Sp

HG

a

ARRAA

KACNEJ

1

pben[(osiorfnr

p(nttk

0h

Fluid Phase Equilibria 356 (2013) 201– 208

Contents lists available at ScienceDirect

Fluid Phase Equilibria

j ourna l ho me page: www.elsev ier .com/ locate / f lu id

tudy of the ionic equilibriums in aqueous solutions and coordinationroperties of Adefovir and Cidofovir used as antiviral drugs

asan Atabey, Hayati Sari ∗

aziosmanpas a University, Science and Arts Faculty, Chemistry Department, 60250 Tokat, Turkey

r t i c l e i n f o

rticle history:eceived 10 March 2013eceived in revised form 8 July 2013ccepted 15 July 2013vailable online 26 July 2013

a b s t r a c t

The dissociation constants of Adefovir ({[2-(6-amino-9H-purine-9-l)ethoxi]methyl}phosphoric acid)used in the treatment of hepatitis B and Cidofovir ({[2-(6-amino-9H-purine-9-l)ethoxi]methyl}phosphoric acid), used in the treatment of cytomegalovirus retinitis in AIDS patients, together with thestability constants of its Cu2+, Ni2+, Zn2+, Co2+, Ca2+, and Mg2+ complexes were studied by potentiometrictitration. Adefovir and Cidofovir were abbreviated to PMEA and HPMPC. All measurements were obtained

−3

eywords:defoviridofovirucleotide analogquilibrium constantsob’s method

under two sets of conditions: at 298 K and ionic strength (I) of 0.1 mol dm (NaCl) and at 310 K and I of0.16 mol dm−3 (NaCl), corresponding to the conditions of human blood. Equilibrium constants in aque-ous solution were calculated using the program SUPERQUAD. Also, solvent effects on the equilibriumconstants of the ligands were examined. Moreover, all coordination studies were carried out with the lig-and/metal molar ratios of 1:1. This ligand/metal stoichiometric ratio was spectrometrically determinedusing Job’s method.

. Introduction

Viruses cause many diseases such as AIDS, hepatitis, Ebola, her-es, influenza and rabies. Treatment of such diseases is difficult;ecause, antibiotics do not affect viruses. Today, the some viral dis-ases are treated immune system stimulation with interferon orucleotide analogs in the form of suppression of viral replication1]. The basic chemical structure of the acyclic nucleotide analogANP) compounds consists of a purine base (i.e., adenine, guanine,r 2,6-diaminopurine) or pyrimidine base, attached to an acyclicide chain that ends in a phosphonate group. The strong C P bonds chemically and enzymatically stable, thus preventing hydrolysisf the ANP compounds in biological systems. Based on the antivi-al activity spectrum, the ANP compounds can be divided in theollowing subclasses [2]. The highly anionic charge of the phospho-ate moiety of the ANP compounds makes their cellular uptakeather inefficient.

Adefovir [{[2-(6-amino-9H-purine-9-yl)ethoxi]methyl}hosphoric acid] (PMEA) and Cidofovir (S)-1-[3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) (see Fig. 1) areucleoside phosphonate analogs, a class of novel antivirals struc-

urally related to natural nucleotides [3,4] and they were showno enter the cells by endocytosis, a process that is marked by slowinetics and temperature dependence [5,6]. Adefovir dipivoxil,

∗ Corresponding author. Tel.: +90 356 252 16 16x3015; fax: +90 356 252 15 85.E-mail address: hayati.sari@gop.edu.tr (H. Sari).

378-3812/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.fluid.2013.07.033

© 2013 Elsevier B.V. All rights reserved.

an orally available prodrug of PMEA, is currently undergoingclinical evaluation as an anti-HIV and anti-hepatitis B virus agent[7–11]. HPMPC is an antiviral nucleotide analog [12–14] withpotent in vitro and in vivo activity against human cytomegalovirus(HCMV) retinitis in AIDS patients [15–17] and other herpesviruses[18–21].

