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Compostos híbridos da artemisinina:direccionamento para as fases sanguínea e hepática do parasita da malária
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A terapia combinada na administração de artemisininas (ACT) é a principal estratégia no controlo da malária sugerida pela OMS. Esta terapia tem como fundamento a combinação de um derivado da artemisinina com outro fármaco antimalárico que tenha maior tempo de semi-vida, com o objectivo de evitar a recrudescência e o desenvolvimento de estirpes resistentes de Plasmodium falciparum. Fármacos híbridos combinam dois farmacóforos diferentes numa única entidade química com modos de acção distintos. Esta abordagem surgiu recentemente como uma estratégia de modo a desenvolver novos fármacos eficazes e pode representar uma alternativa para a terapia de combinação clássica. Nesta tese, descreve-se o design, síntese e avaliação biológica de compostos híbridos baseados na artemisinina, com o objectivo de actuarem nas fases sanguínea e/ou hepática da infecção. Foram sintetizados compostos híbridos de modo a actuar nas fases sanguíneas da infecção, contendo dipeptidilvinilsulfonas covalentemente ligadas à artemisinina através de uma ligação C10-O ou C10-C. As vinilsulfonas são inibidores reconhecidos de proteases de cisteína do P. falciparum, as falcipaínas, envolvidas no processo de degradação da hemoglobina do hospedeiro, que ocorre no vacúolo alimentar do parasita. Foram sintetizadas dipeptidilvinilsulfonas contendo as sequências Gly-Phe, Phe-Phe, Phe-hPhe e Leu-hPhe e acopladas aos ácidos artelínico e artesúnico, originando os compostos híbridos C10-oxa. Estes híbridos apresentaram actividade antimalárica potente, com valores de IC50 entre 2,08 e 4,81 nM para a estirpe W2 do P. falciparum resistente à cloroquina e actividade inibitória na falcipaína-2 na ordem dos μM. Os híbridos contendo a sequência Leu-hPhe foram os inibidores mais potentes desta enzima, onde o derivado ácido artelínico-LeuhPhe- VSMe apresentou um valor de IC50 de 350 nM para a falcipaína-2. Estes compostos também foram capazes de inibir as proteases de cisteína de Plasmodium chabaudi e Babesia bigemina. Os derivados do ácido artesúnico também exibiram elevada actividade antimalárica e inibição da falcipaína-2, mas reduzida estabilidade química e enzimática. Os análogos C10-carba contendo a sequência Leu-hPhe ligada a uma vinilsulfona, vinilsulfonamida ou vinilsulfonato também foram sintetizados. Estes híbridos exibiram igualmente elevada potência antimalárica e inibitória da falcipaína-2, aliada à alta estabilidade em tampão fosfato pH 7,4 e plasma humano. A única excepção foi o derivado sulfonato de etilo, que em tampão fosfato pH 7,4 apresentou um tempo de semivida de 32 horas. Foram também sintetizados compostos híbridos artemisinina C10-oxa e C10-carba contendo a primaquina com o objectivo de actuar nas fases sanguíneas e hepáticas da infecção. Os derivados C10-oxa e C10-carba foram acoplados à primaquina através de uma função amida ou amina. Estes compostos foram capazes de inibir o desenvolvimento da estirpe W2 de P. falciparum com valor de IC50 reduzido na ordem dos nM. Quando testado in vivo num modelo de ratinhos infectados com P. berghei, o híbrido C10-carba com ligação amida foi capaz de suprimir a parasitémia por 15 dias, quando administrado por via intraperitoneal. A actividade inibitória in vitro na fase hepática foi determinada utilizando células Huh7 infectadas com esporozoítos de P. berghei. Todos os compostos híbridos mostraram valores de IC50 entre 5,1-12,5 nM, superiores aos obtidos para a primaquina (IC50= 3,3 μM). Quando testados in vivo, em ratinhos infectados com esporozoítos de P. berghei, os híbridos 4.2 e 4.3 demonstraram reduzir significativamente a infecção no fígado, quando comparados com a primaquina.Artemisinin Combination Therapy (ACT) is now the cornerstone of the strategy for malaria control suggested by WHO. ACTs involve the combination of an artemisinin derivative with a partner drug that clear parasites at a slower rate than arteminisin, in order to avoid recrudescence and development of resistant Plasmodium falciparum strains. Hybrid drugs combine two different pharmacophores in a single chemical entity with a dual mode of action. This approach has recently emerged as a strategy to develop new efficient drugs, and in the case of malaria therapy, may represent an attractive alternative to classical ACTs. In this thesis we describe the design, synthesis and biological evaluation of hybrid compounds based on the artemisinin scaffold, targeting either (i) the blood-stage of infection or (ii) both the blood- and liver-stages of infection. Hybrid compounds targeting the blood-stage of infection were designed to contain dipeptidyl vinyl sulfones linked to the artmesinin scaffold via C10-O or C10-C bonds. Vinyl sulfones are well known inactivators of cysteine proteases from P. falciparum, called falcipains, that are involved in the degradation process of the host hemoglobin that take place in the digestive vacuole of the parasite. Dipeptidyl vinyl sulfones containing the Gly-Phe, Phe-Phe, Phe-hPhe and Leu-hPhe sequences were synthesized and coupled to artelinic and artesunic acids to give rise to C10-oxo hybrid compounds. These hybrids displayed potent antiplasmodial activity, with IC50 values