Biological evaluation of substituted quinolines

Several quinolines were synthesized and evaluated in vitro against several parasites (Trypanosoma brucei, T. cruzi, Leishmania infantum, L. amazonensis, Plasmodium falciparum). Then, they were evaluated in vitro (at 10 microM), against HTLV-1 transformed cells. A few of them displayed interesting activities, comparable to the reference drugs.     

Quinol derivatives as potential trypanocidal agents

Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counter-screen. In this paper, we report screening of further examples of quinols against T. brucei. We were able to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moieties are known to be selectively concentrated in the parasite by transporter proteins. In general these transporter motif-containing analogues showed increased selectivity; however they also showed reduced levels of potency against T. brucei.     

Antileishmanial activity, cytotoxicity and QSAR analysis of synthetic dihydrobenzofuran lignans and related benzofurans

A series of synthetic dihydrobenzofuran lignans and related benzofurans were evaluated for their cytotoxicity in a screening panel consisting of various human tumour cell lines, and for their antiprotozoal activity against L. donovani (axenic amastigotes), chloroquine resistant Plasmodium falciparum (strain K1), Trypanosoma brucei rhodesiense and T. cruzi, and for cytotoxicity on L6 cells. No promising cytotoxicities against human tumour cell lines were observed for newly synthesised compounds, but the dimerisation product of some lipophylic esters of caffeic acid, such as compound 2g, showed a high activity against chloroquine-resistant P. falciparum (strain K1) (IC50 0.43 microg/mL) and L. donovani (axenic amastigotes) (IC50 0.12 microg/mL), which was confirmed in an infected macrophage assay (IC50 0.19 microg/mL). QSAR models for the cytotoxic and antileishmanial activity were generated using Quasar receptor surface modelling.     

Discovery of nitroheterocycles active against African trypanosomes. In vitro screening and preliminary SAR studies

A selection of 76 nitroheterocycles and related compounds from our in-house compound library was screened in vitro against the parasite Trypanosoma brucei rhodesiense, causative agent of human African trypanosomiasis (HAT). The unspecific cytotoxicity of the compounds was also evaluated against rat myoblast L6-cells to measure the selectivity of the compounds towards the parasite. This screening revealed some preliminary structure-activity relationships (SAR) among the series, and six hit compounds showing interesting activity (IC(50)≤10μM) and fair selectivity (SI>17). The 7-nitroquinoxalin-2-one and 5-nitroindazole scaffold derivatives 58 and 35, respectively, are particularly interesting because of their established oral bioavailability in mice. These hits represent interesting starting points for a medicinal project aimed at identifying the SAR behind this class of compounds.     

Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis.

The ethanolic extracts of a number of Turkish freshwater macrophytes (Potamogeton perfoliatus, Ranunculus tricophyllus and Cladophora glomerata) and marine macroalgae (Dictyota dichotoma, Halopteris scoparia, Posidonia oceanica, Scinaia furcellata, Sargassum natans and Ulva lactuca) were assayed for their in vitro antiprotozoal activity. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum were used as test organisms. The cytotoxicity of the extracts was also assessed against primary rat skeletal myoblasts (L6 cells). Whereas none of the extracts were active against T. cruzi, all crude extracts displayed appreciable trypanocidal activity against T. brucei rhodesiense, with S. natans being the most active one (IC(50) 7.4microg/ml). Except for the marine alga H. scoparia, all extracts also possessed leishmanicidal potential. The best antileishmanial activity was exerted by U. lactuca and P. oceanica (IC(50)'s 5.9 and 8.0microg/ml, respectively). Five extracts that demonstrated inhibitory activity towards P. falciparum (IC(50)'s 18.1-48.8microg/ml) were simultaneously assayed against FabI, a crucial enzyme of the fatty acid system of P. falciparum, to find out whether FabI was their target. The extracts of C. glomerata and U. lactuca efficiently inhibited the FabI enzyme with IC(50) values of 1.0 and 4.0microg/ml, respectively. None of the extracts were cytotoxic towards mammalian L6 cells. This work reports for the first time antiprotozoal activity of some Turkish marine and freshwater algae, as well as a target-based antiplasmodial screening for the identification of P. falciparum FabI inhibitors from aquatic and marine macrophytes.     

Identification of selective tubulin inhibitors as potential anti-trypanosomal agents

The potency of a series of sulfonamide tubulin inhibitors against the growth of Trypanosoma brucei (T. brucei), as well as human cancer and primary fibroblast cells were evaluated with the aim of determining whether compounds that selectively inhibit parasite proliferation could be identified. Several compounds showed excellent selectivity against T. brucei growth, and have the potential to be used for the treatment of Human African trypanosomiasis. A T. brucei tubulin protein homology model was built based on the crystal structure of the bovine tubulin. The colchicine-binding domain, which is also the binding site of the tested sulfonamide tubulin inhibitors, showed clear differences between the tubulin structures and presumably explained the selectivity of the compounds.     

