A Virtual Screening Study of the 18 kDa Translocator Protein using Pharmacophore Models Combined with 3D‐QSAR Studies

In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D‐QSAR studies. This hypothesis was used in a ligand‐based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 μM , and among them two compounds exhibited sub‐micromolar K_i values. These results validated our applied methodologies, and the two compounds with sub‐micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.     

Synthesis and Antiplasmodial Activity of Novel Chloroquine Analogues with Bulky Basic Side Chains

Chloroquine is commonly used in the treatment and prevention of malaria, but Plasmodium falciparum, the main species responsible for malaria‐related deaths, has developed resistance against this drug. Twenty‐seven novel chloroquine (CQ) analogues characterized by a side chain terminated with a bulky basic head group, i.e., octahydro‐2H‐quinolizine and 1,2,3,4,5,6‐hexahydro‐1,5‐methano‐8H‐pyrido[1,2‐a][1,5]diazocin‐8‐one, were synthesized and tested for activity against D‐10 (CQ‐susceptible) and W‐2 (CQ‐resistant) strains of P. falciparum. Most compounds were found to be active against both strains with nanomolar or sub‐micromolar IC₅₀ values. Eleven compounds were found to be 2.7‐ to 13.4‐fold more potent than CQ against the W‐2 strain; among them, four cytisine derivatives appear to be of particular interest, as they combine high potency with low cytotoxicity against two human cell lines (HMEC‐1 and HepG2) along with easier synthetic accessibility. Replacement of the 4‐NH group with a sulfur bridge maintained antiplasmodial activity at a lower level, but produced an improvement in the resistance factor. These compounds warrant further investigation as potential drugs for use in the fight against malaria.     

Orally active, antimetastatic, nontoxic diphenyl ether-derived carbamoylphosphonate matrix metalloproteinase inhibitors

Seven 4‐phenoxybenzenesulfonamidopolymethylene carbamoylphosphonates (CPOs) bearing two to eight methylene units in the polymethylene chain were synthesized and evaluated as matrix metalloproteinase (MMP) inhibitors. The five lowest homologues [(CH₂)_2?6] are selective MMP‐2 inhibitors, whereas the two with the longest linkers [(CH₂)_{7, 8}] lack inhibitory activity. The most potent homologues are those with (CH₂)_{5, 6}; these two were evaluated for antimetastatic activity in a murine melanoma model and showed good potency both by oral and intraperitoneal administration without any toxic—including musculoskeletal—side effects. In contrast to the previously reported cis‐ACCP, which was shown to inhibit MMP‐2 for ～30 min, the new compounds inhibit MMP activity for the duration of measurement, lasting several hours. Pharmacokinetic evaluation revealed, on the one hand, low oral bioavailability; on the other hand, a relatively large calculated volume of distribution, consistent with the observed reversible absorption of CPO 5 to hydroxyapatite, as a model for bone.     

Synthetic and Biological Studies of Tubulin Targeting C2‐Substituted 7‐Deazahypoxanthines Derived from Marine Alkaloid Rigidins

C2‐aryl‐ and C2‐alkyl‐7‐deazahypoxanthines as analogues of marine alkaloid rigidins were prepared utilizing novel synthetic methods developed for the construction of the pyrrolo[2,3‐d]pyrimidine ring system. The new compounds exhibited sub‐micromolar to nanomolar antiproliferative potencies against a panel of cell lines including in vitro models for drug‐resistant tumors, such as glioblastoma, melanoma and non‐small‐cell lung cancer. A selected representative C2‐methyl‐7‐deazahypoxanthine was found to inhibit microtubule dynamics in cancer cells, lending evidence for tubulin targeting as a mode of action for these compounds in cancer cells. The results of the docking studies utilizing the colchicine site on β‐tubulin were consistent with the observed structure–activity relationship data, including an important finding that derivatization at C2 with linear alkyl groups leads to the retention of activity, thus permitting the attachment of a biotin‐containing linker for the subsequent proteomics assays. Because many microtubule‐targeting compounds are successfully used to fight cancer in the clinic, the reported antitubulin rigidin analogues have significant potential as new anticancer agents.     

Discovery of a Class of Diketopiperazines as Antiprion Compounds

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d , which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism‐of‐action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP^Sc)‐like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.     

Synthesis and Evaluation of a Series of Bis(pentylpyridinium) Compounds as Antifungal Agents

A series of bis(4‐pentylpyridinium) compounds with a variety of spacers between the pyridinium headgroups was synthesised, and the antifungal activity of these compounds was investigated. Lengthening the alkyl spacer between the pentylpyridinium headgroups from 12 to 16 methylene units resulted in increased antifungal activity against C. neoformans and C. albicans, but also resulted in increased hemolytic activity and cytotoxicity against mammalian cells. However, inclusion of an ortho‐substituted benzene ring in the centre of the alkyl spacer resulted in decreased cytotoxicity and hemolytic activity, while maintaining antifungal potency. Replacement of the alkyl and aromatic‐containing spacers by more hydrophilic ethylene glycol groups resulted in a loss of antifungal activity. Some of the compounds inhibited fungal PLB1 activity, but the low correlation of this inhibition with antifungal potency indicates PLB1 inhibition is unlikely to be the predominant mode of antifungal action of this class of compounds, with preliminary studies suggesting they may act via disruption of fungal mitochondrial function.     

