ES2525714A1 - Therapeutic application of perimidinone analogs against leishmaniosis (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Therapeutic application of perimidinone analogues against leishmaniasis. The present invention relates to compounds of structure I and II for the cutaneous and visceral treatment of Leishmaniasis. It specifically refers to the treatment against some species of the genus Leishmania, in particular, L. amazonensis, L. infantum, L. braziliensis and L. guyanensis. The cytotoxicity of these compounds was determined against macrophages of the 774 cell line.

Description

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THERAPEUTIC APPLICATION OF PERIMIDINONE ANALOGUES AGAINST LEISHMANIOSIS

TECHNICAL SECTOR OF THE INVENTION

The present invention relates to compounds of structure I and II for the cutaneous and visceral treatment of Leishmaniasis. It specifically refers to the treatment against some species of the genus Leishmania, in particular, L. amazonensis, L. infantum, L. braziliensis and L. guyanensis. The cytotoxicity of these compounds was determined against macrophages of the 774 cell line.

STATE OF THE ART

Leishmania is an infection caused by a protozoan belonging to the Trypanosomatidae family and the Leishmania genus. This parasitic disease is considered one of the most relevant and influential of tropical diseases and affects millions of people around the world, according to the World Health Organization registry.

This disease can originate a wide repertoire of clinical symptoms, which can differ according to the different diseases that the species that are included within the genus Leishmania can cause. In this sense, it should be noted that the genus Leishmania encompasses geographically differentiated species such as L. major and L. infantum (Old World species), or L. brasiliensis (New World species), which can be identified using molecular techniques.

Among the different diseases caused by this protozoan, cutaneous leishmaniasis is the most common and widespread form of human leishmaniasis. Visceral Leishmaniasis (VL, also known as Kala-azar) is the most severe and fatal form if not properly treated. Leishmania generates high mortality in many countries, being the cause of a wide spectrum of diseases that are grouped into three main forms: cutaneous, mucocutaneous and visceral leishmaniasis (Desjeux, P. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. 2004, 27, 305-318). In the absence of effective vaccines, the control of the disease is based mainly on chemotherapy, among which drugs include pentavalent antimonials such as sodium stibogluconate and meglumine antimoniate, developed more than five decades ago as the first antimonate drugs. high effectiveness. In cases where there is a relapse, patients need treatment with second generation drugs such as Amphotericin B or pentamidine (Mireille Basselin, Hubert Denise, Graham H. Coombs and Michael P. Barrett. Antimicrob. Agents Chemother. 2002, 46, 3731- 3738; Purkait B, Kumar A, Nandi N, Sardar AH, Das S, Kumar S, Pandey K, Ravidas V, Kumar M, De T, Singh D, Das P. Antimicrob. Agents Chemother. 2012, 56, 1031-1041 ).

Although currently the administration of Amphotericin B is currently based on a liposomal formulation that makes the side effects less, the production costs of this drug are very high for the availability and real treatment of the affected population. Furthermore, the current costs of the therapies against Leishmania, the cytotoxicity of the drugs used commercially and the increase in the resistance of the parasites make it necessary to search for new synthetic alternatives or natural sources for the treatment of the disease (Croft, SL; Coombs, GH Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol. 2003, 19, 502-508).

In this sense, alkaloids of chemical structure 7H-dibenzo [de, h] quinolin-7-one called "oxoisoaporphins" were highly effective in in vitro and in vivo tests against L. amazonensis, L. infantum, L. braziliensis and L. guyanensis with an inhibition of parasite growth, in the liver and spleen, of 98 and 77% respectively (Eduardo Sobarzo-Sánchez, Florencio Martínez Ubeira, Humberto González-Díaz, María Auxiliadora Dea-Ayuela, Francisco Bolás Fernández, Pablo Bilbao Ramos. "Use of oxoisoaporphins in the treatment against Leishmaniosis", International Patent (PCT) 2013, WO2013045730 A1; Eduardo Sobarzo-Sánchez, Pablo Bilbao-Ramos, Maria Dea-Ayuela, Humberto González-Díaz, Matilde Yañez, Eugenio Uriarte, Lourdes Santana, Victoria Martínez-Sernández, Francisco Bolás Fernández, Florencio M. Ubeira. Synthetic Oxoisoaporphine Alkaloids: In Vitro, In Vivo and In Silico Assessment of Antileishmanial Activities. Plos One, 2013, 8 (10), e77560).

Thus, oxoisoaporphin derivatives showed low cytotoxicity in in vitro and in vivo tests, which would not mask the possible antiparasitic activity, and an interesting effectiveness and selectivity against certain Leishmania species, which enhanced them as novel chemical structures that could be exploited as possible antiparasitic drugs.