As a result of the above, numerous studies have been done onthe antiviral activities of PMEA and HPMPC. However, theirs ionicbehaviors in aqueous solution and coordination properties havenot been explained sufficiently, whereas many articles have beenpublished related to the electro-analytical properties of similarcompounds using potentiometric methods [22–26]. Proton trans-fer reactions of PMEA and HPMPC are very important for explainingits coordination properties and biological activity. Thus, in thiswork, the dissociation constants of the ligands, and the stabilityconstants of its complexes with Cu2+, Ni2+, Zn2+, Co2+, Ca2+, andMg2+ ions were studied potentiometrically. This is a very usefulelectro-analytical method [27].

2. Materials and methods

2.1. Reagents

CuCl2, NiCl2, ZnCl2, CoCl2, CaCl2, MgCl2, and NaCl used in

this research were purchased from Merck. All reagents were ofanalytical quality (≥98%) and were used without further purifica-tion. Potassium hydrogen phthalate (KHP) and borax (Na2B4O7)were purchased from Fluka. For calibration of the electrode,

202 H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208

tructu

0((HiCtscuawdatbw

2

p0ApaipswaCccsiaadu(o

m

Fig. 1. Chemical s

.05 mol kg−1 KHP and 0.01 mol kg−1 borax were prepared. PMEApurity 99%) is purchased from Watson International Ltd. HPMPCpurity 98%), ethyl alcohol, 0.1 mol dm−3 NaOH, and 0.1 mol dm−3

Cl as standard were purchased from Aldrich. Solutions of metalsons (1 × 10−3 mol dm−3) were prepared from CuCl2, NiCl2, ZnCl2,oCl2, CaCl2, and MgCl2 and standardized with ethylenediamine-etraacetic acid (EDTA) [28]. A PerkinElmer Lambda 35 UV/vispectrometer was used for determination of the HPMPC-metal stoi-hiometric ratio. CO2-free double-distilled deionized water wassed throughout the experiments, which had been obtained usingn aquaMAXTM-Ultra water purification system (Young Lin Inst.)hose resistivity was 18.2 M� cm−1. All experiments were con-ucted with a Molspin pH meterTM that was connected to anutomatic buret with an Orion 8102BNUWP ROSS Ultra combina-ion pH electrode. Temperature of the titration cell was controlledy a thermostat (DIGITHERM 100, SELECTA) and the cell solutionas stirred constantly during the experiments.

.2. Procedures

A 1 × 10−3 mol dm−3 solution of the ligands in water wasrepared and 0.01 mmol of each was placed in the cell. Then.1 mol dm−3 NaOH and 0.1 mol dm−3 HCl as standard were used.n ionic background of 1.0 mol dm−3 NaCl stock solution wasrepared to maintain constant ionic strength. Then 0.03 mmolcid solution was added from 0.1 mol dm−3 HCl for withdraw-ng to an initial pH of approximately 3. Nitrogen gas (99.9%) wasurged through the cell solutions constantly to exclude atmo-pheric CO2 during all experiments. Solutions of complex titrationere prepared with the same quantities of components. Addition-

lly, 0.01 mmol of each metal ion, including CuCl2, NiCl2, ZnCl2,oCl2, CaCl2, and MgCl2 solutions, was added. All experiments werearried out at the molar ratio of 1:1 ligand to metal and they werearried out at both 298 K, I: 0.1 ionic strength and 310 K, I: 0.16 ionictrength. The electrode was calibrated according to the instructionsn the Molspin manual [29], with buffer solutions of pH 4.005 (KHP)nd pH 9.180 (borax) at 298 K, and pH 4.022 (KHP) and 9.088 (borax)t 310 K [30] in aqueous solution. The summary of the titration con-itions are given in Table 1. The program SUPERQUAD [31] wassed for the determination of equilibrium constants. The pH data

115–135) were obtained after the addition of 0.03 cm3 incrementsf 0.025 mol dm−3 NaOH solutions.

