N-Pyrazolyl- and N-Triazolylamines and -ureas as Antileishmanial and Antitrypanosomal Drugs.

Three types of modifications of antileishmanial pyrazole lead compounds 7 and 8 were conducted to expand the relationships between structural features and antileishmanial/antitrypanosomal activity: (1) the pyrazole core was retained or replaced by a 1,2,4-triazole ring; (2) various aryl moieties including 2-fluorophenyl, pyridin-3-yl and pyrazin-2-yl rings were attached at 3-position of the core azole; (3) either arylmethylamino or ureido substituents were introduced at 5-position of the azole core. The synthesis followed established routes starting with esters 9 or 15 and anhydride 21. The synthesized 3-arylpyrazoles and 3-aryl-1,2,4-triazoles showed only very low antileishmanial activity. The 2-fluorophenyl-substituted pyrazole 18c revealed the highest antileishmanial activity of this series of compounds, but its IC50 value (20 µM) still indicates low activity. However, low micromolar antitrypanosomal activity was detected for the pyridin-3-yl-substituted pyrazoles 12b (IC50 = 4.7 µM) and 14a (IC50 = 2.1 µM). Their IC50 values are comparable with the IC50 values of the reference compounds benznidazole and nifurtimox. Whereas only low unspecific cytotoxicity at the primary peritoneal mouse macrophages (PMM) was detected, considerable cytotoxicity at MRC-5 human fibroblast cells was found for both pyrazoles 12b an 14a. The activity of pyrazole 12b against T. cruzi is 4-fold higher than its unspecific MRC-5 cytotoxicity.

Total synthesis of (±)-auranthine confirmed its refined structure.

Auranthine, isolated in 1986 from Penicillium aurantiogriseum, is a fungal benzodiazepine. Through the successful total synthesis of (±)-auranthine, we confirmed the refined structure of natural (-)-auranthine. We established that natural (-)-auranthine is a fused quinazolino benzodiazepine dione 1 featuring an acyclic aliphatic nitrile moiety, thereby disproving the proposed structure 2.

An automated analysis method enabling the screening of covalent thrombin and factor XIIa inhibitors via liquid chromatography-mass spectrometry.

An automated sample preparation and separation method for the analysis of various enzyme-inhibitor combinations using liquid chromatography (LC) coupled to mass spectrometry (MS) is presented. As conventional anticoagulants have several drawbacks, the most severe being the elevated risk of internal bleedings, it is necessary to develop new-generation anticoagulants with reduced side effects. Therefore, the screening of potential inhibitors against anticoagulation targets like thrombin and FXIIa is important to design a potent and selective inhibitor. To facilitate the analysis of numerous enzyme-inhibitor covalent complexes, automation of the analysis using an LC system with a user-defined injection sequence is helpful. The developed method ensures comparable reaction conditions like reaction time and temperature for all enzyme-inhibitor complexes. Furthermore, it prevents time-consuming manual sample preparation and potential manual errors. To achieve good reproducibility with relative standard deviation of approximately 3% for three-fold determination, multiple cleaning steps were added to the automated sample preparation. Subsequently, this method was applied to screen a variety of 15 aminopyrazole- and aminotriazole-based inhibitors with a covalent mechanism of action against thrombin and to test two covalent inhibitors for FXIIa. Successful complex formation and acylation of the catalytic center of the enzymes was monitored using deconvoluted mass spectra and the matching mass shifts of the acyl moiety of the analyzed inhibitors. The inhibitors' structure directly influenced reaction yields. Sterically demanding aminotriazoles and acyl moieties both affected the product formation negatively. However, the screening yielded several promising candidates for new covalent thrombin inhibitors, which might find their application as prospective anticoagulants.

Functional-Group-Tolerant Pd-Catalyzed Carbonylative Negishi Coupling with Aryl Iodides.