Even so, given the relevance of this parasitic disease, it is still necessary to develop new compounds with high specificity and low toxicity for its treatment.

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DESCRIPTION OF THE INVENTION

The present inventors designed and developed new alkaloids highly effective against different Leishmania species. These compounds differ in the chemical structure of oxoisoaporphins because an additional nitrogen atom has been introduced forming a pyrimidine ring in these alkaloids.

5 In this way, compounds have been obtained with high selectivity and efficiency against some of the four Leishmania species (L. braziliensis, L. amazonensis, L. guyanensis and L. infantum) and a very low cytotoxicity, with the advantage in addition to presenting a high absorption of the same in the body without being excreted, due to its high lipophilicity, and its easy to obtain by synthetic means.

The present invention describes the compounds of structure I and II that have low toxicity and that are

10 capable of effectively and selectively inhibiting the growth of the parasite in in vitro experiments against promastigotes of the species Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis and Leishmania infantum.

In one aspect, the present invention is directed to the use of the compounds of formula I and II, their pharmaceutically acceptable salts, hydrates, solvates, tautomers, stereoisomers and N-oxides, for the preparation

15 of a drug for the treatment of leishmaniasis.

image 1

Ra and Rb are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl substituted, or, Ra and Rb together form a 4- to 7-membered substituted or unsubstituted heterocycle ring containing 0-2 heteroatoms

Additional 25 independently selected from oxygen, sulfur and N-Rc, where Rc is selected from hydrogen, substituted or unsubstituted alkyl, or -C (O) Rb.

In another aspect the invention is directed to a pharmaceutical composition comprising the compounds of formula I or II as described above, and a pharmaceutically acceptable carrier.

In another aspect of the invention it is directed to the use of a pharmaceutical composition comprising a compound of formula I and II, as described above, and a pharmaceutically acceptable vehicle for the preparation of a medicament for the treatment of leishmaniasis.

In another aspect, the invention is directed to compounds of formula II, as described above.

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Detailed description of the invention

"Alkyl" refers to a straight or branched hydrocarbon chain containing no unsaturation, of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, optionally substituted with one to three substituents selected from halogen, cyano, -ORb , -NRaS (O) mRb where m is selected between 1 and 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selected between 1 and 2, -NRaRb, -C (O) Rb , -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl; where Ra and Rb are as previously defined.

"Cycloalkyl" refers to a cyclic hydrocarbon chain containing no unsaturation, of 3 to 10 carbon atoms, preferably 5 to 6 carbon atoms. Cycloalkyl can be monocyclic, bicyclic

or tricyclic and can include fused rings. Optionally the cycloalkyl can be substituted with one to three substituents selected from halogen, cyano, -ORb, -NRaS (O) mRb where m is selected between 1 and 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selected between 1 and 2, -NRaRb, -C (O) Rb, -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb , -CF3, -OCF3, alkyl, aryl, and heteroaryl; where Ra and Rb are as previously defined.

"Alkenyl" refers to a straight or branched cyclic or acyclic hydrocarbon chain containing at least one unsaturation, from 2 to 10 carbon atoms, preferably from 2 to 5 carbon atoms, optionally substituted with one to three substituents selected from halogen, cyano, -ORb, -NRaS (O) mRb where m is selected from 1 to 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selected from 1 to 2, -NRaRb, -C (O) Rb, -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3, alkyl, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl; where Ra and Rb are as previously defined.

"Cycloheteroalkyl" refers to a cycloalkyl containing at least one heteroatom selected from oxygen, nitrogen, or sulfur, for example: pyrrolidinyl, morpholinyl, piperazinyl, and piperidinyl. Optionally the cycloheteroalkyl can be substituted with one to three substituents selected from -ORb, -NRaS (O) mRb where m is selected from 1 to 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selects from 1 to 2, -NRaRb, C (O) Rb, -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, - OCF3, alkyl, aryl, and heteroaryl; where Ra and Rb are as previously defined.

"Aryl" refers to an aromatic hydrocarbon of 6 to 10 carbon atoms, for example: phenyl or naphthyl; optionally the aryl may be substituted with one to three substituents selected from -ORb, NRaS (O) mRb where m is selected from 1 to 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selected between 1 and 2, -NRaRb, -C (O) Rb, -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, - OCF3, alkyl, alkenyl, aryl, and heteroaryl; where Ra and Rb are as previously defined.

