BR102015014710B1 - Antimony organometallic compounds (v) containing quinolone and fluoroquinolone ligands as leishmanicidal chemotherapeutic agents - Google Patents

Abstract

ANTIMONIUM(V) ORGANOMETALLIC COMPOUNDS CONTAINING QUINOLONIC AND FLUOROQUINOLONIC BINDINGS AS CHEMOTHERAPY AGENTS LEISHMANICIDES This is an invention relating to the process of obtaining potential antimony organometallic metallodrugs based on antimony(V), containing quinoline and fluoroquinolinic ligands, as well as the use of these compounds in the treatment of leishmaniasis. The results show that the compounds prepared are obtained in a simple way and with high yields and are effective against different forms of leishmania, and can be used as prototypes for the development of new leishmanicidal drugs.

Description

[001] The object of the invention refers to the development of new prototypes of easily obtainable, high-yield, potent and effective drugs for the treatment of leishmaniasis.

PROBLEM THAT THE INVENTION IS PROPOSED TO SOLVE

[002] Leishmaniasis is the third most important vector-borne disease, after malaria and African sleeping sickness, and ranks ninth in terms of the global burden of all infectious and parasitic diseases (STOCKDALE, L.; NEWTON, R. A Review of Preventative Methods against Human Leishmaniasis Infection. PLOS Negl. Trop. Dis. v. 7, no. 6, p. 1-15, 2013). It is part of the group of extremely neglected diseases, among which are also other diseases caused by protozoa of the Trypanosomatidae family, such as Chagas disease and African sleeping sickness (CAVALLI, A.; BOLOGNESI, M. L.; Neglected Tropical Diseases: Multi-Target -Directed Ligands in the Search for Novel Lead Candidates against Trypanosoma and Leishmania. J. Med. Chem. 2009). Currently, about 310 million people live in areas at risk of Leishmania infection. Leishmaniasis affects 98 countries and it is estimated that approximately 2 million new cases occur annually, of which 1.5 million are cutaneous leishmaniasis and 500,000 are visceral leishmaniasis. A substantial number of cases are not reported, as in only 32 of the 98 countries where the disease is endemic, reporting is mandatory (DESJEUX, P. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. v. 27, p. 305-3018, 2004; SANTOS, D.O. et al. Leishmaniasis treatment - a challenge that remains: a review. Parasitol. Res., v. 103, p. 1-10, 2008; GONZÁLEZ, U. et al. Interventions for American cutaneous and mucocutaneous leishmaniasis (Review). 2. ed. New York: JohnWiley & Sons, 2009; MONZOTE, L. Current Treatment of Leishmaniasis: A Review. Open Antimicrob. Agents. J., v. 1. , p. 9-19, 2009; WHO: World Health Organization, Leishmaniasis. Available at: < http://www.who.int/leishmaniasis/en/>. Accessed 21 Oct 2013). About 220,000 cases of cutaneous leishmaniasis and 58,000 cases of visceral leishmaniasis are officially reported each year (STOCKDALE, L.; NEWTON, R. A Review of Preventative Methods against Human Leishmaniasis Infection. PLOS Negl. Trop. Dis. v. 7, no. 6, pp. 1-15, 2013).

[003] The most common form of cutaneous leishmaniasis is characterized by ulcerative skin lesions that develop at the site of the sandfly vector bite. Secondary bacterial infections are common, causing severe pain and disability (GONZÁLEZ, U. et al. Interventions for American cutaneous and mucocutaneous leishmaniasis (Review). 2nd ed. New York: JohnWiley & Sons, 2009). Mucocutaneous leishmaniasis never heals spontaneously, and secondary infections that are difficult to treat are common. If untreated mucocutaneous leishmaniasis is left untreated, it can cause disfigurement and in some cases death (GONZÁLEZ, U. et al. Interventions for American cutaneous and mucocutaneous leishmaniasis (Review). 2. ed. New York: JohnWiley & Sons, 2009; PIRES, A.M. et al. Immunological and Clinical Aspects of American Cutaneous Leishmaniasis: A REVIEW. Rev. Ciênc. Saúde, v. 14, n. 1, p. 30-39, 2012). In addition, González et al. (GONZÁLEZ, U. et al. Interventions for American cutaneous and mucocutaneous leishmaniasis (Review). 2. ed. New York: JohnWiley & Sons, 2009) reported the occurrence of suicide attempts due to the associated social stigma. the disease. Visceral leishmaniasis is the most serious, fatal in 85-90% of untreated cases, and up to 50% of treated cases (ASSIS, R.R. et al. Glycoconjugates in New World species of Leishmania: Polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts. Biochim. Biophys. Acta., v. 1820, p. 1354-1365, 2012; STANLEY, A.C.; ENGWERDA, C.R. Balancing immunity and pathology in visceral leishmaniasis. Immunol Cell Biol, v. 85, p. 138- 147, 2007; GRIENSVEN, J. V.; DIRO, E. Visceral Leishmaniasis. Infect Dis Clin N Am. v. 26, p. 309-322, 2012; STOCKDALE, L; NEWTON, R. A Review of Preventative Methods against Human Leishmaniasis Infection PLOS Negl. Trop. Dis. v. 7, no. 6, p. 1-15, 2013).

[004] In addition to the facts presented above that demonstrate the worldwide expansion and severity of the disease, another factor that justifies the study and development of new drugs for the treatment of leishmaniasis is based on the fact that the drugs available on the market are in their totally toxic and trigger serious adverse effects.

STATUS OF THE TECHNIQUE

[005] Basically the treatment of leishmaniasis is divided into two groups of drugs, antimony and non-antimony. In general, the drugs available on the market present several problems, as they are toxic and cause many adverse reactions. Among the non-antimonial drugs are: pentamidine, amphotericin B, miltefosine and paromomycin (SANTOS, D.O. et al. Leishmaniasis treatment - a challenge that remains: a review. Parasitol. Res., v. 103, p. 1-10, 2008; GRIENSVEN, J. V.; DIRO, E. Visceral Leishmaniasis. Infect Dis Clin N Am. v. 26, p. 309-322, 2012).

[006] Pentamidine is administered exclusively intravenously or intramuscularly, being widely used in patients unresponsive to antimonials. Treatment usually causes application site pain, myalgias, nausea, headaches, hypotension, tachycardia, and pancreatitis (MONZOTE, L. Current Treatment of Leishmaniasis: A Review. Open Antimicrob. Agents. J., v. 1, p. 9-19, 2009; MITROPOULOS, P. et al. New World cutaneous leishmaniasis: Updated review of current and future diagnosis and treatment. J. Am. Acad. Dermatol, p. 309-322, 2010). Due to the toxicity of the treatment with amphotericin B, the patient must be hospitalized for its performance. The main adverse effects related to the use of amphotericin B are: fever, chills, thrombophlebitis, myocarditis, severe hypokalemia, renal dysfunction, which can lead to death (MITROPOULOS, P. et al. New World cutaneous leishmaniasis: Updated review of current and future diagnosis and treatment. J. Am. Acad. Dermatol, p. 309-322, 2010; GRIENSVEN, J. V.; DIRO, E. Visceral Leishmaniasis. Infect Dis Clin N Am. v. 26, p. 309-322, 2012). Miltefosine, the only drug available for oral administration, can cause gastrointestinal disorders, kidney disorders, in addition to being teratogenic (MONZOTE, L. Current Treatment of Leishmaniasis: A Review. Open Antimicrob. Agents. J., v. 1, p. 9-19, 2009; MITROPOULOS, P. et al. New World cutaneous leishmaniasis: Updated review of current and future diagnosis and treatment. J. Am. Acad. Dermatol, p. 309-322, 2010; GRIENSVEN, J. V.; DIRO , E. Visceral Leishmaniasis. Infect Dis Clin N Am. v. 26, p. 309-322, 2012 ).

[007] Despite being the drugs of choice for the treatment of all forms of leishmaniasis, the use of pentavalent antimonials has a number of limitations. The treatment lasts for at least three weeks, and the drugs are administered parenterally daily, which makes it difficult for the patient to adhere to the treatment. Several systemic adverse effects such as nausea, vomiting, myalgia, diarrhea, skin rashes, hepatotoxicity and cardiotoxicity are reported, requiring medical follow-up. In addition, the emergence of resistance to commercially available antimonials constitutes another serious problem, especially in India (FRÉZARD, F. et al. Pentavalent Antimoniais: New Perspectives for Old Drugs. Molecules, v. 14, p. 2317-2336, 2009 ; GRIENSVEN, J. V.; DIRO, E. Visceral Leishmaniasis. Infect Dis Clin N Am. v. 26, p. 309-322, 2012). Due to these limitations, the WHO recommends and supports the investigation of new drugs against leishmaniasis. One strategy is to modify existing drugs in order to obtain new, more active and less toxic molecules (FRÉZARD, F. et al. Pentavalent Antimonials: New Perspectives for Old Drugs. Molecules, v. 14, p. 2317-2336, 2009) .

