Electrochimica Acta 51 (2006) 5103–5111

Molecular modeling of the voltammetric oxidation at a glassy carbonelectrode of the antimalarial drug primaquine and its prodrugs

succinylprimaquine and maleylprimaquine

Mauro A. La-Scalea a,∗, Carla M.S. Menezes a,∗∗, Guilherme C. Matsutami a,Michelle C. Polli a, Sılvia H.P. Serrano b, Elizabeth I. Ferreira a

a Lapen, Laboratorio de Planejamento e Sıntese de Quimioterapicos Potencialmente Ativos Contra Endemias Tropicais, Departamento de Farmacia,Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Av. Prof. Lineu Prestes, 580 Bl. 13 sup., 05508-900 Sao Paulo, Brazil

b Departamento de Quımica Fundamental, Instituto de Quımica, Universidade de Sao Paulo,Av. Prof. Lineu Prestes, 748 Bl. 2 sup., 05508-90 Sao Paulo, Brazil

Received 29 May 2005Available online 11 May 2006

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bstract

The 8-aminoquinoline primaquine (PQ) is the only antimalarial drug used as tissue schizonticide and relapsing malaria. Antichagasic activityas also reported. Nevertheless, as it also shows serious side effects, prodrugs such as succinyl and maleyl derivatives have been proposed toecrease its toxicity. Although PQ mechanism of action has not been completely elucidated, the promotion of oxidative stress is an advancedypothesis that could explain its activity in both plasmodia and trypanosome parasites. The oxidation of PQ and its prodrugs, maleylprimaquineMPQ) and succinylprimaquine (SPQ), was studied by cyclic voltammetry using glassy carbon electrode. All compounds were oxidized in aqueousedium, with the charge transfer process being pH-dependent in acidic medium and pH-independent in a weak basic medium, being the neutral

orm more easily oxidized. This indicated that the protonation of the nitrogen atoms displays a determinant role in the voltammetric oxidation,eing both prodrugs more easily oxidized than PQ protonated forms, in the order: SPQ < MPQ < PQ. For a better understanding of this behavior,molecular modeling study was performed using the AM1 semi-empirical method from Spartan 04 for Linux (v.119, Wavefunction Inc.). Theedium pH showed to be fundamental not only to the electronic density of the quinoline ring but also to the rearrangement of the nitrogen side

hain. The electronic density of primaquine non-protonated quinoline ring is higher than that in its protonated and diprotonated species. Also, these of prodrugs and the degree of saturation of the carriers (maleic or succinic acid) interfere with this feature. SPQ and MPQ have a slight increasen the quinoline electronic density in comparison to PQ. Nevertheless, the carrier in the side chain of SPQ is closer to the quinoline ring than it isn MPQ, which accounts for the higher electronic density in the former. The most significant effect occurs in the correspondent protonated formsf the nitrogen quinoline. The application of molecular modeling study associated to voltammetric techniques showed to be an important way tonderstand the redox mechanism of electroactive drugs. These results may be related to a biological activity and can be useful to future primaquineerivatives design.

2006 Elsevier Ltd. All rights reserved.

eywords: Primaquine; Primaquine prodrugs; Electrooxidation; Deprotonation; Molecular modeling

. Introduction

The electrooxidation of primaquine diphosphate was studiedy cyclic voltammetry using glassy carbon [1,2] and platinum [3]

∗ Corresponding author. Tel.: +55 11 3091 3793.∗∗ Corresponding author.

E-mail addresses: scalea@usp.br (M.A. La-Scalea),asmenezes@yahoo.com (C.M.S. Menezes).

electrodes. The cyclic voltammogram of this drug showed onlyone irreversible anodic wave controlled by diffusion, involv-ing two electrons in the rate-determining step of the electrodereaction [1]. Theoretical and experimental results indicated theexistence of a relationship between the primaquine dissociationequilibrium and its electrooxidation process, and the quinolinering displayed a fundamental role in both phenomena [1]. More-over, the diprotonated form of primaquine was readily oxidizedto a radical where the unpaired electron was delocalized over the

013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2006.03.085

5104 M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111

aromatic ring [2–4] and, working in alkaline medium, a cationradical was obtained as an intermediate in primaquine oxidation[2].

