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    • 专利摘要:
    Compounds for the treatment of diseases caused by parasites of the genus leishmania. The present invention relates to a family of compounds which are effective against parasites of the genus leishmania, preferably caused by l. Infantum, l. Major or l. Tropica, and which have a low toxicity even at high doses of the compound. The invention also relates to pharmaceutical compositions, preferably suitable for oral or topical administration, comprising these compounds and their use as a medicament for the treatment of visceral, canine or cutaneous leishmaniasis. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2608476A1
    申请号:ES201730208
    申请日:2017-02-20
    公开日:2017-04-11
    发明作者:José Antonio GÓMEZ VIDAL；Mónica DÍAZ GAVILÁN；Francisco FRANCO MONTALBÁN；Francisco MORILLAS MÁRQUEZ；Victoriano CORPAS LÓPEZ；Joaquina MARTÍN SÁNCHEZ；Margarita LÓPEZ-VIOTA GALLARDO；Julián LÓPEZ-VIOTA GALLARDO
    申请人:Universidad de Granada；
    IPC主号:
    专利说明:



    COMPOUNDS FOR THE TREATMENT OF DISEASES CAUSED BY PARASITES OF THE LEISHMANIA GENDER
    SECTOR OF THE TECHNIQUE
    The present invention is generally framed in the field of biomedicine and in particular refers to a new compound useful as a medicament, preferably useful for the treatment of diseases caused by parasites of the genus Leishmania.
    STATE OF THE TECHNIQUE
    Leishmaniasis
    15 Leishmaniasis is a vector-borne disease caused by parasitic protozoa of the genus Leishmania (Kinetoplastida, Trypanosomatidae) and transmitted by the bite of the female phlebotomes (Insecta, Diptera, Phlebotomidae) belonging to the genera Phlebotomus and Lutzomyia. It is one of the most important diseases within tropical diseases and
    20 is included in the “Neglected Tropical Diseases” or forgotten tropical diseases. Despite this, it is not limited to tropical and subtropical areas, and it is also endemic in temperate zones such as our country. The clinical spectrum of leishmaniasis is broad and encompasses from a strict cutaneous form that can heal spontaneously leaving a scar,
    25 to a serious and deadly form without treatment such as visceral leishmaniasis. According to the World Health Organization, up to 350 million people are at risk of leishmaniasis in 98 countries in temperate and tropical areas of the planet. Approximately 12 million people are considered to be currently infected, and 2 million people are produced every year.
    30 new cases, of which 500,000 cases are visceral.
    Leishmania species show two morphological phases during their life cycle:  Promastigote, with elongated and mobile shape with an anterior scourge, in the intestine of the vector.


    3
     Amastigote, with a spherical or ovoid shape and without a visible scourge under an optical microscope, which reproduces in a forced manner within the cells of the endothelial reticulum system of the vertebrate host (man and various animal species depending on the Leishmania species).
    Of the twenty species responsible for leishmaniosis, only two, L. donovani and L. infantum (synonym L. chagasi) cause visceral leishmaniasis. L. infantum is the only species identified that causes both visceral and cutaneous disease. In addition, it has also been identified as the causative agent of cutaneous-mucosal and mucosal leishmaniasis. While in visceral leishmaniasis the parasite proliferates in the bone marrow, liver and spleen, organs in which cells of the endothelial reticulum system abound, in the cutaneous form the parasite remains at the site of the phlebotome bite, which can cause Long-lasting ulcers that end up leaving disfiguring scars. As we said 15 previously, the disease in its visceral form is fatal if it is not treated correctly. L. infantum is the most widely distributed species being present in 61 of the 98 endemic countries of leishmaniosis. L. infantum is endemic in all the countries of the Mediterranean Basin. Unlike L. donovani, leishmaniosis due to L. infantum is a zoonosis, the dog being the main reservoir. In Spain, the prevalence of canine leishmaniasis in random samples using serological techniques varies between 5 and 30%, and can reach 100% in some foci if the PCR technique is used [Martín-Sánchez J, Morales-Yuste M , Acedo-Sánchez C, Barón S, Díaz V, Morillas-Márquez F. Canine leishmaniasis in southeastern Spain. Emerg Infect Dis. 2009 May 25; 15 (5): 795-8. doi: 10.3201 / eid1505.080969]. These high prevalence of infection are a considerable risk factor for the emergence of the disease in humans. In dogs, L. infantum causes canine leishmaniasis, which is a multisystemic syndrome that is very difficult to cure due to the lack of collaboration of the animal's immune system. Until now, none of the drugs 30 used for the treatment of canine leishmaniasis (which are the same used in man) allows the complete elimination of the parasite in the animal. In Spain there can be between 6 and 13 million dogs. Although the prevalence of canine leishmaniasis varies between regions and according to the technique used, we can estimate that 30% of them are infected; this would give us 2.8 million dogs 35 with canine leishmaniasis as potential drug receptors against the