"Heteroaryl" refers to an aryl containing at least one heteroatom selected from oxygen, nitrogen, or sulfur, for example: pyridyl, pyrazolyl, triazolyl, pyrimidyl, isoxazolyl, indolyl, and thiazolyl; optionally the heteroaryl may be substituted with one to three substituents selected from -ORb, -NRaS (O) mRb where m is selected from 1 to 2, -SRb, -S (O) mRb, -S (O) mNRaRb where m is selects between 1 and 2, -NRaRb, -C (O) Rb, -CO2Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3, alkyl, alkenyl, aryl, and heteroaryl; where Ra and Rb are as previously defined. "Halogen" is fluorine, chlorine, bromine, or iodine.

According to a particular embodiment, the values R1, R2, R3, R4, R5, R6, R7, and R8 in the compounds of formula (I), as defined above, are independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, alkenyl, formyl, halogen, -ORb and -NRaRb; where Ra and Rb are as defined above.

According to another particular embodiment, in the compound of formula (I) R1 is selected from hydrogen, methyl, formyl and hydroxymethyl and R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of hydrogen, methyl, halogen, and -ORb; where Rb is selected from hydrogen and substituted or unsubstituted alkyl

According to a particular embodiment, the values R1, R1 ', R2, R2', R3, R3 ', R4, R4', R5, R5 ', and R6, R6' in the compounds of formula (II), as stated defined above, are independently selected from the group consisting of hydrogen, halogen, formyl, substituted and unsubstituted alkyl, alkenyl, -ORb, and -NRaRb; where Ra and Rb are as defined above.

According to another particular embodiment, in the compound of formula (II) R2 and R2 'are independently selected from hydrogen, halogen, methyl and -ORb; where Rb is selected from hydrogen and substituted or unsubstituted alkyl, and R1, R1 ', R3, R3', R4, R4 ', R5, R5', R6, and R6 'are hydrogen.

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According to another particular embodiment, the compounds of formula I and II are selected from:

1.

7H-benzo [e] perimidin-7-one

2.

8-chloro-7H-benzo [e] perimidin-7-one

3.

6-hydroxy-7H-benzo [e] perimidin-7-one

Four.

6-methoxy-7H-benzo [e] perimidin-7-one

5.

4-methyl-7H-benzo [e] perimidin-7-one

6.

2-methyl-7H-benzo [e] perimidin-7-one

7.

7-oxo-7H-benzo [e] perimidine-2-carbaldehyde

8.

6-hydroxy-2-methyl-7H-benzo [e] perimidin-7-one

9.

8-chloro-6-hydroxy-7H-benzo [e] perimidin-7-one

10.

8-chloro-6-hydroxy-2-methyl-7H-benzo [e] perimidin-7-one

eleven.

2- (hydroxymethyl) -7H-benzo [e] perimidin-7-one

12.

8-chloro-2-methyl-7H-benzo [e] perimidin-7-one

13.

8-chloro-7-oxo-7H-benzo [e] perimidine-2-carbaldehyde

14.

7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione

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4,4'-dimethyl-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione

16.

4,4'-dibromo-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione

The activity of the compounds of the invention was tested against different species of Leishmania, and it was found that the compounds of the invention are useful for the treatment against the species Leishmania amazonensis, L. infantum, L. braziliensis and L. guyanensis. Thus, in a particular embodiment, the invention relates to the use of any of the compounds described above for the preparation of a medicament for the treatment of Leishmania amazonensis, L. infantum, L. braziliensis and L. guyanensis.

The compounds of the invention are suitable for preparing pharmaceutical compositions for the preparation of a medicament for the treatment of leishmaniasis.

The use of the pharmaceutical compositions of the invention is directed to oral, injectable or topical administration. Among the pharmaceutical compositions that can be prepared, compositions useful for topical administration are of special interest to the invention. Thus, in a particular embodiment, the use of any of the pharmaceutical compositions described above is directed to the topical route. Compositions used topically can be in the form of a gel or ointment. In a particular embodiment, the compositions administered topically also comprise carbopol®.