Primaquine is the only antimalarial drug used as tissue sch-izonticide and in relapsing malaria. It acts on latent or hipno-zoite forms of Plasmodium ovale and Plasmodium vivax, in theliver of the host, and its combination with blood schizonticidecan be completely curative [5]. On the other hand, primaquineshows serious side effects and its mechanism of action has notbeen totally elucidated [6–8]. One of the advanced hypothe-ses to explain the mechanism of action of primaquine involvesfree radicals formation, that would increase the oxidative stressin the parasite [4,9]. This oxidative action was based on thecycle primaquine redox, where some of its metabolites indi-cated the formation of hydrogen peroxide and the correspondingquinone-imine derivatives as the main products under phys-iologic conditions. Simultaneously, drug-derived radicals andhydroxyl radicals were detected [4,9].

Since the toxicity of primaquine could be related to the oxi-dation of NADPH [10], Thornaley et al. [11] concluded thatthis drug complex specifically with the cofactor, facilitates theelectron transfer to oxygen generating free radicals. Bisby [12]studied the formation of this complex and the primaquine reduc-tion involving one electron by pulse radiolysis. Augusto et al.[13] observed this same behavior with NADH and they detectedfree radicals during the enzymatic oxidation.

tloTssrimppL

[14]. Besides the improvement of the antichagasic activity, themolecular modification (defined as latentiation) of primaquinewas proposed to decrease its toxicity.

The electrochemical methods have been quite useful in quan-titative determination of drugs, as well as in the study of theirmechanism of action [16]. This paper introduces a complemen-tary approach to our previous work with primaquine [1], compar-ing voltammetric behavior of the drug with two of its prodrugs,maleyl (MPQ) and succinyl (SPQ) (Fig. 1), and determiningthe influence of pH medium in the potential values obtainedby cyclic voltammetry. Furthermore, a molecular modeling wasemployed for explaining the structural and electronic differencesas a consequence of the molecular modifications of primaquineand the deprotonation process of the studied compounds. In fact,molecular modeling has been a valuable tool for the proposal ofuseful models for studying structure features and forecastingtheir relationships with the biological activity [17]. The associ-ation between molecular modeling methods and voltammetrictechniques has shown to be an important way to understand theredox mechanism of electroactive drugs and the results hereindescribed may be extensive to biological activity and useful tofuture primaquine derivatives design.

2. Experimental

2.1. Chemicals

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Primaquine showed also antichagasic activity, probablyhrough oxidative stress and/or oxidative action of its metabo-ites [14]. Based on that and considering the inhibitory activityf nitrofurazone on trypanotione reductase, an enzyme found in. cruzi but not in the host, mutual prodrugs of both drugs wereynthesized either directly or by using a spacer group, such asuccinyl, and dipeptides as well [15]. Therefore, trypanotioneeductase would be inhibited by nitrofurazone and an increasen the oxidative stress provoked by primaquine could be much

ore effective, resulting in the death of the parasite. Primaquineeptide prodrugs, synthesized as intermediates of these mutualrodrugs, were also assayed and most were active in vitro inLC-MK2 cell cultures infected with T. cruzi trypomastigotes

Fig. 1. Primaquine (PQ) and its prodrugs succin

The stock solution (0.02 mol/l) of primaquine diphosphatePQH2

2+) (Itaca Laboratorios) and its prodrugs were preparedy direct dissolution in deionized water. The pH study wasccomplished with universal buffer starting from the misturef phosphoric, acetic and boric with NaOH [18]. All solutionsere prepared using analytical grade reagents from Merck andurified water from a Barnted Nanopure UV system.