    leishmaniosis, only in Spain. To them should be added those existing in other endemic countries such as Portugal, France, Italy, Brazil, etc., in addition to other non-endemic. For example, in Germany, despite not being an endemic country of leishmaniasis, it is estimated that there are 20,000 dogs with acquired canine leishmaniasis
    5 during holidays in the countries of the Mediterranean Basin that would also be potential recipients.
    On the other hand, cutaneous leishmaniasis is endemic in more than 70 countries, with an incidence of 1.2 million cases per year. It is caused by various species 10 of Leishmania, mainly by L. tropica and L. major. Also L. infantum (synonym L. chagasi) causes cutaneous leismaniosis although in the latter case, leishmaniasis usually visceralizes in animals. L. tropica is the leading cause of cutaneous leishmaniasis in the Middle East and some areas of North Africa. In Morocco, it is the most geographically widespread species and is a frequent cause of
    15 epidemic outbreaks.
    Leishmaniosis Treatment
    The drugs currently used for the treatment of leishmaniasis
    20 present various problems, including host toxicity, adverse effects, the emergence of resistance and high cost. Among them, pentavalent antimony derivatives, such as meglumine antimoniate or methylglucamine antimony (Glucantime®) have been considered first choice for a long period of time, despite their high toxicity. However, the
    25 meglumine antimoniato is not suitable for oral administration because it is poorly absorbed and is extremely irritating to the gastrointestinal tract, so it must be administered parenterally or by local injection.
    Pentamidine, amphotericin B and paromomycin have been considered as
    30 drugs of second choice, although lately liposomal amphotericin B is replacing antimony derivatives as a drug of choice in some geographical areas. However, pentamidine and amphotericin B have a high toxicity that can be reduced with the latter's lipid formulations but raising the price considerably. On the other hand, the
    35 paromomycin is also not absorbed orally, so it should be administered by


    5
    parenteral or topical route; it is excreted by the kidney without metabolizing and induces nephrotoxicity and ototoxicity, as well as other aminoglycosides [Ramos, J. and Segovia, M.-Spanish Journal of Chemotherapy, 1997. p 26].
    The last drug incorporated into the therapeutic arsenal of leishmaniasis is miltefosine, which initially conceived as an antineoplastic drug, has the advantage of allowing oral administration but is not exempt from toxicity, has resistance problems [Mondelaers A, and others. "Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes." PLoSOne. 2016 Apr 28; 11 (4): e0154101. doi: 10.1371 / journal.pone.0154101] and causes gastrointestinal adverse effects, in addition to being potentially teratogenic and abortive.
    As for cutaneous leishmaniasis, there is currently no satisfactory treatment. The evolution of the lesions tends to chronicity, healing spontaneously in several months although leaving a scar that in some cases can lead to disfigurement or require surgery since the most affected area is usually the face. Among other existing measures, pentavalent, parenteral or intralesional antimonials are used, with numerous side effects, or cryotherapy applying liquid nitrogen on the lesion. Recently, topical paromomycin and topical amphotericin B are being tested. Amphotericin B deoxycholate is active against Leishmania, but adverse effects are common (hyperpyrexia, malaise, hypotension, thrombophlebitis, kidney damage, hypokalemia, anemia and hepatitis). Liposomal amphotericin B is less toxic although its cost has limited its use in cutaneous leishmaniasis. On the other hand, topical paromomycin (Leshcutan®) is not available in Spain or the United States and should be reserved for cases without risk of mucosal involvement.
    HDAC inhibitors with activity against Leishmaniosis
    The drug vorinostat (also called SAHA) (Formula I) is a histone deacetylase (HDAC) inhibitor that has activity against Leishmania [9, 10].


    6
    Vorinostat has as its main disadvantage its toxicity at high concentrations.
    On the other hand, patent ES 2 402 252, requested by the University of Granada, describes a structure with formula II that has activity against Leishmania.
    As with meglumine antimoniato, compound II is not suitable for
    10 oral administration since it is not chemically stable at low pH. The trityl chemical group, present in compound II, is a known unstable protective group at acidic pH (4 and less).
    There is therefore a need to find a medication for the treatment of
    15 Leishmania infection, which solves the problems described in the state of the art, especially that provides greater efficacy in the elimination of the parasite, less toxicity and adverse effects for the patient (host) and allows the possibility of oral and topical administration .
    20 The use of vehiculization and release systems such as nanoparticles can solve problems of solubility of hydrophobic compounds whose use in vivo would be unfeasible, in addition to improving efficacy and reducing adverse effects. 25 BRIEF DESCRIPTION OF THE INVENTION
    The present invention provides a family of compounds effective against Leishmaniosis or diseases caused by parasites of the genus Leishmania, preferably caused by L. infantum, L. major or L. tropica.


    7
    Thus, in a first aspect, the present invention relates to the compound of formula III:
    In a second aspect, the present invention relates to a pharmaceutical composition comprising the compound of formula III as described above and pharmaceutically acceptable excipients. In a particular embodiment, the invention relates to a pharmaceutical composition comprising the compound of formula III supported on pharmaceutically nanoparticles.
    10 acceptable, preferably gold nanoparticles.
    In a third aspect the present invention relates to the use of the compound of formula III or of the pharmaceutical compositions comprising it as a medicament. In particular, the invention relates to its use as a medicament for the treatment of Leishmaniosis or diseases caused by parasites of the genus Leishmania,
    15 more preferably L. infantum, L. major or L. tropica.
    In a particular embodiment, the invention relates to the use of the compound of formula III or of the pharmaceutical compositions comprising it for the treatment or for the manufacture of a medicament for the treatment of cutaneous leishmaniasis.
    In another particular embodiment, the invention relates to the use of the compound of formula III or of the pharmaceutical compositions comprising it for the treatment or for the manufacture of a medicament for the treatment of visceral leishmaniosis or canine leismaniosis.
    In another aspect, the invention relates to combined preparations of compounds
    25 of formula III and other compounds or drugs used for the treatment of diseases caused by parasites of the genus Leishmania, as well as a method of treatment of said diseases which employs one of these combined preparations.


