 / 37 / 15 sporotrichosis and American cutaneous leishmaniasis, with emphasis on pregnant women. 2016. Doctoral Thesis. Accessed January 2, 2025.

[004] Today, more than 1 billion people live in areas endemic for leishmaniasis and are at risk of infection. It is estimated that 30,000 new cases of VL and more than 1 million new cases of CL occur annually. Leishmaniasis continues to be a major health problem in 4 eco-epidemiological regions of the world: the Americas, East Africa, North Africa, and West and Southeast Asia (WHO - WORLD HEALTH ORGANIZATION. 2022. Global health observatory (GHO) data. Leishmaniasis. Situation and trends. Geneva, Switzerland: World Health Organization. Accessed April 5, 2023).

[005] In Brazil, the treatment of leishmaniasis traditionally used pentavalent antimonials, amphotericin B, and pentamidines. These medications are toxic, expensive, difficult to administer, and can induce parasite resistance (Rath, S., Trivelin, LA, Imbrunito, TR, Tomazela, DM, Jesús, MN de & Marzal, PC. Antimoniais usados ​​no tratamento da leishmaniose: estado da arte. Quim. Nova, 26(4): 550-555, 2003). Currently, leishmaniasis therapy involves the use of five drugs: pentavalent antimonials, amphotericin B and its liposomal formulation, miltefosine, paromomycin, and pentamidine. With the exception of the antimonial compounds, all other medications were not initially developed with leishmanicidal action.

[006] The Unified Health System (SUS) has offered specific and free treatment for LT since 2021. This treatment consists of the use of specific medications and rest. The SUS provides miltefosine capsules, the first oral medication for the disease, which allows treatment in the patient's own home through its use.

[007] Current drug therapy for all forms of Leishmaniasis aims to ensure patient adherence to treatment, as well as relief from symptoms and discomfort caused by the disease. Another factor to be considered is the safe administration of the indicated medications, and the control and / or minimization of the occurrence of adverse effects. All drugs currently available for the treatment of this disease are unsatisfactory in terms of efficacy, cost, and ease of administration. Petition 870250014195, dated 21 / 02 / 2025, page 16 / 37 3 / 15 administration and / or safety (Hendrickx, S., Caljon, G., & Maes, L. Need for sustainable approaches in antileishmanial drug discovery. Parasitol Res. 118(10):2743-2752, 2019.

[008] Antimonial drugs, widely used in the treatment of Leishmaniasis, have a therapeutic regimen administered via intramuscular (IM) injections. This method of administration can cause local pain, requiring alternating injection sites to minimize patient discomfort. Despite the difficulties associated with treatment, antimonials remain a crucial option in combating the disease due to their proven efficacy in eliminating the Leishmania parasite (De Oliveira, M P. Evaluation of recombinant chimeric protein plus adjuvant as a candidate vaccine against Visceral Leishmaniasis. Doctoral thesis. 2023. Accessed January 2, 2025).

[009] Amphotericin B is a drug with low water solubility and limited membrane permeability, resulting in poor absorption and low oral bioavailability. Due to these characteristics, amphotericin B must be administered intravenously (IV). This factor represents a significant limitation, especially in poor and developing countries where access to intravenous treatments is more restricted. Even so, amphotericin B remains an important therapeutic option in the fight against leishmaniasis, despite its practical limitations (Rath, S., Thvelin, LA, Imbrunito, TR, Tomazela, DM, Jesús, MN de & Marzal, PC Antimoniais usados ​​no tratamento da leishmaniose: estado da arte. Quim. Nova, 26(4): 550-555, 2003).

[0010] Miltefosine is the only orally administered drug for the treatment of Leishmaniasis. Despite its convenience, miltefosine has toxicity that can cause gastrointestinal and renal disorders. These adverse effects frequently lead to premature interruption of treatment, which contributes to the increase in cases of parasite resistance. Therefore, while miltefosine offers a practical alternative to injectable treatments, its toxicity is an important consideration in the management of the disease (Ministry of Health, INFORMATIVE NOTE No. 13 / 2020CGZV / DEIDT / SVS / MS).

