Structure–Activity Relationship Studies of Pyrrolone Antimalarial Agents

Previously reported pyrrolones, such as TDR32570, exhibited potential as antimalarial agents; however, while these compounds have potent antimalarial activity, they suffer from poor aqueous solubility and metabolic instability. Here, further structure–activity relationship studies are described that aimed to solve the developability issues associated with this series of compounds. In particular, further modifications to the lead pyrrolone, involving replacement of a phenyl ring with a piperidine and removal of a potentially metabolically labile ester by a scaffold hop, gave rise to derivatives with improved in vitro antimalarial activities against Plasmodium falciparum K1, a chloroquine‐ and pyrimethamine‐resistant parasite strain, with some derivatives exhibiting good selectivity for parasite over mammalian (L6) cells. Three representative compounds were selected for evaluation in a rodent model of malaria infection, and the best compound showed improved ability to decrease parasitaemia and a slight increase in survival.     

Activity of Fluorine‐Containing Analogues of WC‐9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine‐containing derivatives, the 3‐(3‐fluorophenoxy)‐ and 3‐(4‐fluorophenoxy)phenoxyethyl thiocyanates, exhibited half‐maximal effective concentration (EC₅₀) values of 1.6 and 4.9 μm , respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC‐9 against intracellular T. cruzi (EC₅₀ values of 5.4 and 5.7 μm , respectively). In addition, 2‐[3‐ (phenoxy)phenoxyethylthio]ethyl‐1,1‐bisphosphonate, which is a hybrid inhibitor containing 3‐phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub‐micromolar levels (EC₅₀=0.7 μm ), which suggests a combined inhibitory effect of the two functional groups.     

Structural Design,Synthesis and Structure–Activity Relationships of Thiazolidinones with Enhanced Anti‐Trypanosoma cruzi Activity

Pharmacological treatment of Chagas disease is based on benznidazole, which displays poor efficacy when administered during the chronic phase of infection. Therefore, the development of new therapeutic options is needed. This study reports on the structural design and synthesis of a new class of anti‐Trypanosoma cruzi thiazolidinones ( 4 a – p ). (2‐[2‐Phenoxy‐1‐(4‐bromophenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one ( 4 h ) and (2‐[2‐phenoxy‐1‐(4‐phenylphenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one ( 4 l ) were the most potent compounds, resulting in reduced epimastigote proliferation and were toxic for trypomastigotes at concentrations below 10 μM , while they did not display host cell toxicity up to 200 μM . Thiazolidinone 4 h was able to reduce the in vitro parasite burden and the blood parasitemia in mice with similar potency to benznidazole. More importantly, T. cruzi infection reduction was achieved without exhibiting mouse toxicity. Regarding the molecular mechanism of action, these thiazolidinones did not inhibit cruzain activity, which is the major trypanosomal protease. However, investigating the cellular mechanism of action, thiazolidinones altered Golgi complex and endoplasmic reticulum (ER) morphology, produced atypical cytosolic vacuoles, as well as induced necrotic parasite death. This structural design employed for the new anti‐T. cruzi thiazolidinones ( 4 a – p ) led to the identification of compounds with enhanced potency and selectivity compared to first‐generation thiazolidinones. These compounds did not inhibit cruzain activity, but exhibited strong antiparasitic activity by acting as parasiticidal agents and inducing a necrotic parasite cell death.     

Metronidazole–Deoxybenzoin Derivatives as Anti‐Helicobacter pylori Agents with Potent Inhibitory Activity against HPE‐Induced Interleukin‐8

A series of new metronidazole–deoxybenzoin derivatives were synthesized and evaluated for their antimicrobial activity against Helicobacter pylori. Highly selective anti‐H. pylori activity was also observed in synthesized compounds. Compound 34 exhibited the most potent activity, similar to the positive control amoxicillin. Furthermore, compounds 17 and 34 were able to significantly decrease H. pylori water extract (HPE)‐induced production of interleukin‐8 (IL‐8) in gastric mucosal cells, which did not show any effect on the cell viability.     

DNA Binding Ellipticine Analogues: Synthesis,Biological Evaluation,and Structure–Activity Relationships

Novel angular and branched ellipticine‐correlated anticancer agents were developed. In particular, compound 24 , with two basic side chains on opposite sides of the molecule, exhibits cytotoxicity in the nanomolar range, acting as a DNA intercalator and topoisomerase II inhibitor. SAR studies with pyridocarbazole derivatives in comparison with corresponding smaller pyrroloquinolines are discussed.

     

Biological Evaluation and X‐ray Co‐crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase,an Enzyme from the Redox Metabolism of Trypanosoma

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure‐based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K_i) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half‐maximal inhibitory concentration, IC₅₀) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X‐ray co‐crystal structures confirmed the binding of the ligands to the hydrophobic wall of the “mepacrine binding site” with the new, solubility‐providing vectors oriented toward the surface of the large active site.     