.2. Succinylprimaquine synthesis

Succinylprimaquine (SPQ) synthesis was carried out inethanol using 0.038 mol of primaquine diphosphate and tri-

maquine (SPQ) and maleylprimaquine (MPQ).

M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111 5105

ethylamine (0.07 mol). Succinic anhydride (0.052 mol) was thenadded and the reaction was maintained for one hour under reflux.The reaction was cooled and the product formed after water addi-tion was filtered, washed and re-crystallized from acetone. Yield:59%. Melting point: 142–145 ◦C. NMR analysis: 1H (DMSO-d6, 300 MHz, δ ppm): 1.08 (m, 2H, H16); 1.19 (d, 3H, H15); 1.51(m, 2H, H17); 2.28 (t, 2H, H22); 2.41 (t, 2H, H23); 3.02 (t, 2H,H18); 3.60 (m, 1H, H14); 3.81 (s, 3H, H12); 6.29 (s, 1H, H5); 6.49(s, 1H, H7); 7.40 (t, 1H, H3); 8.05 (d, 1H, H4); 8.51 (d, 1H, H2);12.23 (s, 1H, H26). IR analysis (KBr, ν cm−1): 3451 (strong,ν O–H), 3266 (strong, ν N–H), 3088–3006 (weak, ν C–H aro-matic), 2958–3926–2863 (weak, ν C–H aliphatic), 1715 (strong,ν C O acid), 1670–1648 (strong, ν C O amide).

2.3. Maleylprimaquine synthesis

Maleylprimaquine (MPQ) was carried out in ethanolusing 0.005 mol of primaquine diphosphate and triethylamine(0.01 mol). Maleic anhydride (0.0063 mol) was then addedand the reaction was maintained for 2 h under reflux andthe product formed after water addition was filtered, washedand re-crystallized from acetone. Yield: 40%. Melting point:118–121 ◦C. NMR analysis: 1H (DMSO-d6, 300 MHz, δ ppm):1.27 (d, 3H, H15); 1.72 (d, 4H, H16, H17); 3.35 (m, 2H, H18);3.61 (q, 1H, H14); 3.88 (s, 3H, H12); 6.02 (d, 1H, H22); 6.21 (d,1H, H23); 6.31 (d, 1H, H5); 6.37 (d, 1H, H7); 7.34 (dd, 1H, H3);7s31Ca

2

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tion Inc.) on a personal computer with the Red Hat version 7.2operational system. The neutral form of PQ and its protonatedspecies, PQH+ and PQH2

2+, generated according to R and S iso-mers, besides the R isomers of the prodrugs, SPQ and MPQ, andthe respective protonated forms (SPQH+ and MPQH+) had thegeometry optimized by MMFF94 molecular mechanical method[19], followed by the AM1 semi-empirical method [20], selectedfor this study. The Monte Carlo method was used for conforma-tional search, assuming the rotation of dihedral angles of theside chain in 120◦ steps for single bonds involving sp3 carbonsand in 180◦ steps for bonds around the sp2 carbon characteristicof maleyc acid derivatives. The R isomer obtained consideringthe aqueous system according to the SMS94 method [21], wasselected as the minimal energy conformer for PQH2

2+ species,after analysis of the 100 first lowest energy conformers andcomparison to the primaquine parameters extracted from theX-ray crystal structure of the corresponding primaquine diphos-phate salt [22]. Thus, the R isomer was adopted for all studiedcompounds, being the minimal energy conformers in aqueoussystem selected. Partial electrostatic charge, HOMO eigenval-ues and stereoelectronic surfaces of the selected conformerswere reevaluated by single point calculation. In this work, themolecular electrostatic potential maps (MEP) were calculatedaccording default parameters implemented in Spartan and super-imposed onto a constant electron density of 0.002 e/au3. TheMEPs graphic representation follows the variation of colorsit

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.63 (s, 1H, H19); 7.95 (dd, 1H, H4); 8.54 (dd, 1H, H2). IR analy-is (KBr, ν cm−1): 3382 (strong, ν N–H), 3244 (strong, ν O–H),066 (weak, ν C–H aromatic), 2951 (strong, ν C–H aliphatic),898 (umbrella, ν C–H aromatic substituted), 1708 (strong, ν

O acid), 1640 (strong, ν C O amide), 1614 (strong, δ N–Hmine), 1605 (δ C C aromatic).

