Accepted Manuscript Title: Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis Author: Paweł Nowak Paulina Olechowska Mariusz Mitoraj Michał Wo´zniakiewicz Paweł Ko´scielniak PII: DOI: Reference:

S0731-7085(15)00260-5 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.027 PBA 10063

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

23-12-2014 17-4-2015 18-4-2015

Please cite this article as: P. Nowak, P. Olechowska, M. Mitoraj, M. Wo´zniakiewicz, P. Ko´scielniak, Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of acid dissociation constants of warfarin and

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hydroxywarfarins by capillary electrophoresis

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Paweł Nowaka, Paulina Olechowskaa, Mariusz Mitorajb, Michał Woźniakiewicza*, Paweł

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Kościelniaka

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a Jagiellonian

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Kraków, Poland

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b

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Chemistry, Kraków, Poland

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Corresponding Author:

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University in Kraków, Faculty of Chemistry, Department of Analytical Chemistry,

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Jagiellonian University in Kraków, Faculty of Chemistry, Department of Theoretical

*Michał Woźniakiewicz, PhD, Jagiellonian University in Kraków, Faculty of Chemistry,

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Department of Analytical Chemistry, Kraków, Poland, Ingardena St. 3, 30-060 Kraków,

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Poland, [email protected], tel./fax: +48 12 663 20 84

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Highlights:

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o Two acid dissociation equilibria have been described

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o Thermodynamic pKa values have been determined

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o CE, CE-DAD and IS-CE methods have been employed

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o Pharmacological relevance has been presented and discussed

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o Theoretical explanations of pKa values have been attempted

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an

19 20 Abstract

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In this work the acid dissociation constants – pKa of warfarin and its all important

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oxidative metabolites have been determined by capillary electrophoresis-based

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methods. It has resulted in a complete description of two acid-base dissociation

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equilibria, yet not investigated experimentally for phase I metabolites of warfarin.

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The capillary electrophoresis (CE) method based on the relation between effective

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electrophoretic mobilities and pH has proven to be a suitable tool for pKa

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determination, while the spectrophotometric (CE-DAD) and the internal standard

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methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD

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approach based on the change in absorbance spectra between the acidic and basic

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forms is a combination between capillary electrophoresis and spectrophotometric

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titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables

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an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate

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than CE and CE-DAD. The Debye-Hückel model has been confirmed experimentally as

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a good predictor of pKa values at various ionic strengths. Therefore, it has been used

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in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic

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strength. The results are important from the analytical, pharmacological, and

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theoretical points of view.

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Keywords: acid dissociation constant, capillary electrophoresis, Debye-Hückel model, drug

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metabolism, hydroxywarfarins, warfarin

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Abbreviations:

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DMSO - dimethyl sulfoxide

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EOF – electroosmotic flow

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WAR – warfarin

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W3 – 3’-hydroxywarfarin

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W4 – 4’-hydroxywarfarin

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W6 – 6-hydroxywarfarin

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W7 – 7-hydroxywarfarin

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W8 – 8-hydroxywarfarin

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W10 – 10-hydroxywarfarin

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51 1. Introduction

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Warfarin (WAR) [3-(α-acetonylbenzyl)-4-hydroxycoumarin] is a widely used drug

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exhibiting anticoagulant activity, applied in the prevention of thrombosis and

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thromboembolism, i.e. formation of blood clots and vascular occlusions [1,2]. Its routine

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application as an effective anticoagulant is however quite problematic owing to complex

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oxidative metabolism, involving several different isoforms of cytochrome P450 enzyme

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(CYP). The main oxidative metabolites of WAR are hydroxywarfarins: 3’-hydroxywarfarin

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(W3), 4’-hydroxywarfarin (W4), 6-hydroxywarfarin (W6), 7-hydroxywarfarin (W7), 8-

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hydroxywarfarin (W8) and 10-hydroxywarfarin (W10). CYP-mediated metabolism of WAR

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is highly regio- and enantioselective, the most popular hydroxywarfarins found in vivo are

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(S)-W7, (R)-W4 and (R)-W10 [1].

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The acid dissociation constant, usually expressed as its pKa value, is one of the most

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important parameters describing physicochemical properties of drugs [3,4]. It characterizes

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acid-base equilibrium of the respective ionizable groups in solution, thus, indicates their

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dissociation (deprotonation) potential at a given pH. The acidic and basic forms of drugs

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differ in charge – at least one of them is ionized, therefore, they also may differ in other key

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properties like water solubility, membrane permeability, affinity of association with

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proteins, and finally, therapeutic activities. As it was demonstrated in the literature,

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therapeutic activity of WAR is also dependent on its ionization level [2]. For that reason, the

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exact value of pKa for WAR is important from the pharmacological point of view, and it was

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determined many times in the past [5-9]. Another issue is acid-base properties of WAR

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metabolites, yet not fully recognized. Hydroxywarfarins, contrary to the parent drug, are

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supposed to be capable of a double dissociation, and this fact complicates predictions of

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their ionization-related properties. Therefore, experimental determination of their exact

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pKa1 and pKa2 values is undeniably necessary for the holistic understanding of WAR

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pharmacokinetics.

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There are many various analytical techniques that allow for accurate estimation of pKa

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values in a sound and rapid way [3,10]. Capillary electrophoresis (CE) belongs to the

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subgroup of separation techniques, and in the recent years, has gained particular interest as

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an efficient tool for pKa determination [11-17]. Its growing popularity originates from the

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clear analytical benefits: (i) very small consumption of samples and buffers; (ii) relatively

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high throughput and simple automation; (iii) possibility to separate the sample component

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from each other, including impurities; (iv) and the accuracy, typically within 0.05 pH units.

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In a standard approach based on CE, pKa values are calculated from a curve presenting

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dependence of electrophoretic mobility on pH. For comparative purposes, the use of

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thermodynamic pKa (pKaT) corresponding to the zero ionic strength and standard

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temperature 25 ̊C is more suitable than the apparent pKa, determined experimentally in

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specific conditions of ionic strength and temperature [18,19].