But, in Section 3.2, mixed solution was used in the experi-ents. Therefore, the potentiometric cell was calibrated before

res of the ligands.

each experiment to obtain the pH values for the solvent mixturestudied [32,33]. For this purpose, the HCl solutions prepared in eachmedium with titrated NaOH solutions. For all the solvent mixturesexamined, reproducible values of the autoprotolysis constants Kw

were calculated from several series of [H+] and [OH−] measure-ments in 0.10 mol dm−3 NaCl [34,35]. During each titration the ionicstrength was maintained at 0.1 mol dm−3 NaCl and a potential read-ing were taken after a suitable time (normally 2–3 min) for eachequilibration.

3. Results and discussion

3.1. Dissociation constants

All measurements for the calculation of dissociation constantsof the ligands were obtained potentiometrically in about room (I:0.1 mol dm−3 of NaCl at 298 K) and human blood (I: 0.16 mol dm−3

of NaCl at 310 K) conditions. If LH75+ and LH4

2+ denotes the fullyprotonated forms of PMEA and HPMPC (see Fig. 10a), theirs depro-tonation equilibriums are as follows:

LHn + H2O � LHn−1 + H3O+ (1)

In each stage, one proton dissociates and dissociation constants(Kn; n = 1–7) are given as;

Kn =[LHn−1] .

[H3O+]

[LHn](2)

Hence, the titration curves for the ligands in water with NaOHas a titrant are shown in Fig. 2.

HPMPC has two parts. One of these parts is cytosine, which isa nucleic base and the most basic atom is 4O within cytosine. It isassumed that high alkalinity causes the protonation of the oxygenatom in the 4 position on the ligand and increases the mobilityof its �-electrons. In solutions more alkaline than pH 9, the oxygenatom in the 4 position begins to be hydrolyzed at appreciable ratios[36,37]. Therefore, electron pairs that are on the sp3 nitrogen ofcytosine participate in resonance and this is caused by the ring ofcytosine having an aromatic character. Consequently, ketone formwas easily transformed into enol form depending on pH (see Fig. 3).

Therefore, cytosine has two ionizable groups: enol in the 4 positionand aniline in the 3 position. Other part of PMEA is adenine that isnucleic base. It has got five protonable nitrogen atoms. Therefore,six pKa values are obtained from this ligand.

H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208 203

Table 1Summary of the experimental parameters for the potentiometric measurements.

System :PMEA and HPMPC with H+, Cu2+, Ni2+, Zn2+, Co2+, Ca2+ and Mg2+ in waterSolution composition :[L] range/mol dm−3 0.001–0.001 [M] range/mol dm−3, 0.1 and 0.16 ionic strength by electrolyte NaClMetal:Ligand ratio :1:1NaOH/mol dm−3 :0.025Calibration electrode :Two point calibration (KHP and Borax)Volume increments/mL :0.03Experimental Method :Titration in range pH 3–11 log ˇ00–1 −13.98 298 K, I: 0.1 and log ˇ00–1 −13.64 310 K, I: 0.16 ionic strength by NaClT/◦C : 298 K and 310 Kntot

a : 115–135ntit

b : 3Method of calculation : SUPERQUADTitration system : Molspin

a Number of titration points per titration.b Number of titrations per metal:ligand system M, Metal ion; L, ligand; ˇ, overall stabil

Tpapms

lcCvt

3

cs

2+ 2+ 2+ 2+

Fig. 2. Titration curves of the ligands under different conditions.

Another aspect of the ligands involves phosphoric acid systems.he phosphoric acid contains two acidic oxygen atoms. Therefore,hosphoric acid has two pKa values. While one of the pKa values isbout 7, the other is very low; in other words, it occurs at a very lowH level (pKa < 2) [38–42]. Therefore, only one pKa value was deter-ined for phosphoric acid in our experimental conditions since we

tudied the pH 3–12 range.In light of the above, three pKa values are obtained from this

igand. pKa1 (LH3) and pKa3 (LH) values are related to 3N and 4O inytosine [43,44]; pKa2 value is related to 1O in phosphate in HPMPC.onsequently, the pKa values of the ligands were calculated withery low standard deviation [31]. Comparisons of the pKa values ofhe ligands are given in Table 2.