    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1.-Synthesis scheme of compound III.
    Figure 2.-Image obtained by microscopy of the nanoparticles. In the image above, only the gold nuclei without compound are observed; in the lower part, the nanoparticles are vehiculizing the compound and a crown is observed around the gold nuclei that almost doubles the hydrodynamic size of the
    10 particles of the upper image.
    DETAILED DESCRIPTION OF THE INVENTION
    Definitions
    15 The term quot; nanoparticlesquot; as described in the present invention it refers to particles of less than 1 micrometer in any of its dimensions, preferably in a range of 10 to 900 nanometers.
    The term "treatment" or "treat" in the context of this document refers to the administration of a compound or a composition according to the invention for
    20 to improve or eliminate a disease, pathological condition or one or more symptoms associated with said disease or condition in a mammal, preferably in canines or humans, more preferably in dogs. "Treatment" also covers the improvement or elimination of the physiological sequelae of the disease. Specifically, the concept "treat" can be interpreted as:
    25 i. Inhibit the disease or pathological condition, that is, stop its development;
    ii. Relieve the disease or the pathological condition, that is, it causes the regression of the disease or the pathological condition;
    iii. Stabilize the disease or pathological condition.
    Throughout the description and the claims the term "comprises" which may also be construed as "consists of", and its variants are not intended to exclude other technical characteristics, additives, components or steps. For the


    9
    Those skilled in the art, other objects, advantages and features of the invention will emerge in part from the description and in part from the practice of the invention.
    Compounds of the invention
    In a first aspect of the invention, the present invention relates to compounds ("compounds of the invention"), of formula III
    Where Ph is a phenyl of formula:
    Ph
    Being:
    R1 = H, F, Cl, Br, I, Me, OMe or NO2.
    R2 = H, F, Cl, Br, I, Me, OMe or NO2
    R3 = H, F, Cl, Br, I, Me, OMe or NO2
    In a particular embodiment, the compounds of the invention are presented in crystalline form as free or solvated compounds (for example hydrates) or as enantiomers, isomers, or salts of said compounds, all of these forms being within the scope of protection of the present invention . Solvation methods are generally known in the state of the art.
    Also included within the scope of the present invention are the prodrugs of the compounds of the invention.
    In a particular embodiment the compounds of the invention are compounds of formula III characterized in that R1 = H or R2 = H or R3 = H.


    10
    In an even more particular embodiment, the compounds of the invention are compounds of formula III characterized in that at least two of the three phenyl radicals are formed by hydrogen. That is, compounds of formula III characterized in that R1 = R2 = H or R1 = R3 = H or R2 = R3 = H.
    In a preferred embodiment, the invention relates to compounds of formula IIIa consisting of compounds of formula III characterized in that the three phenyl radicals are formed by hydrogen. That is, compounds of formula III characterized in that R1 = H, R2 = H and R3 = H.
    IIIa
    Pharmaceutical compositions
    In a second aspect, the invention provides pharmaceutical formulations, forms or compositions, hereinafter "compositions of the invention" comprising as active ingredient a therapeutically effective amount of at least one compound of the invention, in particular a compound of those detailed in the Preferred embodiments of the first aspect of the invention. Said formulations may contain any other active ingredient in the treatment of leishmaniosis or be characterized as containing only one compound of the invention or a combination of compounds of the invention as active ingredient.
    In the sense used in this description, the term "therapeutically effective amount" refers to that amount of a compound of the invention that when administered to a mammal, preferably canids and humans, and among canines, more preferably dogs, is sufficient. to produce the treatment, as defined below, of a disease or pathological condition of interest in the mammal, preferably canids and humans, and among canines, more preferably dogs. The amount of a compound of the invention that


    constitutes a therapeutically effective amount will vary, for example, depending on the activity of the specific compound employed; the metabolic stability and duration of action of the compound; the species (preferably human or canine), the clinical form of the human disease (preferably visceral, cutaneous or mucosal), age, body weight, general state of health, sex and diet of the patient; the route of administration, given the possibility of oral or systemic administration; the mode and time of administration; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject who undergoes therapy, but can be determined by
    10 a specialist in the art according to his own knowledge and that description.
    On the other hand, in accordance with the present invention, the "pharmaceutical form" is the individualized arrangement to which drugs (active ingredients) and excipients (pharmacologically inactive matter) are adapted to constitute a
    15 medication
    The compounds of the invention, III, by their hydrophobic nature, make their use in vivo unfeasible unless they are vehiculized. In particular, compound III in aqueous medium quickly adds, making it impossible to
    20 parenteral administration.
    Thus, said pharmaceutical compositions comprise one or more pharmaceutically acceptable carriers.
    The "pharmaceutically acceptable carriers" that can be used in the formulations of the invention are the vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions. An example of a pharmaceutically acceptable carrier is a gold nanoparticle with a size suitable for administration.
    Optionally, the pharmaceutical composition may comprise another active ingredient. In addition to the requirement of therapeutic efficacy, which may require the use of therapeutic agents, in addition to the compounds of the invention, there may be additional fundamental reasons that compel or strongly recommend
    The use of a combination of a compound of the invention and another agent is measured