The compounds of the invention (A1-A13) can be prepared by techniques commonly used in chemistry and known to a person skilled in the art. Some of the compounds of formulas (I) were described in: [Synthesis, structure, geometrical, and spectral characteristics of the (HLn) 2 [CuCl4] complexes. Crystal and molecular structure of bis (2-methylimidazolium) tetrachlorocuprate (II). Kovalchukova, O. V .; Palkina, K. K .; Strashnova, S. B .; Zaitsev, B. E. Russ. J. Coord. Chem. 2008, 34, 830-835 .; Method of obtaining highly pure 7Hbenzo [e] perimidin-7-one. Golubski, Zbigniew; Kowal, Ryszard. Polish Patent 2006, PL 192093 B1 2006083; Tetracycle formation from the reaction of acetophenones with 1-aminoanthraquinone, and further annulation of pyridine and diazepine rings. Deady, L. W .; Smith, C. L. Aust. J. Chem. 2003, 56 (12), 1219-1224; Benzoperimidine-carboxylic acids and derivatives as antagonists of corticotropin releasing factor receptors. Rabinovich, A. K .; Dhanoa, D. S .; Luthin, D. R .; Bychowski, R. A .; Bhumralkar, Dilip R. PCT Int. Appl. 1998, WO 9808821 A1 19980305; Synthesis of anthrapyrimidine derivatives. Nishio, Kazuo; Kasai, Toshiyasu; Tsuruoka, Shinzo. Kogyo Kagaku Zasshi 1968, 71 (12), 2026-2033; Preparation of 6-hydroxy-7H-benzo [e] perimidin-7-one (4hydroxyanthrapyrimidine). Kowal, Ryszard; Golubski, Zbigniew E. Polish Patent 2001, PL 180370 B1 20010131; Anthrapyrimidines. Weidinger, H .; Eilingsfeld, H .; Haese, G. Deutsche Patent 1963, 1159456 (B); Transformations of 4-methylpyrimidinoanthrone under the action of alkaline agents and amines. Kazankov, M. V .; Zotova, O. A. Russ. J. Org. Chem. 1999, 35 (11), 1706-1710; Dimerization mechanism of 4-methylpyrimidanthrone. Kazankov,

M. V .; Zotova, O. A .; Ulanova, L. A .; Pykhtina, E. V. Russ. J. Org. Chem. 2000, 36 (2), 272-277; Synthesis and Cytotoxic Activity of 7-Oxo-7H-dibenz [f, ij] isoquinoline and 7-Oxo-7H-benzo [e] perimidine Derivatives. Bu, X .; Deady, L. W .; Finlay, G. J .; Baguley, B. C .; Denny, W. A. J. Med. Chem. 2001, 44 (12), 2004-2014; Synthesis and cytotoxic activity of N - [(alkylamino) alkyl] carboxamide derivatives of 7-oxo-7H-benz [de] anthracene, 7-oxo-7Hnaphtho [1,2,3-de] quinoline, and 7-oxo-7H -benzo [e] perimidine. Bu, X .; Chen, J .; Deady, L. W .; Smith, C. L .; Baguley, B. C .; Greenhalgh, D .; Yang, S .; Denny, W. A. Bioorg. Med. Chem. 2005, 13 (11), 3657-3665; A easy and efficient method for hydroxylation of azabenzanthrone compounds. Ning, F. X .; Weng, X .; Huang, S. L .; Gu,

L. J .; Huang, Z. S .; Gu, L. Q. Chin. Chem. Lett. 2011, 22 (1), 41-44].

The compounds of formula (II) (DIME14-DIME16) can be obtained from the substituted derivatives of 1-amino-4-bromo-anthraquinone by using N, N-dimethylformamide dimethyl acetal in refluxing MeCN, generating in situ the loss of the bromine atom at position 4 of anthraquinone. The dimerization of the initial brominated derivatives of 1-amino-anthraquinone is generated in high yield and without further purification.

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The following examples illustrate the invention of compounds with anti-leishmanicidal activity, and should be considered for a better understanding of the same without implying a limitation

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1. SYNTHESIS AND CHARACTERIZATION OF NEW ANALOGUES OF BIS-PERIMIDINONES (DIME14-DIME16)

Example 1

10 7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione (DIME14)

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1-amino-4-bromo-anthraquinone (A) (16.55 mmol,

5g) and N, N-dimethylformamide dimethylacetal (N, N-DFDA) (83.3 mmol, 12 mL) was added dropwise and refluxed for 3 hours. Subsequently, the resulting solution was concentrated in vacuo and the generated residue was reacted with NH4OAc (99 mmol, 8 g) in EtOH (100 mL). After the elapsed time, the obtained orange solid was filtered under vacuum and washed repeatedly with cold EtOH. The compound was crystallized from a CH2Cl2 / EtOH mixture to give DIME14 as orange crystals [4.1g, 53% yield].

1H-NMR (DIME14) (300 MHz, DMSO-d6, ppm) δ 7.82 (m, 5H), 8.11 (m, 2H), 8.29 (m, 1H), 8.47 (m, 2H), 8.93 (m, 2H). 9.35 (s, 1H), 9.54 (s, 1H). 13C-NMR (DIME14) (300 MHz, DMSO-d6, ppm) δ 121.9, 122.1, 122.4, 122.7, 123.8, 124.0, 128.4, 129.8, 131.9, 132.4, 132.6, 133.1, 134.1, 134.7, 135.1, 135.3, 135.8 , 136.0, 136.5, 140.1, 140.5, 146.7, 146.8, 147.1, 147.8, 148.0, 154.4, 155.2, 180.1, 180.7. EA: calculated for C30H14N4O2: C, 77.91; H, 3.05; N, 12.12. found, C, 77.83; H, 3.11; N, 12.09. CI-MS (m / z): 462.8 (M +, 100). IR (KBr, υ, cm-1): 1665

25 (C = O).