.4. Electrochemical assays

The cyclic voltammograms were recorded using an AutolabGSTAT20 potentiostat/galvanostat, from Eco-Chimie, Utrecht,he Netherlands, coupled with cell of 20 ml with system of threelectrodes: glassy carbon (GCE, Analion, ∅ = 2 mm) as worklectrode, Ag/AgCl as reference and Pt as auxiliary electrode.he pH control was carried out with pH-meter Metrohm 654 andombined glass electrode from Metrohm. All the experimentsere performed at room temperature. The GCE was manuallyolished with 3.0 �m diamond suspension on metalographicloth (Arotec S/A, Brazil) and rinsed with water afterwards.

.5. Data treatment

The Pallas 2.1 software, module pKalc 3.2, was used for theKa calculations. The acquisition of voltammetric data were per-ormed using the software GPES 4.9 of the Eco-Chimie. Thereatment data and graphic studies were performed using therigin 7.5 software (OriginLab Corp.).

.6. Molecular modeling

The molecular modeling study was performed by usingPARTAN O2 software for Linux, version 119a (Wavefunc-

ntensity from intense red (electron-rich regions), orange, greeno intense blue (electron-poor regions).

. Results and discussion

.1. Voltammetric behavior of primaquine and its prodrugs

The cyclic voltammogram of primaquine showed only onexidation wave in acidic medium. At pH 7.58 and scan rate (ν) of.1 V/s the oxidation wave is irreversible with anodic peak poten-ial (Ep,a) = 0.654 V. The scan rate variation from 0.05 to 2.0 V/sauses increase in the height of the oxidation wave and Ep,as shifted towards positive potential. The relationship betweennodic current values (Ip,a) and ν1/2 is linear, indicating that thelectrodic reaction was controlled by diffusion, confirming pre-ious results [1–3].

SPQ and MPQ prodrugs display the same voltmmetric behav-or of primaquine. Both compounds showed one irreversiblexidation wave also controlled by diffusion. Fig. 2 shows theyclic voltammograms of MPQ and SPQ in acidic medium.n this experimental condition, a quasi-reversible redox couplepeaks 1 and 2) was recorded at less positive potential in theeverse scan. These results indicate a new species was formedn the first anodic peak, being more easily oxidized than the 8-minoquinolines derivatives themselves. This quasi-reversibleave was shifted to negative potential region as pH increases.his behavior is completely in accordance with the results pre-iously registered to primaquine [1]. Table 1 shows all potentialalues recorded for primaquine and its prodrugs. According tohe literature [23] for quasi-reversible systems, �Ep values canhange between 120 and 60/n mV, suggesting the involvement

5106 M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111

Fig. 2. Cyclic voltammograms of 1.0 mmol/l SPQ and MPQ at pH 2.15 usingGCE. Scan rate = 0.1 V/s.

Table 1Peak potential values obtained by cyclic voltammetry for primaquine and itsprodrugs

pH 2.15 pH 7.58

Ep,a (V) Ep,1 (V) Ep,2 (V) Ep,a (V) Ep,1 (V) Ep,2 (V)

PQ 0.788 0.625 0.584 0.654 −0.135 −0.176MPQ 0.764 0.614 0.572 0.621 −0.124 −0.166SPQ 0.734 0.596 0.566 0.607 −0.120 −0.150

Table 2The pKa values of primaquine diphosphate and its prodrugs

Methods pKa1 pKa2 Reference

Theoretically calculateda,b 5.55 10.2 –Cyclic voltammetry 4.25 9.17 [1]13C NMR spectroscopy 3.20 10.4 [24]1H NMR spectroscopy 3.2 9.90 [25]SPQ by cyclic voltammetryb 4.45 – –MPQ by cyclic voltammetryb 4.91 – –

a Calculated by program Pallas 2.1, module pKalc 3.2.b This work.

of two electrons for peaks 1 and 2. For the two pH values pre-sented, SPQ derivative is easier to be oxidized, and a reactivityorder can be settled as follows: SPQ < MPQ < PQ.