[0011] Paromomycin is a drug administered parenterally, which may hinder its adoption in poor and developing countries due to the need for Petition 870250014195, dated 21 / 02 / 2025, page 17 / 37 / 15 adequate infrastructure for application. Pentamidine, although effective in the treatment of Leishmaniasis, presents the side effect of developing insulin-dependent diabetes mellitus (Rath, S., Trivelin, LA, Imbrunito, TR, Tomazela, DM, Jesús, MN de & Marzal, PC Antimoniais usados ​​no tratamento da leishmaniose: estado da arte. Quim. Nova, 26(4): 550-555, 2003). These limitations highlight the need to consider both the efficacy and practicality, as well as the adverse effects of medications in combating Leishmaniasis.

[0012] Currently, two vaccines are available for dogs: Leishmune® and Leish-Tec®. Even with this great advance in the veterinary field, there is still a long way to go before a vaccine for human use against Leishmaniasis is produced. A viable vaccine for humans must be safe, effective, stable, and low-cost. As Leishmaniasis mainly affects developing countries, large financial, infrastructure, and research investments are necessary to achieve this goal (Palatnik de Sousa, CB et al. Decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with Leishmune in Brazilian endemic areas. Vaccine, 27, 3.505-12, 2009). The implementation of an effective human vaccine could significantly reduce the incidence of the disease and improve the quality of life in endemic areas.

[0013] Considering the difficulties in treatment and the absence of effective vaccines for Leishmaniasis, there is a growing urgency in the search for new therapeutic drugs. Research and development of new compounds are essential to expand the available therapeutic arsenal and improve treatment options for this debilitating disease. Continuous investment in scientific studies and clinical trials is fundamental to discovering innovative treatments that can meet the needs of patients, especially in developing countries where leishmaniasis is more prevalent.

[0014] The search for new treatments for cutaneous leishmaniasis (CL) aims to accelerate healing and reduce scarring in areas such as the face, which helps to reduce psychological trauma with repercussions in the social and economic fields, as well as prevent the spread of the disease, since CL, or cutaneous leishmaniasis, is an infectious, non-contagious disease that causes ulcers on the skin and mucous membranes. In Brazil, there are seven species of leishmania involved in the occurrence of CL cases, notably Leishmania (Leishmania) amazonensis, L. (Viannia) guyanensis and L. Petition 870250014195, dated 21 / 02 / 2025, page 18 / 37 / 15 (V.) braziliensis (Leishmaniasis: Ministry of Health warns about prevention - Ministry of Health. Accessed on April 5, 2023).

[0015] The present invention, in turn, relates to a novel compound derived from dibenzoylmethane, to pharmaceutical compositions comprising dibenzoylmethanes and derivatives, and to their use for the prevention and treatment of Leishmaniasis.

[0016] In a search of the state of the art, no documents were found that use the aforementioned compounds as agents for the treatment of leishmaniasis. However, documents were found that present other compounds with this activity, which do not detract from the novelty or inventive step of the current patent application. By way of example and differentiation, some of them are presented below:

[0017] Patent application BR 10 2019 018355 1 refers to pharmaceutical compositions containing flavonoid derivatives that have selective leishmanicidal activity against promastigote and amastigote forms of different Leishmania species. The compositions described in the present invention can be used to prepare medications for the treatment of CL and VL in humans and dogs. The present invention differs from the current patent application by having different chemical compounds, although also flavonoids.

[0018] Patent application BR 10 20190 19344 1 refers to compositions containing (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy)-cinnamate, a cinnamic acid derivative containing the isobenzofuranone nucleus. This compound exhibits activity against the promastigote and amastigote forms of Leishmania braziliensis and can be applied in the field of medicinal chemistry. The invention described in BR 10 2019 019344 1 also refers to the use of the compound in the preparation of drugs used in antileishmanial therapy, differing from the current patent application by addressing compounds with a different chemical structure.