    therapeutic, as in the treatment of diseases or conditions that directly
    or indirectly modulate the function of the substance.
    The formulations may also contain excipients.
    5 The excipients and vehicles used must be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not exert adverse effects on the treated organism. Pharmaceutical compositions or formulations include
    10 those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best route of administration depends on the patient's condition. The formulations can be in the form of single doses. The formulations are prepared according to methods known in the field of pharmacology. The quantities of active substances
    15 to be administered may vary depending on the particularities of the therapy.
    The compositions of the invention are prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.
    20 Formulation in nanoparticles
    In a preferred embodiment of the compositions of the invention the compound of the invention III is supported on pharmaceutically acceptable nanoparticles,
    25 more preferably on gold nanoparticles.
    In a particular embodiment, the gold nanoparticles used as transport vehicles of the compound of the invention are coated by sodium citrate.
    In another particular embodiment the gold nanoparticles used as transport vehicles of the compound of the invention have an approximate size of between 20 and 30 nm, preferably between 22 and 26 nm.
    The compound III thus functionalized has been shown to have much better solubility characteristics than the compound alone, without diminishing its effectiveness.


    Topical compositions
    In the context of the present invention, "topically"; The route of administration of drugs used by the skin and mucous membranes is understood.
    For topical administration, preferably on the injured tissue or lesions caused by parasites of the genus Leishmania, in particular cutaneous Leishmaniosis, the compositions of the invention are compositions
    10 suitable for topical administration. Preferably, the compositions of the invention comprise a therapeutically effective amount of compound III, preferably a concentration of III equal to or greater than 0.5% w / w), more preferably in a concentration of compound III equal to or greater than 2%.
    Preferably, the compositions of the invention are lipophilic.
    Combined Preparations
    20 Compounds III do not act on cytochrome P450 enzymes, so they should not interact in vivo with other drugs and would admit their use in combination with other drugs in the treatment of leishmaniasis.
    The term "combined preparation"; or also called quot; yuxtapositionquot ;, in
    25 this report means that the components of the combined preparation need not be present as a joint, for example in a true composition, in order to be available for combined, separate or sequential application. In this way, the expression quot; yuxtapuestaquot; implies that it is not necessarily a true combination, in view of the separation
    30 physics of the components.
    Thus, in another aspect, the invention relates to a pharmaceutical composition, preparation or form, hereinafter "combined preparation of the invention", comprising:
    A) a compound of general formula III, and


    b) another active principle against Leishmaniosis.
    In a particular embodiment, the combined preparation of the invention comprises
    a) a compound of general formula III, and
    b) a compound of general formula II.
    Invention kit
    In another aspect, the present invention relates to a kit ("kit-of-parts"; English "kit of parts") for the preparation of the composition or the combined preparation of the invention.
    The term "kitquot;" as used herein, refers to a combination of a set of components suitable for obtaining the composition or the combined preparation of the invention, which may or may not be packaged together, along with its appropriate containers and containers for commercial sale, etc.
    In the present invention, it is understood as "suitable component for obtaining the composition or the combined preparation of the invention", any compound that can be used for obtaining them, and includes, without limitation, aqueous solutions, solid preparations, buffers, syrups, preservation solutions, flavorings, pH correctors, thickeners, etc.
    Kit components can be provided in separate vials (in the form of "kit-of-parts") or in a single vial. Furthermore, it is understood that the kit of the present invention is intended for the preparation of the composition or of the combined preparation or of the pharmaceutical form (for example, of the oral solution, mouthwash, rinse, gargle, elixir, etc.) of the invention. Preferably, the kit components of the present invention are ready to be used to prepare the combined composition or preparation or the pharmaceutical form of the present invention. In addition, the kit preferably contains explanatory instructions on how to prepare the composition or the combined preparation


    fifteen
    or the pharmaceutical form of the present invention. Instructions can be provided to users electronically or on paper.
    Therefore, the invention provides a kit for the preparation of the composition of the invention or of the combined preparation of the invention a container comprising a container with the compound of general formula III in any pharmaceutically acceptable formulation, together with components suitable for obtaining the composition or the combined preparation of the invention.
    In particular, the kit of the invention further comprises a container with the compound of general formula II in any pharmaceutically acceptable formulation.
    In a preferred embodiment, the compound of general formula III is supported on pharmaceutically acceptable nanoparticles, more preferably on gold nanoparticles.
    Use of the compounds of the invention
    A third aspect of the invention consists in the use of the compounds and / or the compositions and / or the combined preparations and / or the kit of the invention as a medicament or for the preparation of a medicament. In a particular embodiment said medicament is suitable for the treatment of Leishmaniosis or diseases caused by parasites of the genus Leishmania, more preferably
    L. infantum, L. major or L. tropica., And more preferably for the treatment of visceral Leishmaniosis, canine Leishmaniosis and / or cutaneous Leishmaniosis.
    In another aspect, the present invention relates to a method for the treatment of patients affected by Leishmaniosis, hereinafter "method of treatment of the invention" by the use of the compounds and / or the compositions and / or the combined preparations and / or the kit of the invention. The effects of this method of treatment include, but are not limited to, the effects of disease elimination, the increase in disease progression time and the index