Rational Optimization of Dihydropyrimidinone-Quinoline Hybrids as Plasmodium falciparum Glutathione Reductase Inhibitors

A series of dihydropyrimidinone-based antimalarial compounds were designed and synthesised based on the previously identified amide-based quinoline hybrids which showed good resistance reversal ability against the resistant strain of Plasmodium falciparum. The aromatic ring on the dihydropyrimidinone of the original hits was exchanged for a methyl group to bring the molecular weights below 500 Da and also determine the effect of the aromatic ring count on the resistance reversal ability of the hybrids. Apart from the previously used amide bond, the hybrid linker was also extended to the triazole linker. Although the triazole linker is synthetically easier to access, the use of an amide linker seems to have an activity advantage. The synthesised compounds in addition to the previously identified hits were subjected to molecular docking particularly targeting the orthosteric site of Plasmodium falciparum glutathione reductase (PfGR) protein. The ligand with the best binding interaction was rationally optimised to increase its suitability as a competitive inhibitor against the cofactor of the PfGR. Two of the optimised ligands showed better binding affinities than the cofactor while one of the two ligands displayed hydrophobically packed correlated hydrogen-bond which is very important in maintaining the ligand stability within the protein. In silico ADME predictions of the synthesised compounds indicate that these compounds possess good pharmacokinetic properties.     

Synthesis and Structure–Activity Relationship Studies of Derivatives of the Dual Aromatase–Sulfatase Inhibitor 4‐{[(4‐Cyanophenyl)(4H‐1,2,4‐triazol‐4‐yl)amino]methyl}phenyl sulfamate

4‐{[(4‐Cyanophenyl)(4H‐1,2,4‐triazol‐4‐yl)amino]methyl}phenyl sulfamate and its ortho‐halogenated (F, Cl, Br) derivatives are first‐generation dual aromatase and sulfatase inhibitors (DASIs). Structure–activity relationship studies were performed on these compounds, and various modifications were made to their structures involving relocation of the halogen atom, introduction of more halogen atoms, replacement of the halogen with another group, replacement of the methylene linker with a difluoromethylene linker, replacement of the para‐cyanophenyl ring with other ring structures, and replacement of the triazolyl group with an imidazolyl group. The most potent in vitro DASI discovered is an imidazole derivative with IC₅₀ values against aromatase and steroid sulfatase in a JEG‐3 cell preparation of 0.2 and 2.5 nM , respectively. The parent phenol of this compound inhibits aromatase with an IC₅₀ value of 0.028 nM in the same assay.     

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.     

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

A series of iminoxylitol derivatives carrying a C‐linked di‐O‐acyl or di‐O‐alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β‐glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di‐O‐hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di‐O‐decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM ); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat‐stress studies, the diether was found to be much more effective in stabilizing the wild‐type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di‐C₁₀‐ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.     

Structure-activity relationship refinement and further assessment of indole-3-glyoxylamides as a lead series against prion disease

Structure–activity relationships within the indole‐3‐glyoxylamide series of antiprion agents have been explored further, resulting in discovery of several new compounds demonstrating excellent activity in a cell line model of prion disease (EC₅₀ <10 nM ). After examining a range of substituents at the para‐position of the N‐phenylglyoxylamide moiety, five‐membered heterocycles containing at least two heteroatoms were found to be optimal for the antiprion effect. A number of modifications were made to probe the importance of the glyoxylamide substructure, although none were well tolerated. The most potent compounds did, however, prove largely stable towards microsomal metabolism, and the most active library member cured scrapie‐infected cells indefinitely on administration of a single treatment. The present results thereby confirm the indole‐3‐glyoxylamides as a promising lead series for continuing in vitro and in vivo evaluation against prion disease.     

Glucocerebrosidase Enhancers for Selected Gaucher Disease Genotypes by Modification of α‐1‐C‐Substituted Imino‐D‐xylitols (DIXs) by Click Chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM . Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.     

Synthesis and biological evaluation of acridine derivatives as antimalarial agents

New N‐alkylaminoacridine derivatives attached to nitrogen heterocycles were synthesized, and their antimalarial potency was examined. They were tested in vitro against the growth of Plasmodium falciparum, including chloroquine (CQ)‐susceptible and CQ‐resistant strains. This biological evaluation has shown that the presence of a heterocyclic ring significantly increases the activity against P. falciparum. The best compound shows a nanomolar IC₅₀ value toward parasite proliferation on both CQ‐susceptible and CQ‐resistant strains. The antimalarial activity of these new acridine derivatives can be explained by the two mechanisms studied in this work. First, we showed the capacity of these compounds to inhibit heme biocrystallization, a detoxification process specific to the parasite and essential for its survival. Second, in our search for alternative targets, we evaluated the in vitro inhibitory activity of these compounds toward Sulfolobus shibatae topoisomerase VI‐mediated DNA relaxation. The preliminary results obtained reveal that all tested compounds are potent DNA intercalators, and significantly inhibit the activity of S. shibatae topoisomerase VI at concentrations ranging between 2.0 and 2.5 μM .     

