Exploring the unexplored chemical space: Rational identification of new Tafenoquine analogs with antimalarial properties.

Patents tend to define a huge chemical space described by the combinatorial nature of Markush structures. However, the optimization of new principal active ingredient is frequently driven by a simple Free Wilson approach. This procedure leads to a highly focused study on the chemical space near a hit compound leaving many unexplored regions that may present highly biological active reservoirs. This study aims to demonstrate that this unveiled chemical space can hide compounds with interesting potential biological activity that would be worth pursuing. This underlines the value and necessity of broadening an approach beyond conventional strategies. Hence, we advocate for an alternative methodology that may be more efficient in the early drug discovery stages. We have selected the case of Tafenoquine, a single-dose treatment for the radical cure of P. vivax malaria approved by the FDA in 2018, as an example to illustrate the process. Through the deep exploration of the Tafenoquine chemical space, seven compounds with potential antimalarial activity have been rationally identified and synthesized. This small set is representative of the chemical diversity unexplored by the 58 analogs reported to date. After biological assessment, results evidence that our approach for rational design has proven to be a very efficient exploratory methodology suitable for the early drug discovery stages.

Evaluation of Free Online ADMET Tools for Academic or Small Biotech Environments.

For a new molecular entity (NME) to become a drug, it is not only essential to have the right biological activity also be safe and efficient, but it is also required to have a favorable pharmacokinetic profile including toxicity (ADMET). Consequently, there is a need to predict, during the early stages of development, the ADMET properties to increase the success rate of compounds reaching the lead optimization process. Since Lipinski's rule of five, the prediction of pharmacokinetic parameters has evolved towards the current in silico tools based on empirical approaches or molecular modeling. The commercial specialized software for performing such predictions, which is usually costly, is, in many cases, not among the possibilities for research laboratories in academia or at small biotech companies. Nevertheless, in recent years, many free online tools have become available, allowing, more or less accurately, for the prediction of the most relevant pharmacokinetic parameters. This paper studies 18 free web servers capable of predicting ADMET properties and analyzed their advantages and disadvantages, their model-based calculations, and their degree of accuracy by considering the experimental data reported for a set of 24 FDA-approved tyrosine kinase inhibitors (TKIs) as a model of a research project.

Synthesis of Chalcones: An Improved High-Yield and Substituent-Independent Protocol for an Old Structure.

Chalcones are a type of molecule that can be considered as easily synthesizable through aldol condensation or that can be readily purchased from habitual commercial vendors. However, on reviewing the literature, one realizes that there are no standard procedures for such aldol condensations, that there exists a wide range of alternative methods for the aldol condensation (indicating that such a condensation is not always simple), and that, in many cases, low yields are obtained that involve purifications by recrystallization or column chromatography. To develop a robust standard protocol independent of the nature of the substituents present on the acetophenone or the benzaldehyde involved in the aldol condensation leading to the chalcone, we made a comparison between an aldol condensation in KOH/EtOH and a Wittig reaction between the corresponding ylide and benzaldehyde in water. We describe an improved procedure for the Wittig reaction and a protocol for the elimination of the Ph3P=O byproduct (and the excess of ylide used) by filtration of the crude reaction product through a silica gel plug. We thus demonstrate that such an improved procedure can be a general method for the synthesis of chalcones in high yield and excellent purity and is clearly an improvement on the classical aldol condensation.

1H-Pyrazolo[3,4-b]pyridines: Synthesis and Biomedical Applications.

Pyrazolo[3,4-b]pyridines are a group of heterocyclic compounds presenting two possible tautomeric forms: the 1H- and 2H-isomers. More than 300,000 1H-pyrazolo[3,4-b]pyridines have been described which are included in more than 5500 references (2400 patents) up to date. This review will cover the analysis of the diversity of the substituents present at positions N1, C3, C4, C5, and C6, the synthetic methods used for their synthesis, starting from both a preformed pyrazole or pyridine, and the biomedical applications of such compounds.

A captured room temperature stable Wheland intermediate as a key structure for the orthogonal decoration of 4-amino-pyrido[2,3-d]pyrimidin-7(8H)-ones.

