Klara Valko

Rapid Method for the Estimation of Octanol / Water Partition Coefficient (Log Poct) from Gradient RP-HPLC Retention and a Hydrogen Bond Acidity Term (Sigma alpha2H)

We propose a rapid method for the measurement of octanol/water partition coefficients (log P(oct)) via fast gradient reversed phase retention and the calculation of the hydrogen bond acidity of the compounds. The cycle time of the generic gradient HPLC method is 5 minutes. The general solvation equation obtained for the log Poct values and the fast gradient Chromatographic Hydrophobicity Indices with acetonitrile (CHI(ACN)) and methanol

Rapid-Gradient HPLC Method for Measuring Drug Interactions with Immobilized Artificial Membrane: Comparison with Other Lipophilicity Measures

A fast-gradient high-performance liquid chromatographic (HPLC) method has been suggested to characterize the interactions of drugs with an immobilized artificial membrane (IAM). With a set of standards, the gradient retention times can be converted to Chromatographic Hydrophobicity Index values referring to IAM chromatography (CHI(IAM)) that approximates an acetonitrile concentration with which the equal distribution of compound can be achieved between the mobile phase and IAM. The CHI(IAM) values are more suitable for interlaboratory comparison and for high throughput screening of new molecular entities than the log k(IAM) values (isocratic retention factor on IAM). The fast-gradient method has been validated against the isocratic log k(IAM) values using the linear free energy relationship solvation equations based on the data from 48 compounds. The compound set was selected to provide a wide range and the least cross-correlation between the molecular descriptors in the solvation equation: (2) where SP is a solute property (e.g., logarithm of partition coefficients, reversed-phase (RP)-HPLC retention parameters, such as log k, log k(w), etc.) and the explanatory variables are solute descriptors as follows: R(2) is an excess molar refraction that can be obtained from the measured refractive index of a compound, pi(2)(H) is the solute dipolarity/polarizability, summation operatoralpha(2)(H) and summation operatorbeta(2)(0) are the solute overall or effective hydrogen-bond acidity and basicity, respectively, and V(x) is the McGowan characteristic volume (in cm(3)/100 mol) that can be calculated for any solute simply from molecular structure using a table of atomic constants. It was found that the relative constants of the solvation equation were very similar for the CHI(IAM) and for the log k(IAM). The IAM lipophilicity scale was quite similar to the octanol/water lipophilicity scale for neutral compounds. The effect of charge on the interaction with IAM was studied by varying the mobile phase pH.

Unique selectivity of perfluorinated stationary phases with 2,2,2-trifluoroethanol as organic mobile phase modifier

The selectivity of Luna C18 Xterra C18 and Fluophase (perfluorinated C6) stationary phases has been investigated with aqueous acetonitrile, methanol and 2,2,2-trifluoroethanol mobile phases using linear solvation equations. The gradient retention times of a set of 60 compounds with known molecular descriptors have been determined. Linear solvation equations have been set up to describe the relationship between the gradient retention times and the molecular properties. The selectivity of the stationary phase/mobile phase systems was characterised by the regression coefficients of the molecular descriptors. The perfluorinated stationary phase showed very different selectivity using 2,2,2-trifluoroethanol (TFE) as co-solvent. Compounds with H-bond donor functionality were retained much less than in the other investigated high-performance liquid chromatography (HPLC) systems. This unique selectivity can be explained by the stronger adsorption of trifluoroethanol on the perfluorinated stationary phase surface, than on the hydrocarbon surface. It suggests the importance of the adsorbed organic modifiers in the separation mechanism during reversed-phase HPLC.

Calculation of Abraham descriptors from experimental data from seven HPLC systems; evaluation of five different methods of calculation

Solvation equations have been obtained for seven high performance liquid chromatographic (HPLC) systems, generated in the reverse phase (RP) mode with fast gradient elution. A training set of 40 compounds was used for each system. The seven equations were then used to calculate Abraham descriptors for a completely separate 40-compound test set. In this way the three descriptors dipolarity/polarizability S, hydrogen bond acidity A, and hydrogen bond basicity B were obtained. Five different procedures were used to calculate the descriptors, (i) Microsoft ‘Solver’, (ii) a program that uses a set of three simultaneous equations, and which we denote as ‘TripleX’, (iii) a program similar to Solver that we denote as ‘Descfit’, (iv) a series of regression equations developed from compounds with known descriptors and (v) a series of modified regression equations. We show that RP-HPLC data for a given compound in seven systems can be used to calculate the three Abraham descriptors reliably. We compare descriptors, and errors in the method, with those obtained from water–solvent partition systems.

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.