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In this work, the acid-base equilibria of WAR and six hydroxywarfarins have been

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investigated and characterized by determination of the respective pKa values. To the best of

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our knowledge, this is the first experimental study of all pharmacologically important

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hydroxywarfarins, and in addition, it covers both dissociation equilibria. The standard CE-

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based method has been used to obtain the most accurate results. Afterwards, usefulness of

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two alternative approaches utilizing CE have been investigated: the spectrophotometric

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method (CE-DAD), based on the changes in molecular spectra recorded by DAD detection

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system; and the internal standard-based method (IS-CE), restricted only to two analytical

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runs and the known pKa value of an internal standard. The variation of pKa1 with changing

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ionic strength has been studied experimentally, and compared with the values predicted by

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the Debye-Hückel model. Finally, pKa1T and pKa2T values have been found for each

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compound, which correspond to the standardized temperature and zero ionic strength. It

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enables creation of a more complete picture of pharmacologically-important properties of

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WAR metabolites, theoretical considerations referring to structure-property relationships,

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and better understanding of their migration profile observed during CE separations.

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2. Material and methods

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2.1. Instrumentation

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WAR (racemic mixture) was supplied by Sigma-Aldrich (St. Louis, MO, USA), W3, W4, W6,

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W7, W8, and W10 (all racemic mixtures) by LGC Standards (Teddington, UK), while all 5 Page 5 of 36

other chemicals by Avantor Performance Materials Poland. S. A. (Gliwice, Poland). All

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standard solutions were prepared in the deionized water (MilliQ, Merck-Millipore Billerica,

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MA, USA) and filtered through the 0.45 μm regenerated cellulose membrane, then degassed

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by centrifugation. Standard concentration of analytes was 0.1 mg/mL, all analytes were

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dissolved in water/methanol (1:1 v/v) mixture. Dimethyl sulfoxide (DMSO) was used as the

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electroosmotic flow (EOF) marker, in 0.2% (v/v) concentration.

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The P/ACE MDQ Capillary Electrophoresis System (Brea, CA, USA) equipped with a diode

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array detector was used for all CE-based experiments, using the bare fused-silica capillary

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of 60 cm total length and 75 μm internal diameter. Separations were conducted in a short-

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injection mode [20], unless stated otherwise, using a 10 cm long outlet capillary part. Owing

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to this fact, the total separation time could be vastly reduced. Sample injection was

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conducted using forward pressure 0.4 psi for 4 s. During separations, 15 kV voltage (anode

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at injection end) and the additional forward pressure of 0.4 psi were applied. The current

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measured during separations was between 25 – 125 µA. Capillary was conditioned at 25 ̊C,

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using the liquid cooling system. Every time, DAD detector collected the whole spectra

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within 200-600 nm. Signal recorded at 200, 280 and 308 nm was used for the further

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analysis.

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Capillary rinsing was conducted between runs applying pressure of 137,9 kPa (20 psi). The

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particular steps of rinsing were: deionized water for 1 min, 0.1 M NaOH for 2 min, and

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background electrolyte (BGE) for 2 min. During the first use of the capillary at a working

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day: methanol for 5 min, 0.1 M HCl for 2 min, deionized water for 2 min, 0.1 M NaOH for 10

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min, and BGE for 10 min were applied. For the fresh capillary conditioning, the latter

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sequence was used, but the duration of each individual step was doubled.

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2.2. Buffering solutions

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Composition of the given BGEs was calculated with the use of PHoEBuS 1.3 software by

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Analis (Namur, Belgium), setting 100 mM as the selected value of ionic strength. The buffers

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of lower ionic strength were prepared by dilution with deionized water. The obtained

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recipes for BGEs preparation have been presented in Table 1. The values of pH were

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verified experimentally prior to CE separations.

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Table 1

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2.3. Methods for pKa determination

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2.3.1. Standard electrophoretic method (CE)

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In the standard CE approach, pKa values have been estimated by applying the non-linear

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regression model to the obtained dependency between effective electrophoretic mobility

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(µeff) and pH. According to the Eq.1, µeff can be calculated from the respective migrations

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times of analyte and EOF marker:

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(1)

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where μeff and μobs are the effective and observed electrophoretic mobilities of analyte (m2 V-

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1 s-1),

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total and effective capillary lengths (m), 0.60 and 0.10 m, respectively; V is the separation

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voltage (V); tobs is the measured migration time of analyte (s), while teof is the time

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measured for neutral marker of EOF – DMSO (s).

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The relation between µeff and pH is described by Eq.2 and Eq.3, for the monoprotic and

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diprotic acid, respectively:

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respectively; μeof is the mobility of electroosmotic flow (m2 V-1 s-1); Ltot and Leff are the

(2)

(3)

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where α values are fitting constants equal to electrophoretic mobility of the deprotonated

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form of the acid with subscript 1 and 2 equal to the order of dissociation. The pKa values are

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equal to the pH of inflection points of the fitted sigmoidal-shape curves.

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2.3.2. Spectrophotometric method (CE-DAD)

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To enable determination of pKa1 by CE-DAD method [21,22], the absorbance of WAR and its

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derivatives was measured at the maximum of electrophoretic peaks at 280 and 308 nm.

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These wavelengths are close to maxima of absorbance of the acidic and basic forms,

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respectively. Afterwards, the parameter β was calculated according to the three different,

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arbitrary proposed definitions:

(5)

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(6)

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where β1, β2 and β3 present different calculation methods, while A280 and A308 are the values

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of absorbance at the given wavelength (nm). As it has been shown in Fig.1, there is a strong

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shift in absorbance at these wavelength between the acidic and basic forms of WAR. Most

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importantly, the direction of this shift is opposite for 280 and 308 nm. These wavelength

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are applicable also for all hydroxywarfarins, whose spectra differ between each other,

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mostly in the case of W7 (not shown).

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Figure 1

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The changing value of β together with changing pH is observed, if the ratio of molar

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absorptivity coefficients at 280 and 308 nm is considerably different for acidic and basic

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form. In that case, an every partially ionized state corresponds to the value of β from the

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range between the two limiting values, related to the protonated and deprotonated forms,

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respectively. In other words, such parameter is then sensitive on a changing contribution of

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the acidic and basic forms, characterized by different shape of absorption spectrum. The

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plot of β against pH gives the curve of similar sigmoidal shape as in the case of the CE

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method, and analogously, the pKa value is indicated by inflection point located on this curve.