Synthesis and Biological Evaluation of Ferrocenylquinoline as a Potential Antileishmanial Agent

The emergence of resistance against antileishmanial drugs in current use necessitates the search for new classes of antileishmanial compounds. Herein we report the design, synthesis, and evaluation of a novel ferrocenylquinoline for activity against Leishmania donovani. 7‐Chloro‐N‐[2‐(1H‐5‐ferrocenyl‐1,2,3‐triazol‐1‐yl)ethyl]quinolin‐4‐amine ( 1 ) was generated by coupling an iron(II) ethynylferrocene species with 4‐(2‐ethylazido)amino‐7‐chloroquinoline using click chemistry. The synthesized compound 1 was tested for its antileishmanial activity using both promastigote and amastigote stages of L. donovani. Compound 1 showed promising anti‐promastigote activity, with an IC₅₀ value of 15.26 μM and no cytotoxicity toward host splenocytes. From the battery of tests conducted in this study, it appears that this compound induces parasite death by promoting oxidative stress and depolarizing the mitochondrial membrane potential, thereby triggering apoptosis. These results suggest that ferrocenylquinoline 1 is a suitable lead for the development of new antileishmanial drugs.     

Enantiopure Indolizinoindolones with in vitro Activity against Blood‐ and Liver‐Stage Malaria Parasites

Malaria continues to be a major cause of morbidity and mortality to this day, and resistance to drugs like chloroquine has led to an urgent need to discover novel chemical entities aimed at new targets. Here, we report the discovery of a novel class of potential antimalarial compounds containing an indolizinoindolone scaffold. These novel enantiopure indolizinoindolones were synthesized, in good‐to‐excellent yields and excellent diastereoselectivities, by cyclocondensation reaction of (S)‐ or (R)‐tryptophanol and 2‐acyl benzoic acids, followed by intramolecular α‐amidoalkylation. Interestingly, we were able to synthesize for the first time 7,13b‐cis indolizinoindolones in a two‐step route. The novel compounds showed promising activity against erythrocytic stages of the human malaria parasite, Plasmodium falciparum, and liver stages of the rodent parasite Plasmodium berghei. In particular, an (S)‐tryptophanol‐derived isoindolinone was identified as a promising starting scaffold to search for novel antimalarials, combining excellent activity against both stages of the parasite′s life cycle with low cytotoxicity and excellent metabolic and chemical stability in vitro.     

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.     

N‐Benzyl‐4‐((heteroaryl)methyl)benzamides: A New Class of Direct NADH‐Dependent 2‐trans Enoyl–Acyl Carrier Protein Reductase (InhA) Inhibitors with Antitubercular Activity

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug‐sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug‐resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH‐dependent 2‐trans enoyl–acyl carrier protein reductase (InhA) inhibitors based on an N‐benzyl‐4‐((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG‐related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure–activity relationships. Furthermore, a co‐crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.     

Templated Chemistry for Sequence‐Specific Fluorogenic Detection of Duplex DNA

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple‐helix formation at adjacent positions on a specific purine‐rich target sequence of duplex DNA. One fluorescein‐labeled probe contains an α‐azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double‐stranded DNA, at least for homopurine–homopyrimidine target sites, under native and nondenaturing conditions.     

Novel Monocyclam Derivatives as HIV Entry Inhibitors: Design,Synthesis, Anti‐HIV Evaluation,and Their Interaction with the CXCR4 Co‐receptor

The CXCR4 receptor has been shown to interact with the human immunodeficiency virus (HIV) envelope glycoprotein gp120, leading to fusion of viral and cell membranes. Therefore, ligands that can attach to this receptor represent an important class of therapeutic agents against HIV, thus inhibiting the first step in the cycle of viral infection: the virus–cell entry/fusion. Herein we describe the in silico design, synthesis, and biological evaluation of novel monocyclam derivatives as HIV entry inhibitors. In vitro activity testing of these compounds in cell cultures against HIV strains revealed EC₅₀ values in the low micromolar range without cytotoxicity at the concentrations tested. Docking and molecular dynamics simulations were performed to predict the binding interactions between CXCR4 and the novel monocyclam derivatives. A binding mode of these compounds is proposed which is consistent with the main existing site‐directed mutagenesis data on the CXCR4 co‐receptor. Moreover, molecular modeling comparisons were performed between these novel monocyclams, previously reported non‐cyclam compounds from which the monocyclams are derived, and the well‐known AMD3100 bicyclam CXCR4 inhibitors. Our results suggest that these three structurally diverse CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor.