Steering New Drug Discovery Campaigns: Permeability, Solubility, and Physicochemical Properties in the bRo5 Chemical Space.

An increasing number of drug discovery programs concern compounds in the beyond rule of 5 (bRo5) chemical space, such as cyclic peptides, macrocycles, and degraders. Recent results show that common paradigms of property-based drug design need revision to be applied to larger and more flexible compounds. A virtual event entitled "Solubility, permeability and physico-chemical properties in the bRo5 chemical space" was organized to provide preliminary guidance on how to make the discovery of oral drugs in the bRo5 space more effective. The four speakers emphasized the importance of the bRo5 space as a source of new oral drugs and provided examples of experimental and computational methods specifically tailored for design and optimization in this chemical space.

Updating the portfolio of physicochemical descriptors related to permeability in the beyond the rule of 5 chemical space.

Beyond rule of 5 (bRo5) molecules are attracting significant interest in modern drug discovery mostly because many novel targets require large and more flexible structures. The main aim of this paper is the identification of ad hoc bRo5 physicochemical descriptors of ionization, lipophilicity, polarity and chameleonicity and their measurement. We used different methods to collect ionization (pKa measures and log k'80 PLRP-S trends), lipophilicity (in octanol/water, in apolar systems and in biomimetic environments), polarity (Δlog Poct-tol, EPSA and Δlog KWIAM) and chameleonicity (ChameLogD) descriptors for 26 bRo5 drugs. A second aim was to check the relationship between physicochemical descriptors and permeability for a subset of compounds for which solid permeability values are reported in the literature. Results showed that the physicochemical profile in the bRo5 chemical space is often experimentally accessible, albeit more tools are required to overcome limitations of individual methods. For the investigated compounds, permeability is governed by Δlog Poct-tol and preliminary data support that chameleonicity could also have an impact.

Entry of therapeutics into the brain: Influence of exposed polarity calculated in silico and measured in vitro by supercritical fluid chromatography.

The present work proposes a novel application of EPSA (not an acronym but found to be referred to by many as Exposed Polar Surface Area), a supercritical fluid chromatography (SFC) polarity readout for assisting in the prediction of the extent of drug permeation through the blood-brain barrier (BBB). For this purpose, EPSA values for 69 structurally unrelated acidic, basic, neutral and amphoteric compounds were determined by a validated SFC method. Additionally, water-accessible surface area (WASA) values for the whole dataset were calculated in silico and compared to experimentally determined EPSA values. All these indexes were used to model the uptake of drugs through the BBB. Highly significant statistical models (r2 (n-1) = 0.81) were achieved by using WASA and/or EPSA values along with other experimentally determined (e.g. phospholipophilicity) and in silico calculated descriptors.

Determining molecular properties with differential mobility spectrometry and machine learning.

The fast and accurate determination of molecular properties is highly desirable for many facets of chemical research, particularly in drug discovery where pre-clinical assays play an important role in paring down large sets of drug candidates. Here, we present the use of supervised machine learning to treat differential mobility spectrometry - mass spectrometry data for ten topological classes of drug candidates. We demonstrate that the gas-phase clustering behavior probed in our experiments can be used to predict the candidates' condensed phase molecular properties, such as cell permeability, solubility, polar surface area, and water/octanol distribution coefficient. All of these measurements are performed in minutes and require mere nanograms of each drug examined. Moreover, by tuning gas temperature within the differential mobility spectrometer, one can fine tune the extent of ion-solvent clustering to separate subtly different molecular geometries and to discriminate molecules of very similar physicochemical properties.

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide-Peptoid Hybrid CXCR7 Modulators.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide-peptoid hybrids with high CXCR7 binding affinities (Ki < 100 nM) and measurable passive permeability (Papp > 5 × 10-6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

Comparative pharmacokinetic profile of cyclosporine (CsA) with a decapeptide and a linear analogue.

The synthesis and in vivo pharmacokinetic profile of an analogue of cyclosporine is disclosed. An acyclic congener was also profiled in in vitro assays to compare cell permeability. The compounds possess similar calculated and measured molecular descriptors however have different behaviors in an RRCK assay to assess cell permeability.

Relationship between Passive Permeability and Molecular Polarity Using Block Relevance Analysis.

EPSA is an experimental descriptor of molecular polarity obtained from chromatographic retention in supercritical fluid chromatography (SFC) systems, previously shown by Goetz et al. to correlate with passive permeability of cyclic peptides. The present study focuses on EPSA in relation to passive permeability of small molecules. We applied block relevance (BR) analysis to interpret the relative significance of mechanistic forces prevailing in EPSA. The BR analysis is a computational tool that allows the interpretation of the balance of intermolecular interactions governing systems such as the aforementioned chromatographic retention in EPSA. EPSA and passive permeability determined by Ralph Russ canine kidney cells (RRCK) or low efflux Madin Darby canine kidney cells (MDCK-LE) and human epithelial colorectal adenocarcinoma cells (Caco-2), studied on a data set of commercial drugs, indicated that EPSA is relevant in describing permeability of hydrophilic drugs (CLogP < 1). We then verified, on a data set of 1699 Rule of 5 compliant Pfizer compounds, that when CLogP < 1, a value of EPSA < 100 significantly increases the likelihood of high permeability.