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2.3.3. Internal-standard method (IS-CE)

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The IS-CE method was proposed several years ago by Rosés’ scientific group [23-27]. It is

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based on the use of internal standard of known pKa value, similar to the predicted pKa of

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analyte, and requires only two runs at two distinct pH values. The first pH should provide

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total dissociation of both the internal standard and the analyte, while the second one, only

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partial dissociation. Then, the sought pKa can be calculated according to the following

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equation:

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given that, (8)

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(7)

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where pKaIS refers to the internal standard, μA- is the effective electrophoretic mobility

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measured when both compounds are totally deprotonated, while in the case of μeff,

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supposed to be partially ionized. To enable the accurate analysis, the analyte and the

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internal standard are injected together from the same vial. The values of Q are calculated

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for the analyte and the internal standard independently.

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Further information on the determination of pKa values has been presented and discussed

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in the next section of this work.

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2.3.4. Debye-Hückel model

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The estimations of thermodynamic pKa were done by using the following equation [18,19]:

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(9)

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where γ denotes activity coefficient of the particular species of an analyte: acidic – HA, and

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basic – A, with a given charge (z). Activity coefficients were calculated based on the

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extended law of Debye-Hückel [28]:

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(10)

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where A and B are the parameters dependent on dielectric constant and temperature, in

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water at 25°C they are 0.509 and 0.33, respectively, z is the charge of the ion, I is the ionic

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strength, while a denotes the radius of hydrated ion. The approximate value of a = 5 Å was

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used for calculations.

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3. Results and discussion

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3.1. Application of the CE method

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At the beginning, pKa1 and pKa2 of WAR and all hydroxywarfarins have been determined by

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the standard CE method. The measurements of μeff in buffers with gradually increasing pH,

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but constant ionic strength of 100 mM and temperature of 25 ̊C, have been conducted

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separately for each compound. Then, the non-linear regression model has been applied,

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resulting in the fitted curves characterized by one or two inflection points indicating the

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respective pKa values, according to Eq.2 and Eq.3. All data obtained in this stage have been

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presented in Fig.S-1 (left part).

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As it can be seen, both pKa1 and pKa2 have been found. W10 however, in spite of possessing

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two hydroxyl groups, does not exhibit the second dissociation, similarly as WAR. It

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originates from alcoholic character of the second hydroxyl group. Interestingly, pKa1 of W10

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is nearly 1.0 pH unit higher than in the case of the other compounds. A slight difference, in

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the range of 0.1 – 0.2 pH units, is noted also between W7 and the rest of compounds, except

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W10. The variation of pKa1 between WAR, W3, W4, W6 and W8 is rather minor, from 4.87

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to 4.93, generally within the range of method errors. As far as pKa2 is concerned, its

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fluctuation turns out to be substantially greater. In enables to classify the compounds

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according to their pKa2 value into three groups. W3 and W4 belong to the first group, pKa2

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amounts to 11.23 and 11.22, respectively. W6 of the second group exhibits pKa2 10.69,

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whereas W7 and W8 of the third group show values 9.39 and 9.50, respectively. The

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uncertainty of pKa2 determination is notably greater than pKa1, nevertheless, qualitative

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differences between pKa2 of hydroxywarfarins are unambiguously clear.

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One shall admit that an effective temperature inside capillary could be higher than 25 ̊C due

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to inefficient cooling, especially in the inlet part. Therefore, the results may be burdened by

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some intrinsic error related to changes in ionization caused by temperature increase.

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Our results reported herein provide the first experimental and complete picture of the acid-

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base properties of WAR and all hydroxywarfarins existing in vivo. One must admit that pKa1

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of WAR was determined oftentimes in the past (4.85 – 5.15) [5-9], while pKa1 of W7 and

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W10 was estimated by us in our previous work (5.19 and 6.06, respectively) [9]. The

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experiments described in our previous work however, were conducted in other conditions

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of temperature and ionic strength, and were devoted mainly to investigation of the

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supramolecular pKa shifts upon complexation with cyclodextrins. In general, the current

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results are consistent with the previous reports [5-9]. Further comments and conclusions

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related to the determined pKa values can be found in Section 3.4.

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3.2. Application of CE-DAD method

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In some cases the CE-DAD instrument gives a possibility to estimate pKa by totally different

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method than aforementioned one [21,22]. In general, it is the case when the acidic and basic

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forms of analyte exert different UV-Vis spectra. Then, the advantages of two techniques, CE

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and spectrophotometric titration, can be combined within one approach. It is worth

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highlighting that application of voltage is in fact not mandatory, however, it enables

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separation of sample components from each other and allow to decrease analysis time. As it

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has been shown in the literature, the only variable parameters used for pKa calculation are

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absorbance at the given wavelength and analyte concentration. Additionally, a constant

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value of a molar absorptivity coefficient is required, determined separately for the acidic

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and basic forms.

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Our method presented herein for the first time, however, require neither any values of

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analyte concentration nor absorptivity coefficient. It is instead based on the measurements

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of absorbance at two distinct wavelengths. Accordingly, the absorbance of WAR or any

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other compound is measured at the maximum of electrophoretic peaks at 280 and 308 nm,

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respectively. The outcomes have been shown in Fig.S-1 (see Supplementary Material) and

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Table 2.

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Table 2

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Table 2 demonstrates that the results are the same for β2 and β3, but differ for β1.

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Remarkably, the excellent accordance is noted between β2, β3, and the reference CE method.

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The less consistent results observed for β1 stem probably from its mathematical definition,

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since the value of β1 changes in a far broader range than β2 and β3,. Such good outcome

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proves that the CE-DAD method is fully prospective and gives response consistent with the

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typical CE approach. The model is based on the stepwise changes in the shape of

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absorbance spectra, instead of ionization entailing the shifts in migration times in CE. It is

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worth highlighting that CE-DAD method has its own advantage, since EOF marker is not

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necessary. Its limitation, in turn, is that the suitable accuracy of measurements can be easily

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obtained only for relatively high concentration of analyte. Notwithstanding, CE and CE-DAD

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methods can be applied interchangeably, however in this case, only for pKa1 determination.