    of survival. The effects of treatment include longer term disease control.
    This treatment consists of the administration to the individuals affected by these 5 diseases of therapeutically effective amounts of a compound of the invention, or a pharmaceutical composition that includes it.
    In a preferred embodiment, the disease is treated using compounds and / or the compositions and / or the combined preparations and / or the kit of the invention
    10 and / or the method of treatment of the invention is visceral leishmaniosis, canine leishmaniosis and / or cutaneous leishmaniosis.
    In another preferred embodiment, the disease that is treated using the compounds and / or the compositions and / or the combined preparations and / or the kit of the invention and / or the method of treatment of the invention is cutaneous leishmaniosis.
    Preferably, a composition suitable for topical administration comprising a therapeutically effective amount of compound III, preferably a concentration, is used for the treatment of cutaneous leishmaniasis.
    20 of III equal to or greater than 0.5% w / w), more preferably in a concentration of compound III equal to or greater than 2%. Preferably said composition suitable for topical use is lipophilic.
    The administration of the compounds of the invention, in pure form or in an appropriate pharmaceutical composition, or in combination with other compounds, compositions or medicaments, can be carried out by means of the administration modes of agents accepted to serve the like. utilities
    Synthesis procedure of the compounds of the invention
    The method used to synthesize the compounds of the invention comprises [Figure 1] reacting in an solution an acid of formula IV, with
    35 benzylhydroxy amine of formula V.


    17
    Where Ph is a phenyl of formula
    Ph
    Being: R1 = H, F, Cl, Br, I, Me, OMe or NO2 10 R2 = H, F, Cl, Br, I, Me, OMeó NO2 R3 = H, F, Cl, Br, I, Me, OMe or NO2
    Preferably the solution will be carried out in anhydrous methanol / THF maintaining the reaction in an argon atmosphere.
    In a preferred embodiment, the compound of general formula III is obtained from a solution with the carboxylic acid derivative of general formula IV (1 equiv.) Ethyl chloroformate (1.35 equiv.), Triethylamine (3.5 equiv.) And Benzylhydroxy amine (1.7 equiv.) in THF / methanol. The reaction should be maintained at 0 ° C and
    20 argon atmosphere.
    Functionalization of III with gold nanoparticles
    In a preferred embodiment, the compositions of the invention comprise compound III functionalized with gold nanoparticles.
    To carry out the functionalization of the gold particles with compound III, the suspension of gold particles is added dropwise to the solution of the dimethylsulfoxide compound (DMSO), preferably 0.5%, and Triton X100, preferably 30 0.1% Then, the suspension obtained is added dropwise onto a solution.


    18
    Bovine Serum Albumin (Bovine Serum Albumina or BSA), preferably 1%. After incubation for at least 2 hours, the particles are concentrated by centrifugation and resuspended in phosphate buffered saline or phosphate buffered saline (Phosphate Buffered Saline or PBS) pH 7.2.
    REALIZATION MODES
    The following examples are provided by way of illustration, and are not intended to be limiting of the present invention:
    Synthesis Example of Compounds of the Invention
    The compound O-benzylhydroxamate, IIIa, was obtained according to a reaction scheme (Figure 1) derived from the process for synthesis of hydroxamic acids. Thus, the compound of formula IIIa is obtained from a solution with the carboxylic acid derivative of formula IV (1 equiv.), In anhydrous THF under argon. The solution is cooled and ethyl chloroformate is added.
    (1.35 equiv.) Followed by triethylamine (3.5 equiv.). The mixture is stirred at low temperature under argon. The precipitated solid obtained is filtered and the filtered liquid is added to a solution containing the compound of formula Va (1.7 equiv.) Dissolved in anhydrous methanol.
    It is kept under stirring for 1.5 h at room temperature, after which time the reaction is diluted with ethyl acetate and washed with distilled water (3x 15 mL). The resulting organic phase is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue obtained is purified by flash chromatography using a mixture of dichloromethane and methanol as eluent to obtain IIIa (white solid, 28 mg, 40% yield).


    19
    IIIa
    Mp 130-131 ° C. 1H NMR (500 MHz, CD3OD) δ 7.54 (m, 2H), 7.33-7.25 (m, 7H),
    5 7.08 (m, 1H), 4.75 (s, 2H), 2.52 (m, 2H), 2.36 (m, 2H), 1.70-1.65 (m, 4H), 1.41 (m, 4H); 13C NMR (125 MHz, CD3OD) δ 176.1, 174.6, 139.9, 138.0, 129.7, 129.5, 129.3, 128.5, 125.1, 121.3, 52.9, 37.9, 33.1, 30.1, 30.0, 26.8, 25.8; HRMS (ESI) m / z calculated for C21H27N2O3 [M + H] + 355.2022, found 355.2025. HPLC purity (λ = 254): 100%, t R = 11.14 min.
    10 HPLC Method: Solution A (H2O: ACN 70:30 + 0.1% HCOOH), solution B (100% ACN + 0.1% HCOOH); isocratic solution 2 min .; gradient from A B 13 min; isocratic solution B 5 min.
    Conservation of the compounds Compound IIIa can be stored at 4 ° C for at least 6 months without apparent changes in its physical-chemical characteristics or loss of activity.
    On the other hand, compound IIIa is chemically stable at pH 2 and pH 5, which allows oral administration.
    Synthesis of inorganic gold-drug particles of compound IIIa
    Initially, gold nanoparticles were synthesized according to the method described by Turkevich [J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Trans. Faraday Soc. 11 (1951) 55], based on the reduction of gold salts in the presence of citrate at high temperatures.
    According to it, a solution of 4.5mg of sodium tetrachloroaurate (III) dihydrate in a volume of 25 mL was prepared. Under constant magnetic stirring, the solution was subjected to a temperature of 180 ° C under reflux. Reached this condition,


