- Email: [email protected]
Measurement of pKa values of newly synthesized heteroarylaminoethanols by CZE
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Measurement of pKa values of newly synthesized heteroarylaminoethanols by CZE ˇ ˇ Anna Liskov a´ a,∗ , Andrea Slampov a´ b a b
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic Institute of Analytical Chemistry, Academy of Science of the Czech Republic, Brno, Czech Republic
a r t i c l e
i n f o
a b s t r a c t
Article history:
Heteroarylaminoethanol derivates are drugs which affect sympathetic nervous system and
Received 31 October 2006
are used for medications of hypertensis. In solutions they behave like weak bases and
Received in revised form
their pKa values represent important information on their potential biological uptake, phar-
20 December 2006
macological activity and in vivo biodisponsibility. This article brings the measurement of
Accepted 27 December 2006
pKa values of the series of seven new important heteroarylaminoethanols, compounds
Published on line 9 January 2007
with potential vasodilating, -adrenolytic and antioxidant activity, by capillary zone electrophoresis (CZE) with diode-array detection. It has been shown that capillary zone
Keywords:
electrophoresis measurements of pKa can be easily performed with very small quantities of
Capillary zone electrophoresis
studied substances, and, due to CZE separation power, the purity of samples is not of key
Constant ionic strength
importance. Moreover, the CZE method is fast and reliable, providing that suitable opera-
Dissociation constant
tional conditions are selected. The method is based on the measurement of the effective
Heteroarylaminoethanol derivates
mobility curves within a suitable pH range and related regression analysis where pKBH + and electrophoretic mobility of BH+ are explicitly involved. The selection of sufficient operational buffers is of key importance for accurate and reproducible results, and, this article brings step by step the consideration procedure involved in this process. Further, this paper brings principles of least square regression analysis of non-linear function corresponding to exact explicit formula for mobility curve of monovalent weak base. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Increased blood pressure is one of the major diseases of the cardiovascular system, leading to various organ dysfunctions, for example, left ventricular hypertrophy, ischemic heart disease, renal failure and cerebrovascular damage (Pinkney and Yudkin, 1994). Studied organic salts (Fig. 1) were designed and synthesized as a potential antihypertensive drug combining -adrenolytic, vasodilating and antioxidant activities
∗
¨ (Kurfurst et al., 2004). They belong to the group of heteroarylaminoethanol derivates that are known as drug affecting the sympathetic nervous system (Ruffolo et al., 1995). Structurally, compounds are also analogues of aryloxypropanolamines (Mokry´ et al., 2003) in which the oxymethylene group of linking chain becomes a part of furan ring. As revealed in recent years, reactive oxygen species (ROS) could play an important role in pathogenesis of hypertension (Friedman et al., 2003). Active compound that, besides the direct hypotensive effect,
Corresponding author. Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, ´ Palackeho 1-3, CZ-612 42, Brno, Czech Republic. Tel.: +42 0541 562924 ´ E-mail address: [email protected] (A. Liˇskova). Abbreviations: CZE, capillary zone electrophoresis; ROS, reactive oxygen species; MO, mesityloxide; MES, 4-morpholineethanesulfonic acid; MOPSO, -hydroxy-4-morpholinepropanesulfonic acid; TES, 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid; TAPS, [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid 0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.12.005
376
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
• Sodium hydroxide (NaOH) and mesityloxide were from Fluka (Buchs, Switzerland). • Tested compounds were produced in Department of Chemical Drugs (Faculty of Pharmacy, VFU Brno, Czech Republic) ¨ (Kurfurst et al., 2004). • Deionized water prepared by using aqua purificator G 7749 ¨ (Miele, Gutesloh, Germany) was used for preparation of all solutions.
2.2.
Fig. 1 – Structure of studied substances.
also have the ability to scavenge ROS should be more effective and useful in long-term antihypertensive therapy. Preliminary findings show promising vasodilating and -adrenolytic activity of tested compounds, as well as significant antioxidant ˇ potency (Racansk a´ et al., 2004). The knowledge of ionization constants during the discovery phase of a new drug provides information about biological uptake, pharmacological activity or in vivo biodisponibility. Acidity constants knowledge of new compounds is also an essential goal for further analytical studies such as enantiomeric separations or affinity constant determinations. Hence the discovery of new molecules requires accurate determination of pKa values. Tested compounds displayed low aqueous solubility and they are available only in small quantities. These facts make difficult the determination of dissociation constants by traditional methods such potentiometric titration, spectroscopy, conductometry or isotachophoresis. In this work CZE as a powerful separation technique widely applied in physicochemical profiling of pharmaceuticals has been used for pKa determination. CZE has emerged as a convenient and precise method for determination of dissociation constant of ionogenic group of various types compounds including amino acids (Zuskova´ ˇ akov ´ et al., 2006) (Vcel a´ et al., 2004), phosphinate group in phosphinic pseudopeptides (Koval et al., 2002), oligonucleotides (McKeown et al., 2001), alkaloids (Gong et al., 2003) or fluorinated propranolol derivates (Upthagrove and Wendel, 2001).
2.
Experimental
2.1.
Chemicals
All chemicals used were of the highest analytical purity. • Formic acid (HCOOH) p.a >99%, sodium formate (HCOONa), sodium acetate-trihydrate (CH3 COONa·3H2 O), acetic acid (CH3 COOH) p.a >99% were from Lachema (Brno, Czech Republic). • MES, MOPSO, TES, HEPES, TAPS and CHES were from Sigma–Aldrich (St. Louis, MO, USA).
Apparatus
• CZE experiments were carried out with a Beckmann P/ACE System 5000 (Beckmann Instruments, Fullerton, CA, USA) equipped with a UV detector set to direct detection at 214 nm. The P/ACE Station software was used for instrument control, data acquisition and data analysis. • Electrophoretic separations were performed in fused-silica capillaries of 57 cm total length, 50 cm effective length and 0.075 mm ID (Composite Metal Services, The Chase, Hallow, Worchester, UK). Before first use, a new capillary was conditioned as follows: 10 min with 1 M NaOH, 10 min with 0.1 M NaOH, 10 min with water and 30 min with the running buffer. Between runs, the capillary was rinsed with water (1 min) and background electrolyte (BGE) (3 min). • The pH values of used buffers were measured with pH meter WTW pH 526 (WTW, Prague, Czech Republic) equipped with a combined electrode SenTix 41.
2.3.
Electrophoretic measurements
• Electrophoretic measurements were performed in electrolyte systems of constant ionic strength 0.01 M within the pH range 3.01–8.96. The PeakMaster computer program (http://www.natur.cuni.cz/gas/pm51setup.exe) was employed to calculate the composition of the series of these BGEs with various pH. Their composition is given in Table 1. • Stock solutions of 1 × 10−3 M of all tested compounds were prepared in deionized water. Working solutions were prepared by diluting the stock solutions in each corresponding BGE to final concentrations 0.5 × 10−4 M. Solutions of samples and buffers were filtered through a 0.22 m syringe filter (Whatman, Maidstone, Kent, UK). • Sample introduction was made by applying pressure of 0.5 psi (3.5 kPa) for 10 s. Measurements were performed at a constant voltage 20 kV. The temperature of capillary was maintained at 25 ◦ C. • EOF was calculated from the migration time of mesityloxide injected as an aqueous solution 1 ml/1000 ml for 10 s at 0.5 psi (3.5 kPa). Mobility measurements were done in triplicate and all values (n = 30) were used in calculations of pKa . • The calculation of EOF mobility was carried out by using Eq. (1), EOF =
Ltot Leff VtEOF
(1)
where Ltot is the total length of the capillary and Leff the length to the detection window. V is the applied voltage and
377
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
Table 1 – Composition and buffering capacity of electrolyte systems
1 2 3 4 5 6 7 8 9 10
BGE system
pKa
pH
Co-ion
Counterion
Ionic strength (M) × 10−3
Buffering capacity (M) × 10−3
Formic acid + NaOH Formic acid + NaOH Acetic acid + NaOH MES + NaOH MES + NaOH MOPSO + NaOH TES + NaOH HEPES + NaOH TAPS + NaOH CHES + NaOH
3.75 3.75 4.76 6.10 6.10 6.90 7.40 7.50 8.40 9.30
3.01 4.01 5.01 5.80 6.21 6.70 7.20 7.46 8.91 8.96
Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium
Formate Formate Acetate MES MES MOPSO TES HEPES TAPS CHES
10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
22.03 7.97 7.85 15.01 9.57 13.72 13.72 11.64 5.09 15.59
tEOF the observed migration time of mesityloxide serving as the EOF marker. • The reproducibility of migration times was studied by replicate injections at the same pH. The relative standard deviations (RSD) of migration times were 1.32–1.45% (intraday) and 1.63–2.04% (inter-day). • The effective electrophoretic mobilities of the analytes were calculated from observed migration times of each analyte and migration times of the mesityloxide (MO) as an EOF marker according to the equation: eff = app − EOF
Ltot Leff = V
1 tapp
−
1
tEOF
(2)
where eff is the effective electrophoretic mobility (m2 V−1 s−1 ), app is the apparent electrophoretic mobility and EOF the mobility of the EOF. • The sets of the experimentally obtained effective mobilities at different pH were fitted by the curve described by the definitive equation for effective mobility, eff , of monovalent weak base, 10−pH
eff = BH +
(3)
KBH+ + 10−pH
see Eqs. (1) and (2) where the model mixture injected contained 0.4 l/ml MO and 0.5 × 10−4 M substance 4/1. Mixture was separated in 10 mM sodium acetate buffer (pH 5.01) and conditions above. For the selection of the pH range and composition of the related buffers we have made the following considerations. To measure reliable mobility curve of a weak base, a pH range used should cover solutions where the lowest pH guarantees practically full protonation ([BH+ ] > 99.9%) of the base, BH+ , and the highest pH brings no protonation, i.e., neutral species, B ([BH+ ] ≤ 0.1%). By using well known HendersonHasselbach equation we get the pH range ±3 pH units around expected pKBH + of substances in question. In our case the expected pKBH + were around six and, therefore, the pH range used should cover pH 3–9. Further experimental factors which were taken into consideration were ionic strength and temperature. Here dilute buffers are recommended, since they allow one to use higher voltages and reach fast analyses while keeping the Joule heat at low levels. This reduces the elevation of the temperature inside the capillary above the thermostating temperature. It is known in capillary electrophoresis practice that electric powers less than 0.4 W per meter capillary bring negligible rise in temperature (less than 0.3 ◦ C) (Foret et al., 1993). Diluted
where BH + is ionic mobility of fully protonated base, BH+ , and KBH + is the dissociation constant of BH+ . • Two unknown quantities in Eq. (3), BH + and KBH + were determined by non-linear least square regression analysis, defined by the condition, n
i −
BH+
i=1
10−pHi KBH+ + 10−pHi
2
= minimum
(4)
where pHi and i are related experimentally set pH value and related measured eff . Condition Eq. (4) lead to a set of two non-linear equations which were solved by numerical iterations and yielded the best fit values of BH + and KBH + . It has also provided the regression coefficient residual variance and standard deviations of BH + and KBH + .
3.
Results and discussions
Fig. 2 brings an example of a typical experimental analysis record used for measuring the primary date tEOF and tapp ,
Fig. 2 – Electropherogram of a model mixture: (1) 0.5 × 10−4 M compound 4/1 + (2) 0.4 l/ml MO in 10 mM sodium acetate buffer (pH 5.01). Analyte was injected by applying a pressure 0.5 psi (3.5 kPa) for 10 s into fused-silica capillary of 57 cm total length, 50 cm effective length and 0.075 mm ID, separated at 20 kV and 25 ◦ C, and detected at 214 nm.
378
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
Table 2 – Power inside the capillary
1 2 3 4 5 6 7 8 9 10
BGE system
pH
I (A)
Formic acid + NaOH Formic acid + NaOH Acetic acid + NaOH MES + NaOH MES + NaOH MOPSO + NaOH TES + NaOH HEPES + NaOH TAPS + NaOH CHES + NaOH
3.01 4.01 5.01 5.80 6.21 6.70 7.20 7.46 8.91 8.96
41.1 31.4 26.1 22.7 21.8 20.7 21.5 21.2 21.6 21.9
buffers also facilitate the possibility to use the concentrations instead of activities of the species involved in calculations. On the other hand, diluted buffers have also some limits and considerations. First of all, according to principles of zone migration in CZE (Foret et al., 1993), the prerequisite of the symmetric peaks and high separation efficiency are negligible pH changes in the zones of analytes, and, negligible contribution of the analytes to the overall conductivity of the solution in the capillary. Hence, the concentration of a buffer used as the BGE should be at least by two orders of magnitude higher than the maximum concentration (apex of the peak) of migrating analyte.Here, the sensitivity of a detector used comes into game, too. The UV absorbance detectors in commercial instrumentation and with usual capillary window (100 m), require the analyte concentrations 10−5 M and higher to provide reliable detection of substances with aromatic rings (which is our case). Hence, our BGEs should have concentrations on the level of milimolar solutions and higher. Another object of consideration in preparation the buffer series was buffer capacity. Too low buffer capacity would result in not sufficient pH control in the analyte zone, and then there would be a bias in the final pKa determination. Hence, the buffers should be selected in such a way that the operational pH is within pKa ± 1, where pKa corresponds to the buffering species. Based on the above considerations, we have selected a series of 10 buffers. They are listed in Table 1. For measuring the mobility curves we have made some tentative analyses and then we have set contrast voltage regime with +20 kV at capillary inlet. This regime provided reasonable fast analyses and sufficiently low Joule heating. The operational data are listed in Table 2. Fig. 3 brings an example of the set of experimental date measured for one selected substance (4/1) and the calculated best fit mobility curve. In this way the data on all substances in question were determined.Statistic processing of the results of measuring the mobility curve is not an easy problem. The usual approach, based on the linearization of above function, is problematic. It has been described already (Dobos et al., 2004). The linearized equation in the procedure proposed uses the expression, b /e , where the symbols represent mobility of the fully ionized species determined at low pH, and electrophoretic mobility at actual experimental pH. It is obvious that this expression gives obscure results at high pH values, where the species are negligibly protonated and their
U (kV)
P (W)
20 20 20 20 20 20 20 20 20 20
0.469 0.358 0.298 0.259 0.249 0.236 0.245 0.242 0.246 0.251
Fig. 3 – Mobility curve of selected substance (4/1).