[008] In the available patent databases, one can find a series of patents that describe the application of a series of molecules that are purely organic or containing metals as pharmacological agents with leishmanicidal activity: BR1020120190958 (Hydrazide-N-Acylhydrazones Compounds, Obtaining Process Of Compounds Hydrazide-N-Acylhydrazones, Use Of Compounds From Hydrazine-N-Acylhydrazones For Treatment Of Leishmaniasis And Chagas Disease And Pharmaceutical Compositions Obtained), PH 104710-0 (Pharmaceutical Formulations Containing Extract And/or Active Fractions Of The Leaves Of Tephrosia Cinerea and Its Use in the Treatment of Leishmaniasis), PI0817954-9 (Use of 5-Hydroxy-2-Hydroxymethyl-(Gamma)-Pirone as a Macrophage Activating Agent in Combating Cutaneous Leishmaniasis), PI0604319-4 (Process of Obtaining Derivatives of N, N-diarylbenzamidines and Use of These Derivatives in the Treatment of Leishmaniasis), PI0602371-1 (Antimonium-based compounds in dissociated state for the treatment of D and Leishmaniasis and Other Diseases, Their Procurement Processes and Pharmaceutical Compositions), PI0513549-4 (Pharmaceutical Compositions for the Treatment of Leishmaniasis), PI0502172-3 (Process of Formulation of the 3-(4'-Bromo-[1] Inclusion Complex) ,T-Biphenyl]-4-IL)-3-(4-Bromophenyl)-N,N-Dimethyl-2-Propen-1-Amine and β-Cyclodextrin and Trypanosomicidal, Antileishmaniasis and Antimycobacterial Activity), PI0409995-8 (Compound , and, Anti-Leishmania Agent), PI 9814815-0 (Agent For The Treatment Of Leishmaniasis With A Glycopyranose Derivative As An Effective Ingredient). The patent PI0602371 -1 (Antimonium-Based Compounds in a Dissociated State for the Treatment of Leishmaniasis and Other Diseases, Their Obtaining Processes and Pharmaceutical Compositions) proposes a formulation with known antimony drugs from their association with cyclodextrins, minimizing the side effects of these drugs and maximizing the degree of absorption of the same by the organism. A similar patent, W02006000069-A1 {Preparation of compounds used for preparing composition for treating e.g. leishmaniasis, comprises mixing antimony derivatives with cyclodextrins in aqueous solution or in solid state), also describes the use of cyclodextrins as agents that aid in the absorption of antimony salts by the body. Other patents propose the same strategy, however using liposomal technology in the encapsulation of classic antimonial drugs, examples: US4186183A (Liposome encapsulated anti-leishmaniasis antimonial drug - formed by wetting lipid film with drug soln., mixing and removing excess drug soln.), EP72234A (Stable liposome(s) of antimony contg. drug - for treatment of Leishmania infection), W09604890-A1 (Vesicle formulation) and US4594241 (Anti-leishmanial pharmaceutical formulations). Internationally, patent FR2101126-A (Antimony salt of 5-fluoro-8-hydroxyquinoline - with amoebicidal, antibacterial, anti-parasiticidal and anthelmintic activity) describes the application of a prototype trivalent antimony drug containing the anionic form of alcohol 5 -fluor-8-hydroxyquinoline. This compound was active against intestinal nematodes and had good parasitic activity, especially against the parasites that cause leishmaniasis, schistosomiasis and filariasis. Patent US3297531-A (Complexes of trivalent antimony with penicillamine antiprotozoal anthe - Imintic) proposes the use of a trivalent antimony having penicillin as a ligand. The inventors report that the compound has good tolerance and can be used to combat leishmaniasis, filariasis, among others. Patent P11106237-1A2 (Manocarriers Formed By Amphiphilic Antimony Complexes(V), Obtaining Process, Pharmaceutical Compositions and Use) describes the formation of pentavalent antimony complexes that can be understood as nanocarriers depending on the amount of surfactant present in the medium. These nanostructures, in addition to presenting leishmanicidal activity, have the ability to carry other drugs, also with leishmanicidal activity, maximizing their action potential. The patent JP2010254668-A (Water-soluble pentavalent antimony containing compound useful in agent for treating leishmaniasis, comprising sulfate group in axial ligand of phthalocyanine complex) proposes the use of a pentavalent antimony containing phthalocyanines as ligands and sulfate groups. These compounds have a desirable solubility in aqueous media without the aid of surfactants and have a good reduction capacity to their trivalent form with greater leishmanicidal action in a reducing atmosphere. Patent W09006931 (Organo.metallic deriv. contg. arsenic or antimony - for treatment of parasitic disorders esp. trypanosomiasis, leishmaniasis and filarioisis) proposes the use of organometallic compounds of antimony(lll) containing substituted phenyl ligands and chelating ligands through the coordination of sulfur and oxygen atoms. These compounds have antiparasitic activity, having been tested against filariasis, schistosomiasis and leishmania.

[009] Silva-Vergara et al. demonstrated the activity of azithromycin against mucosal leishmaniasis in three elderly patients (SILVA-VERGARA, M.L. et al. Azithromycin in he treatment of mucosal leishmaniasis. Rev Inst Med Trop Sao Paulo, v. 46, n.3 , pp. 175-7, 2004). Ponte-Sucre et al. reported that promastigotes of several Leishmania species were sensitive to potassium channel inhibitors such as glibenclamide and 4-aminopyridine (PONTE-SUCRE, A. et al. Sensitivity of Leishmania spp. to glibenclamide and 4 - aminopyridine: a tool for the study of drug resistance development. Mem Inst Oswaldo Cruz, v.92, n.5, p.601-6, 1997). Alrajhi et al. found that fluconazole reduced lesions in 79% of patients with cutaneous leishmaniasis caused by L. major, and patients who received only placebo had a cure rate of 34%, showing the effectiveness of the treatment (ALRAJHI, A.A. et al. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major. N Engl J Med. v.21, n. 12, p.891-5, 2002).

[010] In a review work by Rocha et al., a series of leishmanicidal activities were described for several extracts of different plants, in addition to several isolated substances such as alkaloids, flavonoids, terpenes, coumarins, among others (ROCHA, L.G. et al. A. review of natural products with antileishmanial activity. Phytomedicine, v.12, n. 6, p.514-535, 2005). Melo et al. described the leishmanicidal activity against L. major of the cassine/spectaline alkaloids obtained from Senna spectabilis (MELO, G.M.A. et al. Leishmanicidal activity of the crude extract, fractions and major piperidine alkaloids from the flowers of Senna spectabilis. Phytomedicine, v. 21, pp. 277-281, 2013).

[011] Metal complexes also showed leishmanicidal activity. Raychaudhury et al. demonstrated the leishmanicidal activity of tin complexes against L. donovani amastigotes and promastigotes (RAYCHAUDHURY, B. et al. Antiparasitic activity of a triphenyl tin complex against Leishmania donovani. Acta Tropica, v. 95, n.1, p. 1-8, 2005). Another group synthesized tin(IV)-containing carboxylated complexes and described the promising activity against L. tropica of these complexes (QURESHI, Q.H. et al. Organotin(IV) complexes of carboxylate derivative as potential chemotherapeutic agents against Leishmania. Inorg Chim Acta. v. 423 , pp. 220-228, 2014).

[012] Iridium and rhodium metal complexes, derived from pentamidine, have been described as active against L. donovani (MBONGO, N. et al. In vitro sensitivity of Leishmania donovani to organometallic derivatives of pentamidine. Parasitol Res. v.83, n. .5, p.515-17, 1997). Khan and colleagues synthesized SbHI-based carboxylated complexes with remarkable leishmanicidal and antifungal activity (KHAN, M.l. et al. In vitro anti-leishmanial and anti-fungal effects of new Sb111 carboxylates. Organic and Medicinal Chemistry Letters, v.1, n. 2, pp. 1-7, 2011). Bismuth(lll) carboxylated complexes demonstrated significant activity against L. major promastigotes at low concentrations (ANDREWS, P.C. et al. Anti-Leishmanial activity of homo- and heteroleptic bismuth(lll) carboxylates. J Inorg Biochem. v.105, n. 3, p.454-61, 2011).

[013] Benítez and collaborators reported the activity against L. major promastigotes of the oxidovanadium(IV) N-acylhydrazone complex, containing the phenyl group in its coordination sphere. The ICso value for the complex was 22.1 ± 0.6 μM (BENÍTEZ, J. et al. New oxidovanadium(IV) N-acylhydrazone complexes: Promising antileishmanial and antitrypanosomal agents. Eur J Med Chem. v. 62, p. 20 - 27, 2013). Palladium(ll) complexes were active against promastigote and amastigote forms of L. amazonensis and ligand activity was improved after complexation (FRANCO, L.P. et al. Palladium(ll) imine ligands cyclometallated complexes with a potential leishmanicidal! activity on Leishmania ( L.) amazonensis. v. 22, p. Med Chem Res. 1049-105, 2013 ). Caballero et al. have suggested that lanthanide complexes can become non-toxic antiparasitic drugs due to their relevant activity against L. infantum, L. braziliensis and T. cruzi (CABALLERO, A.B. et al. Lanthanide complexes containing 5-methyl-1,2, 4 triazolo[1,5-a]pyrimidin-7(4H)-one and their therapeutic potential to fight leishmaniasis and Chagas disease. J Inorg Biochem. v. 138, p.39-46, 2014 ). Gold(l) and gold(lll) complexes exhibited significant activity against promastigote and amastigote forms of L. amazonensis, L. brasiliensis and L. major (MOTA, V.Z. et al. Gold complexes with benzimidazole derivatives: synthesis, characterization and biological studies Biometals, v. 27, p. 183-194, 2014).