A chemoselective Pd-mediated carbonylative Negishi-type catalytic protocol for the synthesis of (hetero)aryl ketones is reported. The protocol employs the PEPPSI-IPr precatalyst and CO gas at atmospheric pressure (balloon) to foster the carbonylative coupling between diverse C(sp3)-hybridized organozinc reagents and a broad range of aryl iodides, including substrates carrying aldehyde, aniline, phenol, or carboxylic acid groups, and heteroaryls.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Microwave-Assisted Synthesis, Structure, and Preliminary Biological Evaluation of Novel 6-Methoxy-5,6-dihydro-5-azapurines.

We herein disclose the microwave-assisted synthesis of previously unreported 6-methoxy-5,6-dihydro-5-azapurines, whose purine-like scaffold is promising for drug discovery. The method is simple, fast, and relies on easily accessible reagents such as trimethyl orthoformate, acetic acid, and aminotriazole-derived N,N'-disubstituted formamidines. The preliminary biological evaluation revealed that selected representatives of synthesized 6-methoxy-5,6-dihydro-5-azapurines dose-dependently reduce the viability of HepG2 and A549 cancer cells having little to no influence on five tested purinergic receptors.

Semisynthetic Approach toward Biologically Active Derivatives of Phenylspirodrimanes from S. chartarum.

The phenylspirodrimanes (PSDs) from Stachybotrys chartarum represent a structurally diverse group of meroterpenoids, which, on the one hand, exhibit a structural exclusivity since their occurrence is not known for any other species and, on the other hand, offer access to chemically and biologically active compounds. In this study, phenylspirodrimanes 1-3 were isolated from S. chartarum and their water-mediated Cannizzaro-type transformation was investigated using quantum chemical DFT calculations substantiated by LC-MS and NMR experiments. Considering the inhibitory activity of PSDs against proteolytic enzymes and their modulatory effect on plasminogen, PSDs 1-3 were used as a starting material for the synthesis of their corresponding biologically active lactams. To access the library of the PSD derivatives and screen them against physiologically relevant serine proteases, a microscale semisynthetic approach was developed. This allowed us to generate the library of 35 lactams, some of which showed the inhibitory activity against physiologically relevant serine proteases such as thrombin, FXIIa, FXa, and trypsin. Among them, the agmatine-derived lactam 16 showed the highest inhibitory activity against plasma coagulation factors and demonstrated the anticoagulant activity in two plasma coagulation tests. The semisynthetic lactams were significantly less toxic compared to their parental natural PSDs.

Amide-functionalized 1,2,4-Triazol-5-amines as Covalent Inhibitors of Blood Coagulation Factor XIIa and Thrombin.

To counteract thrombosis, new safe and efficient antithrombotics are required. We herein report the design, synthesis, and biological activity of a series of amide-functionalized acylated 1,2,4-triazol-5-amines as selective inhibitors of blood coagulation factor XIIa and thrombin. The introduction of an amide moiety into the main scaffold of 3-aryl aminotriazoles added certain three-dimensional properties to synthesized compounds and allowed them to reach binding sites in FXIIa and thrombin previously unaddressed by non-functionalized 1,2,4-triazol-5-amines. Among synthesized compounds, one quinoxaline-derived aminotriazole bearing N-butylamide moiety inhibited FXIIa with the IC50 value of 28 nM, whereas the N-phenylamide-derived aminotriazole inhibited thrombin with the IC50 value of 41 nM. Performed mass-shift experiments and molecular modeling studies proved the covalent mechanism of FXIIa and thrombin inhibition by synthesized compounds. In plasma coagulation tests, developed aminotriazoles showed anticoagulant properties mainly affecting the intrinsic blood coagulation pathway, activation of which is associated with thrombosis but is negligible for hemostasis.

Pyrazole-Based Thrombin Inhibitors with a Serine-Trapping Mechanism of Action: Synthesis and Biological Activity.