actual mobilities are approached the zero. Here, the related ratio goes to infinity and the linearization procedure completely fails. Therefore, we have adopted the procedure which is based on direct application of least square method to nonˇ linear expression for mobility curve (Bocek, in preparation, 2006). The parameters of the mobility curves calculated by non-linear least square regression analysis are summarized in Table 3. The pKa values of compounds 4/2, 4/3 a 4/4 refer to evident R2 substituent influence on acidity of the molecule. Fluorine, as the most electronegative substituent, increases acidity of the molecule due to its negative inductive effect, while methyl and methoxy substituent decreases acidity. Compound 4/1 (with fluorine in position four of benzene ring) has similar
Table 3 – Calculated parameters of the mobility curves, ionic mobility of protonated base BH + , acidic dissociation constant of protonated base pKBH + Compound
BH + (m2 V−1 s−1 ) × 10−8
4/1 4/2 4/3 4/4 4/5 4/6 4/6E a
95% confidents limits.
1.84 1.80 1.86 1.86 1.89 1.90 1.81
± ± ± ± ± ± ±
0.17 0.20 0.18 0.22 0.14 0.13 0.21
pKBH + 6.13 6.57 6.11 6.38 6.27 6.19 6.11
± ± ± ± ± ± ±
0.04a 0.05 0.04 0.05 0.03 0.03 0.05
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
acidity as substance 4/3 with fluorine in position 2. Influence of R1 substituents (methyl or ethyl) on pKa value was not observed.
4.
Conclusion
The measurement of dissociation constants and ionic mobilities of protonated bases, seven new compounds, with potential vasodilating, -adrenolytic and antioxidant activity have been performed for the first time. The data obtained provide information useful for physico-chemical studies of the drug candidates as well as for their pharmacological characterization. The CZE method developed in this work based on electrophoretic mobility curves is simple, effective, inexpensive and shows acceptable reproducibility and accuracy for determination of dissociation constant of newly synthesized heteroarylaminoethanols.
Acknowledgements This work was supported by Grants nos. A 400310609 and IAA 4031401 of the Grant Agency of Academy of Science of the Czech Republic. The authors thank to Assoc. Prof. Ludmila ´ ˇ Kˇrivankov a´ and Prof. Petr Bocek, the Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, for interest in this work and valuable hints and advices.
references
ˇ Bocek, unpublished results, 2006. ´ and, ´ ´ Dobos, Zs., Lor T., Hollosy, F., 2004. Determination of the basicity of Mannich ketones by capillary electrophoresis. J. Chromatogr. B 799, 179–183. Friedman, J., Peleg, E., Kagan, T., Shnizer, S., Rosenthal, T., 2003. Oxidative stress in hypertensive, diabetic, and diabetic hypertensive rats. Am. J. Hypertens. 16 (12), 1049–1052.