against the chloroquineresistant P. falciparum W2 strain ranging from 2.08 to 4.81 nM, and falcipain-2 inhibitory activity in the low microM region. Those hybrids containing the Leu-hPhe sequence were the most potent enzyme inhibitors, with the artelinic acid-Leu-hPhe-VSMe derivative presenting an IC50 of 350 nM against falcipain-2. These compounds were also able to inhibit the cysteine proteases from P. chabaudi and Babesia bigemina. Artesunic acid counterparts also showed high antiplasmodial and falcipain-2 inhibitory potencies, but reduced chemical and enzymatic stability. The C10-carba analogues containing the Leu-hPhe sequence and a vinyl sulfone, sulfonamide and sulfonate moieties were also synthesized. These hybrids also displayed high antiplasmodial and falcipain-2 inhibitory potencies, coupled to high stability in pH 7.4 phosphate buffer and human plasma. The only exception was the ethyl sulfonate counterpart, that hydrolyzed with an half-life of 32 h in pH 7.4 phosphate buffer. C10-oxo and C10-carba hybrid compounds containing primaquine were designed to target both the blood- and liver-stages of infection. The C10-oxo and C10-carba derivatives were linked to primaquine with either an amide or amine group. All compounds were able to inhibit the development of P. falciparum W2 in low nM region. When tested in vivo in a P. berghei infected mice model, the C10-carba hybrid was able to suppress parasitaemia for 15 days when given intraperitonealy. The in vitro inhibitory activity against the liver stage was determined using Huh7 cells infected with sporozoites of P. berghei. All hybrid compounds showed IC50 values within the range of 5.1-12.5 nM, that compare favourably to that displayed by primaquine, with a IC50 of 3.3 microM. When tested in vivo, using mice infected with P. berghei sporozoite, hybrids 4.2 and 4.3 were shown to significantly reduce liver infection, when compared to primaquine.
Artemisinin Combination Therapy (ACT) is now the cornerstone of the strategy for malaria control suggested by WHO. ACTs involve the combination of an artemisinin derivative with a partner drug that clear parasites at a slower rate than arteminisin, in order to avoid recrudescence and development of resistant Plasmodium falciparum strains. Hybrid drugs combine two different pharmacophores in a single chemical entity with a dual mode of action. This approach has recently emerged as a strategy to develop new efficient drugs, and in the case of malaria therapy, may represent an attractive alternative to classical ACTs. In this thesis we describe the design, synthesis and biological evaluation of hybrid compounds based on the artemisinin scaffold, targeting either (i) the blood-stage of infection or (ii) both the blood- and liver-stages of infection. Hybrid compounds targeting the blood-stage of infection were designed to contain dipeptidyl vinyl sulfones linked to the artmesinin scaffold via C10-O or C10-C bonds. Vinyl sulfones are well known inactivators of cysteine proteases from P. falciparum, called falcipains, that are involved in the degradation process of the host hemoglobin that take place in the digestive vacuole of the parasite. Dipeptidyl vinyl sulfones containing the Gly-Phe, Phe-Phe, Phe-hPhe and Leu-hPhe sequences were synthesized and coupled to artelinic and artesunic acids to give rise to C10-oxo hybrid compounds. These hybrids displayed potent antiplasmodial activity, with IC50 values against the chloroquineresistant P. falciparum W2 strain ranging from 2.08 to 4.81 nM, and falcipain-2 inhibitory activity in the low microM region. Those hybrids containing the Leu-hPhe sequence were the most potent enzyme inhibitors, with the artelinic acid-Leu-hPhe-VSMe derivative presenting an IC50 of 350 nM against falcipain-2. These compounds were also able to inhibit the cysteine proteases from P. chabaudi and Babesia bigemina. Artesunic acid counterparts also showed high antiplasmodial and falcipain-2 inhibitory potencies, but reduced chemical and enzymatic stability. The C10-carba analogues containing the Leu-hPhe sequence and a vinyl sulfone, sulfonamide and sulfonate moieties were also synthesized. These hybrids also displayed high antiplasmodial and falcipain-2 inhibitory potencies, coupled to high stability in pH 7.4 phosphate buffer and human plasma. The only exception was the ethyl sulfonate counterpart, that hydrolyzed with an half-life of 32 h in pH 7.4 phosphate buffer. C10-oxo and C10-carba hybrid compounds containing primaquine were designed to target both the blood- and liver-stages of infection. The C10-oxo and C10-carba derivatives were linked to primaquine with either an amide or amine group. All compounds were able to inhibit the development of P. falciparum W2 in low nM region. When tested in vivo in a P. berghei infected mice model, the C10-carba hybrid was able to suppress parasitaemia for 15 days when given intraperitonealy. The in vitro inhibitory activity against the liver stage was determined using Huh7 cells infected with sporozoites of P. berghei. All hybrid compounds showed IC50 values within the range of 5.1-12.5 nM, that compare favourably to that displayed by primaquine, with a IC50 of 3.3 microM. When tested in vivo, using mice infected with P. berghei sporozoite, hybrids 4.2 and 4.3 were shown to significantly reduce liver infection, when compared to primaquine.