Wheland intermediates are usually unstable compounds and only a few have been isolated at very low temperatures. During our work on tyrosine kinase inhibitors, we studied the bromination of 7 in order to obtain a dibromo substituted pyrido[2,3-d]pyrimidin-7(8H)-one which could be orthogonally decorated. Surprisingly, treatment of 7 with 3 equiv. of Br2 in acetic acid (AcOH) afforded 12, a captured room temperature stable Wheland bromination intermediate stabilized by the bromination of the imino tautomer of the amino group at C4 of the pyridopyrimidine skeleton. The structure was confirmed by crystal structure determination from powder X-ray diffraction data. Treatment of 12 with DMSO afforded the dibromo substituted compound 13 presenting a bromine atom at C6 and C5-C6 unsaturation. 13 was directly accessed by treating 7 with N-bromosuccinimide (NBS), a protocol extended to other compounds using NBS or N-iodosuccinimide (NIS) to afford 6-halo substituted systems. 26, bearing an iodine at C6 and a p-bromophenylamino at C2, allows the orthogonal decoration of pyridopyrimidines.

C4-C5 fused pyrazol-3-amines: when the degree of unsaturation and electronic characteristics of the fused ring controls regioselectivity in Ullmann and acylation reactions.

Pyrazol-3-amine is a scaffold present in a large number of compounds with a wide range of biological activities and, in many cases, the heterocycle is C4-C5 fused to a second ring. Among the different reactions used for the decoration of the pyrazole ring, Ullmann and acylation have been widely applied. However, there is some confusion in the literature regarding the regioselectivity of such reactions (substitution at N1 or N2 of the pyrazole ring) and no predictive rule has been so far established. As a part of our work on 3-amino-pyrazolo[3,4-b]pyridones 13, we have studied the regioselectivity of such reactions in different C4-C5 fused pyrazol-3-amines. As a rule of thumb, the Ullmann and acylation reactions take place, predominantly, at the NH and non-protonated nitrogen atom of the pyrazole ring respectively, of the most stable initial tautomer (1H- or 2H-pyrazole), which can be easily predicted by using DFT calculations.

Pyrido[2,3-d]pyrimidin-7(8H)-ones: Synthesis and Biomedical Applications.

Pyrido[2,3-d]pyrimidines (1) are a type of privileged heterocyclic scaffolds capable of providing ligands for several receptors in the body. Among such structures, our group and others have been particularly interested in pyrido[2,3-d]pyrimidine-7(8H)-ones (2) due to the similitude with nitrogen bases present in DNA and RNA. Currently there are more than 20,000 structures 2 described which correspond to around 2900 references (half of them being patents). Furthermore, the number of references containing compounds of general structure 2 have increased almost exponentially in the last 10 years. The present review covers the synthetic methods used for the synthesis of pyrido[2,3-d]pyrimidine-7(8H)-ones (2), both starting from a preformed pyrimidine ring or a pyridine ring, and the biomedical applications of such compounds.

Stable 5,5'-Substituted 2,2'-Bipyrroles: Building Blocks for Macrocyclic and Materials Chemistry.

The preparation and characterization of a family of stable 2,2'-bipyrroles substituted at positions 5 and 5' with thienyl, phenyl, TMS-ethynyl, and vinyl groups is reported herein. The synthesis of these new bipyrroles comprises three steps: formation of the corresponding 5,5'-unsubstituted bipyrrole, bromination, and Stille or Suzuki coupling. The best results in the coupling are obtained using the Stille reaction under microwave irradiation. The new compounds have been fully characterized by UV-vis absorption, fluorescence, and IR spectroscopies and cyclic voltammetry. X-ray single-crystal analysis of four of the synthesized bipyrroles indicates a trans coplanar geometry of the pyrrole rings. Furthermore, the substituents at positions 5,5' remain coplanar to the central rings. This particular geometry extends the π-conjugation of the systems, which is in agreement with a red-shifting observed for the λmax of the substituted molecules compared to the unsubstituted bipyrrole. All of these new compounds display a moderate fluorescence. In contrast with unsubstituted bipyrroles, these bipyrroles are endowed with a high chemical and thermal stability and solubility in organic solvents.

Nitrogen positional scanning in tetramines active against HIV-1 as potential CXCR4 inhibitors.

The paradigm, derived from bicyclams and other cyclams, by which it is necessary to use the p-phenylene moiety as the central core in order to achieve high HIV-1 antiviral activities has been reexamined for the more flexible and less bulky structures 4, previously described by our group as potent HIV-1 inhibitors. The symmetrical compounds 7{x,x} and the non-symmetrical compounds 8{x,y} were designed, synthesized and biologically evaluated in order to explore the impact on the biological activity of the distance between the phenyl ring and the first nitrogen atom of the side chains. EC50 exactly followed the order 7{x,x} < 8{x,x} < 4{x,x} indicating that, for such flexible tetramines, the presence of two methylene units on each side of the central phenyl ring increases the biological activity contrary to AMD3100. A computational study of the interactions of 4{3,3}, 7{3,3} and 8{3,3} with CXCR4 revealed interactions in the same pocket region with similar binding modes for 4{3,3} and 7{3,3} but a different one for 8{3,3}.