[0019] Patent document PI 0602640 0 refers to the use of substituted cyclohexanone penta-1,4-dien-3-ones and derivatives, compounds that exhibit strong antitumor activity and outstanding antiparasitic action, proving to be practically non-toxic in laboratory tests and histopathological studies. The invention described in PI0602640 0 also refers to a pharmaceutical composition comprising said compounds, Petition 870250014195, dated 21 / 02 / 2025, page 19 / 37 / 15 method for treating cancers and parasitic diseases. This technology differs from the current patent application in the types of compounds evaluated, which, although presenting similar chemical structures, are totally different compounds.

[0020] Patent document PI 0204079 relates to a new class of chalcone compounds analogous to 2-39-6-39-dihydroxy-4-9-methoxychalcone, as well as to the synthesis of such analogs, the use of the chalcone and such synthesized analog compounds, pharmaceutical compositions containing the same, and pharmaceutical application in the treatment of diseases caused by parasites, in particular protozoa such as leishmania and trypanosoma. The cited document, although addressing the use of a chalcone as a leishmanicidal agent, differs from the current patent application because it deals with a compound with a chemical structure distinct from the current application being claimed.

[0021] Patent BR 10 2017 003112 8 comprises processes for obtaining, isolating, and purifying the isoflavonoid bolusanthol (3r,4r)-3-(2-hydroxy-4-methoxyphenyl)-3,4dihydro-2h-chromene-4,7-diol, as well as the use of the bolusanthol compound in pharmaceutical compositions and its use as a leishmanicidal agent. The cited document, despite addressing the use of a flavonoid, a product of plant extraction, as a leishmanicidal agent, differs from the current patent application because it deals with a compound with a chemical structure distinct from that of the current application.

[0022] Patent BR 10 2013 026621 3 refers to a pharmaceutical composition containing 1,3-Diphenyl-2-benzyl-1,3-propanedione, a dibenzoylmethane derivative. This composition is used in the prevention and control of melanoma cells, and is applied in the pharmaceutical industry as an antitumor agent in the treatment of melanoma. The compound described in BR 10 2013 026621 3 is identical to one of the four presented in the current patent application. However, its use for the treatment of leishmaniasis has never been described in the literature; therefore, the current application refers to the use of this compound for this purpose, which, although not a novel compound, has never had its action as a leishmanicidal agent described in the prior art.

[0023] In the prior art, no technology comprising the derivatives defined in the present technology and its use in the treatment of leishmaniasis has been found.

[0024] For the purposes of abbreviation and definition, the compounds of the present invention, named (A) 1-(2-(benzyloxy)phenyl)-3-phenylpropane-1,3-dione; (B) 1,3-diphenyl-2-benzyl Petition 870250014195, dated 21 / 02 / 2025, p. 20 / 37 / 15 1,3-propanedione, (C) 1-(2-hydroxyphenyl)-3-phenylpropane-1,3-dione and (D) 1,3-diphenyl-2propargyl-1,3-propanedione, could be indicated throughout the text respectively as (A) DBMOBz; (B) DPBP; (C) DBMOH6 and (D) DBP. Brief description of the drawings