Example 2

4,4'-dimethyl-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione (DIME15)

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O NH2

image3 POCl3 / N, N-DFDA / CH3CN NH4OAc / EtOH

O Br

(B)

image4

POCl3 (12.6 mmol, 1.2 mL) was added dropwise to a solution of N, N-dimethylformamide dimethylacetal (N, N-DFDA) (15.80 mmol, 2.1 mL) in CH3CN (40 mL) ) on an ice-water bath at 0 ° C. Subsequently

5 allowed the mixture to react with constant stirring until reaching room temperature, 1 amino-2-meyl-4-bromo-anthraquinone (B) (6.3 mmol, 2g) being added in one portion. The resulting mixture was stirred at room temperature for 30 min. and at 80ºC for a day. The following day the formation of a yellow solid was observed, which was filtered under vacuum and treated with NH4OAc (2g) in EtOH (100 mL) at reflux for 2 hr. Finally DIME15 was generated as a dark orange crystalline solid [2.89g, 94% yield].

10 1H-NMR (DIME15) (300 MHz, DMSO-d6, ppm) δ 2.85 (s, 6H), 7.79 (m, 4H), 8.10 (s, 2H), 8.42 (m, 2H), 8.85 (m, 2H), 9.50 (s, 2H). 13C-NMR (DIME15) (300 MHz, DMSO-d6, ppm) δ 17.6, 17.8, 121.4, 121.7, 123.6, 123.8, 125.1, 125.2, 125.3, 126.5, 126.6, 126.8, 127.9, 128.0, 128.4, 132.7, 133.0 , 133.6, 133.8, 134.0, 134.5, 141.1, 141.3, 141.4, 145.3, 148.1, 148.3, 154.6, 155.2, 155.3, 180.3, 180.4. EA: calculated for C32H18N4O2: C, 78.36; H, 3.70; N, 11.42. found, C, 78.31; H, 3.75; N, 11.31. CI-MS (m / z): 490.8 (M +, 100). IR (KBr, υ, cm -1): 1658 (C = O).

fifteen

Example 3

4,4'-dibromo-7H, 7'H- [6,6'-bibenzo [e] perimidin] -7,7'-dione (DIME16)

image5

POCl3 (16.4 mmol, 1.6 mL) was added dropwise to a solution of N, N-dimethylformamide dimethylacetal (N, N-DFDA) (14.09 mmol, 2 mL) in CH3CN (70 mL) over an ice-water bath at 0 ° C. Subsequently, the mixture was allowed to react with constant stirring until reaching room temperature, being added 1-amino

2,4-dibromo-anthraquinone (C) (4.7 mmol, 1.79g) in one serving. The resulting mixture was stirred at room temperature for 30 min. and at 50ºC for a day. The following day the formation of a yellow solid was observed, which was filtered under vacuum and treated with NH4OAc (2g) in EtOH (100 mL) at reflux for 2 hr. Finally DIME16 was generated as a dark orange crystalline solid [1.81g, 62% yield].

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1H-NMR (DIME16) (300 MHz, DMSO-d6, ppm) δ 7.86 (m, 4H), 8.47 (m, 2H), 8.65 (m, 2H), 8.92 (m, 2H), 9.61 (s, 2H ). 13C-NMR (DIME16) (300 MHz, DMSO-d6, ppm) δ 121.9, 122.5, 123.4, 123.8, 124.0, 124.5, 125.1, 125.3, 126.0, 126.8, 128.2, 128.8, 133.1, 133.3, 133.8, 134.0, 134.4 , 135.7, 142.0, 142.4, 142.8, 144.4, 146.3, 147.4, 154.4, 155.3, 159.8, 160.2, 181.3, 181.6. EA: calculated for C30H12Br2N4O2: C, 58.09; H, 1.95; N, 9.03. found, C, 58.03; H, 1.97; N, 9.02. CI-MS (m / z): 620.8 (M +, 100). IR (KBr, υ, cm -1): 1665 (C = O).