The electrooxidation of the studied compounds is pH-dependent, as it also may be observed by the results presentedin Table 1. In fact, the plots shown in Fig. 3, indicating clearlythat protons are involved in the oxidation process, confirm thisbehavior.

The Ep,a value for the anodic wave of primaquine is shiftedto less positive potential increasing pH, suggesting the existenceof acid–base equilibrium before electrochemical reaction. The(A) plot in Fig. 3, previously published [1], is presented forcomparison and shows three different regions, with intersec-tions corresponding to the experimental pKa values. The firstpKa1 represents the acid–base equilibrium between the dipro-tonated (PQH2

2+) and the monoprotonated (PQH+) forms andpKa2 between this latter and the molecular primaquine form(PQ). However, a slight increase of Ep,a values between the pHvalues 4 and 9 was registered. This behavior might be related tothe conformational molecular alterations during the deprotona-tion process, which approximates the side chain to the quinolinering due to the PQH+ formation (Table 4 and Fig. 4). There-fore, despite the fact that this conformational alteration would beresponsible for the small displacement (�Ep,a/�pH = 7.4 mV)of Ep,a to more positive values, the deprotonation process facil-itates the voltammetric oxidation of the primaquine. Table 2slsi

Fig. 3. Plot of Ep,a vs. pH for 1.0 mmol/l of: (

hows the experimental and theoretical pKa values reported initerature. Although they do not fit completely because they areupported by different methodologies, the same behavior is reg-stered (Scheme 1).

A) primaquine and (B) SPQ and MPQ.

M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111 5107

Fig. 4. AM1 minimal energy conformers in the aqueous system of the primaquine species PQH22+, PQH+ and PQ. Atoms color: carbon (gray), nitrogen (blue),

oxygen (red) and hydrogen (white); molecular electrostatic potential maps (B), corresponding to the orientations in (A), in the ranges of 60 to 180 kcal/mol (PQH2+),5 to 135 kcal/mol (PQH+) and −55 to 23 kcal/mol (PQ), superimposed onto total electron density of 0.002 eV/au3.

For all compounds, the pH influence is more significantuntil pH 5. After this value, primaquine and its prodrugs can beconsidered pH-independent, which confirms the deprotonationof nitrogen quinoline as a determinant step, facilitating theoxidative process and showing that the neutral forms aremore electroactive. Comparing plots (A) and (B) (Fig. 3),the influence of molecular modification in primaquine sidechain is evidenced. The linkage of maleyl and succinyl groupschanged the original behavior of primaquine in the second stepdeprotonation, because the amide derivative obtained precludesthe protonation of the aliphatic nitrogen in acidic medium.Moreover, both prodrugs showed Ep,a values less positivethan primaquine in the same studied pH range, indicatingthat the molecular modification had contributed to facilitatethe drug electrooxidation. Table 2 presents the correspondent

experimental pKa1 values for the nitrogen quinoline depro-tonation of prodrugs. The estimate pKa1 values theoreticallyindicate these values as being the same for all compoundsand the lack of pKa2 values for the prodrugs confirms themolecular modification performed by the carrier groups inprimaquine.