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3.3. Application of the IS-CE method

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The method based on the use of internal standard of known pKa value was presented as a

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fully prospective alternative for other methods used in high throughput pKa determination,

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especially, in drug discovery studies [23-27]. At the moment, however, its true potential is

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still unknown. In particular, consistency between IS-CE and CE methods applied jointly for

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the same analytes seems to be an interesting issue to be investigated.

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To this end, IS-CE has been tested in the numerous variants. Firstly, all the analytes have

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been used individually as the internal standards for calculation of pKa of the others. As the

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pKa of internal standards, the values obtained from CE method have been used. In this way,

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a given pKa1 value has been estimated from six various reference pKa1 values. Secondly, the

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calculations have been done for three distinct μeff values corresponding to partial ionization,

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attributed to three distinct pH values. As the value of μeff related to the fully deprotonated

286

species, the value indicated by a bottom horizontal asymptote during function fitting has

287

been applied (see Fig.S-1). In consequence, particular pKa could be determined by IS-CE

288

many times, using different approaches. In addition, pKa2 has been determined for all

289

compounds except WAR and W10. All pKa values have been gathered in Table 3.

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Table 3

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In general, as far as pKa1 values are concerned, satisfactory accordance is noticed between

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the IS-CE and CE methods. In majority, discrepancy is less than 0.1 pH units, and this fact

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highlights the potential of IS-CE method. The application of W10 as the internal standard

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gives the worst predictions, and also, the values obtained for W10 as the analyte are mostly

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poorer than the values calculated for other compounds. It seems to be logical that this

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situation results from the particularly high pKa1 value of W10. Nevertheless, the calculations

297

performed for μeff obtained at pH 5.25 are relatively consistent with CE. It prompts us that a

298

choice of pH from the range between pKa of internal standard and analyte, is actually, the

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most profitable. It can be also noted that the results obtained for WAR at pH around 6.0 are

300

worse than for hydroxywarfarins. In this case, putatively, possession of the second hydroxyl

301

group might be the main factor affecting accuracy of predictions.

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In the case of pKa2 the discrepancy between the CE and IS-CE methods is significantly

303

higher, mostly over 0.2 pH units. As it can be easily concluded, the satisfactory results are

304

noted only if pKa2 of analyte is close to pKa2 of internal standard, like for W3 and W4, or W7

305

and W8. Nevertheless, the right qualitative trend between particular compounds is

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indicated in each column (see Table 3). Owing to this qualitative accordance one can

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speculate that only two analytical runs and the obtained very approximate pKa2 values

308

should suffice for proper identification of metabolites based on their migration order in CE

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(compare with Section 3.6).

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An overall evaluation of the IS-CE method is not straightforward. On the one hand, an

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accurate pKa value is rather difficult to be obtained when properties of an internal standard

312

and an analyte differ from each other. On the other hand, for compounds of more similar

313

nature the outcomes are appreciably amended. Secondly, a rough pKa value or a direction of

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its shift with regard to a reference molecule or state, can be obtained very fast. Even if IS-CE

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is not a perfect tool, its potential of time saving merits attention during development of new

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high throughput methods for pKa determination.

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3.4. Thermodynamic pKa

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Determination of thermodynamic pKa, contrary to apparent values discussed above, enables

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an easy comparison between different experimental conditions. pKaT refers to the zero ionic

320

strength, and thus, it is defined by the activities of respective species, in this state equal to

321

their concentrations.

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In order to investigate the usefulness of the extended Debye-Hückel model for prediction of

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pKaT values, the variation of apparent pKa1 with changing ionic strength has been studied

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experimentally. For that purpose, the CE procedure has been repeated using diluted BGEs,

325

to 25 and 10 mM ionic strength, respectively. It allowed us to compare possible differences

326

between particular ionic strengths obtained experimentally with the values calculated from

327

Debye-Hückel model (see Section 2.3.4). The results have been collected in Table 4.

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Table 4

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They suggest that the current version of Debye-Hückel model suitably predicts the variation

330

of pKa1 with respect to changes in ionic strength. The differences between the theoretical

331

model and the values verified experimentally are in the order of only 0.01 pH units.

332

Therefore, the Debye-Hückel correction has been applied for calculation of pKa1T, and also

333

pKa2T. It is worth mentioning that the scale of this correction is greater for pKa2T than for

334

pKa1T, what can be derived from Eq.9 and Eq.10, due to the fact that second dissociation

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occurs for the singly ionized species.

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3.5. Theoretical and pharmacological considerations

337

We have further performed preliminary DFT/TZP/BLYP-D3 study using ADF program [29]

338

in order to characterize the structures of all compounds (WAR, W3, W4, W6, W7, W8 and

339

WAR-10) in gas phase. They are presented in Fig.2. It is clearly visible that all compounds

340

contain intramolecular hydrogen bonding of the type O…HO. More importantly, it has been

341

found that the bond lengths within the O…HO bridge are very similar (~1.0 Å for O–H and

342

~1.78 Å for H…O) in all compounds except W10, for which the shortest H…O distance (1.61

343

Å) has been found, see Fig.2. It shows that the O–H proton of W10 is engaged in the

344

strongest intramolecular interaction. It correlates with the trend in the pKa1 values, i.e.

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markedly larger value of pKa1 is noted for W10 with respect to the remaining regio-isomers.

346

Accordingly, one could suggest that the characteristics of O…HO bonding could directly

347

determine the trend in pKa1 values.

348

Figure 2

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14 Page 14 of 36

As far as the second pKa2 values are concerned, one shall note that changes in stability when

350

going from monoanion to dianion are of vital importance. We see that W3 and W4 exhibit

351

larger pKa2 values as compared to W6, W7 and W8. It is noticeable that in the former case

352

the hydroxyl is introduced at the single phenyl ring, whereas in the latter one various O–H

353

positions at coumarin moiety are considered. It could be the case that delocalization of

354

negative charge, leading to resonance and induction effects, is stronger for the two aromatic

355

coumarin rings than for the single phenyl ring. Coumarin moiety, in addition, contains the

356

electron withdrawing units, carbonyl and heteroatom. An important role of carbonyl unit in

357

enhancement of anion stability is known, it is reflected e.g. by dissociation of carboxyl acids.