2. IN VITRO EFFECTIVENESS TESTS AGAINST LEISHMANIA SPP

Promastigotes in the logarithmic growth phase of the different Leishmania species (L. infantum, L. braziliensis, L. amazonensis and L. guyanensis) were arranged at a concentration of 1.25 x 106 promastigotes / mL, in a final volume of 200 µL of Schneider's medium in 96-well culture plates. For the determination of drug susceptibility, serial dilutions of the compounds were prepared in Schneider culture medium (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 µg / mL), which were added in triplicate to the plates of culture. They were left in incubation at 26 ° C in contact with the compounds and after 48 hours, 20 µl of a solution of 2.5 mM resazurin in PBS was added, leaving incubation for 3 h at 26 ° C. The fluorescence intensity was then determined in a spectrofluorimeter (Infinite Tecan I-control) at 535 nm (excitation) / 590 nm (emission) wavelength. Finally, by comparison with the control wells, the percentage of viable parasites was determined. The mean inhibitory concentration (IC50) was calculated using the Probit Regression method using the SPSS v.15.0 statistical package.

3. CELLULAR CYTOTOXICITY

J774 macrophages were placed in log growth phase at a concentration of 2.5x10 5 cells / ml, in a final volume of 200 µl in 96-well culture plates. Serial dilutions of the compounds were prepared in the same way as in the determination of the in vitro efficacy of RPMI-1640 culture medium, which were added in triplicate to the microtiter plates. They were left in incubation at 37 ° C in contact with the compounds and after 24 hours, 20 µl of a solution of 2.5 mM resazurin in PBS was added, leaving incubation for 3 h at 37 ° C. Then, the intensity of fluorescence in a spectrofluorimeter (Infinite Tecan I-control) at 535 nm (excitation) -590 nm (emission) wavelength. The mean cytotoxic concentration (CC50) was determined by the Probit Regression method using the SPSS v.15.0 statistical package.

COMPOUND ASSESSMENT

The compounds evaluated in the present invention are based on the following general formulas, corresponding to the general formula (I) and (II):

A. 7H-BENZO [e] PERIMIDIN-7-ONA (3-AZAOXOISOAPORPHINE), General Formula (I):

R2

R3

image6 R1 R4

(I)

R8

R7

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In what:

a) where if: - R1, R2, R3, R4, R5, R6, R7 and R8 is hydrogen; it is 7H-benzo [e] perimidin-7one, hereinafter called A1; B) where if: - R1, R2, R3, R4, R6, R7 and R8 is hydrogen and R5 represents a chlorine; it is 8-chloro-7H-benzo [e] perimidin-7-one, hereinafter called A2; c) where if: - R1, R2, R3, R5, R6, R7 and R8 is hydrogen, R4 represents a hydroxyl; it is 6-hydroxy-7H-benzo [e] perimidin-7-one, hereinafter called A3; d) where if: - R1, R2, R3, R5, R6, R7 and R8 is hydrogen and R4 represents a methoxy; it is 610 methoxy-7H-benzo [e] perimidin-7-one, hereinafter referred to as A4; e) where if: - R1, R3, R4, R5, R6, R7 and R8 is hydrogen and R2 represents a methyl; it is 4-methyl-7H-benzo [e] perimidin-7-one, hereinafter called A5; f) where if: - R2, R3, R4, R5, R6, R7 and R8 is hydrogen and R1 represents a methyl; it is 2-methyl-7H-benzo [e] perimidin-7-one, hereinafter called A6; 15 g) where if: - R2, R3, R4, R5, R6, R7 and R8 is hydrogen and R1 represents an aldehyde; it is 7oxo-7H-benzo [e] perimidine-2-carbaldehyde, hereinafter called A7;

h) where if: - R2, R3, R5, R6, R7 and R8 is hydrogen; R1 represents a methyl and R4 represents a hydroxyl; it is 6-hydroxy-2-methyl-7H-benzo [e] perimidin-7-one, hereinafter called A8;

I) where if: - R1, R2, R3, R6, R7 and R8 is hydrogen; R4 represents a hydroxyl and R5 represents a chlorine; it is 6-hydroxy-8-chloro-7H-benzo [e] perimidin-7-one, hereinafter called A9;

j) where if: - R2, R3, R6, R7 and R8 is hydrogen; R1 represents a methyl, R4 represents a hydroxyl and R5 represents a chlorine; it is 2-methyl-6-hydroxy-8-chloro-7H-benzo [e] perimidin25 7-one, hereinafter called A10; k) where if: - R2, R3, R4, R5, R6, R7 and R8 is hydrogen and R1 represents a hydroxymethyl; it is 2- (hydromethyl) -7H-benzo [e] perimidin-7-one, hereinafter called A11; l) where if: - R2, R3, R4, R6, R7 and R8 is hydrogen; R1 represents a methyl and R5 represents a chlorine; it is 8-chloro-2-methyl-7H-benzo [e] perimidin-7-one, hereinafter called A12; and

m) where if: - R2, R3, R4, R6, R7 and R8 is hydrogen; R1 represents an aldehyde and R5 represents a chlorine; it is 8-chloro-7H-benzo [e] perimidine-2-carbaldehyde, hereinafter called A13;

