Identification of (4-(9H-fluoren-9-yl) piperazin-1-yl) methanone derivatives as falcipain 2 inhibitors active against Plasmodium falciparum cultures

Background

Falcipain 2 (FP-2) is the hemoglobin-degrading cysteine protease of Plasmodium falciparum most extensively targeted to develop novel antimalarials. However, no commercial antimalarial drugs based on FP-2 inhibition are available yet due to the low selectivity of most FP-2 inhibitors against the human cysteine proteases.

Design, synthesis and biological evaluation of pyrazolyl-thiazolinone derivatives as potential EGFR and HER-2 kinase inhibitors

A series of pyrazolyl-thiazolinone derivatives (E1-E36) have been designed and synthesized and their biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Thirty-four of the 36 compounds were reported for the first time. Among them, compound 2-(5-(4-bromophenyl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (E28) displayed the most potent inhibitory activity (IC(50)=0.24μM for EGFR and IC(50)=1.07μM for HER-2). Antiproliferative assay results indicated that compound E28 owned high antiproliferative activity against MCF-7, B16-F10 and HCT-116 in vitro, with IC(50) value of 0.30, 0.54, and 0.70μM, respectively. Docking simulation was further performed to position compound E28 into the EGFR active site to determine the probable binding model. Based on the preliminary results, compound E28 with potent inhibitory activity in tumor growth would be a potential anticancer agent.     

Analysis of non-peptidic compounds as potential malarial inhibitors against Plasmodial cysteine proteases via integrated virtual screening workflow

Falcipain-2 (FP-2) and falcipain-3 (FP-3), haemoglobin-degrading enzymes in Plasmodium falciparum, are validated drug targets for the development of effective inhibitors against malaria. However, no commercial drug-targeting falcipains has been developed despite their central role in the life cycle of the parasites. In this work, in silico approaches are used to identify key structural elements that control the binding and selectivity of a diverse set of non-peptidic compounds onto FP-2, FP-3 and homologues from other Plasmodium species as well as human cathepsins. Hotspot residues and the underlying non-covalent interactions, important for the binding of ligands, are identified by interaction fingerprint analysis between the proteases and 2-cyanopyridine derivatives (best hits). It is observed that the size and chemical type of substituent groups within 2-cyanopyridine derivatives determine the strength of protein–ligand interactions. This research presents novel results that can further be exploited in the structure-based molecular-guided design of more potent antimalarial drugs.     

Design, synthesis and biological evaluation of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as aminopeptidase N/CD13 inhibitors

A series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were designed, synthesized and assayed for their activities against aminopeptidase N (APN/CD13) and MMP-2. The results showed that most compounds exhibited higher inhibitory activities against APN than that of MMP-2. Within this series, compound 12h (IC(50)=6.28 ± 0.11 μM) showed similar inhibitory activities compared with Bestatin (IC(50)=5.55 ± 0.01 μM), and it could be used as novel lead compound for the future APN inhibitors development as anticancer agents.     

Synthesis and evaluation of hybrid drugs for a potential HIV/AIDS-malaria combination therapy

Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target Plasmodium falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by the covalent fusion of azidothymidine (AZT) with dihydroartemisinin (DHA), a tetraoxane or a 4-aminoquinoline derivative; and the small library was tested for antiviral and antimalarial activity. Our data suggests that compound 7 is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC(50)=26nM, SI>3000), a moderate activity against HIV (IC(50)=2.9μM; SI>35) and not toxic to HeLa cells at concentrations used in the assay (CC(50)>100μM). Pharmacokinetics studies further revealed that compound 7 is metabolically unstable and is cleaved via O-dealkylation. These studies account for the lack of in vivo efficacy of compound 7 against the CQ-sensitive Plasmodium berghei N strain in mice, when administered orally at 20mg/kg.     

Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: Synthesis and biological evaluation

A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a-k) and N-benzhydrylpyrimidin-4-amines (7a-k) in conjunction with varying steric and electronic properties at the C-2 position were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. The naphth-1-ylmethyl compound 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine) exhibited optimum dual ChE (AChE IC(50)=8.0 μM, BuChE IC(50)=3.9 μM) and hAChE-promoted Aβ-aggregation inhibition (30.8% at 100 μM), whereas in the N-benzhydryl series, compound 7f (N-benzhydryl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-amine) exhibited optimum combination of dual ChE (AChE IC(50)=10.0 μM, BuChE IC(50)=7.6μM) and hAChE-promoted Aβ-aggregation inhibition (32% at 100 μM). These results demonstrate that a 2,4-disubstituted pyrimidine ring serves as a suitable template to target multiple pathological routes in AD, with a C-2 cyclohexylpiperazine substituent providing dual ChE inhibition and potency whereas a C-4 diphenylmethane substituent provides Aβ-aggregation inhibition.     