358

Apart from these speculations, the increased electrostatic attraction of the proton caused by

359

the negative charge localized on the oxygen of dissociated OH group should be considered

360

for each hydroxywarfarin. Such effect can account for the higher pKa2 noted for W6, than for

361

W7 and W8.

362

We plan in the future to calculate free energy changes for all compounds, as well as the

363

related theoretical pKa values. It will be performed both in the gas phase and in the water.

364

Furthermore, the electronic structures that could help us to understand factors that

365

determine pKa values on a microscopic level will be also calculated.

366

Another important issue is an equilibrium between distinct tautomers of WAR and its

367

derivatives which can coexists in solution: the open form and the two diastereomeric

368

hemiketals, depicted in Fig.3. It is remarkable that different compounds may exhibit

369

different partition between particular tautomers, depending also on the solvent

370

composition [30]. Furthermore, amount and spatial distribution of the potential hydrogen

371

donors and acceptors varies between the open and the hemiketal forms. Therefore, acid-

372

base equilibrium defined by proximity of given molecule regions can be associated with

373

equilibrium between the tautomers. This aspect cannot be omitted during elucidation of

374

structure-related properties of WAR and its metabolites.

375

Figure 3

376

A pharmacological relevance of our results is in fact noticeable. The values of pKa1T and

377

pKa2T should be discussed in two different areas of interest. The first one is elucidation of

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15 Page 15 of 36

WAR pharmacokinetics. It is commonly known that a role of the phase I oxidative

379

metabolism of drugs mediated by CYP enzymes is to increase a drug hydrophilicity and

380

solubility. This increase may stem from the two reasons: appearance of novel ionizable

381

groups yielding charge upon dissociation, or formation of groups which do not dissociate

382

but change an overall character of given molecule sites. One of the decisive factors for the

383

prevalence of one effect over other one is the relation between pKa of this group and pH of

384

environment. Formation of the second OH group during phase I metabolism of WAR, as can

385

be concluded from pKa2T values, does not trigger an increase in overall charge of molecule at

386

physiological pH. Yet more interesting conclusions can be drawn when referring to the

387

pharmacological relevance of pKa1T. Regarding the range of pH of natural intracellular

388

environment or body fluids like blood, urine or bile – generally from 5.5 to 8.0, one can

389

conclude that both parent compound and every hydroxylated metabolite occur rather in the

390

ionized form. The intriguing exception can be however W10 with pKa1≈ 6. In this case the

391

pKa1value may suffice for existence of significant fraction of the non-ionized form, far more

392

hydrophobic, especially in slightly acidic matrices like e.g. urine (pH ≈ 5.5). The conclusion

393

could be that phase I metabolism leading to W10 may cause in fact the adverse effects on

394

solubility in some matrices. This is of special relevance because (R)-W10 is one of the main

395

metabolites found in biological material collected from patients.

396

The second pharmacologically-relevant area of interest is, still enigmatic, the phase II

397

conjugative metabolism of WAR. It is quite likely that the reactions of glucuronidation

398

catalyzed by UDP-glucuronosyltransferases depend on hydroxylation site, thus, exert high

399

regioselectivity [31-33]. This may be in consequence one of the reasons why a diverse

400

profile of WAR phase II metabolites found in vivo is correlated with the expression of given

401

CYP enzymes. The last issue is a hypothetic modulation of WAR metabolism directly by

402

hydroxywarfarins. The two different phenomena should be considered, either a

403

transcriptional modulation of genes expression or a direct inhibition of CYP enzymes e.g. by

404

blocking of the active sites of enzymes. One may predict that these phenomena are

405

somehow related to structure and conformation of the particular metabolites and

406

respective stabilities of O–H bonds. In consequence, their pKa values would be of greater

407

biochemical relevance.

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16 Page 16 of 36

3.6. pKa-related migration profile

409

Apart from the theoretical structure-activity relationships and the pharmacological

410

importance of the data obtained in this work, another interesting issue is a separation of all

411

compounds from each other mixed in a single sample. To this end, the CE method based on

412

differences in already known pKa values seems to be the simplest opportunity. To attempt

413

this approach, CE-based separation has been conducted at pH 10.4. This pH has been

414

selected as optimal for obtaining different ionization levels of the particular compounds,

415

thus also high selectivity. In Fig.4 the migration profile has been depicted.

416

Figure 4

417

It has occurred that the individual migration times properly reflect the order of pKa2 values.

418

In particular, almost total but still slightly different ionization can be predicted from the

419

position of W8 and W7 peaks, the ionization level below 50% from W6 peak, while yet

420

lower but still visible ionization from W3 and W4 peaks. The lack of separation between W3

421

and W4 proves that their pKa2 is very similar. Interestingly, W10 has been separated from

422

WAR, and this is probably caused not by different ionization, but rather by a different

423

hydrodynamic resistance during electromigration. Hypothetically, W10 as capable of

424

forming stronger intramolecular hydrogen bonds may migrate in a more occlusive way, and

425

thus, actually a more mobile form than WAR. This hypothesis is supported by different

426

shape of the W10 molecule presented in Fig.2. Further investigation of these analytically

427

important conundrums will yet be the aim of our future work.

428

4. Conclusions

429

The classical CE method has allowed us to determine thermodynamic pKa values with the

430

aid of extended Debye-Hückel model, upon its experimental verification. The CE-DAD

431

method has occurred to be very useful for determination of pKa1, and provides totally

432

different physicochemical background than CE. Its additional advantage is that information

433

about concentration of analyte and absorptivity coefficient values is not needed. Both

434

methods are complementary and can be applied interchangeably. Accuracy of the IS-CE is

435

strictly dependent on degree of similarity between an internal standard and an analyte, and

436

this method appears to be especially useful for qualitative predictions. Due to vast

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17 Page 17 of 36

economization of time and materials, however, its potential is still worth to be appreciated

438

and further tested.