[0025] The figures presented in this patent application are described below: - Figure 1: Chemical structure of compounds with leishmanicidal activity. (A) DBMOBz; (B) DPBP; (C) DBMOH6 (D) DBP. Figure 2: Graphs of the percentage of cell viability of RAW 264.7 macrophages after treatment with the compounds for 24 hours. Graph (E): treatment with DBMOBz; Graph (F): treatment with DPBP; Graph (G): treatment with DBMOH6 and Graph (H): treatment with DBP. Control +: cells and culture medium without treatment. ANOVA followed by Tukey's post-hoc test was performed. Figure 3: Graphs of the percentage of cell viability of RAW 264.7 macrophages infected with Leishmania after treatment with the compounds for 48 hours. Graph (I): treatment with DBMOBz; Graph (J): treatment with DPBP; Graph (K): treatment with DBMOH6 and Graph (L): treatment with DBP. Macrophages were infected with L. braziliensis and were treated at different concentrations ranging from 9,375 μM to 150 μM. Untreated cells: medium + macrophages + L. braziliensis; Amphotericin B: medium + macrophages + L. braziliensis and Amphotericin B (3mM) and 1% DMSO (ANOVA followed by Tukey's post-hoc test was performed). - Figure 4: Graphs of the evaluation of the cell viability of pro-mastigote L. braziliensis after treatment with the compounds for 48 hours. Graph (M): treatment with DBMOBz; Graph (N): treatment with DBMOH6 and Graph (O): treatment with DBP. Concentrations ranging from 9,375 μM to 150 μM. Untreated cells: medium + macrophages + L. braziliensis; Amphotericin B: medium + macrophages + L. braziliensis and Amphotericin B (3mM) and 1% DMSO (ANOVA followed by Tukey's post-hoc test was performed). Figure 5: Growth curve of L. braziliensis. Petition 870250014195, dated 21 / 02 / 2025, page 21 / 37 / 15 Description of the invention

[0026] The present invention relates to formulations comprising dibenzoylmethane (DBM) and its derivatives (DBMOBz, DPBP, DBMOH6 and DBP) which may be used as therapeutic agents for the topical treatment of Leishmaniasis and as parasiticidal agents for the control of the Leishmaniasis vector. It also relates to the novel compound 1-(2-(benzyloxy)phenyl)-3-phenylpropane-1,3-dione (B), derived from DBM, and to its use and that of the other compounds for these purposes.

[0027] The compounds proved to be stable at room temperature, easy to handle and low cost to produce, and can be used for the production of medicines for the treatment of cutaneous leishmaniasis in different mammalian species, such as dogs and humans.

[0028] The formulations involving the compounds described in Figure 1 are characterized by comprising the individual use or combination thereof, associated with pharmaceutically acceptable ingredients, such as emulsifier, preservative, surfactant, talc, lactose, magnesium stearate, stearic acid, corn starch, humectants, anti-adherents, coating agents, for the treatment of CL, as combined compositions or co-administered with other agents with one or more known medications, such as meglumine antimoniate, sodium stibogluconate (SSG), liposomal amphotericin B, paromomycin sulfate and miltefosine. Furthermore, the compositions can be administered orally, via capsules, tablets, powder, granules, dragees or oral solution, or topically, via ointments, gels, sprays, creams, lotions containing the compound.

[0029] When administered in combination with another known drug, or another combination of known drugs, the compounds of the present invention can be physically combined with these known drugs in an appropriate form, such as a pill, a topical formulation, an injectable solution, or any other suitable form.

[0030] The advantage of this invention lies in the way the product can be used for LC therapy, as it can be prepared for topical application through a transdermal gel formulation that acts on the deeper layers of the dermis, Petition 870250014195, dated 21 / 02 / 2025, page 22 / 37 / 15, potentially reaching the bloodstream. This is a therapeutic alternative to oral medications. Petition 870250014195, dated 21 / 02 / 2025, page 23 / 37 / 15 Demonstration Experiment

[0031] The dibenzoylmethane (DBM) derivatives that are the subject of the present patent application were tested to assess whether they have a cytotoxic effect on the RAW264,7 mammalian cell line using the MTT colorimetric assay. Example 1: In vitro analysis of cell viability using RAW264.7 macrophage by MTT assay.