.2. Solvent effect on dissociation constants of the ligands

Different organic solvents or solvent ratios change the dielectriconstants of solution media. Different organic solvents or differentolvent ratios cause ionic behavior different than the observed ionic

N

N

NH2

HO

OPO

HOHO

O H

H

HO

OPO

HOHO

Fig. 3. Mechanism of keto-enol t

ity constant.

behavior in aqueous solutions because of a greater effect of the elec-trostatic interactions [45,46]. Therefore, dielectric constants play avery important role in proton transfer reactions, and interaction ofmetal/ligand in solution and biological systems [47,48]. Ethyl alco-hol has a dielectric constant lower than that of water. Thus, in thepresent study, the solvent effect on dissociation equilibriums wasinvestigated and pKa values of the ligands are given in Table 3 andFig. 4.

According to Table 3, because of that reason increasing alcoholpercentage decreased ionization and pKa values of PMEA generallyincreased. But, for HPMPC; while pKa2 and pKa3 values generallyincreased, pKa1 value decreased.

Hence, a result of the measurements we can say that acidityof N H and O H bonds in the ligands were generally increasedwhen polarity or dielectric constants of solution were increased.pKa changes of the ligands are given Fig. 4.

3.3. Stability constants

Different metal complex forms were obtained in the solutiondepending on pH under the experimental conditions for each metalion used. The species distribution curves of the complex forms pro-duced in the solution were calculated using the computer programSUPERQUAD.

The cumulative stability constants (ˇmlh) are defined by Eqs. (6)and (7).

mM + lL + hH � MmLlHh (6)

ˇmlh = [MmLlHh]

[M]m[L]l[H]h(7)

where M is Cu2+, Ni2+, Zn2+, Co2+, Ca2+, and Mg2+ ions, L is ligand,and H is proton, and m, l, and h are the respective stoichiomet-ric coefficients. All experiments on complex systems of Cu2+, Ni2+,

Zn , Co , Ca , and Mg with ligands in solution were conductedto determine the stability of various species. Job’s method was usedto determine the stoichiometric ratio for M2+- ligand complex sys-tems in this work. Absorbance–wavelength UV visible absorption

N

N

NH2

HO

OPO

HOHO

O H

H

N

N

NH2

O H

H

utromerism in the HPMPC.

204 H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208

Table 2Dissociation constants of the ligands in room and human blood conditions ((�) standard devision and (�2) chi-square).

Ligand Species 298 K, I:0.1 mol dm−3 NaCl 310 K, I:0.16 mol dm−3 NaCl

log pKa values log pKa values

PMEA LH6 43.92 (6) 3.83 (2) 43.71 (1) 3.64 (3)LH5 40.09 (4) 4.49 (1) 40.07 (1) 4.37 (4)LH4 35.59 (3) 6.64 (1) 35.69 (1) 6.55 (2)LH3 28.96 (4) 7.33 (1) 29.15 (2) 7.26 (1)LH2 21.68 (3) 10.41 (4) 21.89 (2) 10.17 (6)LH 11.22 (2) 11.22 (4) 11.74 (3) 11.74 (8)� 0.53 – 0.40 –�2 23.99 – 22.06 –

HPMPC LH3 22.24 (6) 4.91 (1) 21.69 (4) 4.57 (1)LH2 17.33 (5) 7.02 (3) 17.12 (4) 6.89 (2)LH 10.31 (4) 10.31 (2) 10.23 (3) 10.23 (4)� 0.63 – 0.73 –�2 15.59 – 20.67 –

Table 3Solvent effect on dissociation constants of the ligands at 298 K, I: 0.1 mol dm−3 NaCl ((�) standard devision and (�2) chi-square).

pKa values

Ligand 0% (v/v) 10% (v/v) 20% (v/v) 30% (v/v) 40% (v/v) 50% (v/v)