[014] Studies point to quinolones and fluoroquinolones as possible therapeutic agents in the fight against leishmaniasis. In a study carried out by Romero et al., they showed that seven types of quinolones and fluoroquinolones, including nalidixic acid, ciprofloxacin and norfloxacin, showed selective inhibitory effects on intracellular amastigotes of L. panamensis (ROMERO, I.C. et al. Selective Action of Fluoroquinolones Against Intracellular Amastigotes of Leishmania (Viannia) panamensis in vitro. J. Parasitol. v. 91, p. 1474-1479, 2005 ). Moxifloxacin was active against L. tropica promastigotes (LIMONCU, M.E. et al. Investigation of in vitro Antileishmanial Activity of Moxifloxacin, Linezolid and Caspofungin on Leishmania tropica Promastigotes. Turkiye Parazitol. Derg. v. 37, p. 1-3, 2013 ). Raether and coauthors described in vivo leishmanicidal activity against L. donovani using ofloxacin, ciprofloxacin and norfloxacin (RAETHER, W. et al. Potent antibacterial fluoroquinolones with marked activity against Leishmania donovani in vivo. Parasitol. Res. v. 75, p. 412 -413, 1989).

[015] Currently, the number of researches on chemical units that have the ability to act on multiple molecular targets has grown, and these compounds are called multitargets. Multi-targeted compounds have several advantages over those that act on single targets as they tend to increase activity, reduce resistance and decrease the necessary doses, consequently reducing the chances of developing side effects. According to Cavalli et al., there is a strong indication that multi-targeted compounds may provide new options for the treatment of neglected parasitic diseases such as leishmaniasis, malaria and Chagas disease (CAVALLI, A.; BOLOGNESI, M. L; Neglected Tropical Diseases: MultiTarget -Directed Ligands in the Search for Novel Lead Candidates against Trypanosoma and Leishmania. J. Med. Chem. 2009). The group also highlighted that the multi-target strategy was used to obtain complexes of steroids with platinum, which showed greater leishmanicidal activity against L. mexicana than the starting compounds (CAVALLI, A.; BOLOGNESI, M. L.; Neglected Tropical Diseases: Multi-Target -Directed Ligands in the Search for Novel Lead Candidates against Trypanosoma and Leishmania. J. Med. Chem. 2009). Based on this strategy, it is clear the possibility of uniting different active compounds against Leishmania, such as quinolones and fluoroquinolones and antimony, in order to obtain a characteristic, more active and less toxic multi-target compound, which is the objective of this invention.

TECHNIQUE DESCRIPTION

[016] The synthesis methodology used for the preparation of organometallic antimony complexes containing ligands derived from quinolones and fluoroquinolones, objects of this patent, is based on classical synthesis procedures to obtain antimony-based organometallic compounds, examples: YIN, H.D. et al. Synthesis, characterizations and crystal structures of new organoantimony(V) complexes with various isomers of fluoromethylbenzoate ligands. Polyhedron, v. 28, p. 2919-2926, 2009; QUAN, L. et al. Synthesis, characterization and crystal structures of tri- and tetraphenylantimony(V) compounds containing arylcarbonyloxy moiety. J. Organomet. Chem. v. 694, p. 3708-3717, 2009; and WEN, L. et al. Triphenyl bis(2,4,5-trifluoro-3-methoxybenzoate) antimony(V). Cryst Minutes. v. 64, p. 1303, 2008. The basic structure of the compounds object of this patent is (RCOOj∑SbRa, where R = methyl or phenial; and RCOO = the deprotonated form of quinolones and fluoroquinolones, such as: Nalidixic acid, Oxolinic acid, Pipemidic acid, Pyromydic acid , Balofloxacino, Besifloxacino, Cinoxacino, Ciprofloxacino, Clinafloxacino, Enoxacino, Fleroxacino, Flumequina, Garenoxacino, Gatifloxacino, Gemifloxacino, Grepafloxacino, Levofloxacino, Lomefloxacino, Moxifloxacino, Nadifloxacino, Norfloxacino, Ofloxacino, Pazufloxacino, Pefloxacino, Prulifloxacino, Rosoxacino, Rufloxacino, Sitafloxacino, Sparfloxacino , Temafloxacin, Tosufloxacin, Trovafloxacin etc.

Examples of synthesis of drug prototypes prepared Triphenyl organometallic derivatives:

[017] (Nal)2SbPh3, b/s[1,4-Dihydro-1-ethyl-7-methyl-1,8-naphthyrinin-4-one-3-carboxylate] Triphenylantimony(V);

[018] (Cip)2SbPh3, triphenylantimony b/s[1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylate](V);

[019] (Nor)2SbPh3, triphenylantimony b/s[1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylate](V).

[020] The synthesis strategy for the (Nal)2SbPh3 complex was the same adopted for all the complexes shown in Figure 1. Thus, only the synthesis of the (Nal)2SbPh3 complex is described below and the others follow the same strategy. In a 100 ml Schlenk flask, 30 ml of dry methanol under argon and 1.4 mmol (0.032 g) of sodium metal were added. After its consumption, 0.5 mmol (0.127 g) of nalidixic acid was added, and the solution was kept under stirring at room temperature for 0.5 h. Then 0.25 mmol (0.106 g) of triphenylantimony(V) dichloride was added. The mixture was kept at room temperature under stirring under an argon atmosphere for 12 h. Finally, the solvent was removed and the material dried in a high vacuum pump, thus obtaining a dark yellow solid that was washed with water and subjected to drying again. Yield: 97%.

[021] The complexes with derivatives of ciprofloxacin and norfloxacin ligands were obtained in the same way, generating two yellow solids and with quantitative yields.

[022] Figure 1 shows the synthesis scheme of the (Nal)2SbPh3, (Cip)2SbPh3 and (Nor)2SbPh3 complexes.

Trimethyl Organometallic Derivatives:

[023] Trimethylantimony(Nal)2Sb(CH3)3,bis[1,4-Dihydro-1-ethyl-7-methyl-1,8-naphthyrinin-4-one-3-carboxylate]( V)

[024] Trimethylantimony (Cip)2Sb(CH3)3,b/s[1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylate](V )

Trimethylantimony [025](Nor)2Sb(CH3)3, b/s[1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylate](V )

[026] For the synthesis of the (Nal)2Sb(CH3)3 complex, 20 mL of dry methanol was added in a Schlenk flask under an argon atmosphere. Subsequently, 0.7 mmol (0.016 g) of sodium metal was added to the flask and waited until it was completely consumed. After this period, 0.25 mmol (0.064 g) of nalidixic acid was added, and the solution was kept under stirring at room temperature for 0.5 h. Then 0.125 mmol (0.041 g) of trimelantimmonium(V) dibromide was added. The mixture was kept at room temperature under stirring under an argon atmosphere for 12 h. Finally, the solvent was removed and the material dried in a high vacuum pump, thus obtaining a light yellow solid. The complexes with the ligands ciprofloxacin and norfloxacin were obtained in the same way (Figure 2), being formed two solids, cream [(Cip)2Sb(CH3)3] and white [(Nor)2Sb(CH3)3], respectively, all with quantitative yield.

[027] Figure 2 shows the synthesis scheme of the (Nal)2Sb(CH3)3, (Cip)2Sb(CH3)3 and (Nor)2Sb(CH3)3 complexes

Tests to evaluate the leishmanicidal activity of some of the prototypes In vitro tests Maintenance of macrophages and parasites

[028] Macrophages of the J774 strain were used for in vitro assays to determine cell viability and infection. These cells were maintained in culture bottles in 5 ml of DMEM (Dulbecco's Modified Eagle Medium) with 10% fetal bovine serum (FBS). At the time of use, cells were counted, adjusted in DMEM medium supplemented with 10% FBS. The species L. braziliensis (strain MHOM/BR/01/BA788), L. amazonensis (strain MHOM/BR/87/BA125) and L chagasi (strain MCAN/BR/89/BA262) were used to evaluate the leishmanicidal activity. The promastigote forms were maintained in culture bottles in 5 ml of Schneider's medium (Sigma) with 10% FBS in a BOD oven at 27°C. At the time of use, the parasites were placed in Falcon tubes and centrifuged at 3000 rpm for 10 minutes. Then, the supernatant was discarded and the pellet formed was resuspended in Schneider medium (Sigma). The parasites were then counted in a Neubauer chamber, for later plating of the cells and carrying out the experiments.