Investigating molecular dynamics-guided lead optimization of EGFR inhibitors.

The epidermal growth factor receptor (EGFR) is part of an extended family of proteins that together control aspects of cell growth and development, and thus a validated target for drug discovery. We explore in this work the suitability of a molecular dynamics-based end-point binding free energy protocol to estimate the relative affinities of a virtual combinatorial library designed around the EGFR model inhibitor 6{1} as a tool to guide chemical synthesis toward the most promising compounds. To investigate the validity of this approach, selected analogs including some with better and worse predicted affinities relative to 6{1} were synthesized, and their biological activity determined. To understand the binding determinants of the different analogs, hydrogen bonding and van der Waals contributions, and water molecule bridging in the EGFR-analog complexes were analyzed. The experimental validation was in good qualitative agreement with our theoretical calculations, while also a 6-dibromophenyl-substituted compound with enhanced inhibitory effect on EGFR compared to the reference ligand was obtained.

When Unsuspected Crystallinity Ruins Biological Testing in Early Discovery: A Case Study.

The impact of the crystalline or amorphous structure of a solid on the solubility and pharmacokinetic properties of a drug candidate is always considered by the pharmaceutical industry during the development of a new drug; however, it is not so frequently considered during the early drug discovery process by organic and medicinal chemists, particularly those working in academia. We want to share, as an example, the false negative obtained in the biological testing of a solid sample of a tyrosine kinase inhibitor due to its unexpected crystallinity and lower solubility with respect to a solid amorphous batch of the same compound and the experimentation carried out to establish the origin of such a discrepancy.

A new and practical method for the synthesis of 6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3Η,8Η)-diones.

A one step general synthetic methodology for the synthesis of 6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (17{[Formula: see text]} ([Formula: see text]) and 20{[Formula: see text]} ([Formula: see text])) is described. This methodology is based on reacting a 2-aryl-substituted acrylate (16{[Formula: see text]}) with the corresponding 6-aminopyrimidin-4(3[Formula: see text]-one (13 ([Formula: see text]; 19 ([Formula: see text])) in presence of a base under microwave irradiation. The resulting pyrido[2,3-[Formula: see text]]pyrimidines present an aryl substituent at position C6, precisely the one directly related to the biological activity of such heterocycles. These protocols have been extended to other 2-alkyl-substituted and 3-alkyl (or aryl)-substituted acrylates but with lower yields.

Synthesis of 2-arylamino substituted 5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-ones from arylguanidines.

A practical protocol was developed for the synthesis of 2-arylamino substituted 4-amino-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones from α,ß-unsaturated esters, malononitrile, and an aryl substituted guanidine via the corresponding 3-aryl-3,4,5,6- tetrahydropyrido[2,3-d]pyrimidin-7(8H)-ones. Such compounds are formed upon treatment of 2-methoxy-6-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles with an aryl substituted guanidine in 1,4-dioxane and are converted to the desired 4-aminopyridopyrimidines with NaOMe/MeOH through a Dimroth rearrangement. The overall yields of this three-step protocol are, generally speaking, higher than the multicomponent reaction, previously developed by our group, between an α,ß-unsaturated ester, malononitrile, and an aryl substituted guanidine.

Deconstructing Markush: Improving the R&D Efficiency Using Library Selection in Early Drug Discovery.

Most of the product patents claim a large number of compounds based on a Markush structure. However, the identification and optimization of new principal active ingredients is frequently driven by a simple Free Wilson approach, leading to a highly focused study only involving the chemical space nearby a hit compound. This fact raises the question: do the tested compounds described in patents really reflect the full molecular diversity described in the Markush structure? In this study, we contrast the performance of rational selection to conventional approaches in seven real-case patents, assessing their ability to describe the patent's chemical space. Results demonstrate that the integration of computer-aided library selection methods in the early stages of the drug discovery process would boost the identification of new potential hits across the chemical space.

Overcoming Paradoxical Kinase Priming by a Novel MNK1 Inhibitor.