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´ ´ L., Bocek, ˇ Foret, F., Kˇrivankov a, P., 1993. Phenomena Accompanying Electrophoresis. Capillary Zone Electrophoresis. VCH Verlagsgesellschaft, Weinheim. Gong, S., Su, X., Bo, T., Zhang, X., Liu, H., Li, K.A., 2003. Determination of dissociation constants of ten alkaloids by capillary zone electrophoresis. J. Sep. Sci. 26, 549–554. ˇ ˇ ´ M., Garrow, T.A., 2002. Koval, D., Kaˇsicka, V., Jira´ cek, J., Collinsova, Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis. J. Chromatogr. B 770, 145–154. ¨ ˇ J., Csollei, ¨ Kurfurst, P., Marek, J., Vanco, J., 2004. (R)-4-(4fluorophenyl)-1-[2-hydroxy-2-(3-methyl-1-benzofuran-2yl)ethyl]-piperazinium chloride propan-1-ol solvate. Acta Cryst. C 60, 494–495. McKeown, A.P., Shaw, P.N., Barret, D.A., 2001. Electrophoretic behaviour of oligonucleotides and mono-, di-, triphosphate nucleotides by capillary zone electrophoresis. Electrophoresis 22, 1119–1126. ´ P., Zemanova, ´ M., Csollei, ¨ ˇ ´ E., Tumova, ´ I., 2003. Mokry, J., Racansk a, Synthesis and pharmacological evaluation of novel potential ultrashort-acting beta-blockers. Pharmazie 58 (1), 18–21. Pinkney, J.H., Yudkin, J.S., 1994. Antihypertensive drugs – issues beyond blood-pressure control. Prog. Cardiovasc. Dis. 36 (5), 397–415. ˇ ˇ ´ E., Kurfurst, ¨ ¨ Racansk a, P., Csollei, J., Svec, P., 2004. Cardiovascular effects of newly sythesized hybrid heteroarylaminoethanols. Acta Fac. Pharm. Univ. Comenianae 51, 182–191. Ruffolo, R.R., Bondinell, W., Hieble, J.P., 1995. Alpha-adrenoceptors and beta-adrenoceptors – from the gene to the clinic. 2. Structure-activity-relationships and therapeutic applications. J. Med. Chem. 38 (19), 3682–3686. Upthagrove, A.L., Wendel, L., 2001. Importance of amine pKa and distribution coefficient in the metabolism of fluorinated propranolol derivates. Preparation, identification of metabolite regioisomers, and metabolism by CYP2D6. Drug Metab. Dispos. 29, 1377–1388. ˇ akov ´ ´ K., Zuskova, ´ I., Kenndler, E., Gaˇs, B., 2004. Vcel a, Determination of cationic mobilities and pKa values of 22 amino acids by capillary zone electrophoresis. Electrophoresis 25, 309–317, http://www.natur.cuni.cz/gas/pm51setup.exe. ´ I., Novotna, ´ A., Vcel ˇ akov ´ ´ K., Gaˇs, B., 2006. Zuskova, a, Determination of limiting mobilities and dissociation constants of 21 amino acids by capillary zone electrophoresis at very low pH. J. Chromatogr. B 841, 129–134.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Measurement of pKa values of newly synthesized heteroarylaminoethanols by CZE ˇ ˇ Anna Liskov a´ a,∗ , Andrea Slampov a´ b a b
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic Institute of Analytical Chemistry, Academy of Science of the Czech Republic, Brno, Czech Republic
a r t i c l e
i n f o
a b s t r a c t
Article history:
Heteroarylaminoethanol derivates are drugs which affect sympathetic nervous system and
Received 31 October 2006
are used for medications of hypertensis. In solutions they behave like weak bases and
Received in revised form
their pKa values represent important information on their potential biological uptake, phar-
20 December 2006
macological activity and in vivo biodisponsibility. This article brings the measurement of
Accepted 27 December 2006
pKa values of the series of seven new important heteroarylaminoethanols, compounds
Published on line 9 January 2007
with potential vasodilating, -adrenolytic and antioxidant activity, by capillary zone electrophoresis (CZE) with diode-array detection. It has been shown that capillary zone
Keywords:
electrophoresis measurements of pKa can be easily performed with very small quantities of
Capillary zone electrophoresis
studied substances, and, due to CZE separation power, the purity of samples is not of key
Constant ionic strength
importance. Moreover, the CZE method is fast and reliable, providing that suitable opera-
Dissociation constant
tional conditions are selected. The method is based on the measurement of the effective
Heteroarylaminoethanol derivates
mobility curves within a suitable pH range and related regression analysis where pKBH + and electrophoretic mobility of BH+ are explicitly involved. The selection of sufficient operational buffers is of key importance for accurate and reproducible results, and, this article brings step by step the consideration procedure involved in this process. Further, this paper brings principles of least square regression analysis of non-linear function corresponding to exact explicit formula for mobility curve of monovalent weak base. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Increased blood pressure is one of the major diseases of the cardiovascular system, leading to various organ dysfunctions, for example, left ventricular hypertrophy, ischemic heart disease, renal failure and cerebrovascular damage (Pinkney and Yudkin, 1994). Studied organic salts (Fig. 1) were designed and synthesized as a potential antihypertensive drug combining -adrenolytic, vasodilating and antioxidant activities
∗
¨ (Kurfurst et al., 2004). They belong to the group of heteroarylaminoethanol derivates that are known as drug affecting the sympathetic nervous system (Ruffolo et al., 1995). Structurally, compounds are also analogues of aryloxypropanolamines (Mokry´ et al., 2003) in which the oxymethylene group of linking chain becomes a part of furan ring. As revealed in recent years, reactive oxygen species (ROS) could play an important role in pathogenesis of hypertension (Friedman et al., 2003). Active compound that, besides the direct hypotensive effect,
Corresponding author. Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, ´ Palackeho 1-3, CZ-612 42, Brno, Czech Republic. Tel.: +42 0541 562924 ´ E-mail address: [email protected] (A. Liˇskova). Abbreviations: CZE, capillary zone electrophoresis; ROS, reactive oxygen species; MO, mesityloxide; MES, 4-morpholineethanesulfonic acid; MOPSO, -hydroxy-4-morpholinepropanesulfonic acid; TES, 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid; TAPS, [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid 0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.12.005
376
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
• Sodium hydroxide (NaOH) and mesityloxide were from Fluka (Buchs, Switzerland). • Tested compounds were produced in Department of Chemical Drugs (Faculty of Pharmacy, VFU Brno, Czech Republic) ¨ (Kurfurst et al., 2004). • Deionized water prepared by using aqua purificator G 7749 ¨ (Miele, Gutesloh, Germany) was used for preparation of all solutions.