Cytotoxicity evaluation assay against promastigote forms

[029] Promastigote forms of L. braziliensis, L. amazonensis or L. chagasi at a concentration of 105 parasites/well in a volume of 200 μL were cultured in triplicates in a 96-well plate with Schneider's medium supplemented with 10% FBS, 2 mM of L-glutamine, 2% human urine. Different concentrations of the complexes: (Nal)2SbPh3, (Nal)2Sb(CH3)3, (Cipj∑SbPhs, (Cip)2Sb(CH3)3, (Nor)2SbPh3e(Nor)2Sb(CH3)3; of the ligands: acid nalidixic, ciprofloxacin and norfloxacin; PhaSbCh and antimony salts (CHsjsSbBr∑ and meglumine antimoniate, the standard drug used as a reference), were added to the wells containing the promastigotes and the plate was incubated in a BOD oven at 27 °C for 48 hours. After this period, the parasites were homogenized and 100 μL of the suspension was transferred to a tube containing 10 mL of isoton.The number of parasites was determined in an automatic cell counter model CC530 (ÁVILA, J.L. et al. Specific inhibitory effect of 3- deazaneplanocin a against several leishmania mexicana and leishmania braziliensis strains. Am. J. Trop. Med. Hyg, V. 57, N. 4, P. 407-412, 1997; MELO, G.M.A. et al. Leishmanicidal activity of the crude extract, fractions and major piperidine alkaloids from the flowers of Senna spectabilis Phytomedicine, v. 21, p . 277-281, 2013).

Cytotoxicity evaluation assay against intracellular amastigotes

[030] Macrophages were counted and adjusted in DMEM medium supplemented with 10% FBS at a concentration of 105 cells/mL and 500 μL of this suspension was distributed in a 24-well plate (Nunc, Denmark). Macrophages were infected with promastigote forms of L. amazonensis, in the stationary phase of growth, at a ratio of 10 parasites: 1 macrophage on coverslips. After 4 hours, the wells were washed with Hank's to remove non-internalized leishmania. The cells were treated with solutions of the complexes (Nal)2SbPh3, (Cip)2SbPh3, (NorJ∑SbPhs and the standard drug meglumine antimoniate at concentrations of 100 μM, 10 pM and 1 pM and were kept for 48 hours in an oven at 37 °C with a humid atmosphere containing 7% CO2. After this period, the wells were washed with Hank's to remove the substances. The macrophages were fixed with methanol, then the slides were stained with panoptic and mounted on slides. of the number of infected macrophages and the number of amastigotes in 100 macrophages was performed using an optical microscope with a 100x objective (immersion oil) (NUNES, M.P. et al. Cd40 Signaling Induces Reciprocal Outcomes In Leishmania-infected macrophages; roles of host genotype and cytokine milieu. Micro. Infect, v. 7, p. 78-85, 2005).

Cell viability assay

[031] The macrophage viability study against (Nal)2SbPh3, (Cip)2SbPh3, (NorJ∑SbPhs and meglumine antimoniate) complexes was performed using the MTT method (3-[4,5-dimethyl-thiazole bromide) -2-yl]-2,5-diphenyltetrazolium) In this assay, macrophages of the J774 strain were plated (5 x 10 5 /per well) and exposed to different concentrations of the compounds (100, 10 and 1 pM) for a period of 48 hours. Control wells contained cells cultured only with culture medium and cells cultured in the presence of the DMSO substances diluent. After the incubation period, the supernatant was discarded and then 200 μL of the MTT solution (10% in DMEM medium) in each well. The plates were then reincubated for 4 hours in an oven at 37 °C and 5% CO2 and the reading was performed in a spectrophotometer at 490 nm. The results were expressed as percentage of cell death, considering as control 100% viable cell count in unstimulated cultures (MOSMANN , T. Rapid colorimetric assay for cellular growth and survival. J. Immunol. Methods, v.65, p. 55-63, 1983). in vivo tests

Animals

[032] BALB/C mice obtained from the University of Campinas and Golden Hamsters from Fiocruz-RJ were used to determine the therapeutic effect of the selected compounds in the in vitro models. To evaluate the in vivo effect against L. amazonensis, young adult male BALB/c mice (20-25 g) were used, aged 6 to 8 weeks. To evaluate the in vivo effect against L. chagasi, young adult male Golden Hamsters (20-25 g) were used, aged 6 to 8 weeks. All animal tests used in this work were previously approved by the Ethics Committee for the Use of Laboratory Animals at UFAL (CEP N° 2.2013).

In vivo assay of infection with L. amazonensis in the ear of BALB/c mice

[033] Promastigote forms of L. amazonensis (MHOM/BR/87/BA125) (105 cells in 10 μL) were inoculated subcutaneously in the ear of Balb/c mice. Five animals were used per group, these are summarized into groups treated with the standard drug meglumine antimoniate, and with the complexes (Nal)2SbPh3 and (NorJaSbPha. In addition, the infected/untreated group and the uninfected group. After one week of infection, the animals started to be treated with the complexes (NalJ∑SbPha and (No^SbPha, previously selected from in vitro viability assays on promastigote and amastigote forms of L. amazonensis and with the standard drug meglumine antimoniate by via ip at a dose of 30 μmol/kg. (adapted from PEREIRA, J.C.M. et al. Antileishmanial activity of ruthenium(ll)tetraammine nitrosyl complexes. Eur. J. Med. Chem. v. 45, p. 4180-4187, 2010) .

[034] Treatment was given in a single daily dose per day for 28 days. Lesion size was measured in mm twice a week during the treatment period using a digital caliper and expressed as the difference between the infected and uninfected ear. In addition, the animals were weighed during the treatment period to verify if there was a significant change in the animal's weight. After four weeks of treatment, the animals were anesthetized for cardiac puncture, the collected blood was centrifuged at 1500 rpm for five minutes to obtain the serum, and with this, to analyze possible enzymatic alterations, the dosage of serum concentrations was performed. of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine and urea according to the procedures described in the Doles kits for each of the aforementioned enzymes. Subsequently, the infected ears and draining lymph nodes were aseptically removed and macerated in Schneider (Sigma) to determine the parasite load through limiting dilution analysis (adapted from PEREIRA, J.C.M. et al. Antileishmanial activity of ruthenium(ll)tetraammine nitrosyl complexes. Eur.J.Med.Chem.v.45, p.4180-4187, 2010).

[035] To quantify the parasites in the infected ears and draining lymph nodes, the limiting dilution technique was used (TASWELL, C. Limiting dilution assays for the separation, characterization and quantification of biologically active particles and their clonal progeny. In: T.C. Pretlow, T.P. Pretlow (Eds.), Cell Separation: Methods and Selected Applications, Academic Press, New York, 1986, pp. 109-145). The infected ears and draining lymph nodes were aseptically removed and macerated in 1 mL of Schneider medium (Sigma) with 10% fetal bovine serum until a suspension was formed. Subsequently, 200 μL of the macerate from each sample was transferred to a 96-well flat-bottomed culture plate, in duplicate, representing a 1:5 dilution. 11 serial dilutions of the macerate were performed, using the proportion of 1:10. The plates were incubated in a BOD oven at 26 °C for 14 days. Up to the fourteenth day of culture, the number of parasites was calculated by multiplying the dilution factor 5 by 10 raised to the power referring to the number of the last dilution that had at least one parasite present (LIMA, 1997).

In vivo visceral infection assay of golden hamsters with L. chagasi

[036] In order to evaluate the leishmanicidal activity of the compounds, eight-week-old male golden hamsters were infected intraperitoneally with 107 L. chagasi promastigotes in the stationary phase (DENISE, H. et al. Studies on the CPA). cysteine peptidase in the Leishmania infantum genome strain JPCM5.BMC Mol. Biol. v. 7, p. 42-48, 2006). After 45 days of infection, the animals were randomly separated into experimental groups of 5 hamsters each. The animals received daily intraperitoneal treatment at doses of 30 μmol/kg/day of the complex (NorjaSbPha and 150,100 and 30 μmol/kg/day of meglumine antimoniate for 15 days. The control group received the same number of injections with water for After the end of the treatment, the animals were sacrificed and the parasite load in the spleen was evaluated using the limiting dilution method, as previously described (LIMA, H.C. et al. A simple method for quantifying Leishmania in tissues of infected animals. Parasitol Today, v. 13, pp. 80-82, 1997) The toxicity of the compounds was also determined through biochemical assays in the plasma of the animals, using commercial kits (Doles Reagentes e Equipamentos para Laboratórios, Ltda, Brazil).

Statistical analysis

[037] The data obtained were expressed as mean ± standard error of the mean (M ± S.P.M.) after statistical analysis using one-way analysis of variance (ANOVA) followed by Dunnett's post-test, where the differences between the means were considered significant when p < 0.05 when compared to the control group. It was determined for the complexes, ligands, salts of antimony and antimoniate of meglumine, the inhibitory concentration 50 (Ciso), the lethal concentration 50 (LC50) and the selectivity index (IS) leishmania/macrophage J774.