Artemisinin Combination Therapy (ACT) is now the cornerstone of the strategy for malaria control suggested by WHO. ACTs involve the combination of an artemisinin derivative with a partner drug that clear parasites at a slower rate than arteminisin, in order to avoid recrudescence and development of resistant Plasmodium falciparum strains. Hybrid drugs combine two different pharmacophores in a single chemical entity with a dual mode of action. This approach has recently emerged as a strategy to develop new efficient drugs, and in the case of malaria therapy, may represent an attractive alternative to classical ACTs. In this thesis we describe the design, synthesis and biological evaluation of hybrid compounds based on the artemisinin scaffold, targeting either (i) the blood-stage of infection or (ii) both the blood- and liver-stages of infection. Hybrid compounds targeting the blood-stage of infection were designed to contain dipeptidyl vinyl sulfones linked to the artmesinin scaffold via C10-O or C10-C bonds. Vinyl sulfones are well known inactivators of cysteine proteases from P. falciparum, called falcipains, that are involved in the degradation process of the host hemoglobin that take place in the digestive vacuole of the parasite. Dipeptidyl vinyl sulfones containing the Gly-Phe, Phe-Phe, Phe-hPhe and Leu-hPhe sequences were synthesized and coupled to artelinic and artesunic acids to give rise to C10-oxo hybrid compounds. These hybrids displayed potent antiplasmodial activity, with IC50 values against the chloroquineresistant P. falciparum W2 strain ranging from 2.08 to 4.81 nM, and falcipain-2 inhibitory activity in the low microM region. Those hybrids containing the Leu-hPhe sequence were the most potent enzyme inhibitors, with the artelinic acid-Leu-hPhe-VSMe derivative presenting an IC50 of 350 nM against falcipain-2. These compounds were also able to inhibit the cysteine proteases from P. chabaudi and Babesia bigemina. Artesunic acid counterparts also showed high antiplasmodial and falcipain-2 inhibitory potencies, but reduced chemical and enzymatic stability. The C10-carba analogues containing the Leu-hPhe sequence and a vinyl sulfone, sulfonamide and sulfonate moieties were also synthesized. These hybrids also displayed high antiplasmodial and falcipain-2 inhibitory potencies, coupled to high stability in pH 7.4 phosphate buffer and human plasma. The only exception was the ethyl sulfonate counterpart, that hydrolyzed with an half-life of 32 h in pH 7.4 phosphate buffer. C10-oxo and C10-carba hybrid compounds containing primaquine were designed to target both the blood- and liver-stages of infection. The C10-oxo and C10-carba derivatives were linked to primaquine with either an amide or amine group. All compounds were able to inhibit the development of P. falciparum W2 in low nM region. When tested in vivo in a P. berghei infected mice model, the C10-carba hybrid was able to suppress parasitaemia for 15 days when given intraperitonealy. The in vitro inhibitory activity against the liver stage was determined using Huh7 cells infected with sporozoites of P. berghei. All hybrid compounds showed IC50 values within the range of 5.1-12.5 nM, that compare favourably to that displayed by primaquine, with a IC50 of 3.3 microM. When tested in vivo, using mice infected with P. berghei sporozoite, hybrids 4.2 and 4.3 were shown to significantly reduce liver infection, when compared to primaquine.
Descrição
Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2012
Palavras-chave
Teses de doutoramento - 2012