Targeting the kinases MNK1 and MNK2 has emerged as a valuable strategy in oncology. However, most of the advanced inhibitors are acting in an adenosine triphosphate (ATP)-competitive mode, precluding the evaluation of different binding modes in preclinical settings. Using rational design, we identified and validated the 4,6-diaryl-pyrazolo[3,4-b]pyridin-3-amine scaffold as the core for MNK inhibitors. Signaling pathway analysis confirmed a direct effect of the hit compound EB1 on MNKs, and in line with the reported function of these kinases, EB1 only affects the growth of tumor but not normal cells. Molecular modeling revealed the binding of EB1 to the inactive conformation of MNK1 and the interaction with the specific DFD motif. This novel mode of action appears to be superior to the ATP-competitive inhibitors, which render the protein in a pseudo-active state. Overcoming this paradoxical activation of MNKs by EB1 represents therefore a promising starting point for the development of a novel generation of MNK inhibitors.

Improving VEGFR-2 docking-based screening by pharmacophore postfiltering and similarity search postprocessing.

Conventional docking-based virtual screening (VS) of chemical databases is based on the ranking of compounds according to the values retrieved by a scoring function (typically, the binding affinity estimation). However, using the most suitable scoring function for each kind of receptor pocket is not always an effective process to rank compounds, and sometimes neither to distinguish between correct binding modes from incorrect ones. To improve actives from decoys selection, here we propose a three-step VS protocol, which includes the conventional docking step, a pharmacophore postfilter step, and a similarity search postprocess. This VS protocol is retrospectively applied to VEGFR-2 (Kdr-kinase) inhibitors. The resulting docking poses calculated using the Alpha HB scoring function implemented in MOE are postfiltered according to defined pharmacophore interactions (structure based). The selected poses are again ranked according to their molecular similarity (MACCS fingerprint) to the cognate ligand. Results show that both the overall and early VS performance improve the application of this protocol.

A diversity-oriented synthesis of 3-(2-amino-1,6-dihydro-6-oxo-pyrimidin-5-yl)propanoic esters.

The synthesis of dimethyl 2-(methoxymethylene) pentanedioates by an unusual Michael addition of 3,3-dimethoxypropionate to α, ß-unsaturated esters is described. These new intermediates can subsequently be converted to methyl 3-(2-amino-1,6-dihydro-6-oxo-pyrimidin-5-yl)propanoates upon treatment with guanidine carbonate. The resulting pyrimidine derivatives are open-chain analogues of pyrido[2,3-d]pyrimidines with interesting biological activities.

1,6-Naphthyridin-2(1H)-ones: Synthesis and Biomedical Applications.

Naphthyridines, also known as diazanaphthalenes, are a group of heterocyclic compounds that include six isomeric bicyclic systems containing two pyridine rings. 1,6-Naphthyridines are one of the members of such a family capable of providing ligands for several receptors in the body. Among such structures, 1,6-naphthyridin-2(1H)-ones (7) are a subfamily that includes more than 17,000 compounds (with a single or double bond between C3 and C4) included in more than 1000 references (most of them patents). This review will cover the analysis of the diversity of the substituents present at positions N1, C3, C4, C5, C7, and C8 of 1,6-naphthyridin-2(1H)-ones, the synthetic methods used for their synthesis (both starting from a preformed pyridine or pyridone ring), and the biomedical applications of such compounds.

Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3' untranslated region (3'UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3-d]pyrimidin-7-(8H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3-d]pyrimidin-7-(8H)-ones derivatives (1a-e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.

Expanding the Diversity at the C-4 Position of Pyrido[2,3-d]pyrimidin-7(8H)-ones to Achieve Biological Activity against ZAP-70.

Pyrido[2,3-d]pyrimidin-7(8H)-ones have attracted widespread interest due to their similarity with nitrogenous bases found in DNA and RNA and their potential applicability as tyrosine kinase inhibitors. Such structures, presenting up to five diversity centers, have allowed the synthesis of a wide range of differently substituted compounds; however, the diversity at the C4 position has mostly been limited to a few substituents. In this paper, a general synthetic methodology for the synthesis of 4-substituted-2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones is described. By using cross-coupling reactions, such as Ullmann, Buchwald-Hartwig, Suzuki-Miyaura, or Sonogashira reactions, catalyzed by Cu or Pd, we were able to describe new potential biologically active compounds. The resulting pyrido[2,3-d]pyrimidin-7(8H)-ones include N-alkyl, N-aryl, O-aryl, S-aryl, aryl, and arylethynyl substituents at C4, which have never been explored in connection with the biological activity of such heterocycles as tyrosine kinase inhibitors, in particular as ZAP-70 inhibitors.