    1mL of a 1% solution of sodium citrate was added to the gold solution. The particles were allowed to grow in the presence of citrate ions for a time never less than 20 minutes. Subsequently, the particle suspension was allowed to reach room temperature before being used. The result is a
    5 stable suspension of gold particles, wine red.
    In order to carry out the functionalization of the gold particles with compound IIIa, the compound was first dissolved at a concentration of 0.25% in a solution composed of 0.5% Dimethylsulfoxide (DMSO) and 0.1% Triton X100. Once the compound IIIa dissolved, 20 mL of gold particle suspension is added dropwise. Immediately afterwards, the suspension of gold particles functionalized with compound IIIa on a 1% BSA solution is added dropwise. It is allowed to incubate for a minimum of 2 hours in the presence of BSA, and then afterwards, it is centrifuged at 14000 rpm to remove the excess BSA present in the
    15 medium of the conjugated particles. Once the particles have been centrifuged, the supernatant is removed and resuspended in PBS pH 7.2 prior to use.
    It has been found that the initial suspensions that have a pinkish color tend to change color towards blue, when the volume in which the particles are resuspended is low. For this reason, in order to avoid aggregation of the particles, it is resuspended in volumes greater than 5 mL.
    By means of measurements made by light scattering techniques and by microphotographs obtained by Transmission Electron Microscopy of 25 electrons such as those shown in the image below, an approximate size has been determined for the gold particles used as drug transport vehicles IIIa, of (24 ± 2) nm. The presence of IIIa and BSA is clearly observed by light scattering techniques, such as a crown around the gold nuclei, doubling the hydrodynamic size of the particles (47 ± 8) nm
    30 [Figure 2]. The suspensions have a pinkish color.
    The adsorption of the drug IIIa on the surface of the gold particles shows changes in the surface electric charge of the gold particles. Gold particles at physiological pH show surface electric charge (-36 ± 7) mV, while the 35 gold particles coated with IIIa and BSA under the same pH conditions show


    twenty-one
    a surface electric charge of (-10 ± 4) mV. From the electrokinetic point of view, any difference determined on the surface electric charge is attributed to the adsorption of any entity on the surface of the particles, in this case, the drug IIIa and the BSA.
    Examples of tests with the compounds of the invention
    Evaluation of the in vitro activity of compound IIIa on infection caused by L. infantum
    To test the percentage of infection by L. infantum, bone marrow derived macrophages (BMDM) of Swiss ICR (CD-1) mice were used. These cells were removed as established by [Zamboni, D, S,; Rabinovitch, M. Nitric oxide partially controls Coxiellaburnetii phase II infection in mouse primary macrophages. Infect Immun 2003, 71 (3), 1225-1233]. BMDMs were grown on coverslips in wells of 24-well microtiter plates, at a concentration of 4x105 BMDM in each well containing RPMI-1640 medium with 10% fetal bovine serum (FCS) and 5% L929 cell medium. The BMDM plates were left overnight at a temperature of 37 ° C at 5% CO2 for the cells to adhere. Infections were performed as established by [Zauli-Nascimento, R C., Miguel, D. C., Yokoyama-Yasunaka, J. K .; Pereira, L. I., PeIIi de Oliveira, M. A., Ribeiro-Dias, F., Doria, M. L. Uliana, S. R. In vitro sensitivity of Leishmania (Viannia) braziliensis isolates to meglumine antimoniate and amphotericin
    B. Trop. Med. Int. Health 2010 15 (1) 68-76] adjusted to the proportion of 5 promastigotes in the stationary phase of L. infantum for each macrophage. Promastigotes were added to the wells by keeping them for 2 hours at 37 ° C and 5% CO2 in RPMI-1640 with 10% FCS. After this time the medium was removed and the macrophages were washed with RPMI-1640 medium to remove the promastigotes.
    A new RPMI-1640 medium enriched with 10% SBF and 5% conditioned medium of L929 cells and containing the drug in its different concentrations was added. After 48 h of incubation at 37oC, 5% CO2, the covers were fixed with methanol and stained with 20% Giemsa. 200 macrophages distributed across all fields of the cover were counted and the percentage of infected and non-infected macrophages was calculated. Macrophages containing at least one amastigote were considered infected. Each experiment was performed in triplicate. After treatment