New antithrombotic drugs are needed to combat thrombosis, a dangerous pathology that causes myocardial infarction and ischemic stroke. In this respect, thrombin (FIIa) represents an important drug target. We herein report the synthesis and biological activity of a series of 1H-pyrazol-5-amine-based thrombin inhibitors with a serine-trapping mechanism of action. Among synthesized compounds, flexible acylated 1H-pyrazol-5-amines 24e, 34a, and 34b were identified as potent 16-80 nM thrombin inhibitors, which showed practically no off-targeting effect against other physiologically relevant serine proteases. To prove that synthesized compounds are covalent thrombin inhibitors, the most potent derivative 24e (FIIa IC50 = 16 nM) was studied in a mass-shift assay, where it has been shown that 24e transfers its acyl moiety (pivaloyl) to the catalytic Ser195 of thrombin. Performed herein docking studies also confirmed the covalent mechanism of thrombin inhibition by synthesized compounds. Acylated aminopyrazoles found during this study showed only limited effects on plasma coagulation in activated partial thrombin time (aPTT) and prothrombin time (PT) in vitro assays. However, such thrombin inhibitors are expected to have virtually no effect on bleeding time and can be used as a starting point for developing a safer alternative to traditional non-covalent anticoagulants.

Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors.

Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N-acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post-screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N-acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N-acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.

Factor XII(a) inhibitors: a review of the patent literature.

Introduction: Blood coagulation factor XII (FXII) is an emerging and potentially safe drug target, which dysregulation is associated with thrombosis, hereditary angioedema, and (neuro)inflammation. At the same time, FXII-deficiency is practically asymptomatic. Industrial and academic institutions have developed a number of potential therapeutic agents targeting either FXII zymogen or its active form FXIIa for the treatment of thrombotic and inflammatory conditions associated with the activity of this enzyme.Areas covered: A short overview of the FXII(a) structure and function, underlining its suitability as a drug target, is given. The article reviews patents reported over the last three decades on FXII(a)-targeting therapeutic agents. These agents include small molecules, proteins, peptides, oligonucleotides, siRNAs, and monoclonal antibodies.Expert opinion: The performed analysis of patents revealed that many FXII(a) inhibitors are in the early preclinical stage, while several already showed efficacy in vivo animal models of thrombosis, sepsis, hereditary angioedema, and multiple sclerosis. Two anti-FXIIa agents namely tick protein Ir-CPI and monoclonal antibody CSL312 are currently in human clinical trials. The results of these trials and further studies of FXII(a) pathophysiological functions will encourage the development of new FXII(a) inhibitors.

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Structure-Based Design, Synthesis, and Biological Evaluation of Triazole-Based smHDAC8 Inhibitors.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, which affects over 200 million people worldwide and leads to at least 300,000 deaths every year. In this study, initial screening revealed the triazole-based hydroxamate 2 b (N-hydroxy-1-phenyl-1H-1,2,3-triazole-4-carboxamide) exhibiting potent inhibitory activity toward the novel antiparasitic target Schistosoma mansoni histone deacetylase 8 (smHDAC8) and promising selectivity over the major human HDACs. Subsequent crystallographic studies of the 2 b/smHDAC8 complex revealed key interactions between the inhibitor and the enzyme's active site, thus explaining the unique selectivity profile of the inhibitor. Further chemical modifications of 2 b led to the discovery of 4-fluorophenoxy derivative 21 (1-[5-chloro-2-(4-fluorophenoxy)phenyl]-N-hydroxy-1H-1,2,3-triazole-4-carboxamide), a nanomolar smHDAC8 inhibitor (IC50 =0.5 µM), exceeding the smHDAC8 inhibitory activity of 2 b and SAHA (vorinostat), while exhibiting an improved selectivity profile over the investigated human HDACs. Collectively, this study reveals specific interactions between smHDAC8 and the synthesized triazole-based inhibitors and demonstrates that these small molecules represent promising lead structures, which could be further developed in the search for novel drugs for the treatment of schistosomiasis.

Proline-based hydroxamates targeting the zinc-dependent deacetylase LpxC: Synthesis, antibacterial properties, and docking studies.