RESULTS OBTAINED Result of the synthesis of the new prototypes of drugs against leishmaniasis

[038] (Nal)2SbPh3 - Molecular formula (F.M.): C42H37N40eSb. Molecular mass (M.M.): 814.18 g/mol. Anal. Elem. Theoretical: C: 61.86; H: 4.57; N: 6.87; O: 11.77; Sb: 14.93. IV (Vmax/cnr1, KBr); 1620 (v C=O); 740-690 (Phoff piano); 451 (v Sb-O). 1H NMR (400MHz, CD3OD, ppm): 1.46 (t, 6H, H12, J = 7.15 Hz); 2.67 (s, 6H, H14); 4.53 (q, 4H, H11, J=7.11 Hz); 7.33 (d, 2H, H6, J=8.24 Hz); 7.63 (m, 9H, H3', H4' and H5'); 7.99 (m, 6H, H2', H6'); 8.60 (d, 2H, H5, J=8.18 Hz); 8.69 (s, 2H, H2). 13 C NMR (100MHz, CD3OD, ppm): 15.60 (C12); 24.78 (C14); 47.40 (C11); 119.66 (C3); 121.84 (C10); 122.11 (C6); 130.72 and 132.64 (Ph); 135.58 (C5); 136.38 and 137.68 (Ph); 149.75 (C2); 150.07 (C7); 170.82 (C9); 172.73 (C13); 178.78 (C4).

[039] (Cip)2SbPh3 - Molecular formula (F.M.): C52H49F2N6OeSb. Molecular mass (M.M.): 1012.27 g/mol. Anal. Elem. Theoretical: C: 61.61; H:4.87; F: 3.75; N: 8.29; O: 9.47; Sb: 12.01. IR (vmax/cnr1, KBr): 1640 (v C=O); 740-690 (Phoff piano); 451 (v Sb-O). 1H NMR (400MHz, CD3OD, ppm): 1.08 (m, 4Hc/s, Cyclopropyl); 1.25 (q, 4Hyrans, Cyclopropyl, J = 6.32 Hz); 2.96 (tap, 8H, H16, H18,); 3.18 (tap, 8H, H15, H19); 3.51 (m, 2Hgen, Cyclopropyl); 7.38 (d, 2H, H8, J = 7.37 Hz); 7.55 (m, 9H, H3', H4' and H5'); 7.86 (d, 2H, H5, J = 13.74 Hz); 7.92 (m, 6H, H2', H6); 8.52 (s, 2H, H2).

[040] (Nor)2SbPh3 - Molecular formula (F.M.): Cso^θF∑NθOθSb. Molecular mass (M.M.): 814.18 g/mol. Anal. Elem. Theoretical: C: 61.86; H: 4.57; N: 6.87; O: 11.77; Sb: 14.93. IR (Vmax/cnr1, KBr): 1620 (v C=O); 740-690 (Phoff piano); 451 (v Sb-O). 1H NMR (400MHz, CD3OD, ppm): 1.49 (t, 6H, H12, J = 7.22 Hz); 3.02 (tap, 8H, H15, H17); 3.24 (tap, 8H, H14, H18); 4.36 (q, 4H, H11, J = 7.22 Hz); 7.02 (d, 2H, H8, J = 7.22 Hz); 7.63 (m, 9H, H3', H4' and H5'); 7.96 (d, 2H, H5, J = 13.74 Hz); 7.99 (m, 6H, H2', H6'); 8.52 (s, 2H, H2). 13 C NMR (100MHz, CD3OD, ppm): 14.85 (C12); 46.60 (C15, C17); 49.87 (d, C14, C18, 4JC-F = 2.15 Hz); 52.07 (C11); 105.88 (d, C8, 3JC-F = 2.70 Hz); 113.65 (d, C5, 2JC-F = 23.08Hz); 118.46 (C3); 124.51 (d, C10, 3JC-F=6.79 Hz); 130.91, 132.97, 134.33 and 136.34 (Ph); 138.25 (C9); 146.67 (d, C7, 2JC-F = 10.87 Hz); 148.99 (C2); 154.76 (d, C6, 1JC-F= 246.63 Hz), 173.08 (C13); 177.04 (d, C4, 4JC-F=2.28 Hz).

[041] (Nal)2Sb(CH3)3 - Molecular formula (F.M.): C27H3iN4θeSb. Molecular mass (M.M.): 628.13 g/mol. Anal. Elem. Theoretical: C: 51.53; H: 4.97; N: 8.90; O: 15.25; Sb: 19.35. IR (Vmax/cnr1, ATR): 1627 (v C=O); 749-703 (Ph outside the piano). 1H NMR (400 MHz, CD3OD, ppm): 1.47 (t, 6H, H12, J = 7.10 Hz); 1.59 (s, 9H, HT); 2.68 (s, 6H, H14); 4.54 (q, 4H, H11, J=7.15 Hz); 7.34 (d, 2H, H6, J=8.14 Hz); 8.61 (d, 2H, H5, J = 8.14 Hz); 8.76 (s, 2H, H2). 13 C NMR (100 MHz, CD3 OD, ppm): 4.99 (CT); 15.64 (C12); 25.31 (C14); 47.44 (C11); 119.45 (C3); 121.88 (C10); 122.04 (C6); 137.68 (C5); 149.87 (C2); 149.95 (C7); 164.51 (C9); 172.79 (C13); 178.79 (C4).

[042] (Cip)2Sb(CH3)3 - Molecular formula (F.M.): Cs∑HAθF∑NeOeSb. Molecular mass (M.M.): 828.23 g/mol. Anal. Elem. Theoretical: C: 61.61; H: 4.87; F: 3.75; N: 8.29; O: 9.47; Sb: 12.01. IV (vmto/cπr1, ATR): 1620 (v C=O); 740-703 (Piano piano). 1H NMR (400 MHz, CD3OD, ppm): 1.15 (m, 4HC/S, Cyclopropyl); 1.34 (q, 4Htrans, Cyclopropyl, J = 6.35 Hz); 1.59 (s, 9H, HT); 3.04 (tap, 8H, H16, H18); 3.27 (tap, 8H, H15, H19); 3.60 (m, 2Hgen, Cyclopropyl); 7.48 (d, 2H, H8 J = 7.47 Hz); 7.97 (d, 2H, H5, J = 13.69 Hz); 8.64 (s, 2H, H2). 13 C NMR (100 MHz, CD3 OD, ppm): 3.96 (CT); 8.65 (C12, C13); 35.77 (C11); 46.61 (C16, C18); 52.05 (d, C15, C19, 4JC-F=4.42 Hz); 106.59 (d, C8, 3JC-F= 2.68 Hz); 113.25 (d, C5, 2JC-F= 23.34 Hz); 117.70 (C3); 123.82 (d, C10, 3JC-F=6.95 Hz); 140.14 (C9); 146.40 (d, C7, 2JC-F=10.83 Hz); 149.08 (C2); 154.78 (d, C6, 1JC-F= 246.67 Hz), 173.13 (C14); 177.34 (d, C4, 4JC-F=2.21 Hz).

[043] (Nor)2Sb(CH3)3 - Molecular formula (F.M.): C5oH49F2N6θθSb. Molecular mass (M.M.): 814.18 g/mol. Anal. Elem. Theoretical: C: 61.86; H: 4.57; N: 6.87; O: 11.77; Sb: 14.93. IR (vmax/crrr1, ATR): 1620 (v C=O); 740-703 (Ph outside the piano). 1H NMR (400 MHz, CD3OD, ppm): 1.50 (t, 6H, H12, J = 7.04 Hz); 1.59 (s, 9H, HT); 3.03 (tap, 8H, H15, H17); 3.25 (tap, 8H, H14, H18); 4.37 (q, 4H, H11, J = 7.05 Hz); 7.02 (d, 2H, H8, J = 7.05 Hz); 7.99 (d, 2H, H5, J = 13.78 Hz); 8.57 (s, 2H, H2). 13 C NMR (100 MHz, CD3 OD, ppm): 4.94 (CT); 14.82 (C12); 46.62 (C15, C17); 49.87 (d, C14, C18, 4JC-F= 2.11 Hz); 52.06 (C11), 105.69 (d, C8, 3JC-F = 2.67 Hz); 113.53 (d, C5, 2JC-F = 23.04Hz); 117.92 (C3); 124.42 (d, C10, 3JC-F = 6.75 Hz); 138.10 (C9); 146.60 (d, C7, 2JC-F=10.76 Hz); 149.43 (C2); 154.49 (d, C6, 1JC-F= 246.41 Hz), 173.16 (C13); 177.25 (d, C4, 4JC-F=2.20 Hz).

Pharmacological results Viability assay of L. braziliensis, L. amazonensis and L. chagasi in vitro

[044] The effect of nalidixic acid, (NaO∑SbPha, (Nal)2Sb(CH3)3, ciprofloxacin, (Cip)2SbPh3, (Cip)2Sb(CH3)3, norfloxacin, (NoΦSbPhs (Nor)2Sb(CH3) 3, in addition to the standard drug meglumine antimoniate, was evaluated on the growth of promastigote forms of L. braziliensis, L. amazonensis and L. chagasi. According to the data described in Table 1, it was observed that the ligands, nalidixic acid and norfloxacin, were able to significantly inhibit the growth of promastigote forms of L. brasiliensis with a maximum effect of 70.5 ± 1.8% and 60.9 ± 1.8 and an IC50 of 0.6 ± 0.02 μM and 0, 2 ± 0.05 μM. Ciprofloxacin was not able to inhibit the growth of promastigote forms of the aforementioned species. The complexes (Nal)2SbPh3, (Cip)2SbPh3 and (Nor)2SbPh3 were potent and effective in inhibiting the growth of promastigote forms of L. brasiliensis with IC50 of 0.03 ± 0.02 pM, 0.2 ± 0.03 pM and 12.3 ± 1.3 pM and maximum effect of 97.1 ± 0.4%, 98, 2 ± 0.4% and 92.8 ± 0.6% respectively. The complexes (Nal)2Sb(CH3)3, (Cip)2Sb(CH3)3 and (Nor)2Sb(CH3)3 did not show leishmanicidal activity against L. brasiliensis.