    22
    at various concentrations of each compound, the response observed in the form of% infection becomes% reduction of infection. Finally, a multiple linear regression analysis (SPSS 150 software) is performed to calculate the IC50 (concentration of the compound that reduces parasite load by 50%) and CC50
    5 (concentration of the compound that kills 50% of the mammalian cells) of the product evaluated. The activity of compound IIIa against the parasite responds to a double inverse pattern with the following formula: RP = 1 / (0.0118124 + 0.00044687 / C) (R2 = 97%), where RP is the reduction in the percentage of infection and C is the concentration of compound IIIa.
    The results obtained for compound IIIa are shown in Table 1, after their in vitro evaluation against intracellular L. infantum (IC50) and its cytotoxicity in mammalian cells (CC50). The drugs vorinostat (I), pentamidine (PI) and meglumine antimony agent (MA) or Glucantime ® were used as control.
    Compound IC50 (µM)CC50 (µM) toCC50 (µM) b
    II 3.21 ± 1.8> 100> 100
    IIIa 0.69 ± 0.1> 100> 100
    Vorinostat (I) 6.3 ± 3.85-20NRc
    PI 1.50 ± 0.15-206.08 ± 0.2
    MA 144.40 ± 41.0200-500273.08 ± 12.2
    Table 1.CI50 and CC50 of compound IIIa: Activity against amastigotes of L. infantum in macrophages and cytotoxicity in mammalian cells.
    (to) CC50 on macrophages derived from bone marrow of mice (BMDM)
    (b)
    CC50 on L929 mouse fibroblasts
    (C)
    NR: Not performed 20
    Efficacy and toxicity in vitro Compound IIIa showed an IC50 against intracellular amastigotes of L. infantum approximately 5 times higher than that of compound (II) and also showing an activity superior to the drugs used as control. This fact coupled with its withdrawal
    Cytotoxicity (CC50> 100 µM), gives it a selectivity index greater than 100. Like compound II, compound IIIa showed no activity against L. infantum promastigotes in the concentration range 1-100 µM. Evaluation of the in vivo activity of compound IIIa in an animal model infected with L. infantum


    2. 3
    In this trial, 6 weeks old female Balb / c mice have been used
    Subacute toxicity in vivo
    5 The compound (IIIa) vehiculized on gold nanoparticles was administered to a group of 10 6-week-old Swiss ICR mice (CD-1) at a dose of 500 mg / kg / day for 10 days. After the treatment, an additional observation of the animals was carried out for two weeks. The appearance of signs of pain, suffering, diarrhea, weight loss, hepato or splenomegaly was evaluated. During treatment and after
    At follow-up, no animal died or any of the signs indicated above appeared, indicating that the compound lacks acute or subacute toxicity (lethal dose 50% (LD50)> 500 mg / kg).
    Infection procedure and treatment
    15 35-week-old female Balb / c mice were infected with 10 million promastigotes of L. infantum in a stationary phase of intraperitoneal growth in a volume of 0.4 mL of isotonic saline. After 28 days post-infection, the animals were divided into five groups of 7 mice each that underwent different daily intraperitoneal treatments for 10 days.
    20 The treatments used in vivo are as follows: control Control group treated with gold nanoparticles without drug; group treated with compound (IIIa) at 10 mg / kg; Tratado Group treated with compound (IIIa) vehiculized in gold nanoparticles at 25 mg / kg; 25 control Reference control group treated with meglumine antimonate at 104 mg SbV / kg;  Group treated with a combination of meglumine antimoniate at 104 mg SbV / kg and compound (IIIa) vehiculized in gold nanoparticles at 10 mg / kg.
    30 For the quantification of the parasitic load on the organs of the mice sacrificed after treatment, 20 mg of tissue is weighed, homogenized and the DNA is extracted with a suitable commercial kit, this being resuspended in a volume of 20 microL. Next, a quantitative PCR (in real time) is performed. This PCR uses TaqMan probes and is specific to L. infantum. The number of parasites is calculated
    35 present in each sample interpolating the Ct obtained in a standard curve


    24
    previously established using serial dilutions of parasites. From the number of parasites present in the sample and knowing the weight of the organ used for DNA extraction, the number of parasites / mg of organ is estimated.
    5 Results obtained in vivo Compound IIIa vehiculized in gold nanoparticles was very effective at 25 mg / kg, reducing parasitic loads by 99% in liver and 93% in spleen, compared to 83% reductions in liver and 52 % in spleen using meglumine antimony agent as control drug.
    10 Table 2 shows a comparison, in terms of reduction of parasitic load, of compounds II and IIIa, both vehiculized in gold nanoparticles.
    Compound II Compound IIIaMA
    Reduction of parasitic load in spleen 96%93%52%
    Reduction of parasitic load in liver 68%99%83%
    Table 2. Reduction in the percentage of infection by L. infantum with respect to the control without treatment after 14 days of treatment at a dose of 25 mg / kg / day of compound II or 10 days
    15 treatment at the same dose of compound IIIa. The dose of meglumine antimony (MA) was 390 mg / kg (equivalent to 104 mg / kg / day of SbV antimony) and the treatment lasted 14 days.
    Toxicity assessment in infected animals
    In the treatment with compound IIIa of animals experimentally infected with L. infantum no gastrointestinal adverse effects, weight loss, pain or other general symptoms were observed. Nor were alterations in the biochemical and enzymatic values evaluated in serum or blood, such as transaminases (GPT and GOT), urea, alkaline phosphatase and creatinine. Nor was hepato or
    25 splenomegaly associated with treatment. Therefore, the drug also shows no toxicity in infected mice.
    Compound IIIa has turned out to be a potent drug against the amastigotes of L. infantum being the dose-dependent activity. There is a positive relationship between the concentration administered and the percentage of infection reduction found.


    In addition, it has not shown activity on the promastigote form at the doses evaluated. The dose at which cytotoxic effects are evidenced is much higher than the antiparasitic dose. The index of selectivity is also higher than that found for pentamidinaisetianato (IS = 4) and the megumin antinoniato (IS = 2) that constitute the
    5 control drugs (drugs commonly used in the treatment of leishmaniosis). Evaluation of the in vivo activity of compound IIIa in an animal model infected with L. tropica
    In another trial the efficacy of compound IIIa against cutaneous leishmaniasis was evaluated using 80-100g Syrian hamsters infected by Leishmania tropica.
    15 The hamsters were inoculated in the sole of the left foot with 3 × 107 parasites / 50 µL phosphate buffer saline (PBS).
    The compound was administered using two different types of ointments, one more hydrophilic and one more lipophilic (indicate specific composition), and with two different concentrations of IIIa 20 (0.5% and 2% w / w). The efficacy in the treatment of cutaneous leishmaniasis was measured by determining the reduction of the parasitic load and the size of the lesion.
    As a comparative treatment, Glucantime was administered by intralesional injections of 100 mg SbV / kg. The reduction in parasite load of the 25 hamsters treated with this drug was 61.6%
    Although the 4 compositions tested reduced the parasitic load, the most effective ointment was the most lipophilic with the highest concentration of compound III.
    30 2.5% compound III ointment reduces the parasitic load by up to 75% and the size of the lesions is progressively reduced without showing any toxicity or side effects in animals. The activity was higher than the reference compound (pentavalent antimony) which only showed a reduction of the parasite load of 62%.


    Type of ointment IIIa concentrationParasitic load reduction
    Hydrophilic 0.5%41.5%
    Hydrophilic 2%59.7%
    Lipophilic 0.5%55%
    Lipophilic 2%74.8%
    Drug Interaction Test
    Potential drug interactions can be predicted by evaluating in vitro cytochrome P450.
    It has been found that compound IIIa, as previously
    10 proved for compound II, it does not act on cytochrome P450 enzymes, so it should not interact in vivo on other drugs and would admit its combined use with other drugs in the treatment of leishmaniasis. In particular, the combination between drugs II and III is viable from this point of view.

    权利要求:
    Claims (18)
    [1]
    1. A compound of general formula III
    as well as racemic compounds, stereoisomers, salts or solvates thereof; where Ph is a phenyl of formula
    Ph
    And where R1 = H, F, Cl, Br, I, Me, OMe or NO2. R2 = H, F, Cl, Br, I, Me, OMe or NO2
    15 R3 = H, F, Cl, Br, I, Me, OMe or NO2
    [2]
    2. Compound according to previous claim characterized in that R1 = H or R2 = H or R3 = H.
    Compound according to the preceding claim characterized in that at least two of the three phenyl radicals are formed by hydrogen
    [4]
    4. Compound according to previous claim characterized in that R1 = H,
    R2 = H and R3 = H (Compound IIIa). 25
    IIIa

    28
    [5]
    5. Pharmaceutical composition comprising at least one compound according to
    any one of claims 1 to 4 as an active ingredient and a pharmaceutically acceptable carrier.
    [6]
    6. Pharmaceutical composition comprising as the only active ingredient a compound, or a combination of compounds, according to any of claims 1 to 4, together with a pharmaceutically acceptable carrier.
    [7]
    7. Pharmaceutical composition according to any of claims 5 or 6 characterized in that the compounds according to any of claims 1 to 4 are functionalized in gold nanoparticles.
    Composition according to the preceding claim characterized in that the gold nanoparticles are composed of a gold core and coated with sodium citrate.
    [9]
    9. Composition according to any of claims 7 or 8 characterized
    20 because the nanoparticles have a diameter between 20 and 30 nm, preferably between 22 and 26 nm.
    [10]
    10. Composition according to any of claims 5 to 9 suitable for
    oral administration 25
    [11]
    eleven. Composition according to any of claims 5 to 9 suitable for topical administration
    [12]
    12. Composition according to the preceding claim wherein the concentration of
    Compound III is equal to or greater than 0.5% w / w, preferably equal to or greater than 2%.
    [13]
    13. Combined preparation comprising:
    a) a compound of general formula III, and b) another active ingredient against Leishmaniosis.

    29
    [14]
    14. Combined preparation according to the preceding claim comprising a) a compound of general formula III, and b) a compound of general formula II
    [15]
    fifteen. Combined preparation according to claims 13 or 14 characterized in that the compound of general formula III is supported on pharmaceutically acceptable nanoparticles, more preferably on gold nanoparticles.
    [16]
    16. Kit for the preparation of the composition according to any of claims 5 to 12 or of the combined preparation according to any of claims 13 to 15.
    17. Use of a compound according to any of claims 1 to 4 or composition according to any of claims 5 to 12, or combined preparation according to any of claims 13 to 15 or the kit according to claim 16 for the preparation of a medicament
    Use according to the preceding claim for the preparation of a medicament for the treatment of Leishmaniosis.
    [19]
    19. Use according to the preceding claim characterized in that the Leishmaniosis
    It is canine leishmaniasis or visceral leishmaniasis. 25
    [20]
    20. Use according to claim 18 characterized in that the Leishmaniosis is cutaneous Leishmaniosis.
    [21]
    21. Use according to any of claims 18 to 20 characterized in that the Leishmaniosis is caused by L. Infantum.
    [22]
    22. Use according to any of claims 18 or 20 characterized in that the Leishmaniosis is caused by L. tropica.

    31
    Figure 1
    Figure 2
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    WO2013045734A1|2011-09-26|2013-04-04|Universidad De Granada|Compounds having antileishmanial activity|
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