The Zn2+-dependent deacetylase LpxC is an essential enzyme in Gram-negative bacteria, which has been validated as antibacterial drug target. Herein we report the chiral-pool synthesis of novel d- and l-proline-derived 3,4-dihydroxypyrrolidine hydroxamates and compare their antibacterial and LpxC inhibitory activities with the ones of 4-monosubstituted and 3,4-unsubstituted proline derivatives. With potent antibacterial activities against several Gram-negative pathogens, the l-proline-based tertiary amine 41g ((S)-N-hydroxy-1-(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)pyrrolidine-2-carboxamide) was found to be the most active antibacterial compound within the investigated series, also showing some selectivity toward EcLpxC (Ki = 1.4 µM) over several human MMPs.

Development of Novel Quinoxaline-Based κ-Opioid Receptor Agonists for the Treatment of Neuroinflammation.

Neuroinflammatory disorders, such as multiple sclerosis or experimental autoimmune encephalomyelitis (EAE), an established mouse model mimicking part of the human pathology, are characterized by inflammatory infiltrates containing T helper 1 (TH1) and TH17 cells, which cause demyelination and neurodegeneration. Disease onset and perpetuation are mediated by peripherally generated autoreactive T cells infiltrating into the central nervous system, where they are restimulated by antigen-presenting cells. Here, we show that newly designed peripherally active, potent, and selective κ-opioid receptor (KOR) agonists comprising the ethylenediamine KOR pharmacophore in a perhydroquinoxaline scaffold exhibit potent anti-inflammatory capacities in primary antigen presenting cells as well as T cells. In the EAE model, the secondary amine 12 and the triazole 14 were able to ameliorate disease severity and to delay disease onset by blocking effector T cell activation. Importantly, the beneficial effects were mediated via signaling through KOR because off-target effects were excluded by using KOR-deficient mouse mutants.

Auranthine, a Benzodiazepinone from Penicillium aurantiogriseum: Refined Structure, Absolute Configuration, and Cytotoxicity.

The structure of the known Penicillium aurantiogriseum-derived secondary metabolite auranthine was refined using a combination of synthetic, spectroscopic, and X-ray diffractometric approaches. Thus, auranthine was shown to be a fused quinazolino benzodiazepinedione (2) bearing an acyclic aliphatic nitrile moiety, thereby significantly differing from the originally proposed structure 1 published in 1986. Its absolute configuration was confirmed by CD spectroscopy and DFT calculations. The cultivation of P. aurantiogriseum was optimized, allowing high production of auranthine. The cytotoxicity profile of auranthine and its semisynthetic analogues is reported. The refined structure of auranthine provides a valid target for the total synthesis of this underexplored natural product and its derivatives.

Characterization of Histone Deacetylase 8 (HDAC8) Selective Inhibition Reveals Specific Active Site Structural and Functional Determinants.

Metal-dependent histone deacetylases (HDACs) are key epigenetic regulators that represent promising therapeutic targets for the treatment of numerous human diseases. Yet the currently FDA-approved HDAC inhibitors nonspecifically target at least several of the 11 structurally similar but functionally different HDAC isozymes, which hampers their broad usage in clinical settings. Selective inhibitors targeting single HDAC isozymes are being developed, but precise understanding in molecular terms of their selectivity remains sparse. Here, we show that HDAC8-selective inhibitors adopt a L-shaped conformation required for their binding to a HDAC8-specific pocket formed by HDAC8 catalytic tyrosine and HDAC8 L1 and L6 loops. In other HDAC isozymes, a L1-L6 lock sterically prevents L-shaped inhibitor binding. Shielding of the HDAC8-specific pocket by protein engineering decreases potency of HDAC8-selective inhibitors and affects catalytic activity. Collectively, our results unravel key HDAC8 active site structural and functional determinants important for the design of next-generation chemical probes and epigenetic drugs.

Replacement of the Benzylpiperidine Moiety with Fluorinated Phenylalkyl Side Chains for the Development of GluN2B Receptor Ligands.