[045] Table 1 shows the determination of potency (IC50) and maximum effect of nalidixic acid, (Nal)2SbPh3, (Nal)2Sb(CH3)3, ciprofloxacin, (Cip)2SbPh3, (Cip)2Sb(CH3)3 , norfloxacin, (Nor)2SbPh3, (Nor)2Sb(CH3)3, PhsSbCh, (CH3)3SbBr2 and meglumine antimoniate (all at concentrations of 100, 30, 10, 3, 1, 0.3, 0.03 and 0.01 pM) on Leishmania braziliensis promastigotes.

[046] The results refer to: the Inhibitory Concentration of 50 (Ciso) of the growth of promastigotes calculated through concentration-response curves and expressed as mean ± standard error of the mean; b Maximum effect (ME) which is expressed as the mean of the maximum effect ± standard error of the mean in triplicates of a representative experiment. The values of E.M. they were considered significant when *p < 0.05, **p < 0.01 up to a concentration of 100 μM in relation to the DMSO group.

[047] In the viability assay of promastigote forms of L. amazonensis, none of the ligands, nalidixic acid, ciprofloxacin and norfloxacin, were able to inhibit the growth of promastigote forms of the parasite at the concentrations tested. The complexes (NaQ∑SbPha, (Cip)2SbPh3 and (Nor)2SbPh3, however, showed potent leishmanicidal activity with Ciso of 3.9 ±1.6 μM, 4.4 ± 0.4 pM and 13.7 ± 2, 3 pM and maximum effect of 95.3 ± 0.9%, 96.3 ± 0.2% and 95.9 ± 0.1% respectively.(Nal)2Sb(CH3)3, (Cip)2Sb( CH3)3 and (Nor)2Sb(CH3)3 and meglumine antimoniate did not inhibit the growth of promastigote forms of L. amazonensis, as observed for L. brasiliensis (Table 2).

[048] Table 2 shows the determination of potency (Ciso) and maximum effect of nalidixic acid, (NalJ∑SbPhs, (Nal)2Sb(CH3)3, ciprofloxacin, (Cip)2SbPh3, (Cip)2Sb(CH3)3 , norfloxacin, (Nor)2SbPh3, (Nor)2Sb(CH3)3, PhaSbCh, (CH3)3SbBr2 and meglumine antimoniate (all at concentrations of 100, 30, 10, 3, 1 and 0.3 μM) on promastigotes of Leishmania amazonensis.

[049] The results refer to: the Inhibitory Concentration of 50 (Ciso) of the growth of promastigotes calculated through concentration-response curves and expressed as mean ± standard error of the mean; b Maximum effect (ME) which is expressed as mean of maximum effect ± standard error of the mean in triplicates of a representative experiment. The values of E.M. they were considered significant when *p < 0.05, **p < 0.01 up to a concentration of 100 μM in relation to the DMSO group.

[050] The effect on the growth of promastigote forms of L. chagasi of nalidixic acid, ciprofloxacin, norfloxacin, (Nal)2SbPh3, (Cip)2SbPh3, (Nor)2SbPh3β meglumine antimoniate was evaluated at concentrations of 100, 30, 10 , 3, 1 and 0.3 μM (Table 3). As observed for promastigotes of L. braziliensis and L. amazonensis species, meglumine antimoniate did not inhibit the growth of promastigotes of L. chagasi and L. chagasi at any of the concentrations tested. The ligands, nalidixic acid, ciprofloxacin and norfloxacin, did not show leishmanicidal activity against L. chagasi, as observed for L. amazonensis promastigotes.

[051] The (Nal)2SbPh3,(Cip)2SbPh3e(Nor)2SbPh3 complexes were able to significantly inhibit the promastigote forms of the parasite. All had similar efficacy, with a maximum effect of 96.7 ± 0.3%, 97.6 ± 0.8% and 91.5 ± 2.3% and a Ciso of 17.5 ± 0.6 μM, 8.4 ± 0.2 μM and 9.5 ± 0.3 μM, respectively. It is observed that complexes with the second generation fluoroquinolones ciprofloxacin and norfloxacin were more potent than the complex with nalidixic acid. (Nal)2Sb(CH3)3, (Cip)2Sb(CH3)3 and (Nor)2Sb(CH3)3 did not show leishmanicidal activity, as observed for the other Leishmania species tested.

[052] Table 3 shows the determination of potency (IC50) and maximum effect of nalidixic acid, (NalJ∑SbPhs, (Nal)2Sb(CH3)3, ciprofloxacin, (Cip)2SbPh3, (Cip)2Sb(CH3)3 , norfloxacin, (Nor)2SbPh3. (Nor)2Sb(CH3)3, Ph3SbCI2, (CH3)3SbBr2 and meglumine and pentamidine antimoniate (all at concentrations of 100, 30, 10, 3, 1 and 0.3 μM) on of Leishmania chagasi promastigotes.

[053] The results refer to: the Inhibitory Concentration of 50 (Ciso) of the growth of promastigotes calculated through concentration-response curves and expressed as mean ± standard error of the mean; b Maximum effect (ME) which is expressed as the mean of maximum effect ± standard error of the mean in triplicates of a representative experiment. The values of E.M. they were considered significant when *p < 0.05, **p < 0.01 up to a concentration of 100 μM in relation to the DMSO group.

[054] It was also evaluated the leishmanicidal potential of ligands, complexes and the standard drug meglumine antimoniate on the amastigote forms of L. amazonensis. For this, the intracellular infection assay was carried out in a coverslip with macrophages of the J774 lineage. Due to the relevant leishmanicidal activity against promastigote forms of L. amazonensis, quinolone and fluoroquinolone complexes with triphenylantimony(V) were selected for testing against amastigote forms. By analyzing the data in Table 4, it is observed that the complexes were also able to significantly inhibit the growth of amastigote forms of L. amazonensis having similar potencies and efficacies. They presented the following values of Ciso and maximum effect: (Nal)2SbPh3 (9.4 ± 0.3 pM, 77.3 ± 2.4%), (Cip)2SbPh3 (2.4 ± 0.1 pM, 70, 1 ± 2.6%) and (Nor)2SbPh3 (36.3 ± 8.2 pM, 70.1 ± 2.9%). At the concentrations tested, nalidixic acid, ciprofloxaxin, norfloxacin and the standard drug meglumine antimoniate did not show leishmanicidal activity.

[055] Table 4 shows the determination of potency (Ciso) and maximum effect of nalidixic acid, (Nall∑SbPha, ciprofloxacin, (Cip)2SbPh3, norfloxacin, (Nor)2SbPh3, PhaSbCh and meglumine antimoniate (all at concentrations of 100, 10 and 1 μM) on the replication of Leishmania amazonensis amastigotes.

[056] The results refer to: the Inhibitory Concentration of 50 (Ciso) of the growth of amastigotes calculated through concentration-response curves and expressed as mean ± standard error of the mean; b Maximum effect (ME) which is expressed as the mean of the maximum effect ± standard error of the mean in triplicates of a representative experiment. The values of E.M. they were considered significant when *p < 0.05, **p < 0.01 up to a concentration of 100 μM in relation to the DMSO group.

[057] The present invention demonstrates a significant increase in the leishmanicidal activity of the complexes (Nal)2SbPh3, (Cip)2SbPh3 and (Nor)2SbPh3 in all assays against different species and evolutionary forms of Leishmania when they were compared to free acid ligands. nalidixic, ciprofloxacin and norfloxacin.

Cell viability assay: MTT reduction

[058] The determination of cell viability was performed using the MTT reduction assay. For this, cultures of J774 macrophages treated or not with the test substances were used for a period of 48 hours. The MTT reduction assay is a colorimetric assay that measures the reduction of the yellow compound, MTT, by the mitochondrial enzyme succinate dehydrogenase. MTT enters cells, where within the mitochondria it is reduced to an insoluble purple product (formazan). The reduction of MTT only occurs in metabolically active cells, and its conversion to formazan is a measure of cell viability (SOFLAEI, S. et al, Anti-leishmanial activities of selenium nanoparticles and selenium dioxide on Leishmania infantum. Comp. Clin. Pathol, v. 23, pp. 15-20, 2014). The ligands, triphenylantimony(V) dichloride and the complexes that exhibited leishmanicidal activity, (Nal)2SbPh3, (Cip)zSbPh3 and (Nor)2SbPh3, were selected for this assay.

[059] It is observed through the results shown in Table 5. that only ciprofloxacin did not exhibit toxicity at the concentrations tested. Nalidixic acid, (NalJ∑SbPhs, norfloxacin, (Noij∑SbPhs and meglumine antimoniate when compared to control (DMSO 0.1%) induced toxicity in a statistically significant way, however all had LC50 above 100 μM. (Cip) 2SbPh3 and the antimony salt used for the synthesis of the complexes, PhaSbCh, induced significant toxicity with a percentage of cell death of 100 ± 0.1% and LC50 of 19.3 ± 6.4 μM and 42.4 ± 5.9 μM respectively.

[060] Table 5 shows the determination of the lethal concentration (CLso) of nalidixic acid, (Nal)2SbPh3, ciprofloxacin, (Cip)2SbPh3, norfloxacin, (Nor)2SbPh3, PhsSbCh and meglumine antimoniate (all at concentrations of 100, 10 and 1 μM) for macrophages of the J774 lineage in the MTT assay.

[061] The results refer to: the 50% Lethal Concentration (LC50) calculated through toxic concentration-response curves. bMean ± standard error of mean maximum cytotoxicity in triplicates of a representative experiment. Maximum effect values were considered significant when *p < 0.05, **p < 0.01 in relation to the DMSO 0.1% group. NT: substance does not show significant lethal activity for cells up to a concentration of 100 μM in relation to the DMSO group.

[062] The selectivity index (IS), which consists of the ratio between the 50% lethal concentration (LC50) for macrophage cells of the J774 lineage and 50% inhibitory concentration (IC50) for the parasite cells was calculated for the ligands, meglumine complexes and antimoniate (Table 6) (NAKAMURA, 2006).

[063] The (Nal)2SbPh3 complex was the most selective for L. brasiliensis promastigotes, with a selectivity index greater than 3333, which implies that the complex was about 3333 times more toxic to promastigotes than to macrophages of the lineage. J774. The ligands, nalidixic acid and norfloxacin, and the (Cip)2SbPhse (Nor)2SbPh3 complexes also exhibited relevant selectivity indices. It is worth noting that, even with good selectivity indices, the (Cip)2SbPh3β complex and the antimony salt PhsSbChin induced a maximum toxicity of 100% while the (Nal)2SbPh3 and (Norj∑SbPhs) complexes induced maximum toxicities of only 43.88 ± 3 .8% and 48.8 ± 3.8% respectively.

[064] All complexes showed greater inhibitory effects on L. brasiliensis and L. amazonensis promastigotes and L. amazonensis amastigotes than against macrophages, with the order of selectivity being maintained for the different species and evolutionary forms tested. The (Nal)2SbPh3 complex was the most selective, followed by the (Cip)zSbPh3 and (Nor)zSbPh3 complexes respectively. Among the compounds studied, those with the highest selectivity rates for L. chagasi promastigotes were (Nal)2SbPhs (IS > 5.7) and (Nor)2SbPh3 (IS > 10.5). The (Cip)2SbPh3 complex showed the lowest selectivity index, which was 2.4 times more cytotoxic to L chagasi promastigotes than J774 macrophages.

[065] Table 6 shows the selectivity indices for promastigote forms of L. brasiliensis, L. amazonensis and L. chagasi and amastigote forms of L. amazonensis.

[066] the selectivity index between J774 macrophages and L. brasiliensis promastigotes; b selectivity index between J774 macrophages and L. amazonensis promastigotes;c selectivity index between J774 macrophages and L. brasiliensis amastigotes; d selectivity index between J774 macrophages and L. chagasi; ND: not determined; IS: selectivity index.

In vivo infection assay with L. amazonensis

[067] The combined results of the MTT reduction assay performed with J774 macrophages and the viability assays against promastigote forms and replication of intracellular amatigote forms with L. amazonensis, allowed the selection of the complexes (NaQ∑SbPhs and (Nor)2SbPh3 for the in vivo infection assay with this species, as they were potent and effective in inhibiting the replication of the parasite's evolutionary forms, presented a satisfactory macrophage/leishmania selectivity index and maximum toxicity below 50%. The susceptibility of BALB/c mice to Leishmania infection is associated with the development of a Th2-type immune response, with macrophage deactivation and production of IL-4 and IL-10 cytokines (SACKS , D., NOBEN-TRAUTH, N. The Immunology of susceptibility and resistance to Leishmania Major in mice, Nature Reviews Immunology, v. 2, p. 8 45-858, 2002; LORÍA-CERVERA, E.N.; ANDRADE-NARVÁEZ, F.J. - Animal models for the study of leishmaniasis immunology. Rev. Inst. Med. trop. Sao Paulo, v. 56, n.1, p.1-11, 2014).

[068] It was observed during the course of infection of BALB/c mice with L. amazonensis that the complex (NalJ∑SbPha was not able to significantly control lesion development, inducing ear thickness reduction only at the 17th day of treatment (Figure 3) (Noij∑SbPhs significantly inhibited ear growth throughout the treatment period, consequently preventing the development of the lesion. This result was superior to that shown by the standard drug meglumine antimoniate, which from on the 24th day of treatment, it no longer controlled the growth of the infected ear, not preventing the appearance of the lesion (Figure 3).

[069] Figure 3 shows the in vivo effect of intraperitoneal (i.p.) treatment with (Nal)2SbPh3, (Nor)2SbPh3 and meglumine antimoniate at a dose of 30 μmol/kg/day x 28 days on the course of injury in mice BALB/c infected with L. amazonensis. Lesions were monitored weekly. The results are represented by the mean ± e.p.m. of the lesion thickness of five animals in each experimental group.

[070] The results of the analysis of the parasite load of the ears and lymph nodes of the infected animals showed that the (Nor)2SbPh3 complex significantly reduced the amount of parasites in the lymph node, with an inhibition percentage of 61.1 ± 9.2%, as well as in the ear with an inhibition percentage of 29.2 ± 4.2%. On the other hand, the (Nal)2SbPh3 complex and the meglunin antimoniate were not able to reduce the L. amazonensis burden in the lymph node and ear of infected BALB/c mice at the concentrations tested (Figure 4A and 4B).

[071] Figure 4 shows the in vivo effects of i.p. with (Nal)2SbPh3, (Nor)2SbPh3 and meglumine antimoniate (AM) at a dose of 30 μmol/kg/day x 28 days on the parasite load in lymph nodes (A) and ear (B) of BALB/c mice infected with L. amazonensis. Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the control group.

[072] Although the (Nal)2SbPh3 complex showed excellent potency against L. amazonensis promastigotes and amastigotes in in vitro assays, this effect was not observed in vivo. The (Nor)2SbPh3 complex maintained the response profile observed in the in vitro assays, demonstrating a significant in vivo leishmanicidal activity, superior to that observed for the standard drug, meglumine antimoniate, at the same dose.

[073] In order to evaluate possible hepatotoxic effects caused by meglumine antimoniate complexes and standard drug, the dosage of liver toxicity markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, was performed in the serum of BALB/c mice. infected with L. amazonensis. ALT or glutamic-pyruvic transaminase (TGP) is predominantly located in the liver (about 90% in the cytoplasm), and can be found in moderate concentration in the kidneys and in smaller amounts in the heart and skeletal muscles. The enzyme AST or glutamic-oxalacetic transaminase (TGO) is found in high concentration in the liver, cardiac muscle and skeletal muscle and in smaller amounts in the kidneys and pancreas. In hepatocytes it is predominantly found in mitochondria (80%). Any tissue injury or disease affecting the liver parenchyma will release a greater amount of these enzymes into the bloodstream, raising the serum levels of ALT and AST (MESSIAS, J.B. et al. Evaluation of hematological and biochemical parameters of rats in the second third of pregnancy submitted to action of the methanolic extract of Cereus jamacaru DC., Cactaceae. Rev. Bras. Farmacogn. v. 20, p. 478-483, 2010).

[074] It is observed in the results shown in Figure 5A and 5B that after treatment for 28 days with a daily dose of 30 μmol/kg of (NorJzSbPha there was a statistically significant increase in serum levels of ALT, but not of AST. the ALT enzyme is more rapidly released, as it is predominantly found in the cytoplasm, probably treatment with (NorJ∑SbPha did not cause liver cell damage severe enough to increase serum AST levels. Messias et al. reported that in hepatocellular damage the predominant form in serum is cytoplasmic (ALT), while in severe lesions, mitochondrial enzyme (AST) is released, increasing the AST/ALT ratio (MESSIAS, J.B. et al. Evaluation of hematological and biochemical parameters of rats in the second third of pregnancy submitted to the action of Cereus jamacaru DC. of meglumine, (Nal)2SbPh3 and (NorJ∑SbPhs were respectively 3.2, 3.6, 2.8 and 1.5. Treatment with the complex (NaQ∑SbPhs and meglumine antimoniate did not significantly increase the serum levels of ALT and AST, however, a higher value of the AST/ALT ratio was observed for these substances and for the infected control than for the complex ( Nor)2SbPh3, which suggests that this complex was not significantly toxic to the liver.

[075] Figure 5 shows the in vivo effect of i.p. with the (Nal)2SbPh3 and (Nor)2SbPh3 complexes and the standard drug meglumine antimoniate on the levels of ALT and AST in the serum of animals infected with L. amazonensis at a dose of 30 μmol/kg/day x 28 days. Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the control group.