Docking Studies of Structurally Diverse Antimalarial Drugs Targeting PfATP6: No Correlation between in silico Binding Affinity and in vitro Antimalarial Activity.

PfATP6, a calcium‐dependent ATPase of Plasmodium falciparum, is considered the putative target of the antimalarial drug artemisinin and its derivatives. Herein, the 3D structure of PfATP6 was modeled on the basis of the crystal structure of SERCA 1a, the mammalian homologue. Model validation was achieved using protein structure checking tools. AutoDock4 was used to predict the binding affinities of artemisinin (and analogues) and various other antimalarial agents for PfATP6, for which in vitro activity is also reported. No correlation was found between the affinity of the compounds for PfATP6 predicted by AutoDock4 and their antimalarial activity.     

Synthesis and Biological Evaluation of Papain‐Family Cathepsin L‐Like Cysteine Protease Inhibitors Containing a 1,4‐Benzodiazepine Scaffold as Antiprotozoal Agents

Novel papain‐family cathepsin L‐like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure–activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3‐bromoisoxazoline moiety. Several rhodesain and falcipain‐2 inhibitors displaying single‐digit micromolar or sub‐micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.     

Antichagasic,Leishmanicidal, and Trichomonacidal Activity of 2‐Benzyl‐5‐nitroindazole‐Derived Amines

Three different series of new 5‐nitroindazole derivatives—1‐(ω‐aminoalkyl)‐2‐benzylindazolin‐3‐ones (series A ; ten compounds), 3‐(ω‐aminoalkoxy)‐2‐benzylindazoles (series B ; four compounds) and 3‐alkylamino‐2‐benzylindazoles (series C ; five compounds)—have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A , B , and C were efficient against T. cruzi. Some compounds in series A , after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C . With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole‐sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole‐sensitive and resistant isolates, showing the absence of cross‐resistance between these derivatives and the reference drug.     

Comparing the Antileishmanial Activity of Gold(I) and Gold(III) Compounds in L. amazonensis and L. braziliensis in Vitro

Abstract : A series of mononuclear coordination or organometallic Au^I/Au^III complexes ( 1 – 9 ) have been comparatively studied in vitro for their antileishmanial activity against promastigotes and amastigotes, the clinically relevant parasite form, of Leishmania amazonensis and Leishmania braziliensis. One of the cationic Au^I bis-N-heterocyclic carbenes ( 3 ) has low EC₅₀ values (ca. 4 μM) in promastigotes cells and no toxicity in host macrophages. Together with two other Au^III complexes ( 6 and 7 ), the compound is also extremely effective in intracellular amastigotes from L. amazonensis. Initial mechanistic studies include an evaluation of the gold complexes′ effect on L. amazonensis’ plasma membrane integrity.     

Structure–Activity Relationships and Molecular Docking of Novel Dihydropyrimidine‐Based Mitotic Eg5 Inhibitors

Dihydropyrimidine‐based compounds belong to the first discovered inhibitors of the human mitotic kinesin Eg5. Although they are used by many research groups as model compounds for chemical genetics, considerably less emphasis has been placed on the improvement of this type of inhibitor, with the exception of two recent studies. Dihydropyrimidines can be divided into class I (analogues that bind in the S configuration) and class II type inhibitors, which bind in the R configuration. Herein we report the synthesis and optimization of novel class II type dihydropyrimidines using a combination of in vitro and docking techniques.     

Synthesis and Quantitative Structure–Activity Relationships of Selective BCRP Inhibitors

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ABC transporter superfamily. This protein has a number of physiological functions, including protection of the human body from xenobiotics. The overexpression of BCRP in certain tumor cell lines causes cross‐resistance against various drugs used in chemotherapeutic treatment. In a previous work we showed that a new class of compounds derived from XR9576 (tariquidar) selectively inhibits BCRP. In this work we synthesized more members of this class, with modification on the second and third aromatic rings. The inhibitory activities against BCRP and P‐gp were assayed using a Hoechst 33342 assay for BCRP and a calcein AM assay for P‐gp. Finally, quantitative structure–activity relationships for both aromatic rings were established. The results obtained show the importance of the electron density on the third aromatic ring, influenced by substituents, pointing to interactions with aromatic residues of the protein binding site. In the second aromatic ring the activity of compounds is influenced by the steric volume of the substituents.     