35

B. 7H, 7'H- [6,6'-BIBENZO [e] PERIMIDIN] -7,7'-DIONA (BIS-3-AZAOXOISOAPORPHINE), General Formula (II):

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image7

image8

In what:

a) where if: - R1, R1 ', R2, R2', R3, R3 ', R4, R4', R5, R5 ', and R6, R6' is hydrogen; it is 7H, 7'H5 [6,6'-bibenzo [e] perimidin] -7,7'-dione, hereinafter called DIME1;

b) where if: - R1, R1 ', R3, R3', R4, R4 ', R5, R5', and R6, R6 'is hydrogen and R2, R2' represents a methyl; it is 4,4'-dimethyl-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione, hereinafter called DIME2; and

c) where if: - R1, R1 ', R3, R3', R4, R4 ', R5, R5', and R6, R6 'is hydrogen and R2, R2' represents a bromine; it is 4,4'-dibromo-7H, 7'H- [6,6'-bibenzo [e] perimidin] -7,7'-dione, hereinafter called DIME3;

RESULTS OF LEISHMANICIDAL ACTIVITY OF PERIMIDINONES

15 Considering the perimidinone analogs and dimers obtained by the experimental procedures mentioned above in the present invention, and to determine the leishmanicidal activity of these derivatives, the in vitro efficacy against promastigotes in the logarithmic growth phase of four Leishmania species was determined. ; L. amazonensis, L. braziliensis, L. infantum and L. guyanensis and the cytotoxicity of each of them was verified against macrophages of the J774 line. In this way, the IC50 values can be appreciated.

20 expressed in μg / mL in the following table:

Table 1. In vitro evaluation of the promastigote form of various Leishmania species and in vitro cytotoxicity on macrophages of the J774 line, expressed in IC50 (μg / mL).

Compounds

Anti-Leishmania activities J774 [CC50] cytotoxicity

L. braziliensis

L. amazonensis L. guyanensis L. infantum

IC50

IC50

IC50

IC50

A1

> 100 > 100 > 100 > 100 91,033

A2

> 100 > 100 > 100 > 100 > 100

A3

0.41 3.12 1,415 0.78 3,586

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A4

9,581 7.47 2.97 14.39 > 100

TO 5

66,843 59.70 28.50 > 100 > 100

A6

> 100 > 100 > 100 > 100 > 100

A7

56,796 53.50 23.94 23.89 > 100

A8

3.27 8.14 1.50 3.01 > 100

A9

28,443 77.18 13.77 53.48 > 100

A10

52,655 61.22 49.40 78.28 > 100

A11

36,915 34.87 21.24 36.42 > 100

A12

> 100 > 100 > 100 > 100 > 100

A13

> 100 > 100 > 100 > 100 > 100

DIME1

0.501 2.74 2.44 3.70 19,244

DIME2

14,712 35.78 16.67 69.08 > 100

DIME3

40,857 > 100 > 100 27.39 > 100

According to the experimental data, it can be concluded that both A4, A8 and A11 represent very interesting analogues in leishmanicidal activity against the four species tested. Furthermore, the activities of some derivatives can be compared with respect to the degree of substitution, with A8 being the compound that acts best.

5 against the different strains, mainly against L. guyanensis, responsible for generating skin and mucocutaneous lesions in animals and in man, and L. infantum, responsible in man for generating visceral lesions. The cytotoxicities of most of these compounds tested against the J774 cell line are very low (> 100), which allows us to infer that the leishmanicidal activities are not being masked by the toxicity of the compound.

Finally, we can highlight the antiparasitic activity of the perimidinone dimers obtained and evaluated in this invention, where the DIME2 derivative has an outstanding and selective anti-leishmania activity against L. brasiliensis and L. guyanensis. These antecedents allow us to ensure the use of these derivatives as a good alternative without the undesirable side effects such as cytotoxicity that regularly accompanies the compounds used commercially for the treatment of the disease, and a better tolerance at the moment.

15 to administer them through the different formulations mentioned in this invention.

In another aspect, the present invention provides possible pharmaceutical formulations for the preparation of a medicament, based on the compounds presented herein, for the therapeutic and selective treatment against leishmaniasis.

The doses in which the compound could be administered vary within a wide range, adjusting to the requirements of each particular case. The different pharmaceutical compositions of the invention can be administered by oral, topical and injectable routes according to the different pharmaceutical formulations described in Tables 2-6.

25 EXAMPLE A (Tablet)

Table 2. Pharmaceutical formulation and weight of the active ingredient plus the excipients of a tablet.

image9

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Active ingredient

150

Microcrystalline cellulose

100

Starch

100

Sodium croscarmellose

75

Magnesium stearate

75

Tablet Weight

500

EXAMPLE B (Capsule) Table 3. Pharmaceutical formulation and weight of the active ingredient plus the excipients of a capsule.

Component

mg / Capsule

Active ingredient

80

Pre-gelatinized corn starch

100

Cellulose microcrystals

fifty

Opadry OYS 96-14

twenty

Capsule Weight

250

EXAMPLE C (Gel) Table 4. Pharmaceutical formulation and weight of the active ingredient plus the excipients of a gel.

Component

Quantity

Active ingredient

10 mg

Carbopol-940

5 mg

Propylene glycol

1 ml

1M HCl

10 l

Triethanolamine

20 l

H2O

c.s.p. 10 ml

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EXAMPLE D (Ointment) Table 5. Pharmaceutical formulation and weight of the active ingredient plus the excipients of an ointment.

Component

Quantity

Active ingredient

3 mg / g ointment

Zinc oxide

2 g

Vaseline

10 g

EXAMPLE E (Injectable solution) Table 6. Pharmaceutical formulation and weight of the active ingredient of an injectable solution.

Component

Quantity

Active ingredient

10 mg

1M HCl

20 l

NaCl

9 mg

H2O

c.s.p. 1 ml

Claims (8)

P201431466 03-10-2014 1. Use of compounds of formula I and II, their pharmaceutically acceptable salts, hydrates, solvates, tautomers, stereoisomers and N-oxides, for the preparation of a medicine for the treatment of leishmaniasis, image 1 substituted, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or, Ra and Rb together 15 form a 4- to 7-membered substituted or unsubstituted heterocycle ring containing 0-2 additional heteroatoms independently selected from oxygen, sulfur, and N-Rc, where Rc is selected from hydrogen, substituted or unsubstituted alkyl, or -C ( O) Rb. 2. Use of compounds of formula (I), according to claim 1, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, substituted and unsubstituted alkyl, alkenyl, formyl, halogen, -ORb and -NRaRb; where Ra and Rb are as defined in claim 1. 3. Use of compounds of formula (I), according to claim 2, wherein R1 is selected from hydrogen, methyl, formyl and hydroxymethyl and R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of hydrogen, methyl, halogen, and -ORb; where Rb is selected from hydrogen and alkyl 25 substituted or unsubstituted. 4. Use of compounds of formula (II), according to claim 1, wherein R1, R1 ', R2, R2', R3, R3 ', R4, R4', R5, R5 ', and R6, R6' are selected from independently from the group consisting of hydrogen, halogen, formyl, substituted and unsubstituted alkyl, alkenyl, -ORb; where Rb is as defined in claim 1. 30 5. Use of compounds of formula (II), according to claim 4, wherein R2 and R2 'are independently selected from hydrogen, halogen, methyl and -ORb; where Rb is preferably selected from hydrogen and substituted or unsubstituted alkyl, and R1, R1 ', R3, R3', R4, R4 ', R5, R5', and R6, R6 'are hydrogen. Use of compounds of formula I and II, according to claim 1, selected from: -7H-benzo [e] perimidin-7-one -8-chloro-7H-benzo [e] perimidin-7-one - 6-hydroxy-7H-benzo [e] perimidin-7-one 14 P201431466 03-10-2014 -6-methoxy-7H-benzo [e] perimidin-7-one -4-methyl-7H-benzo [e] perimidin-7-one -2-methyl-7H-benzo [e] perimidin-7-one -7-oxo-7H-benzo [e] perimidine-2-carbaldehyde 5-6-hydroxy-2-methyl-7H-benzo [e] perimidin-7-one

-

8-chloro-6-hydroxy-7H-benzo [e] perimidin-7-one

-

8-chloro-6-hydroxy-2-methyl-7H-benzo [e] perimidin-7-one

-2- (hydroxymethyl) -7H-benzo [e] perimidin-7-one - 8-chloro-2-methyl-7H-benzo [e] perimidin-7-one 10 - 8-chloro-7-oxo-7H-benzo [e] perimidine-2-carbaldehyde -7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione

-

4,4'-dimethyl-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione

-

4,4'-dibromo-7H, 7'H- [6,6'-bibenzo [e] perimidine] -7,7'-dione.

7. Use according to any of the preceding claims, wherein the treatment is against Leishmania amazonensis, L. infantum, L. braziliensis and L. guyanensis species. 8. Pharmaceutical composition comprising a compound of formula I or II, as described in any of claims 1 to 6, and a pharmaceutically acceptable carrier. Use of a pharmaceutical composition according to claim 8, for the preparation of a medicament for the treatment of leishmaniasis. 10. Use according to claim 8, for administration orally, injectable or topically. 11. Compound of formula II, as described in claims 1, 4, 5 and 6. fifteen