In Vivo Half-Life Extension of BMP1/TLL Metalloproteinase Inhibitors Using Small-Molecule Human Serum Albumin Binders.

Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer in vivo half-lives are highly desirable. One of the most promising approaches to extend the in vivo half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of AlbuBinder 1, a small-molecule noncovalent HSA binder, to extend the in vivo half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of AlbuBinder 1 with BMP1/TLL inhibitors were prepared. In particular, conjugate c showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to AlbuBinder 1 should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies.

DNA-Encoded Macrocyclic Peptide Library.

DNA-encoded library technology (ELT) is a cutting-edge enabling technology platform for drug discovery. Here we describe how to design and synthesize a macrocyclic DNA-encoded library; how to perform selection, sequencing, and data analysis to identify potential active peptides; and how to synthesize off-DNA peptides to confirm activity. This approach provides an effective tool for pharmaceutical research based on peptides.

Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies.

Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

The Discovery of in Vivo Active Mitochondrial Branched-Chain Aminotransferase (BCATm) Inhibitors by Hybridizing Fragment and HTS Hits.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

Improving the passive permeability of macrocyclic peptides: Balancing permeability with other physicochemical properties.

A number of methods to improve the passive permeability of a set of cyclic peptides have been investigated using 6- and 7-mer macrocyclic templates. In many cases the peptides were designed by molecular dynamics calculations to evaluate the methods. The aim of this study was not only to improve passive permeability, but also to balance permeability with other physicochemical properties with the goal of understanding and applying the knowledge to develop active cyclic peptides into drug candidates. Evaluation of the methods herein suggest that increasing passive permeability often occurs at the expense of solubility and lipophilicity. Computational methods can be useful when attempting to predict and design features to balance these properties, though limitations were observed.

Intracellular drug concentration and disposition--the missing link?

Improved understanding of the concentration of a potential drug molecule at the site of action in physiologically relevant cells or tissues has emerged as a key challenge in the early stages of drug discovery. Improved ability to carry out such studies with label-free methodology has the potential to improve understanding of drug uptake and trafficking and thus contribute to the reduction of rates of attrition in drug discovery.

Switching the stereochemical outcome of 6-endo-trig cyclizations; synthesis of 2,6-cis-6-substituted 4-oxopipecolic acids.

A base-mediated 6-endo-trig cyclization of readily accessible enone-derived α-amino acids has been developed for the direct synthesis of novel 2,6-cis-6-substituted-4-oxo-L-pipecolic acids. A range of aliphatic and aryl side chains were tolerated by this mild procedure to give the target compounds in good overall yields. Molecular modeling of the 6-endo-trig cyclization allowed some insight as to how these compounds were formed, with the enolate intermediate generated via an equilibrium process, followed by irreversible tautomerization/neutralization providing the driving force for product formation. Stereoselective reduction and deprotection of the resulting 2,6-cis-6-substituted 4-oxo-l-pipecolic acids to the corresponding 4-hydroxy-L-pipecolic acids was also performed.

A new general approach for the stereocontrolled synthesis of functionalised γ- and δ-lactams.

A new flexible approach for the stereoselective synthesis of substituted 1H-pyrrol-2(5H)-ones and 3,6-dihydro-1H-pyridin-2-ones has been developed. The general strategy employed the stereoselective reduction of a series of α,ß-unsaturated ketones under chelation control to give the corresponding allylic alcohols. Overman rearrangement to install the key C-N bond followed by conversion to either prop-2-enoyl or but-3-enoyl derivatives and a ring closing metathesis reaction gave the target unsaturated γ- and δ-lactams. The synthetic utility of these compounds as building blocks was demonstrated by the preparation of the N-Boc derivative of (-)-coniine.

Ladder polyether synthesis via epoxide-opening cascades directed by a disappearing trimethylsilyl group.

Epoxide-opening cascades offer the potential to construct complex polyether natural products expeditiously and in a manner that emulates the biogenesis proposed for these compounds. Herein we provide a full account of our development of a strategy that addresses several important challenges of such cascades. The centerpiece of the method is a trimethylsilyl (SiMe(3)) group that serves several purposes and leaves no trace of itself by the time the cascade has come to an end. The main function of the SiMe(3) group is to dictate the regioselectivity of epoxide opening. This strategy is the only general method of effecting endo-selective cascades under basic conditions.