439

One may conclude that the values of pKa1T and pKa2T obtained in the current work are

440

important from the analytical, pharmacological and theoretical points of view. The acid-

441

base properties depend on hydroxylation site. In particular, relatively large variations are

442

observed between pKa2T values. W10 exhibits specific properties, namely it undergoes only

443

one dissociation contrary to other hydroxywarfarins, and its pKa1T surpasses the values

444

noted for any other compounds. These features are related to the specific molecular

445

structure and alcoholic character of the second OH group. It turns out that phase I

446

metabolism leads to creation of more hydrophilic molecules, however still only singly

447

ionized at physiological pH, similarly as the parent drug. The specific conformations and

448

hydrogen binding energies, as well as the actual proximity of the given hydrogen donors

449

and acceptors, may be very important factors for explanation of regioselective phase II

450

metabolism and modulation of CYP-mediated metabolism, not studied in depth so far.

451

Selection of proper pH enables the efficient separation by CE of almost all tested

452

compounds mixed together, based on their pKa2 values.

453

The authors declare no competing financial interest.

454

Acknowledgments

455

Author Paweł Nowak has received the financial support from Krakowskie Konsorcjum

456

“Materia-Energia-Przyszłość” within the subsidy KNOW.

457

The research was carried out with equipment purchased with financial support from the

458

European Regional Development Fund within the framework of the Polish Innovation

459

Economy Operational Programme (contract no. POIG.0 2.01.00-12-0 23/08).

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18 Page 18 of 36

References

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[1] L.S. Kaminsky, Z-Y. Zhang, Human P450 Metabolism of Warfarin, Pharmacol. Ther. 73

462

(1997) 67–74

463

[2] M. Gebauer, Synthesis and structure-activity relationships of novel warfarin derivatives,

464

Bioorg. Med. Chem. 15 (2007) 2414–2420.

465

[3] S. Babić, A.J.M. Horvat, D. Mutavdžić-Pavlović, M. Kaštelan-Macan, Determination of pKa

466

values of active pharmaceutical ingredients, Trac-Trend. Anal. Chem. 26, (2007) 1043–

467

1061.

468

[4] I. Ghosh, W.M. Nau, The strategic use of supramolecular pKa shifts to enhance the

469

bioavailability of drugs, Adv. Drug Deliver. Rev. 64 (2012) 764–783.

470

[5] H. Wan, A. Holmen, M. Någård, W. Lindberg, Rapid screening of pKa values of

471

pharmaceuticals by pressure-aassisted capillary electrophoresis combined with short-end

472

injection, J. Chromatogr. A 979 (2002) 369–377.

473

[6] J.M. Cabot, E. Fuguet, C. Ràfols, M. Rosés, Fast high-throughput method for the

474

determination of acidity constants by capillary electrophoresis. II. Acidic internal standards,

475

J. Chromatogr. A 1217 (2010) 8340–8345.

476

[7] M. Shalaeva, J. Kenseth, F. Lombardo, A. Bastin, Measurement of Dissociation Constants

477

(pKa Values) of Organic Compounds by Multiplexed Capillary Electrophoresis Using

478

Aqueous and Cosolvent Buffers, J. Pharm. Sci. 97 (2008) 2581–2606.

479

[8] Y. Ishihama, M. Nakamura, T. Miwa, T. Kajima, N. Asakawa, A Rapid Method for pKa

480

Determination of Drugs Using Pressure-Assisted Capillary Electrophoresis with Photodiode

481

Array Detection in Drug Discovery, J. Pharm. Sci. 91 (2002) 933–942.

482

[9] P. Nowak, M. Garnysz, M.P. Mitoraj, F. Sagan, M. Woźniakiewicz, P. Kościelniak,

483

Analytical aspects of achiral and cyclodextrin-mediated capillary electrophoresis of

484

warfarin and its two main derivatives assisted by theoretical modelling, J. Chromatogr. A

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1377 (2015) 106–113.

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[10] J. Reijenga, H. van Arno, L. van Antonie, B. Teunissen, Development of Methods for the

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Determination of pKa Values. Anal. Chem. Insights 8 (2013) 53−71.

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[11] P. Nowak, M. Woźniakiewicz, P. Kościelniak, Application of capillary electrophoresis in

489

determination of acid dissociation constant (pKa) values, J. Chromatogr. A 1377 (2015) 1–

490

12.

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[12] S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, Determination of acid

492

dissociation constants by capillary electrophoresis, J. Chromatogr. A 1037 (2004) 445–454.

493

[13] M. Riesová, J. Svobodová, K. Ušelová, Z. Tošner, I. Zusková, B. Gaš, Determination of

494

thermodynamic values of acidic dissociation constants and complexation constants of

495

profens and their utilization for optimization of separation conditions by Simul 5 Complex,

496

J. Chromatogr. A 1364 (2014) 276–288.

497

[14] E.C. Demiralay, Z. Ustun, Y.D. Daldal, Estimation of thermodynamic acidity constants of

498

some penicillinase-resistant penicillins, J. Pharmaceut. Biomed. 91 (2014) 7–11.

499

[15] J.L. Wang, X.J. Xu, D.Y. Chen, Determination of pK(a) values of a triptolide derivative

500

and its impurities by pressure-assisted capillary electrophoresis, J. Pharmaceut. Biomed. 88

501

(2014) 22–26.

502

[16] V. Solínová, V. Kašička, Determination of acidity constants and ionic mobilities of

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polyprotic peptide hormones by CE, Electrophoresis 34 (2013) 2655–2665.

504

[17] H.X. Ren, L.C. Wang, X.S. Wang, X. Liu, S.X. Jiang, Measurement of acid dissociation

505

constants and ionic mobilities of 3-nitro-tyrosine and 3-chloro-tyrosine by capillary zone

506

electrophoresis, J. Pharmaceut. Biomed. 77 (2013) 83–87.

507

[18] K. Vceláková, I. Zusková, E. Kenndler, B. Gaš. Determination of cationic mobilities and

508

pK(a) values of 22 amino acids by capillary zone electrophoresis, Electrophoresis 25 (2004)

509

309–317.