Synthesis,Bioactivity, Docking and Molecular Dynamics Studies of Furan‐Based Peptides as 20S Proteasome Inhibitors

Proteasome inhibitors are promising compounds for a number of therapies, including cardiovascular and eye diseases, diabetes, and cancers. We previously reported a series of furan‐based peptidic inhibitors with moderate potencies against the proteasome β5 subunit, hypothesizing that the C‐terminal furyl ketone motif could form a covalent bond with the catalytic residue, threonine 1. In this context, we describe further optimizations of the furan‐based peptides, and a series of dipeptidic and tripeptidic inhibitors were designed and synthesized, aiming at improved potency and better solubility. Most of the tripeptidic inhibitors demonstrated improved potency and selectivity as β5 subunit inhibitors in both enzymatic and cellular assays, and good antineoplastic activities in various tumor cell lines were also observed. However, no inhibitory effects were observed for the dipeptidic compounds, which led us to presume that a noncovalent binding mode is adopted. Docking studies and molecular dynamics simulations were carried out to verify this presumption, with results showing that the distance between the furyl ketone motif and Thr1 is slightly too long to form covalent bond.     

Anti‐Dengue‐Virus Activity and Structure–Activity Relationship Studies of Lycorine Derivatives

Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. No vaccines or specific therapeutics are currently available for dengue. Lycorine, which is a natural plant alkaloid, has been shown to possess antiviral activities against flaviviruses. In this study, a series of novel lycorine derivatives were synthesized and assayed for their inhibition of dengue virus (DENV) in cell cultures. Among the lycorine analogues, 1‐acetyllycorine exhibited the most potent anti‐DENV activity (EC₅₀=0.4 μM ) with a reduced cytotoxicity (CC₅₀>300 μM ), which resulted in a selectivity index (CC₅₀/EC₅₀) of more than 750. The ketones 1‐acetyl‐2‐oxolycorine (EC₅₀=1.8 μM ) and 2‐oxolycorine (EC₅₀=0.5 μM ) also exhibited excellent antiviral activities with low cytotoxicity. Structure–activity relationships for the lycorine derivatives against DENV are discussed. A three‐dimensional quantitative structure–activity relationship model was established by using a comparative molecular‐field analysis protocol in order to rationalize the experimental results. Further modifications of the hydroxy group at the C1 position with retention of a ketone at the C2 position could potentially lead to inhibitors with improved overall properties.     

Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure–Activity Relationships,Bioisosteric Imidazopyridine Nitriles,and X‐ray Crystal Structure Analysis with Human Cathepsin L

The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure‐based design and resulted in inhibitors with inhibition constants in the single‐digit nanomolar range. Comprehensive structure–activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high‐affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X‐ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub‐micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell‐based assays, the introduction of a basic side chain on the inhibitors resulted in a 35‐fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off‐target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quantitative in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.     

Quinoline-Dihydropyrimidin-2(1H)-one Hybrids: Synthesis,Biological Activity,and Mechanistic Studies

A novel class of quinoline-dihydropyrimidin-2(1H)-one (DHPM) hybrids was synthesized and in vitro antiplasmodial activity was evaluated against chloroquine sensitive (D10) and chloroquine resistant (Dd2) strains of Plasmodium falciparum, the human malaria parasite. The antiplasmodial activity was compared to previously reported DHPM based molecular hybrids. Dual mode of antiplasmodial action of the most active member has been evaluated through heme binding study and in silico docking in the active site of dihydrofolate enzymes (wild-type as well as mutant). Favourable pharmacokinetic parameters were predicted in the ADMET evaluation. The new hybrids were also tested against a number of DNA and RNA viruses. No antiviral activity was found, except for one hybrid that showed mild inhibitory activity against two strains of cytomegalovirus (AD-169 and Davis), The most active hybrid was found to be a selective inhibitor of the growth of P. falciparum as well as a modest inhibitor of varicella zoster virus in HEL cells. Cytotoxicity of all hybrids was assessed in HEL, HeLa, Vero, MDCK, and CRFK cell cultures.     

Non‐natural Peptide Triazole Antagonists of HIV‐1 Envelope gp120

We investigated the derivation of non‐natural peptide triazole dual receptor site antagonists of HIV‐1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG‐156 [R‐I‐N‐N‐I‐X‐W‐S‐E‐A‐M‐M‐CONH₂, in which X=ferrocenyltriazole‐Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co‐receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG‐156, typified by UM‐24 (Cit‐N‐N‐I‐X‐W‐S‐CONH₂) and containing the critical central stereospecific ^LX‐^LW cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM‐24 to the greatest extent possible. The analogue with the critical “hot spot” residue Trp 6 replaced with L ‐3‐benzothienylalanine (Bta) (KR‐41), as well as a completely non‐natural analogue containing D ‐amino acid substitutions outside the central cluster (KR‐42, ^DCit‐^DN‐^DN‐^DI‐X‐Bta‐^DS‐CONH₂), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV‐1 Env gp120.