Direct small-molecule kinase activation: Novel approaches for a new era of drug discovery.

The pharmaceutical industry has traditionally targeted the inhibition of dysregulated kinases to treat diseases such as cancer and inflammatory disorders. In contrast to the human genome sequencing project, which aimed to identify novel biological targets, the possibility of activating kinases uses known targets in a novel manner. In an approach that is similar to other target classes (eg, GPCRs and nuclear receptors), transient upregulation of kinase function using small molecules has been increasingly demonstrated to lead to favorable disease outcomes. This review discusses direct small-molecule kinase activators: specifically, how these molecules were discovered, characterized, evaluated and developed into drug leads. The choice of potential targets, the mechanisms of activation and the common strategies used to discover activators are also highlighted.

Strategic use of nickel(0)-catalyzed enyne-epoxide reductive coupling towards the synthesis of (-)-cyatha-3,12-diene.

Various situations are explored in which the nickel(0)-catalyzed enyne-epoxide reductive coupling was utilized to access key intermediates towards the total synthesis of (-)-cyatha-3,12-diene (1). Enantioenriched 3,5-dien-1-ols with a variety of functionality were obtained in a straightforward manner from easily accessible 1,3-enynes and terminal epoxides.

Glycosylated foldamers to probe the carbohydrate-carbohydrate interaction.

The synthesis of a triglycosylated helical foldamer based on a combination of cyclopentyl- and pyrrolidinyl-based amino acids is described. This structure is stable in water, maintaining as it does a series of carbohydrate units in proximity to one another, and represents the basis of a new approach to the study of carbohydrate-carbohydrate interactions.

Ladder polyether synthesis via epoxide-opening cascades using a disappearing directing group.

The combination of a trimethylsilyl group, a Brønsted base, a fluoride source, and a hydroxylic solvent enables the first construction of the tetrad of tetrahydropyran rings found in the majority of the ladder polyether natural products by way of a cascade of epoxide-opening events that emulates the final step of Nakanishi's proposed biosynthetic pathway. The trimethylsilyl group disappears during the course of the cascade, and thus these are the first epoxide ring-opening cascades that afford ladder polyether subunits containing no directing groups at the end of the cascade.

Synthesis and pharmacological characterization of novel analogues of the nicotinic acetylcholine receptor agonist (+/-)-UB-165.

(+/-)-UB-165 (1) is a potent neuronal nicotinic acetylcholine receptor (nAChR) ligand, which displays functional selectivity between nAChR subtypes. Using UB-165 as a lead structure, two classes of racemic ligands were synthesized and assessed in binding assays for three major nAChR subtypes (alpha4beta2, alpha3beta4, and alpha7). The first class of compounds comprises the three pyridine isomers 4-6, corresponding to the 3-, 2-, and 4-substituted pyridine isomers, respectively. Deschloro UB-165 (4) displayed a 2-3-fold decrease in affinity at alpha4beta2 and alpha3beta4 nAChR subtypes, as compared with (+/-)-UB-165, while at the alpha7 subtype a 31-fold increase in affinity was observed. At each of the nAChR subtypes, high affinity binding was dependent on the presence of a 3-substituted pyridine, and the other isomers, 5 and 6, resulted in marked decreases in binding affinities. The second class of compounds is based on replacing the pyridyl unit of 1 with a diazine moiety, giving pyridazine (7), pyrimidine (8), and pyrazine (9), which retain the "3-pyridyl" substructure. Modest reductions in binding affinity were observed for all of the diazine ligands at all nAChR subtypes, with the exception of 7, which retained potency comparable to that of 4 in binding to alpha7 nAChR. In functional assays at the alpha3beta4 nAChR, all analogues 4-9 were less potent, as compared with 1, and the rank order of functional potencies correlated with that of binding potencies. Computational studies indicate that the 3-substituted pyridine 4 and 2-substituted pyridine 5, as well as the diazine analogues 7-9, all conform to a distance-based pharmacophore model recently proposed for the alpha4beta2 receptor. However, the nicotinic potencies of these ligands vary considerably and because 5 lacks appreciable nicotinic activity, it is clear that further refinements of this model are necessary in order to describe adequately the structural and electronic demands associated with this nAChR subtype. This rational series of compounds based on UB-165 presents a systematic approach to defining subtype specific pharmacophores.