PMEA 3.83 (2) 3.92 (2) 3.97 (1) 3.98 (3) 4.02 (5) 4.37 (6)4.49 (1) 4.30 (2) 4.05 (3) 4.22 (6) 4.80 (5) 4.88 (8)6.64 (1) 6.74 (2) 6.94 (4) 6.92 (7) 7.66 (5) 7.77 (8)7.33 (1) 7.53 (2) 7.72 (5) 7.99 (7) 8.10 (8) 7.95 (8)10.41 (4) 10.77 (2) 10.81 (5) 10.83 (7) 10.93 (9) 10.99 (8)11.22 (4) 11.26 (3) 11.36 (8) 11.44 (9) 11.65 (9) 11.87 (9)� 0.72 0.93 1.53 1.42 1.73�2 13.75 8.00 19.10 96.97 72.20

HPMPC 4.91 (1) 4.80 (2) 4.62 (1) 4.58 (3) 4.56 (2) 4.56 (3)7.02 (3) 7.20 (2) 7.27 (3) 7.45 (1) 7.58 (3) 7.66 (3)10.31(2) 10.55 (3) 10.70 (4) 10.90 (3) 11.01 (3) 11.15 (5)� 1.64 0.98 1.18 1.98 1.58�2 22.56 60.69 40.53 38.56 70.50

Fig. 4. pKa values versus percent ethyl alcohol-water mixtures (a) PMEA (b) HPMPC (298 K, I: 0.1 mol dm−3 NaCl).

Fig. 5. Absorbance-wavelength UV visible absorption spectra diagrams of the ligands and their Cu2+ complexes.

H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208 205

Fig. 6. The Job’s diagrams of Cu2+- L complexes (241–231 nm).

Table 4Overall stability constants in M2+ – PMEA system (298 K, I: 0.1 mol dm−3 NaCl) ((�) standard devision and (�2) chi-square).

Species (mhl) Cu2+ Ni2+ Zn2+ Co2+ Ca2+ Mg2+

log10ˇ

101 12.70 (7) 12.66 (9) 9.88(4) 11.92 (3) 12.46 (5) 13.11 (3)111 22.93 (4) – 20.07 (4) – 22.85 (1) 23.47 (2)121 30.59 (2) 32.19 (3) 28.38 (4) 30.61 (3) 30.20 (3) 31.17 (2)131 37.68 (3) 39.11 (4) 37.22 (2) 37.87 (4) 36.76 (1) 38.01 (1)141 44.08 (4) 43.30 (7) 44.07 (4) 42.29 (7) 41.20 (3) 44.06 (2)151 48.34 (5) – 48.32 (6) – 45.06 (5) 48.73 (3)

–

0.8718.62

sF

o2Mtaaac

MtrM

pbapmroF

TO

1–21 – −8.24 (8)

� 0.76 1.27

�2 24.24 97.45

pectra diagrams of the ligands and the Job’s diagram are given inigs. 5 and 6.

According to Figs. 5 and 6, two maximum absorbencies werebserved as 241 nm and 307 nm in UV-vis. spectrum of PMEA at98 K. Job’s method is used in 241 nm and stochiometric ratio for2+-PMEA complexes systems are determined as 1:1 together with

he complexes formulated as ML, MLH, MLH2, MLH3, MLH4, MLH5nd MLH−2 between the PMEA and the Cu2+, Ni2+, Zn2+, Co2+, Ca2+

nd Mg2+ ions are depending on pH. Respective the obtained over-ll stability constants of these species in room and human bloodonditions are given in Tables 4 and 5.

The complexes species which are ML, MLH, MLH2, MLH3 MLH4,LH5 and MLH−2, were obtained in solution under our experimen-

al conditions. Where LH75+ represents the ligand (PMEA) and M

epresents all metal ions used. The alleged complex structure of2+-PMEA system in this study is illustrated in Fig. 7.PMEA is composed of two main parts: a nucleobase and a

hosphate unit. Each of them has potential proton- or metal ion-inding sites: the nitrogen atoms at the nucleobase, and the oxygentoms at the phosphate group. Structure of M2+- PMEA com-lexes evidence has been provided [49–53] that the metal ion is

ainly located at, the nucleobase and the proton at the phosphate

esidue. Complexation starts when phosphate oxygen and nitrogenf nucleic base that participate in the coordination was dissociated.irstly, phosphate group ionizes and then nitrogen of nucleic base.