Chiral Sulfoxide-Induced Single Turn Peptide α-Helicity.

Inducing α-helicity through side-chain cross-linking is a strategy that has been pursued to improve peptide conformational rigidity and bio-availability. Here we describe the preparation of small peptides tethered to chiral sulfoxide-containing macrocyclic rings. Furthermore, a study of structure-activity relationships (SARs) disclosed properties with respect to ring size, sulfur position, oxidation state, and stereochemistry that show a propensity to induce α-helicity. Supporting data include circular dichroism spectroscopy (CD), NMR spectroscopy, and a single crystal X-ray structure for one such stabilized peptide. Finally, theoretical studies are presented to elucidate the effect of chiral sulfoxides in inducing backbone α-helicity.

Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network.

Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical development of OT as an oral peptide therapeutic has been hindered historically by its unfavorable physicochemical properties, including molecular weight, polarity and number of hydrogen bond donors, which determines poor cell permeability. Here we describe the first systematic study of single and multiple N-methylations of OT and their effect on physicochemical properties as well as potency at the OT receptor. The agonist EC50 and percent effect for OTR are reported and show that most N-methylations are tolerated but with some loss in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through the EPSA chromatographic method and the results validated against NMR temperature coefficient experiments and the determination of NMR solution structures. We found that backbone methylation of residues not involved in IMHB and removal of the N-terminal amine can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR agonist activity. The results of this study also expose the potential challenge of using the N-methylation strategy for the OT system; while exposed polarity is reduced, in some cases backbone methylation produces a significant conformational change that compromises agonist activity. The data presented provides useful insights on the SAR of OT and suggests future design strategies that can be used to develop more permeable OTR agonists based on the OT framework.

A Fast Chromatographic Method for Estimating Lipophilicity and Ionization in Nonpolar Membrane-Like Environment.

This study describes the design and implementation of a new chromatographic descriptor called log k'80 PLRP-S that provides information about the lipophilicity of drug molecules in the nonpolar environment, both in their neutral and ionized form. The log k'80 PLRP-S obtained on a polymeric column with acetonitrile/water mobile phase is shown to closely relate to log Ptoluene (toluene dielectric constant ε ∼ 2). The main intermolecular interactions governing log k'80 PLRP-S were deconvoluted using the Block Relevance (BR) analysis. The information provided by this descriptor was compared to ElogD and calclog Ptol, and the differences are highlighted. The "charge-flush" concept is introduced to describe the sensitivity of log k'80 PLRP-S to the ionization state of compounds in the pH range 2 to 12. The ability of log k'80 PLRP-S to indicate the propensity of neutral molecules and monoanions to form Intramolecular Hydrogen Bonds (IMHBs) is proven through a number of examples.

Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides.

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a ß-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.

EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides.

Most peptides are generally insufficiently permeable to be used as oral drugs. Designing peptides with improved permeability without reliable permeability monitoring is a challenge. We have developed a supercritical fluid chromatography technique for peptides, termed EPSA, which is shown here to enable improved permeability design. Through assessing the exposed polarity of a peptide, this technique can be used as a permeability surrogate.

The discovery of potent nonstructural protein 5A (NS5A) inhibitors with a unique resistance profile-Part†2.

In ongoing studies towards novel hepatitis C virus (HCV) therapeutics, inhibitors of nonstructural protein 5A (NS5A) were evaluated. Specifically, starting from previously reported lead compounds, peripheral substitution patterns of a series of biaryl-linked pyrrolidine NS5A replication complex inhibitors were probed and structure-activity relationships were elucidated. Using molecular modelling and a supercritical fluid chromatographic (SFC) technique, intramolecular H-bonding and peripheral functional group topology were evaluated as key determinants of activity and membrane permeability. The novel compounds exhibited retained potency as compared with the lead compounds, and also showed promising results against a panel of resistance viruses. Together, the results of the study take us a step closer towards understanding the potency of daclatasvir, a clinical candidate upon which the compounds were based, and to designing improved analogues as second-generation antiviral agents targeting NS5A.

High throughput method for the indirect detection of intramolecular hydrogen bonding.

A supercritical fluid chromatography method was developed for the detection of intramolecular hydrogen bonds in pharmaceutically relevant molecules. The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. The technique described here correlates chromatographic retention with the exposed polarity of a molecule. Molecules that can form an intramolecular hydrogen bond can hide their polarity and therefore exhibit lower retention than similar compounds that cannot. By use of a pairwise analysis strategy, intramolecular hydrogen bonds are identified within a test set of compounds with diverse topologies. The chromatographic results are confirmed by NMR chemical shift and temperature coefficient studies.

Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.

Desorption electrospray ionization (DESI) was coupled to an ambient pressure drift tube ion mobility time-of-flight mass spectrometer (IM-TOFMS) for the direct analysis of active ingredients in pharmaceutical samples. The DESI source was also coupled with a standalone IMS demonstrating potential of portable and inexpensive drug-quality testing platforms. The DESI-IMS required no sample pretreatment as ions were generated directly from tablets and cream formulations. The analysis of a range of over-the-counter and prescription tablet formations was demonstrated for amphetamine (methylphenidate), antidepressant (venlafaxine), barbiturate (Barbituric acid), depressant (alprazolam), narcotic (3-methylmorphine) and sympatholytic (propranolol) drugs. Active ingredients from soft and liquid formulations, such as Icy Hot cream (methyl salicylate) and Nyquil cold medicine (acetaminophen, dextromethorphan, doxylamine) were also detected. Increased sensitivity for selective drug responses was demonstrated through the formation of sodiated adduct ions by introducing small quantities of NaCl into the DESI solvent. Of the drugs and pharmaceuticals tested in this study, 68% (22 total samples) provided a clear ion mobility response at characteristic mobilities either as (M + H)(+), (M - H)(-), or (M + Na)(+) ions.

Integrating intramolecular hydrogen bonding (IMHB) considerations in drug discovery using ΔlogP as a tool.

This study demonstrates that ΔlogP(oct-tol) (difference between logP(octanol) and logP(toluene)) describes compounds propensity to form intramolecular hydrogen bonds (IMHB) and may be considered a privileged molecular descriptor for use in drug discovery and for prediction of IMHB in drug candidates. We identified experimental protocols for acquiring reliable ΔlogP(oct-tol) values on a set of compounds representing IMHB motifs most prevalent in medicinal chemistry, mainly molecules capable of forming 6-, 7-member IMHB rings. Furthermore, computational ΔlogP(oct-tol) values obtained with COSMO-RS software provided a good estimate of experimental results and can be used prospectively to assess IMHB. The proposed interpretation method based on ΔlogP(oct-tol) data allowed categorization of the compounds into 2 groups: with high propensity to form IMHB and poor propensity or poor relevance of IMHB. The relative (1)H NMR chemical shift of an exchangeable proton was used to verify presence of IMHB and to validate the IMHB interpretation scheme.

Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (PF-04217903) for the treatment of cancer.

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.

Use of 3D properties to characterize beyond rule-of-5 property space for passive permeation.

The application of conformationally dependent measures of size and polarity to characterize beyond rule-of-5 (Ro5) space for passive permeation was investigated. Specifically, radius of gyration, an alternative to molecular weight, and three-dimensional polar surface area and the generalized Born/surface area dehydration free energy, alternatives to hydrogen-bond donor and acceptor counts, were computed on models of the permeating conformations of over 35 000 molecules. The resulting guidelines for size and polarity, described by the 3D properties, should aid the design of Ro5 violators with passive permeability.

On-column solvent exchange for purified preparative fractions.

On-column solvent exchange, using many of the principles of solid-phase extraction, has been implemented to significantly reduce evaporation cycle time following reverse-phase preparative HPLC. Additional benefits, such as a reduced potential for salt formation, thermal decomposition, and residual solvent, are also described. Fractions obtained from preparative separations, typically in a large volume of acetonitrile:water, are injected into the preparative HPLC and then eluted in acetonitrile, creating a new fraction in a volatile organic solvent. Minimal modification to the instrument was required, and unattended operation is possible. Acetonitrile evaporation is achieved within 3 h, compared with 17 h for aqueous-based fractions; lower temperatures can be used during the evaporation step; mobile-phase additives, likely to form salts with the target compound if concentrated in the fraction, are removed before evaporation; sample recovery and purity are unaffected.

Terpenes and flavonoids from an Egyptian collection of Cleome droserifolia.

Four new sesquiterpene derivatives have been isolated from the aerial parts of Cleome droserifolia. Their structures were established as 6-di(7-hydroxy, 1, 5-epoxy germacrane) (2), 4(15)-guaiane-6-ol (3), 7alpha-germacra-1(10), 4(15)-diene-5beta, 6alpha-diol (4) and 4,7,8-eudesma-triol (5). In addition, a new dolabellane diterpene derivative with the naturally rare peroxy function was identified as methyl ester of 2,18-O-diacetyl-16-O-(3-hydroxy-3-methylglutaryl)-7-hydroperoxydolabella-3,8(17)diene-2,16,18 triol (7). Two known flavonoid derivatives, pinocembrin (6) and quercetin-3-glucoside,7-rhamnoside (1) were isolated from the same source. Structures were established by spectroscopic data.