Synthesis and antimalarial and antituberculosis activities of a series of natural and unnatural 4-methoxy-6-styryl-pyran-2-ones, dihydro analogues and photo-dimers

Previous studies have identified the 3,6-dialkyl-4-hydroxy-pyran-2-one marine microbial metabolites pseudopyronines A and B to be modest growth inhibitors of Mycobacterium tuberculosis and a range of tropical diseases including Plasmodium falciparum and Leishmania donovani. In an effort to expand the structure-activity relationship of this compound class towards infectious diseases, a library of natural product and natural product-like 4-methoxy-6-styryl-pyran-2-ones and a subset of catalytically reduced examples were synthesized. In addition, the photochemical reactivity of several of the 4-methoxy-6-styryl-pyran-2-ones were investigated yielding head-to-head and head-to-tail cyclobutane dimers as well as examples of asymmetric aniba-dimer A-type dimers. All compounds were evaluated for cytotoxicity and activity against M. tuberculosis, P. falciparum, L. donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi. Of the styryl-pyranones, natural product 3 and non-natural styrene and naphthalene substituted examples 13, 18, 21, 22 and 23 exhibited antimalarial activity (IC(50) <10 μM) with selectivity indices (SI) >10. Δ(7) Dihydro analogues were typically less active or lacked selectivity. Head-to-head and head-to-tail photodimers 5 and 34 exhibited moderate IC(50)s of 2.3 to 17 μM towards several of the parasitic organisms, while the aniba-dimer-type asymmetric dimers 31 and 33 were identified as being moderately active towards P. falciparum (IC(50) 1.5 and 1.7 μM) with good selectivity (SI ~80). The 4-tert-butyl aniba-dimer A analogue 33 also exhibited activity towards L. donovani (IC(50) 4.5 μM), suggesting further elaboration of this latter scaffold could lead to the identification of new leads for the dual treatment of malaria and leishmaniasis.     

Synthesis and antiprotozoal activity of oligomethylene- and p-phenylene-bis(methylene)-linked bis(+)-huprines

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.     

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.     

In vitro and in silico studies of polycondensed diazine systems as anti-parasitic agents

Parasitic diseases caused by protozoarian agents are still relevant today more than ever. Recently, we synthesized several polycondensed diazine derivatives by means 1,3-dipolar cycloaddition reactions. A broad selection of these compounds were submitted to in vitro biological screening against Plasmodium falciparum, Leishmania infantum, Trypanosoma brucei, and Trypanosoma cruzi, resulting active at micromolar level. Induced Fit Docking/MM-GBSA studies were performed giving interesting indications about the probable mechanism of action of the most active compounds.     

Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas' disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.     

Biological evaluation of glycosyl-isoindigo derivatives against the pathogenic agents of tropical diseases (malaria, Chagas disease, leishmaniasis and human African trypanosomiasis)

The biological activities of diversely substituted glycosyl-isoindigo derivatives against the causative agents of tropical diseases (malaria, Chagas disease, leishmaniasis and human African trypanosomiasis) are reported. Some of the compounds tested showed interesting activities with good selectivity indices, particularly against Trypanosoma brucei rhodesiense. These results suggested, for the first time, that glycosyl-isoindigo derivatives could be of interest for the discovery of new lead compounds to treat tropical diseases.     

Alkyl-linked bis-THTT derivatives as potent in vitro trypanocidal agents

The effect of several alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) on Leishmania donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum is reported. Most of the compounds exhibited a potent activity against the three parasitic strains but the best in vitro activity profiles were found against T. b. rhodesiense with IC(50) values ranging between 0.3 and 4 microM for the most active compounds.     

Antitrypanosomal and cysteine protease inhibitory activities of alkyldiamine cryptolepine derivatives

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC(50) of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.     

Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4 h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.     

Synthesis of isoquinuclidine analogs of chloroquine: antimalarial and antileishmanial activity

The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring system may be viewed as a semi-rigid boat form of the piperidine ring and, when properly substituted, a scaffold for rigid analogs of biologically active ethanolamines and propanolamines. It is present in natural products (such as ibogaine and dioscorine) that display interesting pharmacological properties. In this study, we have expanded our continuing efforts to incorporate this ring system in numerous pharmacophores, by designing and synthesizing semirigid analogs of the antimalarial drug chloroquine. The analogs were tested in vitro against Plasmodium falciparum strains and Leishmania donovani promastigote cultures. Compounds 6 and 13 displayed potent antimalarial activity against both chloroquine-susceptible D6 and the -resistant W2 strains of P. falciparum. All analogs also demonstrated significant antileishmanial activity with compounds 6 and 13 again being the most potent. The fact that these compounds are active against both chloroquine-resistant and chloroquine-sensitive strains as well as leishmanial cells makes them promising candidates for drug development.     

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC₅₀ value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC₅₀ values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.     

Identification of novel inhibitors of UDP-Glc 4'-epimerase, a validated drug target for african sleeping sickness

Novel inhibitors of Trypanosoma brucei and mammalian UDP-Glc 4'-epimerase were identified by screening a small library of natural products and commercially available drug-like molecules. The inhibitors possess low micromolar potency against the T. brucei and human enzymes in vitro, display a degree of selectivity between the two enzymes, and are cytotoxic to cultured T. brucei and mammalian cells.     

Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.     

Novel 2H-isoquinolin-3-ones as antiplasmodial falcipain-2 inhibitors

A series of 1-aryl-6,7-disubstituted-2H-isoquinolin-3-ones (2–10) was synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2, as well as against cultured P. falciparum strain FCBR parasites. All compounds displayed inhibitory activity against recombinant falcipain-2 and against in vitro cultured intraerythrocytic P. falciparum, with the exception of 9. The new compounds exhibited no selectivity against human cysteine proteases such as cathepsins B and L. The inhibitory activity of the synthesized compounds was also evaluated against another protozoal cysteine protease, namely rhodesain of Trypanosoma brucei rhodesiense.