able 5verall stability constants in M2+ – PMEA system (310 K, I: 0.16 mol dm−3 NaCl) ((�) stan

Species (mhl) Cu2+ Ni2+ Zn

log10ˇ

101 16.73 (2) 15.79 (6) 17111 26.65 (4) 25.65 (3) 25121 33.81 (2) 32.94 (5) 32131 40.55 (1) 39.40 (5) 38141 43.12 (8) 43.70 (9) 43151 – – –1–21 −4.46 (3) −2.08 (6) −2� 0.57 0.93 0� 2 12.34 34.55 26

−7.38 (5) – – 0.82 0.64 0.54 57.22 22.83 12.46

According to Figs. 5 and 6, two maximum absorbencies wereobserved at 231 nm and 320 nm in the UV visible absorption spectraof HPMPC at 298 K. Job’s method was used at 231 nm. Stoichio-metric ratios for M2+-HPMPC complex systems were determinedas 1:1 and the complexes formulated as ML, MLH, MLH2, andMLH−2 between HPMPC and the Cu2+, Ni2+, Zn2+, Co2+, Ca2+, andMg2+ ions depend on pH. The overall stability constants of thesespecies obtained in room and human blood conditions are given inTables 6 and 7.

The complexes obtained in solution under our experimentalconditions were ML, MLH, MLH2, and MLH−2, where LH4

2+ rep-resents the ligand; the other species with protons less than 4 arethe de-protonated species of the (HPMPC) ligand; M is one of thefollowing metal ions Cu2+, Ni2+, Zn2+, Co2+, Ca2+, and Mg2+; and Lrepresents the HPMPC ligand. Complex structures of HPMPC withmetal ions are presented in Fig. 8.

HPMPC is composed of two main parts: a nucleobase and aphosphate unit. Metal ions simultaneously coordinate to potentialbinding centers of the ligand such as the phosphonate group and4O atom of the nucleobase. However, binding of metal ions to 4Oand the phosphonate group is not possible because of steric effects

[54,55]. Another potent binding site is the ether oxygen, which iswithin easy reach of metal ions already coordinated to the phospho-nate group. Alternative species such as macrochelatesevidently donot form. Thus, the first binding site is the phosphonate group and

dard devision and (�2) chi-square).

2+ Co2+ Ca2+ Mg2+

.79 (2) 15.81 (5) 12.14 (6) 14.32 (2)

.48 (2) 24.83 (2) 23.56 (5) 22.07 (3)

.44 (2) 32.10 (3) 33.53 (1) 28.88 (1)

.83 (3) 38.54 (4) 40.33 (4) 35.66 (4)

.35 (4) 43.05 (8) 44.43 (3) 41.49 (2) – 47.33 (6) 45.61 (7).99 (6) −1.051 (4) – –.51 0.96 0.77 0.81.69 25.32 14.90 9.11

206 H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208

NH

NHHN

NH

OP

HO O

HO

NH3

N

NN

N

NH2

O

P

MO

O

O

2+

(a) (b)Fig. 7. (a) Structure of LH7

5+ fully protonated form of PMEA in acidic media. (b) Structure of ML species of M2+-PMEA complexes.

Table 6Overall stability constants in M2+ – HPMPC system (298 K, I: 0.1 mol dm−3 NaCl, 0.03 mmol HCl) ((�) standard devision and (�2) chi-square).

Species (mhl) Cu2+ Ni2+ Zn2+ Co2+ Ca2+ Mg2+

logˇ

101 14.20 (9) 18.99 (4) 16.90(6) 18.58 (7) 17.72 (9) 17.03 (7)111 21.68 (5) 25.87 (6) 24.30 (7) 25.34 (2) 23.91 (9) 23.25 (8)121 26.84 (8) 29.49 (6) 27.83 (8) 29.97 (5) 28.83 (5) 27.90 (3)1–11 – – – – – –1–21 −0.22 (2) −0.168 (8) −0.176 (9) −0.18 (9) – –� 1.45 1.58 1.65 1.35 1.43 1.33�2 29.10 42.49 54.01 57.01 17.80 12.35

Table 7Overall stability constants in M2+ – HPMPC system (310 K, I: 0.16 mol dm−3 NaCl, 0.03 mmol HCl) ((�) standard devision and (�2) chi-square).