Synthesis and antimalarial activity of new haemanthamine-type derivatives

Thirty one derivatives were prepared from the natural alkaloids haemanthamine (1), haemanthidine (2) and 11-hydroxyvittatine (3). They were evaluated for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum and some structure-activity relationships were outlined. For haemanthamine derivatives having a methoxy group at C-3, the presence of a free hydroxyl group at C-11 is important for the activity. The double bond at C-1-C-2 plays also an important role to achieve good inhibitory activity. Compound 35 with two nicotinate groups at C-3 and at C-11 was the most active compound with a IC(50)=0.8±0.06μM.     

Synthesis, biological evaluation, and molecular docking of N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives as xanthine oxidase and tyrosinase inhibitors

Claisen-Schmidt condensation of 3-formyl-9-methylcarbazole with various amides of 3-aminoacetophenone afforded N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives. All compounds were investigated for their in vitro xanthine oxidase (XO), tyrosinase and melanin production inhibitory activity. Most of the target compounds had more potent XO inhibitory activity than the standard drug (IC(50)=4.3-5.6μM). Interestingly, compound 7q bearing cyclopropyl ring was found to be the most potent inhibitor of XO (IC(50)=4.3μM). Molecular modelling study gave an insight into its binding modes with XO. Compounds 7a, 7d, 7e, 7g, and 7k were found to be potent inhibitors of tyrosinase (IC(50)=14.01-17.52μM). These results suggest the possible use of these compounds for the design and development of novel XO and tyrosinase inhibitors.     

Synthesis, biological evaluation, and molecular docking studies of 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety as potential antitumor agents

A series of new 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety were synthesized. Antiproliferative assay results indicated that compounds 6o and 6u exhibited the most potent activity against gastric cancer cell SGC-7901, which was more potent than the positive control. Especially, compound 6o exhibited significant telomerase inhibitory activity (IC(50)=2.3±0.07μM), which was comparable to the positive control ethidium bromide. Docking simulation was performed to position compound 6o into the active site of telomerase (3DU6) to determine the probable binding model.     

Pyrido[1,2-a]pyrimidin-4-ones as antiplasmodial falcipain-2 inhibitors

Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is a promising target for antimalarial chemotherapy and inhibition of this protease affects the growth of parasite adversely. A series of pyrido[1,2-a]pyrimidin-4-ones were synthesized and evaluated for their in vitro FP-2 inhibitory potential. Compounds (14,17) showed excellent FP-2 inhibition and can serve as lead compounds for further development of potent FP-2 inhibitors as potential antimalarial drugs.     

Synthesis and biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one analogues as potent anti-inflammatory agents

In this study, eighteen new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives possessing either a 1,3,4-thiadiazole or a 1,2,4-triazole-5-thione moiety were synthesized and tested for anti-inflammatory activity in vitro (COX-1/COX-2, 5-LOX) and in vivo (rat paw edema assay). Compounds 15, 16, 25, 26 and 28-30 showed dual COX-2 (IC(50)'s in the 2.1-10.9 μM range), and 5-LOX (IC(50)'s in the 6.3-63.5 μM range) inhibitory activity. When administered orally to rats, dual COX-2/5-LOX inhibitors showed higher anti-inflammatory activity in vivo (30-45% reduction of the inflammatory response) than the reference drug ibuprofen (18%). Among dual COX-2/5-LOX inhibitors, the most potent compound (28) exhibited the best anti-inflammatory profile by inhibiting both COX-2 (IC(50)=2.1 μM) and 5-LOX (IC(50)=6.3 μM) enzymes. We investigated the binding interactions of compound 28 by an enzyme-ligand molecular modeling (docking) studies, which showed favorable binding interactions in both COX-2 and 5-LOX active sites. Furthermore, the dual acting COX-2/5-LOX compound 28 exhibited a superior gastrointestinal safety profile (ulcer index=0.25) compared to the reference drug ibuprofen (UI=7.0) when administered orally at the same molar dose. These observations suggest that isoxazolo[4,5-d]pyridazin-4(5H)-one analogs represent a new scaffold to design potent, effective, and safe anti-inflammatory agents possessing dual COX-2/5-LOX inhibitory activity.     

Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.     

Identification of diverse natural products as falcipain-2 inhibitors through structure-based virtual screening

Ten natural compounds are successfully identified as falcipain-2 (FP-2) inhibitors from our in-house natural products database using structure-based virtual screening, which show moderate inhibitory activities against FP-2 with IC₅₀ values ranging from 3.18 to 68.19 μM. While one of the inhibitors (compound 5) also exhibits in vitro antiplasmodial activity against chloroquine sensitive strain (3D7) and chloroquine resistant strain (Dd2) of Plasmodium falciparum in the micromolar range (IC₅₀s = 5.54 μM and 4.05 μM against 3D7 cells and Dd2 cells, respectively). Furthermore, the predicted binding poses are analyzed to explain the structure–activity relationships, which will be helpful for further structural modifications.     

Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies

The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ～90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC(50) values for each drug in both tests were similar, were lowest for posaconazole (<5 μM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.     

Design and synthesis of 4,6-substituted-(diaphenylamino)quinazolines as potent EGFR inhibitors with antitumor activity

A type of novel 4,6-substituted-(diaphenylamino)quinazolines, which designed based on the 4-(phenylamino)quinazoline moiety, have been discovered as potential EGFR inhibitors. These compounds displayed good antiproliferative activity and EGFR-TK inhibitory activity. Especially, 4-((4-(3-bromophenylamino)quinazolin-6-ylamino)methyl)phenol (5b), showed the most potent inhibitory activity (IC(50)=0.28μM for Hep G2, IC(50)=0.59μM for A16-F10 and IC(50)=0.87μM for EGFR) and effectively induces apoptosis in a dose-dependent manner in the Hep G2 cell line. Molecular docking of 5b into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.     

Basis for antifolate action and resistance in malaria

Resistance to antifolates of the malaria parasite Plasmodium falciparum stems from stepwise mutations of the target enzyme dihydrofolate reductase (DHFR). New drugs can be developed against resistant parasites, which are assumed to have limited possibilities in mutations. Mechanisms of resistance other than reduced binding of inhibitors to mutant enzymes may be possible and need to be further explored. New synergistic combinations of drugs targeting DHFR and dihydropteroate synthase may be employed, with new provisions against development of resistance.     

Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan (5a-5q) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhibitors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed the most potent biological activity against HEPG2 cancer cell line (IC(50)=0.81 μM for HEPG2 and IC(50)=0.93 μM for MetAP2), which was comparable to the positive control. Docking simulation by positioning compound 5k into the MetAP2 structure active site was performed to explore the possible binding model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.     

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7μM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1μM) and Trypanosoma brucei rhodesiense (IC(50)=64.5μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.     

Anticancer Properties of Distinct Antimalarial Drug Classes

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC₅₀s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC₅₀s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.     

Design, synthesis and biological evaluation of benzo[1.3.2]dithiazolium ylide 1,1-dioxide derivatives as potential dual cyclooxygenase-2/5-lipoxygenase inhibitors

3-(4-Bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide (5) was discovered as a new prototype for dual inhibitors of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). Thus, the structure-activity relationships of benzo[1.3.2]dithiazolium ylide 1,1-dioxide skeleton were carried out. The 6-NO(2) group played an essential role in the inhibitory activity. In addition, moderate-sized lipophilic substituents at the para-position of the 3-aryl moiety were required for dual COX-2/5-LOX inhibitory activity. Among the identified potent dual inhibitors, 3-(4-tbutylphenyl) derivative 30c (IC(50) values of 0.27 μM and 0.30 μM against COX-2 and 5-LOX, respectively) and 3-(4-biphenyl) derivative 30f (IC(50) values of 0.50 μM and 0.15μM against COX-2 and 5-LOX, respectively) were the most potent dual COX-2/5-LOX inhibitors. Intraperitoneal administration of 30c at 100mg/kg demonstrated potent acute anti-inflammatory activity. As a result, benzo[1.3.2]dithiazolium ylide 1,1-dioxide represented a novel scaffold for the exploitation in developing dual COX-2/5-LOX inhibitors.