2.2.
Fig. 1 – Structure of studied substances.
also have the ability to scavenge ROS should be more effective and useful in long-term antihypertensive therapy. Preliminary findings show promising vasodilating and -adrenolytic activity of tested compounds, as well as significant antioxidant ˇ potency (Racansk a´ et al., 2004). The knowledge of ionization constants during the discovery phase of a new drug provides information about biological uptake, pharmacological activity or in vivo biodisponibility. Acidity constants knowledge of new compounds is also an essential goal for further analytical studies such as enantiomeric separations or affinity constant determinations. Hence the discovery of new molecules requires accurate determination of pKa values. Tested compounds displayed low aqueous solubility and they are available only in small quantities. These facts make difficult the determination of dissociation constants by traditional methods such potentiometric titration, spectroscopy, conductometry or isotachophoresis. In this work CZE as a powerful separation technique widely applied in physicochemical profiling of pharmaceuticals has been used for pKa determination. CZE has emerged as a convenient and precise method for determination of dissociation constant of ionogenic group of various types compounds including amino acids (Zuskova´ ˇ akov ´ et al., 2006) (Vcel a´ et al., 2004), phosphinate group in phosphinic pseudopeptides (Koval et al., 2002), oligonucleotides (McKeown et al., 2001), alkaloids (Gong et al., 2003) or fluorinated propranolol derivates (Upthagrove and Wendel, 2001).
2.
Experimental
2.1.
Chemicals
All chemicals used were of the highest analytical purity. • Formic acid (HCOOH) p.a >99%, sodium formate (HCOONa), sodium acetate-trihydrate (CH3 COONa·3H2 O), acetic acid (CH3 COOH) p.a >99% were from Lachema (Brno, Czech Republic). • MES, MOPSO, TES, HEPES, TAPS and CHES were from Sigma–Aldrich (St. Louis, MO, USA).
Apparatus
• CZE experiments were carried out with a Beckmann P/ACE System 5000 (Beckmann Instruments, Fullerton, CA, USA) equipped with a UV detector set to direct detection at 214 nm. The P/ACE Station software was used for instrument control, data acquisition and data analysis. • Electrophoretic separations were performed in fused-silica capillaries of 57 cm total length, 50 cm effective length and 0.075 mm ID (Composite Metal Services, The Chase, Hallow, Worchester, UK). Before first use, a new capillary was conditioned as follows: 10 min with 1 M NaOH, 10 min with 0.1 M NaOH, 10 min with water and 30 min with the running buffer. Between runs, the capillary was rinsed with water (1 min) and background electrolyte (BGE) (3 min). • The pH values of used buffers were measured with pH meter WTW pH 526 (WTW, Prague, Czech Republic) equipped with a combined electrode SenTix 41.
2.3.