Determination of solute descriptors of tripeptide derivatives based on high-throughput gradient high-performance liquid chromatography retention data

Abstract Recently proposed chromatographic hydrophobicity indices (CHIs) have been calculated from the correlation of fast gradient HPLC retention times with ϕ0 values from isocratic HPLC measurements for 30 oligopeptide derivatives. Measurements were performed on five HPLC columns; an Inertsil ODS (In), a Prodigy ODS (Pro), an immobilised artificial membrane (IAM), a permethylated β-cylcodextrin (CD) and a cyanopropyl (CN) stationary phase. The ranking of CHI values of the tripeptide derivative of the type Z–Ala–Xaa–Val–OMe for the CD, IAM and CN phases is comparable with other amino acid hydrophobicity scales. The CHI values were used for the determination of three molecular descriptors of the solvation equation established by Abraham; these are the effective hydrogen-bond acidity Σα2H, the effective hydrogen-bond basicity Σβ2H and the solute dipolarity/polarisability π2H. The comparison of the solute descriptors of the tripeptide derivatives in terms of the change of the sequence and the chirality of the amino acids shows a strong influence on Σα2H, Σβ2H and π2H.

Rapid method for estimating octanol-water partition coefficient (log POCT) from isocratic RP-HPLC and A hydrogen bond acidity term (A)

The linear solvation equation approach has been used to describe the octanol/water lipophilicity scale (logPoct) and the isocratic retention factors (log k) obtained using reversed phase HPLC with acetonitrile. Both the octanol/water partition coefficients and the RP-HPLC retention data obtained from the literature, showed good correlation with the molecular descriptors such as size, excess molar refractivity, H-bond acidity/basicity, and polarity/dipolarity. However, the impact of the H-bond acidity term was very different on the two lipophilicity scales. The H-bond acidity term was not significant in describing the octanol/water lipophilicity, while the H-bond acidity of the molecules decreased significantly their RP-HPLC retention. As the other terms had very similar impact on the two lipophilicity scales, it made it possible to convert one scale to the other by incorporating only the H-bond acidity of the compounds as is shown by the equation below, where A is the compound H-bond acidity. Using the simpler hydrogen bond donor counts (HBC) also helped to align the two lipophilicity scales to each other. The validity of the above equations was tested using a test set of 41 drug compounds with our measured data. The log Poct values were estimated from isocratic RP-HPLC retention data with the H-bond acidity term and counts, with an error of 0.284 and 0.325 log unit, respectively.

Change of mobile phase pH during gradient reversed-phase chromatography with 2,2,2-trifluoroethanol–water as mobile phase and its effect on the chromatographic hydrophobicity index determination

We have shown previously that using a trifluoroethanol containing mobile phase provides a unique chromatographic selectivity. This is essential to derive molecular descriptors by HPLC which requires retention data from several systems. It also requires that the ionisation is suppressed so that retention times reflect the properties of the neutral molecules. Therefore the pH change of the mobile phase during gradient elution and its effect on the solute ionisation have been studied. During gradient elution of mixtures of ammonium acetate and butylammonium formate with trifluoroethanol as an organic modifier it was found that the pH was almost constant when the gradient started with a low pH. However, when the starting mobile phase pH was above 8 the pH dropped very quickly as the trifluoroethanol concentration increased in the mobile phase. The CHI descriptor (a retention index derived directly from gradient retention times) of several basic compounds as a function of starting mobile phase pH has been measured using trifluoroethanol gradient. The effect of the trifluoroethanol on the pKa change of the compounds has been investigated. The experimental data fit closely to a previously derived equation that describes gradient retention times as a function of mobile phase pH and analyte ionisation constant (pKa). This equation makes it possible to predict the CHI descriptor for ionisable compounds at various pH values. We have used butylamine for high pH mobile phase preparation as is more basic than ammonia and for many basic drugs the retention of the neutral form could be obtained directly (without extrapolation).

In Vitro Measurement of Drug Efficiency Index to Aid Early Lead Optimization

The concepts of drug efficiency (D(eff) ) and Drug Efficiency Index (DEI) have been recently introduced as useful parameters to optimize the absorption, distribution, metabolism, elimination/excretion, and toxicity properties and in vivo efficacy potential of molecules during lead optimization and at pre-clinical stages. The available free drug concentration relative to dose depends on the compounds bioavailability, clearance, and the nonspecific binding to proteins and phospholipids. In this paper, we have demonstrated, using the data of over 115 known drug molecules, that the nonspecific binding can be determined in vitro very efficiently using biomimetic high-performance liquid chromatography measurements. DEI can therefore be estimated from in vitro measurements. The data show that high in vitro DEI values can be associated with lower efficacious dose. A strategy is described of how to use the DEI parameter during early lead optimization. An example is given to highlight the advantages of optimizing on DEI value rather than on potency alone. In order to facilitate the in silico compound design, correlation between in vitro DEI and in silico ligand efficiency parameters such as ligand lipophilicity efficiency has been revealed, suggesting the potential use of these efficiency-related parameters across lead optimization.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.