Species (mhl) Cu2+ Ni2+ Zn2+ Co2+ Ca2+ Mg2+

log10ˇ

101 14.06 (4) 18.19 (3) 16.39 (6) 17.95 (7) 16.99 (8) 16.69 (8)111 21.15 (4) 24.98 (5) 23.99 (7) 24.98 (4) 23.01 (9) 22.83 (9)121 25.94 (7) 29.28 (3) 29.50 (8) 30.02 (1) 27.44 (4) 27.42 (8)1–11 – – – – – –

−0.21.7

58.0

maoi

1–21 −0.23 (1) −0.22 (6)� 0.99 1.63

�2 56.22 28.86

etal ions, and 5-membered chelates containing the ether oxygen

re formed (see Fig. 10b) [56,57]. Additionally, distribution curvesf these species, which are obtained from the equilibrium constantsn room and human blood conditions, are given in Figs. 9 and 10.

Fig. 8. (a) Structure of LH42+ fully protonated form of HPMPC in acidi

3 (9) −0.23 (8) – –8 1.28 1.05 1.186 55.15 9.88 8.88

2+

In Fig. 9, the species distribution diagrams of M -PMEA systemdepend on pH is shown at 25 ◦C, I: 0.1 mol dm−3 NaCl. CuL-CuH5Land ZnL-ZnH5L complexes species were obtained in the solutionunder our experimental conditions between pH 3–12. While at

c media. (b) Structure of ML species of M2+-HPMPC complexes.

H. Atabey, H. Sari / Fluid Phase Equilibria 356 (2013) 201– 208 207

Fig. 9. Species distribution diagrams (a) Cu2+-PMEA (b) Cu2+-HPMPC complexes systems. (298 K, I: 0.1 mol dm−3 NaCl).

u2+-H

afotasac9o

tinatCptSM

uammtphaCs

Fig. 10. Species distribution diagrams (a) Cu2+-PMEA (b) C

bout the pH of 9 approximately 90% of the total Cu2+ turns to CuHLorming the main constituent of the complex species, about the pHf 12 approximately 95% of the total Zn2+ turns to ZnHL forminghe main constituent of the complex species. Besides NiL-NiH4Lnd CoL-CoH4L complexes forms, NiH−2L and CoH−2L hydrolysispecies were obtained at about pH > 10. While at about the pH of 9pproximately 95% of the total Ni2+ turns to NiH4L forming the mainonstituent of the complex species, about the pH of 6 approximately0% of the total Co2+ turns to CoH3L forming the main constituentf the complex species.

As known that kind of complexes species and molecular struc-ure of these species are depended coordination number of metalons additionally to coordination cites of ligands. Coordinationumber of Ca and Mg are two. Since coordination of Ca and Mgre bonded donor atoms of ligands enough, hydrolysed species forhese ions were not observed under experimental conditions. CaL-aH5L and MgL-MgH5L complexes species were obtained betweenH 3–12. But, CaL and CaHL are occurred as main constituent ofhe complex species about the pH of 9 and 12 approximately 97%.imilarity, about the pH of 9 and 12 approximately 98% of the totalg2+ turns to MgL and MgHL forming.All M2+-HPMPC complex forms were obtained in the solution

nder experimental conditions between pH 3 and 10. While atbout pH 6 about 98% of the total Cu2+ turns to CuH2L, forming theain constituent of the complex species, at about pH 5.5 approxi-ately 95% of the total Zn2+ and approximately 95% of the total Ni2+

urn to ZnHL and NiHL, forming the main components of the com-lex species. ZnH2L and NiH2L form at less than pH 3. Furthermore,

ydrolysis species for all M2+-HPMPC complexes were obtained atbout pH > 8. Two main maximum occurrences were observed fora2+-HPMPC (CaL and CaH2L) and Mg2+-HPMPC (MgL and MgH2L)ystems at about pH 3–8 of approximately 98.