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[19] V. Solínová, V. Kašička, D. Koval, M. Cesnek, A. Holý. Determination of acid-base

511

dissociation constants of amino- and guanidinopurine nucleotide analogs and related

512

compounds by capillary zone electrophoresis, Electrophoresis 27 (2006) 1006–1019.

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[20] Z. Glatz, Application of short-end injection procedure in CE, Electrophoresis 34 (2013)

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631–642.

515

[21] X. Wu, S. Gong, T. Bo, Y. Liao, H. Liu, Determination of dissociation constants of

516

pharmacologically active xanthones by capillary zone electrophoresis with diode array

517

detection, J. Chromatogr. A 1061 (2004) 217–223.

518

[22] S.P. Ozkorucuklu, J.L. Beltrán, G. Fonrodona, D. Barrón, G. Alsancak, J. Barbosa,

519

Determination of Dissociation Constants of Some Hydroxylated Benzoic and Cinnamic Acids

520

in Water from Mobility and Spectroscopic Data Obtained by CE-DAD, J. Chem. Eng. Data 54

521

(2009) 807–811.

522

[23] E. Fuguet, C. Ràfols, M. Rosés, A fast high throughput method for the determination of

523

acidity constants by capillary electrophoresis. 3. Basic internal standards, J.

524

Chromatography A 1218 (2011) 3928–3934.

525

[24] J.M. Cabot, E. Fuguet, C. Ràfols, M. Rosés, Determination of acidity constants by the

526

capillary electrophoresis internal standard method. IV. Polyprotic compounds, J.

527

Chromatogr. A 1279 (2013) 108–116.

528

[25] R.S. Gravador, E. Fuguet, C. Ràfols, M. Rosés, Temperature variation effects on the

529

determination of acidity constants through the internal standard–capillary electrophoresis

530

method, Electrophoresis 34 (2013) 1203–1211.

531

[26] J.M. Cabot, E. Fuguet, M. Rosés, Internal Standard Capillary Electrophoresis as a High

532

Throughput Method for pKa Determination in Drug Discovery and Development, ACS Comb.

533

16 (2014) 518–525.

534

[27] J.M. Cabot, E. Fuguet, M. Rosés, Determination of acidity constants of sparingly soluble

535

drugs in aqueous solution by the IS-CE method, Electrophoresis 35 (2014) 3564–3569.

536

[28] P. Debye, P. Huckel, On the theory of electrolytes, Phyz. Z. 24 (1923) 305–325.

537

[29] G. teVelde, F.M. Bickelhaupt, E.J. Baerends, C. Fonseca Guerra, S.J.A. van Gisbergen, J.G.

538

Snijders, T. Ziegler, Chemistry with ADF, J. Comput. Chem. 22 (2001) 931–967.

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[30] B. Castleberry, E.J. Valente, D.S. Eggleston, Open-cyclic warfarin isomerism: 5-

540

hydroxywarfarin, J. Cryst. Spectrosc. 20 (1990) 583–593.

541

[31] C.P. Pugh, D.L. Pouncey, J.H. Hartman, R. Nshimiyimana, L.P. Desrochers, T.E. Goodwin,

542

G. Boysen, G.P. Miller, Multiple UDP-glucuronosyltransferases in human liver microsomes

543

glucuronidate both R- and S-7-hydroxywarfarin into two metabolites, Arch. Biochem.

544

Biophys. 564 (2014) 244–253.

545

[32] D.R. Jones, G.P. Miller, Assays and applications in warfarin metabolism: What we know,

546

how we know it and what we need to know, Expert Opin. Drug Met. 7 (2011) 857–874.

547

[33] D.R. Jones, J.H. Moran, G.P. Miller, Warfarin and UDP-glucuronosyltransferases: Writing

548

a new chapter of metabolism, Drug Met. Rev. 42 (2010) 53–59.

an

us

cr

ip t

539

549

Ac ce p

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M

550

22 Page 22 of 36

550 Fig.1. Absorbance spectra obtained during CE-DAD separation of WAR at pH 2.5 and 8.0,

552

respectively. Under these conditions only one form: the acidic (protonated) or the basic

553

(deprotonated) exists in solution. Vertical dashed lines indicate the wavelengths (280 and

554

308 nm) for which the absorbance values were recorded and further processed.

555

Fig.2. DFT/TZP/BLYP-D3 optimized structures of WAR and hydroxywarfarins together with

556

the selected bond distances (in Å).

557

Fig.3. Tautomeric forms of WAR, potential hydrogen donors and acceptors have been

558

pointed by red and blue colors, respectively.

559

Fig.4. Electropherogram presenting the separation of all analytes mixed together,

560

conducted at pH 10.4 in 25 mM ionic strength (in the

an

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ip t

551

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561

23 Page 23 of 36

561

Table 1. Composition and predicted pH of all buffering solutions prepared for experiments,

562

calculated for 50 mL total volume and 100 mM ionic strength.

564 565

cr

ip t

NaH2PO4 (100 mM) 4.80 CH3COONa (500 mM) 10.00 10.00 Na2HPO4 (100 mM) 4.70 13.29 16.25 NaOH (1 M) 1.02 2.13 2.50 KCl (1 M) 4.36 0.45

an

M

d

Ac ce p

563

H3PO4 (100 mM) 18.52 CH3COOH (500 mM) 14.16 2.52 NaH2PO4 (100 mM) 3.59 1.01 0.12 Na2B4O7·10H2O (50 mM) 39.77 28.67 25.05 NaOH (1 M) 0.64 4.55

te

Phosphate buffer I 2.50 Acetic buffer 4.50 5.25 Phosphate buffer II 6.00 7.00 8.00 Borate buffer 9.40 10.00 11.00 NaOH / KCl 12.00 13.00