[076] Changes in blood urea and creatinine levels are widely used as indicators of glomerular filtration deficiency. Urea is synthesized in the liver from protein catabolism and creatinine is derived from muscle creatine metabolism and both are excreted by glomerular filtration and eliminated in the urine. The accumulation of these substances in the bloodstream is used for the diagnosis of renal diseases (BRITO, M.V.H. et al. Effect of copaiba oil on serum urea and creatinine levels in rats submitted to renal ischemia and reperfusion syndrome. Acta Cir. Bras. v. 20, p. 243-246, 2005). None of the substances used to treat Balb/c mice infected with L. amazonensis was able to induce an increase in blood levels of urea and creatinine. Based on these results, it is possible to suggest that they did not cause kidney damage (Figure 6).

[077] Figure 6 shows the in vivo effect of i.p. with the (Nal)2SbPh3 and (Nor)2SbPh3 complexes and the standard drug meglumine antimoniate on the levels of creatinine and urea in the serum of animals infected with L. amazonensis at a dose of 30 μmol/kg/day x 28 days. Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the control group.

In vivo infection assay with L. chagasi

[078] For the assay of infection of Golden hamsters with L. chagasi, the (Nor)2SbPh3 complex was selected, as it exhibited significant leishmanicidal activity in vivo against L. amazonensis and was the most selective against L. chagasi promastigotes. Golden hamsters are highly susceptible to infection by L. chagasi, which is considered the best experimental model for the study of visceral leishmaniasis, as it reproduces the clinical-pathological characteristics of the disease in humans (GUPTA, S.; NISHI. Visceral leishmaniasis: Experimental models for drug discovery. Indian J Med Res. v. 133, n.1, p. 27-39, 2011; LORÍA-CERVERA, E.N.; ANDRADE-NARVÁEZ, F.J. - Animal models for the study of leishmaniasis immunology. Rev. Inst. Med. Trop. Sao Paulo, v. 56, n.1, p.1-11, 2014).

[079] Through the analysis of Figure 7, it is observed that the complex significantly reduced the number of parasites in the spleen of infected hamsters, this reduction being higher than that observed for the standard drug meglumine antimoniate at the same dose (30 μmol/kg/ day) and similar to that induced by higher doses of this drug. The complex (NoO∑SbPhs was efficient to treat both cutaneous leishmaniasis and visceral leishmaniasis in an animal model that mimics the disease in humans, and can be used as a prototype for the development of new leishmanicidal drugs.

[080] Figure 7 shows the in vivo effect of i.p. with (Nor)2SbPh3 at a dose of 30 μmol/kg/day and meglumine antimoniate (AM) at doses of 150, 100 and 30 μmol/kg/day for 15 days on spleen parasite load of Golden hamsters infected with L. chagasi . Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the control group.

[081] The levels of ALT and AST in the serum of Golden hamsters infected with L. chagasi are shown in Figure 8. It is observed that there was a statistically significant decrease in the serum levels of ALT in all animals treated with different doses of antimoniate of meglumine and the (Nor)2SbPh3 complex. The AST/ALT ratio was calculated for all groups tested, with values 0.89, 2.21, 1.04, 1.01 attributed, respectively, to meglumine antimoniate at doses of 150, 100 and 30 μmol/kg /day and the (Nor)2SbPh3 complex The values for the uninfected and infected controls were 1.7 and 1.01, respectively. In view of the exposed data, it was found that the values of the AST/ALT ratio of (Nor)2SbPh3 and infected control were equal, which indicates that the complex did not induce liver toxicity, which was also observed for meglumine antimoniate.

[082] Figure 8 shows the in vivo effect of i.p. with (Nor)2SbPh3 at a dose of 30 μmol/kg/day and meglumine antimoniate (AM) at doses of 150, 100 and 30 μmol/kg/day for 15 days on ALT (A) and AST (B) levels in the serum of Golden hamsters infected with L. chagasi. Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the infected control group.

[083] According to the results shown in Figure 9, the complex (NorJ∑SbPha and meglumine antimoniate at a dose of 30 μmol/kg/day did not increase the blood levels of urea and creatinine in Golden hamsters infected with L. chagasi. Meglumine antimoniate at doses of 150 and 100 μmol/kg/day induced a significant increase in creatinine levels, which may be an indication of kidney damage caused by the drug.

[084] Figure 9 shows the in vivo effect of i.p. with (Nor)2SbPh3 at a dose of 30 μmol/kg/day and meglumine antimoniate (AM) at doses of 150, 100 and 30 μmol/kg/day for 15 days on creatinine (A) and urea (B) levels in the serum of Golden hamsters infected with L. chagasi. Results refer to the mean ± standard error of the mean of five animals from a representative experiment. Values were considered significant when *p < 0.05 versus the infected control group.

PATENT ADVANTAGES

[085] This invention presents an unprecedented class of antimony(V) complexes with the quinolone nalidixic acid and the fluoroquinolones ciprofloxacin and norfloxacin, effective against different forms of leishmania, active in vivo and in vitro and can be used as prototypes for the development of new leishmanicidal drugs. A fundamental fact for the development of drugs is observed in this invention, since the synthesis of the complexes is simple and efficient, and their formation is confirmed through NMR and infrared techniques. The (Nal)2SbPh3, (Cip)2SbPh3, (Nor)2SbPh3 complexes were potent and effective in inhibiting the growth of promastigotes of L. brasiliensis, L. amazonensis and L. chagasi and amastigotes of L. amazonensis, in addition to demonstrating greater selectivity for the parasites than for the macrophages of the J774 strain. In all tests, it was observed that the standard drug meglumine antimoniate was ineffective against promastigote and amastigote forms of the aforementioned species at concentrations equal to the developed complexes. It was observed that there was an increase in leishmanicidal activity in vitro when comparing the complexes and free ligands, nalidixic acid, ciprofloxacin and norfloxacin, most likely due to the action of the complexes on multiple targets. (Nal)2SbPh3 and (Nor)2SbPh3 presented LC50 values greater than 100 μM and maximum toxicity less than 50%, being chosen for the in vivo infection assay with L. amazonensis. This experiment demonstrated the promising leishmanicidal activity of the (Nor)2SbPh3 complex in an in vivo model of cutaneous leishmaniasis, as it was able to reduce the size of the infected ear, inhibit the appearance of the lesion, and significantly reduce the parasitic load of the ear. and lymph node of infected animals and did not change the serum concentration of urea, creatinine and AST. It only increased the ALT concentration, but the AST/ALT ratio remained within the normal range. A significant in vivo leishmanicidal activity against L. chagasi was also observed for the (Nor)2SbPh3 complex, in a model that mimics the disease in humans. The complex (Noij∑SbPhs was significantly more active than the antimoniate drug meglumine in in vivo models of cutaneous and visceral leishmaniasis.

Claims (5)

1- ANTIMONIUM(V) ORGANOMETALLIC COMPOUNDS CONTAINING QUINOLONIC AND FLUOROQUINOLONIC LINKERS AS CHEMOTHERAPEUTIC AGENTS LEISHMANICIDES characterized by having the general molecular formula R'3Sn(OOCR)2, derived from the reaction between alkyl or aryl tin(IV) chloride and quinoline carboxylates or sodium fluoroquinolines, where, R: alkyl or aryl groups such as methyl (Me) and phenyl (Ph); OOCR: anionic form of quinolinic or fluoroquinolinic acids such as Nalidixic acid, Ciprofloxacin, Norfloxacin, Oxolinic acid, Pipemidic acid, Pyromydic acid, Balofloxacin, Besifloxacin, Cinoxacin, Clinafloxacin, Enoxacin, Fleroxacin, Flumequine, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin , Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Ofloxacin, Pazufloxacin, Pefloxacin, Prulifloxacin, Rosoxacin, Rufloxacin, Sitafloxacin, Sparfloxacin, Temafloxacin, Tosufloxacin, Trovafloxacin. 2- ORGANOMETALLIC COMPOUNDS OF ANTIMONIUM(V) CONTAINING QUINOLONIC AND FLUOROQUINOLONIC BINDINGS AS CHEMOTHERAPY AGENTS LEISHMANICIDES, according to claim 1, characterized in that they are obtained from a solution of triorganoantimony(v) and a sodium salt of quinolinic or fluoroquinolinic acids . 3- ORGANOMETALLIC COMPOUNDS OF ANTIMONIUM(V) CONTAINING QUINOLONIC AND FLUOROQUINOLONIC BINDINGS AS CHEMOTHERAPEUTIC AGENTS LEISHMANICIDES, according to claims 1 and 2, characterized by being effective in the treatment of leishmaniasis. 4- ORGANOMETALLIC COMPOUNDS OF ANTIMONIUM(V) CONTAINING QUINOLONIC AND FLUOROQUINOLONIC BINDINGS AS CHEMOTHERAPEUTIC AGENTS LEISHMANICIDES, according to claims 1, 2 and 3, characterized by being effective in the treatment of leishmaniasis in humans. 5- ORGANOMETALLIC COMPOUNDS OF ANTIMONIUM(V) CONTAINING QUINOLONIC AND FLUOROQUINOLONIC BINDINGS AS CHEMOTHERAPEUTIC AGENTS LEISHMANICIDES, according to claims 1, 2 and 3, characterized by being effective in the treatment of canine leishmaniasis.

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