The 4-benzylpiperidine moiety is a central structural element of potent N-methyl-d-aspartate (NMDA) receptor antagonists containing the GluN2B subunit. To obtain novel GluN2B ligands suitable for positron emission tomography, the benzylpiperidine moiety was replaced with fluorinated ω-phenylalkylamino groups. For this purpose three primary propyl- and butylamines 7 a-c and one butyraldehyde 7 d bearing a fluorine atom and an ω-phenyl moiety were prepared in 3- to 7-step syntheses. Compounds 7 a-d were attached to various scaffolds of potent GluN2B antagonists (scaffold hopping) instead of the original 4-benzylpiperidine moiety. Although benzoxazol-2-ones and indoles with a benzylpiperidine moiety show high GluN2B affinity, the corresponding fluorophenylalkylamine derivatives did not result in high Glu2B affinity. Moderate GluN2B affinity was observed for a 3-(fluoroalkyl)-substituted tetrahydro-1H-3-benzazepine (Ki =239 nm). However, high GluN2B affinity was obtained for the tetrahydro-5H-benzo[7]annulen-7-amines 12 a-c (Ki =17-30 nm). Docking studies resulted in the same binding pose for 12 a as for the lead compound Ro 25-6981. It can be concluded that some GluN2B ligands (benzoxazolones, indoles) do not tolerate replacement of the 4-benzylpiperidine moiety with flexible fluorinated phenylalkyl side chains, but other scaffolds such as tetrahydro-3-benzazepines and -benzo[7]annulenes retain interaction with NMDA receptors containing the GluN2B subunit.

LpxC inhibitors: a patent review (2010-2016).

INTRODUCTION: The Zn2+-dependent deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising target for the development of antibiotics selectively combating Gram-negative pathogens. Researchers from industry and academia have synthesized structurally diverse LpxC inhibitors, exhibiting different LpxC inhibitory and antibacterial activities. Areas covered: A brief introduction into the structure and function of LpxC, showing its suitability as antibacterial target, along with the structures of several reported LpxC inhibitors, is given. The article reviews patents (reported between 2010 and 2016) and related research publications on novel small-molecule LpxC inhibitors. Emphasis is placed on structure-activity relationships within the reported series of LpxC inhibitors. Expert opinion: The performed analysis of patents revealed that the current search for novel LpxC inhibitors is focused on small molecules, sharing common structural features like a Zn2+-chelating group as well as a highly lipophilic side-chain. However, despite the promising preclinical data of many of the reported compounds, besides the recently withdrawn clinical candidate ACHN-975, no other LpxC inhibitor has entered clinical trials. The lack of clinical candidates might be related with undesired effects caused by the common structural elements of the LpxC inhibitors.

Novel Potent Proline-Based Metalloproteinase Inhibitors: Design, (Radio)Synthesis, and First in Vivo Evaluation as Radiotracers for Positron Emission Tomography.

As dysregulation of matrix metalloproteinase (MMP) activity is associated with a wide range of pathophysiological processes like cancer, atherosclerosis, and arthritis, MMPs represent a valuable target for the development of new therapeutics and diagnostic tools. We herein present the chiral pool syntheses, in vitro evaluation, and SAR studies of a series of d- and l-proline- as well as of (4R)-4-hydroxy-l-proline-derived MMP inhibitors possessing general formula 1. Some of the synthesized hydroxamic acids were found to be potent MMP inhibitors with IC50 values in the nanomolar range, also demonstrating no off-target effects toward the other tested Zn2+-dependent metalloproteases (ADAMs and meprins). Utilizing the structure of the (2S,4S)-configured 4-hydroxyproline derivative 4, a selective picomolar inhibitor of MMP-13, the radiolabeled counterpart [18F]4 was successfully synthesized. The radiotracer's biodistribution in mice as well as its serum stability were evaluated for assessing its potential use as a MMP-13 targeting PET imaging agent.