[0032] Macrophages were plated at a density of 1x10⁵ cells at 200 pL per well in 96-well plates, using RPMI medium with 10% Fetal Bovine Serum (FBS) as the culture medium for cell growth. The plates were incubated at 37°C and 5% CO₂ for 24 hours. After the incubation period, 50 pL of the supernatant was removed from the plate. The compounds were diluted in 1% DMSO at concentrations ranging from 9.375 μM to 150 μM. The positive control (amphotericin) was 3.125 μg / mL and the negative control was 0.1% DMSO. Immediately afterwards, the plates were incubated again at 37°C and 5% CO₂ for 48 hours. Two controls were established: the first control containing only cells and RPMI medium, and the second control with RPMI medium, cells, and 1% DMSO, to ensure that the dilution of the compounds in DMSO was not toxic to the cells.After the cell treatment period, 50 pL of supernatant were removed from each well and 50 pL of 3-(4,5-dimethyl-2-thiazolyl)2-,5-diphenyl-2H-trezolic bromide (MTT) solution at a concentration of 0.05 mg / mL was added. After the MTT application, the plates were incubated for 3 h under standard conditions. At the end of the incubation period, the formation of formazan crystals was observed, which were homogenized with the addition of DMSO. The absorbance of the crystals was measured by reading the plate in a spectrophotometer at a wavelength of 570 nm.

[0033] To perform the cell proliferation (CP) calculations, the following equation was used (1): PC(%) = (ABSTrata-ABSbranco^x 100 (ABScontrole-ABSbranco~) Equation (1) PC = Cell proliferation in percentage; ABSTrata = Treatment absorbance; ABSbranco = RPMI medium absorbance; ABScontrole = RPMI medium + RAW cells. 264.7. Petition 870250014195, dated 21 / 02 / 2025, page 24 / 37 / 15 The graphs in Figure 2 show the viability of macrophage cells after treatment with the compounds DBMOBz; DPBP; DBMOH6 and DBP. It can be observed in graphs (E), (F), (G) and (H) that the compounds did not show cytotoxicity at the tested concentrations. Furthermore, it is possible to note that the cells were less sensitive to the compounds compared to the conventional drug used to treat leishmaniasis, Amphotericin B. Example 2: In vitro evaluation of leishmanicidal activity in macrophage cells infected with leishmania.

[0034] This assay was conducted to evaluate the ability of the compounds to inhibit parasite growth in infected macrophage cells.

[0035] Cells were plated in 96-well plates with a density of 1x10⁵ cells per well, in RPMI medium with 10% FBS, and incubated at 37°C with 5% CO₂ for 4 hours to encourage cell adhesion. Three culture bottles containing promastigote forms on the 7th day of parasite replication were used, as they needed to be in the stationary phase for infection to occur in macrophages. The contents of the bottles were transferred to a sterile Falcon tube, centrifuged for 11 minutes at 3500 RPM, the supernatant was discarded, and 5 mL of RPMI medium was added. Parasite counting was performed in a Neubauer chamber, inoculating 1x10⁵ parasites per well. Subsequently, the supernatant from the wells with adhered macrophages was discarded, and 200 µL of solution containing parasites per well was added to induce infection. The plate was incubated for a period of 24 hours under standard culture conditions.

[0036] After this period, the wells were washed to remove the metabolized medium and non-infective parasites. 200 pL of the supernatant were collected and 200 pL of 1x PBS were added, repeating the washing three times. After the process, 200 pL of RPMI medium were added and the plates were incubated under standard culture conditions for 24 h.

[0037] After this period, the compounds were diluted in RPMI and DMSO media at concentrations of 9.375 pM to 150 pM, and controls were prepared. 200 pL of the supernatant from each well was removed, and 200 pL of the compound dilutions was added. For this assay, the following controls were used: control 1 of cells without Petition 870250014195, dated 21 / 02 / 2025, page 25 / 37 / 15 treatment; control 2 with Amphotericin B; control 3 with 1% DMSO. After inoculation of the compounds, the plate was incubated under standard conditions and cultured for 48h.

[0038] After the treatment period, the supernatant was removed and the wells were washed with 1x PBS. Then, 50 pL of incomplete RPMI medium with 0.05% SDS (Sodium Docetyl Sulfate) was added to lyse the macrophage cells. After the addition of SDS, the plate was taken to the microscope for observation and left to stand for 25 minutes until complete cell lysis. Subsequently, 200 pL of Culture Medium (medium 199) was added to stop the lysis.

[0039] Immediately after applying medium 199, the plate was incubated in a BOD incubator at 26°C and 5% CO2 for 5 days, so that parasites not inhibited by the compound could recover growth. After the incubation period, the plate was taken to the spectrophotometer for fluorescence reading at 540 nm. To evaluate the percentage of viable parasites, the same calculations described in Example 1 were performed.

[0040] The graphs in Figure 3 show the cell viability of macrophages infected with L. brasiliensis after treatment with the compounds. All compounds showed leishmanicidal activity, with compound DBP being the most active, Graph (K), reducing infection by 20% (at a concentration of 37.5 pM) and being more effective than the drug amphotericin B. Compound DBMOBz, Graph (J), was the second most efficient, reducing infection by 27% after exposure of infected cells to a concentration of 75 pM of the compound. Compound DPBP, Graph (I), reduced infection by 50% after exposure of infected cells to a concentration of 37.5 pM. Finally, compound DBMOH6, Graph (L), began to show a reduction in infection from 13.75 pM, with more significant leishmanicidal activity after exposure to the highest concentration tested (150 pM).

[0041] The inhibitory concentrations of 50% of the macrophage cell population after treatment with the compounds (infected and uninfected) are shown in Table 1. From these data it was possible to calculate the Selectivity Index (SI) of each compound for infected macrophage cells, indicating how selective a compound is for the target cell; a value greater than 1 indicates that the activity is selective. The SI calculation for each compound was done using Equation (2): Petition 870250014195, dated 21 / 02 / 2025, page 26 / 37 13 / 15 IS EC50 Equation (2) Table 1. Values ​​of the 50% inhibitory concentrations of the human macrophage population (CC50) and the human macrophage population infected with leishmaniasis (ECso) and selectivity index IS. Compound CC50 EC50 IS DPBP 233.0 32.12 7.28 DBMOBZ 318.0 41.80 7.60 DBM0H6 312.0 82.07 3.80 DPB 236.0 48.10 4.9 Example 3: Determination of the anti-promastigote effect in coats of L. brasiliensis M2904-GFP

[0042] Tests to evaluate the action of the compounds on promastigotes were performed in 96-well plates. After the 4th day of culture, the parasites were transferred to a sterile 50 mL conical-bottom tube and centrifuged at 3000 RPM for 10 min at 4°C. The precipitate was suspended in 5 mL of complete 199 medium. These 5 mL were transferred to a single tube, and the cells were counted in a Neubauer chamber (50x dilution in 4% formalin) to determine the number of Leishmania / mL. Three identical 96-well plates were plated to evaluate the action of the compounds during 24, 48, and 72 hours of treatment. After counting, 4 x 10⁶ parasites were plated in each well, in a final volume of 200 µL per well (Leishmania + complete 199 medium). Subsequently, 4 µL of the compounds were added. As controls, DMSO (1%) and 2.5 µL of Amphotethcine B were used, all in quadruplicate. A control was also added to evaluate Leishmania growth in the absence of the compounds.

[0043] After the addition of the compounds, the plates were incubated in a BOD incubator at 25°C. After 23, 47, and 71 hours, the plates were removed from the incubator and 20 pL of resazurin were added to each well. The plates were incubated again for another 1 hour. After this period, the plates were subjected to reading in a microplate reader (Spectramax M5) at 570 nm and 600 nm, at 1-hour intervals, for a total of 3 hours. Petition 870250014195, dated 21 / 02 / 2025, page 27 / 37 / 15

[0044] The graphs in Figure 4 show the evaluation of the leishmanicidal activity of the compounds against pro-mastigote forms of L. brasiliensis. Three of the compounds, DBP, DBMOBz, and DBMOH6, showed activity against the promastigote forms. The compound DBP, Graph (N), showed activity against the parasite at concentrations of 75 and 150 μM, with a 24% reduction in cell viability at both concentrations. The compounds DBMOH6 and DBMOBz, Graphs (M) and (O) respectively, showed a reduction in parasite growth from a concentration of 13.75 μM, being more effective at 150 μM, showing viability of only 33%. This means that these compounds can be used not only for the treatment of the disease but also for the control of the vector Phlebotomus pappatasi (sand fly) as a parasiticide. Example 4: Growth curve of parasites

[0045] The promastigote forms of L. braziliensis, strain M2904, were maintained in Medium 199 culture medium, supplemented with 20% fetal bovine serum (FBS), 1% adenine (1mM), 1% penicillin G (100U / mL) and 0.25% hemin, with pH 7.0 and incubated in a BOD incubator at 26°C.

[0046] Three cell culture flasks were prepared, containing the M2904-GFP strain of L. braziliensis in 10 mL of medium 199, with an initial concentration of 1x10⁵ parasites / mL in the promastigote form. For 15 days, two daily direct counts were performed in a Neubauer chamber. The arithmetic mean of the daily counts was used to calculate the number of parasites. After the counting period, the parasite growth curve was constructed using GraphPad Prism 5 software. Two biological replicates were used for this assay.

[0047] The results obtained for the growth curve can be seen in the graph in Figure 5. Conclusion

[0048] The evaluated compounds exhibit leishmanicidal activity, showing potential for treating the neglected disease cutaneous leishmaniasis, in topical or oral medicinal form. The compounds DBMOBZ, DBMOH6, and DBP also showed potential for inhibiting the promastigote form, and may be Petition 870250014195, dated 21 / 02 / 2025, page 28 / 37 / 15 considered for the prevention of the disease and to be used as a parasiticide. The advantage of this invention lies in the way the product can be used for therapy, being able to be prepared for topical application, in the form of gel, spray, cream, ointment. Being a therapeutic alternative to oral medications.

Claims

1. PHARMACEUTICAL FORMULATIONS with leishmanicidal activity, characterized by comprising at least one of the compounds (A) 1-(2(benzyloxy)phenyl)-3-phenylpropane-1,3-dione, (B) 1,3-diphenyl-2-benzyl-1,3-propanedione, (C) 1-(2-hydroxyphenyl)-3-phenylpropane-1,3-dione and (D) 1,3-diphenyl-2-propargyl-1,3-propanedione, associated with each other or with one or more pharmaceutically acceptable active ingredients and carriers or excipients such as talc, lactose, magnesium stearate, stearic acid, corn starch, humectants, anti-adherents, coating agents, disintegrants.

2. PHARMACEUTICAL FORMULATIONS with leishmanicidal activity, according to claim 1, characterized by comprising at least one of the compounds 1-(2-(benzyloxy)phenyl)-3-phenylpropane-1,3-dione, 1,3-diphenyl-2-benzyl-1,3-propanedione, 1-(2-hydroxyphenyl)-3-phenylpropane-1,3-dione and 1,3-diphenyl-2-propargyl-1,3-propanedione, associated or combined or co-administered with other leishmanicidal agents selected from the group such as meglumine antimoniate, sodium stiboglconate (SSG), liposomal amphotericin B, paromomycin sulfate and miltefosine.

3. PHARMACEUTICAL FORMULATIONS, according to any of claims 1 and 2, characterized by being able to be administered orally, by means of capsules, tablets, powder, granules, dragees or oral solution, or topically, by means of ointments, gels, sprays, creams, lotions containing the compound.

4. USE OF THE SYNTHETIC COMPOUNDS (A), (B), (C) and (D) defined in claims 1 and 2, characterized by being for the preparation of medicaments to treat Cutaneous Leishmaniasis in different species of mammals such as dogs and humans.

5. USE OF THE SYNTHETIC COMPOUNDS (A), (B), (C) and (D) defined in claims 1 and 2, characterized by being for the preparation of parasiticides for the control of the Leishmaniasis vector as a means of preventing leishmaniasis. Petition 870250014195, dated 21 / 02 / 2025, page 30 / 37 2 / 2 6. SYNTHETIC COMPOUND with leishmanicidal action defined in claims 1 and 2, characterized by being a 1-(2-(benzyloxy)phenyl)-3-phenylpropane-1,3-dione (B) and comprising the structure:

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