Electrophoretic measurements
• Electrophoretic measurements were performed in electrolyte systems of constant ionic strength 0.01 M within the pH range 3.01–8.96. The PeakMaster computer program (http://www.natur.cuni.cz/gas/pm51setup.exe) was employed to calculate the composition of the series of these BGEs with various pH. Their composition is given in Table 1. • Stock solutions of 1 × 10−3 M of all tested compounds were prepared in deionized water. Working solutions were prepared by diluting the stock solutions in each corresponding BGE to final concentrations 0.5 × 10−4 M. Solutions of samples and buffers were filtered through a 0.22 m syringe filter (Whatman, Maidstone, Kent, UK). • Sample introduction was made by applying pressure of 0.5 psi (3.5 kPa) for 10 s. Measurements were performed at a constant voltage 20 kV. The temperature of capillary was maintained at 25 ◦ C. • EOF was calculated from the migration time of mesityloxide injected as an aqueous solution 1 ml/1000 ml for 10 s at 0.5 psi (3.5 kPa). Mobility measurements were done in triplicate and all values (n = 30) were used in calculations of pKa . • The calculation of EOF mobility was carried out by using Eq. (1), EOF =
Ltot Leff VtEOF
(1)
where Ltot is the total length of the capillary and Leff the length to the detection window. V is the applied voltage and
377
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
Table 1 – Composition and buffering capacity of electrolyte systems
1 2 3 4 5 6 7 8 9 10
BGE system
pKa
pH
Co-ion
Counterion
Ionic strength (M) × 10−3
Buffering capacity (M) × 10−3
Formic acid + NaOH Formic acid + NaOH Acetic acid + NaOH MES + NaOH MES + NaOH MOPSO + NaOH TES + NaOH HEPES + NaOH TAPS + NaOH CHES + NaOH
3.75 3.75 4.76 6.10 6.10 6.90 7.40 7.50 8.40 9.30
3.01 4.01 5.01 5.80 6.21 6.70 7.20 7.46 8.91 8.96
Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium
Formate Formate Acetate MES MES MOPSO TES HEPES TAPS CHES
10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
22.03 7.97 7.85 15.01 9.57 13.72 13.72 11.64 5.09 15.59
tEOF the observed migration time of mesityloxide serving as the EOF marker. • The reproducibility of migration times was studied by replicate injections at the same pH. The relative standard deviations (RSD) of migration times were 1.32–1.45% (intraday) and 1.63–2.04% (inter-day). • The effective electrophoretic mobilities of the analytes were calculated from observed migration times of each analyte and migration times of the mesityloxide (MO) as an EOF marker according to the equation: eff = app − EOF
Ltot Leff = V
1 tapp
−
1
tEOF
(2)
where eff is the effective electrophoretic mobility (m2 V−1 s−1 ), app is the apparent electrophoretic mobility and EOF the mobility of the EOF. • The sets of the experimentally obtained effective mobilities at different pH were fitted by the curve described by the definitive equation for effective mobility, eff , of monovalent weak base, 10−pH
eff = BH +
(3)
KBH+ + 10−pH
see Eqs. (1) and (2) where the model mixture injected contained 0.4 l/ml MO and 0.5 × 10−4 M substance 4/1. Mixture was separated in 10 mM sodium acetate buffer (pH 5.01) and conditions above. For the selection of the pH range and composition of the related buffers we have made the following considerations. To measure reliable mobility curve of a weak base, a pH range used should cover solutions where the lowest pH guarantees practically full protonation ([BH+ ] > 99.9%) of the base, BH+ , and the highest pH brings no protonation, i.e., neutral species, B ([BH+ ] ≤ 0.1%). By using well known HendersonHasselbach equation we get the pH range ±3 pH units around expected pKBH + of substances in question. In our case the expected pKBH + were around six and, therefore, the pH range used should cover pH 3–9. Further experimental factors which were taken into consideration were ionic strength and temperature. Here dilute buffers are recommended, since they allow one to use higher voltages and reach fast analyses while keeping the Joule heat at low levels. This reduces the elevation of the temperature inside the capillary above the thermostating temperature. It is known in capillary electrophoresis practice that electric powers less than 0.4 W per meter capillary bring negligible rise in temperature (less than 0.3 ◦ C) (Foret et al., 1993). Diluted
where BH + is ionic mobility of fully protonated base, BH+ , and KBH + is the dissociation constant of BH+ . • Two unknown quantities in Eq. (3), BH + and KBH + were determined by non-linear least square regression analysis, defined by the condition, n
i −
BH+
i=1
10−pHi KBH+ + 10−pHi
2
= minimum
(4)
where pHi and i are related experimentally set pH value and related measured eff . Condition Eq. (4) lead to a set of two non-linear equations which were solved by numerical iterations and yielded the best fit values of BH + and KBH + . It has also provided the regression coefficient residual variance and standard deviations of BH + and KBH + .
3.
Results and discussions
Fig. 2 brings an example of a typical experimental analysis record used for measuring the primary date tEOF and tapp ,
Fig. 2 – Electropherogram of a model mixture: (1) 0.5 × 10−4 M compound 4/1 + (2) 0.4 l/ml MO in 10 mM sodium acetate buffer (pH 5.01). Analyte was injected by applying a pressure 0.5 psi (3.5 kPa) for 10 s into fused-silica capillary of 57 cm total length, 50 cm effective length and 0.075 mm ID, separated at 20 kV and 25 ◦ C, and detected at 214 nm.