PMPC complexes systems. (310 K, I: 0.16 mol dm−3 NaCl).

In Fig. 10, the species distribution diagrams of M2+-PMEA sys-tem on depend pH is shown at 37 ◦C, I: 0.16 mol dm−3 NaCl. For allmetal ions, ML-MH4L complexes forms were obtained in the solu-tion under experimental conditions. Moreover MH−2L hydrolysisspecies were obtained at about pH > 10. CuHL formed about 90% ofthe total Cu2+, which revealed its maximum occurrence; while themaximum occurrence of NiHL (about 90%) was at a pH of about 9;while the maximum occurrence of CoHL (about 80%) was at a pHof about 8. Two main complexes were seen in Zn2+-PMEA systemaccording to Fig. 10. About the pH of 5.5 approximately 80% of thetotal Zn2+ turns to ZnH3L and about the pH of 4 approximately 80%of the total Zn2+ turns to ZnH4L forming the main constituent of thecomplex species. CaL and CaH2L are occurred as main constituentof the complex species about the pH of 9 and 13 approximately 95%.But, about the pH of 10 approximately 98% of the total Mg2+.

According to species distribution diagrams of the Cu2+-HPMPCsystem; for all metal ions, ML-MH4L complex forms were obtainedin the solution under experimental conditions. Moreover, MH−2Lhydrolysis species were obtained at about pH > 8. CuH2L formedabout 98% of the total Cu2+, which revealed its maximum occur-rence; while the maximum occurrence of NiH2L (about 98%) was ata pH of about 3; the maximum occurrence of CoH2L and ZnH2L(about 99%) was at a pH of about 3. Similarly, two main maxi-mum occurrences were observed for Ca2+-HPMPC (CaL and CaH2L)and Mg2+-HPMPC (MgL and MgH2L) systems at about pH 3–8 ofapproximately 98% at 310 K.

4. Conclusion

In the present work, the ionic equilibriums of PMEA and HPMPCused as antiviral drugs were investigated. Dissociation constantsof the ligands and the stability constants of its Cu2+, Ni2+, Zn2+,

2 se Equ

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o2+, Ca2+, and Mg2+ complexes were studied using potentiometricitration. The experimental data obtained were refined using therogram SUPERQUAD in aqueous solution. All measurements werearried out in about room (I: 0.1 mol dm−3 of NaCl at 298 K) anduman blood (I: 0.16 mol dm−3 of NaCl at 310 K) conditions. Disso-iation constants of PMEA were calculated as 3.84, 4.49, 6.64 7.33,0.41 and 11.22 at 298 K, I: 0.1 ionic strength and 3.64, 4.37, 6.55,.26, 10.27 and 11.74 at 310 K, I: 0.16 ionic strength. For HPMPC;dissociation constants are 4.91, 7.02, and 10.31 in 298 K, I: 0.1 ionictrength and 4.57, 6.89, and 10.23 in 310 K, I: 0.16 ionic strength inqueous solutions. The ligand/metal stoichiometric ratio was deter-ined according to Job’s method, using a UV/vis spectrometer, and

ll coordination studies were carried out with the ligands/metalolar ratios of 1:1. For PMEA; various formed complexes formu-

ated as ML, MHL, MH2L, MH3L, MH4L, MH5L and MH−2L betweenhe PMEA with Cu2+, Ni2+, Zn2+, Co2+, Ca2+ and Mg2+ ions and moretabile macro-chelate is obtained between phosphate-coordinatedetal ions (M2+) and 1N of the adenine in M2+-PMEA complex sys-

em. Moreover, in this study all main complex species take placet less than pH 3, provided that the formation of the 5-memberedhelates includes the ether oxygen in M2+-HPMPC complex form.

cknowledgments

The author gratefully acknowledges the support of this work bycientific Research Council of Gaziosmanpas a University for finan-ial support (Project number: 2011/35).

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