Buffer composition [mL]

us

pH

24 Page 24 of 36

565

Table 2. Comparison of CE and CE-DAD methods used for pKa1 determination

CE-DAD

CE-DAD

CE-DAD

4.87 ± 0.02

4.71 ± 0.03

4.86 ± 0.03

4.86 ± 0.03

W3

4.87 ± 0.04

4.77 ± 0.02

4.89 ± 0.02

4.88 ± 0.02

W4

4.93 ± 0.04

4.77 ± 0.03

4.91 ± 0.03

4.91 ± 0.03

W6

4.92 ± 0.07

4.62 ± 0.02

4.92 ± 0.02

4.92 ± 0.02

W7

5.09 ± 0.07

5.08 ± 0.04

5.10 ± 0.03

5.10 ± 0.04

W8

4.88 ± 0.06

4.64 ± 0.03

4.84 ± 0.03

4.84 ± 0.03

W10

5.80 ± 0.03

5.68 ± 0.03

5.79 ± 0.02

5.79 ± 0.02

an

566

us

WAR

cr

CE (ref)

ip t

pKa1

M

567

Ac ce p

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d

568

25 Page 25 of 36

Table 3. Apparent pKa values (in 100 mM ionic strength) obtained by the IS-CE method for

569

all possible combinations between potential internal standards and analytes, based on the

570

selected data obtained by the CE method. Internal standard pH of partial

WAR

W3

W4

W6

W7

4.88

4.91

4.90

4.94

4.85

4.84

4.85

4.71

4.71

4.67

ionization 4.87

W6

W7

W8

W10

4.92

4.67

4.66

4.90

4.89

4.93

4.88

5.11

4.87

4.90

4.88

4.95

4.83

4.82

5.06

4.93

4.97

4.92

5.14

4.94

4.94

5.01

4.89

4.89

4.88

5.12

4.96

4.91

5.14

4.94

4.92

4.99

4.93

4.92

5.15

5.04

5.27

5.07

5.14

5.08

5.32

4.86

pH ≈ 5.25

4.89

pH ≈ 6.00

5.03

4.83

pH ≈ 4.50

4.89

4.91

pH ≈ 5.25

4.96

4.93

4.93

4.96

pH ≈ 6.00

5.09

4.93

pH ≈ 4.50

4.89

4.90

4.92

pH ≈ 5.25

4.94

4.92

4.91

pH ≈ 6.00

5.12

4.96

4.96

pH ≈ 4.50

5.02

5.03

5.05

5.05

pH ≈ 5.25

5.09

5.06

5.06

5.07

pH ≈ 6.00

5.29

5.13

5.13

5.08

pH ≈ 4.50

4.86

4.87

4.89

4.89

4.93

pH ≈ 5.25

4.90

4.87

4.87

4.88

4.90

pH ≈ 6.00

5.09

4.93

4.93

4.88

4.89

pH ≈ 4.50

5.55

5.56

5.59

5.58

5.62

5.57

pH ≈ 5.25

5.75

5.72

5.72

5.73

5.75

5.73

pH ≈ 6.00

5.77

5.61

5.61

5.57

5.57

5.56

4.87

4.87 4.87

te

W4

4.85

pH ≈ 4.50

Ac ce p

W3

4.89

4.87

M

pH ≈ 6.00

5.12

us

pH ≈ 5.25

d

WAR

W10

4.89

an

pH ≈ 4.50

W8

cr

Analyte

ip t

568

4.92

5.09

5.11 4.88

4.95 5.12 5.80

26 Page 26 of 36

pH ≈ 10.50

11.23

pH ≈ 11.00

W7

10.90 10.36 10.37

11.23

10.93

pH ≈ 9.90

11.22

10.82 10.23 10.22

pH ≈ 10.50

11.20 11.22 10.87 10.34 10.35

pH ≈ 11.00

11.22

pH ≈ 9.90

11.09

11.09

pH ≈ 10.50

11.02

11.04 10.69 10.15 10.16

pH ≈ 11.00

10.99

11.00

pH ≈ 9.90

10.38

10.38

pH ≈ 10.50

10.26

10.27

pH ≈ 9.90

10.49

10.50

10.10

9.51

pH ≈ 10.50

10.36

10.37

10.03

9.49

10.91

pH ≈ 11.00

9.98 9.93

9.39

9.38 9.40

9.50

d

W8

10.10 10.09

M

pH ≈ 11.00

cr

W6

11.25

us

W4

10.83 10.24 10.24

an

W3

11.23

ip t

pH ≈ 9.90

As pKa of the internal standard, the values calculated from the CE method (in 100 mM ionic

572

strength) have been used.

574

Ac ce p

573

te

571

27 Page 27 of 36

Table 4. Apparent (valid at given ionic strength) and thermodynamic (standardized to zero ionic strength) pKa

pKa2 100 mM

pKa2T

4.87 ± 0.02

4.89 ± 0.04

4.99 ± 0.03

4.99

4.87 ± 0.04

4.89 ± 0.04

4.93 ± 0.03

4.97

11.23 ± 0.06

11.55

W4

4.93 ± 0.04

4.98 ± 0.05

4.97 ± 0.04

5.03

11.22 ± 0.06

11.54

W6

4.92 ± 0.07

4.94 ± 0.06

4.97 ± 0.04

5.01

10.69 ± 0.08

11.01

W7

5.09 ± 0.07

5.09 ± 0.04

5.10 ± 0.04

5.16

W8

4.88 ± 0.06

4.88 ± 0.03

4.93 ± 0.04

4.97

W10

5.80 ± 0.03

5.93 ± 0.03

5.92 ± 0.05

W3

Δcalculated

0.03

Δpredicted

0.04 Δcalculated

581 582

9.71

9.50 ± 0.12

9.82

5.95

0.03

Δpredicted 0.02 has been calculated as an average from three values obtained for three distinct ionic strength by

Ac ce p

576 577 578 579 580

pKa1T

9.39 ± 0.16

d

WAR

cr

pKa1T

us

pKa1 10 mM

an

pKa1 25 mM

M

pKa1 100 mM

ip t

values.

te

574 575

applying respective Debye-Hückel corrections; Δcalculated denotes an average difference between apparent pKa for the same compound and obtained experimentally for two distinct ionic strengths; Δpredicted denotes a difference between apparent pKa for the same compound and predicted for two distinct ionic strengths from Debye-Hückel model.

long-end injection mode).

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*Graphical Abstract

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Figure 1 bw print only

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Figure 1 color web only

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Figure 2 revised color- web only

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Figure 2 revised bw- print only

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Figure 3 bw print only

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Figure 3 color web only

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Figure 4

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