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Table 2 – Power inside the capillary
1 2 3 4 5 6 7 8 9 10
BGE system
pH
I (A)
Formic acid + NaOH Formic acid + NaOH Acetic acid + NaOH MES + NaOH MES + NaOH MOPSO + NaOH TES + NaOH HEPES + NaOH TAPS + NaOH CHES + NaOH
3.01 4.01 5.01 5.80 6.21 6.70 7.20 7.46 8.91 8.96
41.1 31.4 26.1 22.7 21.8 20.7 21.5 21.2 21.6 21.9
buffers also facilitate the possibility to use the concentrations instead of activities of the species involved in calculations. On the other hand, diluted buffers have also some limits and considerations. First of all, according to principles of zone migration in CZE (Foret et al., 1993), the prerequisite of the symmetric peaks and high separation efficiency are negligible pH changes in the zones of analytes, and, negligible contribution of the analytes to the overall conductivity of the solution in the capillary. Hence, the concentration of a buffer used as the BGE should be at least by two orders of magnitude higher than the maximum concentration (apex of the peak) of migrating analyte.Here, the sensitivity of a detector used comes into game, too. The UV absorbance detectors in commercial instrumentation and with usual capillary window (100 m), require the analyte concentrations 10−5 M and higher to provide reliable detection of substances with aromatic rings (which is our case). Hence, our BGEs should have concentrations on the level of milimolar solutions and higher. Another object of consideration in preparation the buffer series was buffer capacity. Too low buffer capacity would result in not sufficient pH control in the analyte zone, and then there would be a bias in the final pKa determination. Hence, the buffers should be selected in such a way that the operational pH is within pKa ± 1, where pKa corresponds to the buffering species. Based on the above considerations, we have selected a series of 10 buffers. They are listed in Table 1. For measuring the mobility curves we have made some tentative analyses and then we have set contrast voltage regime with +20 kV at capillary inlet. This regime provided reasonable fast analyses and sufficiently low Joule heating. The operational data are listed in Table 2. Fig. 3 brings an example of the set of experimental date measured for one selected substance (4/1) and the calculated best fit mobility curve. In this way the data on all substances in question were determined.Statistic processing of the results of measuring the mobility curve is not an easy problem. The usual approach, based on the linearization of above function, is problematic. It has been described already (Dobos et al., 2004). The linearized equation in the procedure proposed uses the expression, b /e , where the symbols represent mobility of the fully ionized species determined at low pH, and electrophoretic mobility at actual experimental pH. It is obvious that this expression gives obscure results at high pH values, where the species are negligibly protonated and their
U (kV)
P (W)
20 20 20 20 20 20 20 20 20 20
0.469 0.358 0.298 0.259 0.249 0.236 0.245 0.242 0.246 0.251
Fig. 3 – Mobility curve of selected substance (4/1).
actual mobilities are approached the zero. Here, the related ratio goes to infinity and the linearization procedure completely fails. Therefore, we have adopted the procedure which is based on direct application of least square method to nonˇ linear expression for mobility curve (Bocek, in preparation, 2006). The parameters of the mobility curves calculated by non-linear least square regression analysis are summarized in Table 3. The pKa values of compounds 4/2, 4/3 a 4/4 refer to evident R2 substituent influence on acidity of the molecule. Fluorine, as the most electronegative substituent, increases acidity of the molecule due to its negative inductive effect, while methyl and methoxy substituent decreases acidity. Compound 4/1 (with fluorine in position four of benzene ring) has similar
Table 3 – Calculated parameters of the mobility curves, ionic mobility of protonated base BH + , acidic dissociation constant of protonated base pKBH + Compound
BH + (m2 V−1 s−1 ) × 10−8
4/1 4/2 4/3 4/4 4/5 4/6 4/6E a
95% confidents limits.
1.84 1.80 1.86 1.86 1.89 1.90 1.81
± ± ± ± ± ± ±
0.17 0.20 0.18 0.22 0.14 0.13 0.21
pKBH + 6.13 6.57 6.11 6.38 6.27 6.19 6.11
± ± ± ± ± ± ±
0.04a 0.05 0.04 0.05 0.03 0.03 0.05
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 0 ( 2 0 0 7 ) 375–379
acidity as substance 4/3 with fluorine in position 2. Influence of R1 substituents (methyl or ethyl) on pKa value was not observed.
4.
Conclusion
The measurement of dissociation constants and ionic mobilities of protonated bases, seven new compounds, with potential vasodilating, -adrenolytic and antioxidant activity have been performed for the first time. The data obtained provide information useful for physico-chemical studies of the drug candidates as well as for their pharmacological characterization. The CZE method developed in this work based on electrophoretic mobility curves is simple, effective, inexpensive and shows acceptable reproducibility and accuracy for determination of dissociation constant of newly synthesized heteroarylaminoethanols.
Acknowledgements This work was supported by Grants nos. A 400310609 and IAA 4031401 of the Grant Agency of Academy of Science of the Czech Republic. The authors thank to Assoc. Prof. Ludmila ´ ˇ Kˇrivankov a´ and Prof. Petr Bocek, the Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, for interest in this work and valuable hints and advices.
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