Based on the above discussed, the Ep,a values presented inTable 1 correspond to the compounds at different protonatedforms. In acidic medium (pH 2.15) the diprotonated form ofprimaquine (PQH2

2+) and monoprotonated forms of prodrugs,SPQH+ and MPQH+, predominate. In these cases, the protona-tion of the N-quinoline ring lead to more positive Ep,a values. AtpH 7.58 the prodrugs are in the neutral form, while primaquineis in monoprotonated (PQH+) form. These differences mightexplain the higher reactivity of the prodrugs, because in alkaline

disso

Scheme 1. Equilibrium ciations of primaquine.

5108 M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111

medium (pH 10), where all compounds are in the neutral forms(SPQ, MPQ and PQ), the Ep,a values do not show significantdifferences: 0.620, 0.614 and 0.630 V, respectively. Thus, theelectroactivity differences observed among the compounds maybe explained in function of the structural and electronic changesproduced by primaquine latentiation and by the different pro-tonated forms of these compounds, as will be discussed in thenext section.

Therefore, it is evident that the quinoline ring displays afundamental role in the dissociation equilibrium and elec-trooxidation process of all compounds, showing that they formthe same products. Previous study [1] showed that the PQoxidation involves two electrons in the rate-determining stepof the electrode reaction. Considering this number of electronsinvolved and based on the oxidative O-dealkylation of the drugby means of its biotransformation [26], the 6,9-quinone-iminederivative would be the main product of PQ oxidation, witha cation radical as an intermediate [2]. The oxidation productcan be reduced and re-oxidized forming species more reactivethan PQ [27]. Therefore, the 6,9-quinone-imine is reduced tothe 6-hydroxyprimaquine derivative (Ep,1), being the latter oxi-dized and the former obtained again (Ep,2). This is a hypothesisbeing advanced, since EPR studies indicated the formation of5,8-quinone-imine derivative as the PQ oxidation product [9].Nevertheless, the molecular modeling approach showed that theelectronic density on the quinoline ring increases with pH. Thisimtodt

is a strong indication that this group plays an important role inthe oxidative stress produced by primaquine and its prodrugs.The complete elucidation of PQ electrooxidation mechanism isa goal for further studies.

3.2. Molecular modeling

As already mentioned, being the primaquine oxidationdirectly related to the protonation state of its nitrogen atoms,in the molecular modeling study, three different situations havebeen established for comparison, following the distribution ofthe predominant form of primaquine and its prodrugs for the pH:

1. acidic medium (pH 2.15): diprotonated form of primaquine(PQH2

2+), protonated form of succinylprimaquine (SPQH+)and protonated form of maleylprimaquine (MPQH+);

2. neutral medium (pH 7.58): monoprotonated form of pri-maquine (PQH+) and neutral forms of succinylprimaquine(SPQ) and maleylprimaquine (MPQ);

3. alkaline medium (pH 10): neutral forms of primaquine(PQ) and succnylprimaquine (SPQ) and maleylprimaquine(MPQ).

Taking the above into consideration, the AM1 optimizedgeometry indicated variations in the conformation of the sidec

A

B

TI roton

I469315763264304493

B

D

ndicates an increase in the drug capacity of donating electrons,ainly in the proximity of the methoxyl oxygen atom in C6,

hus corroborating its possibility to be the electroactive groupf the molecule. Furthermore, if the biological activity of therug is dependent on free radicals formation, it can be inferredhat the necessary lower potential to oxidize the quinoline ring

able 3nteratomic distances in the selected minimal energy conformer of neutral and p

Gas system

R S

nteratomic distance (A)N(1)–C(2) 1.346 1.3N(1)–C(9) 1.393 1.3C(2)–C(3) 1.415 1.4C(3)–C(4) 1.378 1.3O(11)–C(12) 1.432 1.4N(13)–C(14) 1.464 1.4C(14)–C(15) 1.530 1.5C(14)–C(16) 1.544 1.5C(18)–N(19) 1.493 1.4

ond angle (◦)C(2)–N(1)–C(9) 121.7 121.7N(1)–C(2)–C(3) 121.3 121.3C(2)–C(3)–C(4) 119.3 119.3C(6)–O(11)–C(12) 117.1 117.1C(8)–N(13)–C(14) 118.0 118.0N(13)–C(14)–C(16) 109.5 109.4C(17)–C(18)–N(19) 112.9 112.9

ihedral angle (◦)C(9)–C(8)–N(13)–C(14) −163 163N(13)–C(14)–C(16)–C(17) −171 171

hain of primaquine and derivatives due to:

. protonation form of the nitrogen atoms in the side chain,N(19), and in the quinoline ring, N(1), whose numerationcan be observed in Fig. 1;

. primaquine latentiation through amide bond.

ated forms of primaquine and its prodrugs

Aqueous system Raios, X-ray

R S

1.346 1.346 1.3281.394 1.394 1.3731.414 1.414 1.3811.378 1.378 1.3691.433 1.432 1.4301.464 1.464 1.4691.532 1.531 1.5131.542 1.542 1.5061.492 1.492 1.465

121.9 122.0 122.4121.4 121.4 121.1119.2 119.2 119.3117.2 117.2 117.3118.1 118.1 122.9111.1 111.1 109.6113.2 113.2 114.3

177 177 169−84 109 −59

M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111 5109

Table 4Interatomic distances in the selected minimal energy conformer of neutral and protonated forms of primaquine and its prodrugs

Compound Interatomic distances (A)

N(1)–N(19) N(13)–N(19) C(20)–N(1) C(20)–N(13) C(24)–N(1) C(24)–N(13) O(21)–N(1) O(21)–N(13) O(25)–N(1) O(25)–N(13)

PQ 7.37 5.43 – – – – – – – –PQH+ 2.96 2.82 – – – – – – – –PQH2

2+ 7.63 5.68 – – – – – – – –

SPQ 6.42 5.46 7.59 6.56 6.91 6.94 8.29 6.81 6.17 6.42SPQH+ 5.07 3.91 4.15 3.77 3.94 5.03 2.94 3.04 2.92 4.51

MPQ 6.81 5.43 7.83 6.46 9.72 9.31 8.46 6.79 10.52 9.93MPQH+ 4.63 4.98 4.04 5.11 6.75 8.51 2.84 4.39 7.75 9.27

These variations were better evaluated considering the anal-ysis of the 100 first conformers of lower energies. In these, theside chain approximates or distances from the quinoline ringaccording to the protonation state of the species (neutral, monoor diprotonated) of primaquine and its prodrugs. These observa-tions were equally observed such as in gas as in aqueous systems,the last calculated by the SMS94 method [20].

The minimal energy conformers of both R and S isomers ofPQH2

2+ obtained with AM1 and calculated in gas and aqueoussystems, have shown similar geometric features regarding inter-atomic distances, bond angles and dihedral angles as well as theX-ray crystallographic data of the primaquine diphosphate salt[22], as it can be seen in Table 3. However, the highest relation-ship in respect to the dihedral angle N(13)–C(14)–C(16)–C(17)observed between the R configuration of that conformer in aque-ous system, associated to this molecular modeling study tries tosimulate an electrochemical condition, it also conducts to selectthe conformer of R configuration and the aqueous system forbetter represent the side chain of primaquine and its prodrugs.Moreover, based on the observed comparison, the AM1 methodmight be considered as able to reproduce experimental dataof structure considered as minimal energy. Similar analysiswas reported to other quinoline antimalarials as mefloquine[28].

In the selected conformers, the side chain is found to beextended in PQH2

2+ and PQ as well, while approximation totbtT

reflected in the slight increase of the Ep,a values of PQ betweenthe pH values 4 and 9 as it can be seen in Fig. 3A.

Primaquine produgs may also introduce changes in the con-formation of the side chains, showing be closer to the quinolinering. Minor interatomic distances are observed between theamide nitrogen N(19) and the quinoline nitrogen N(1), speciallyfor SPQ, when compared to PQ (Table 4). SPQ also shows theminor distances between the carbonyl carbon C(20) of the amideand N(1), as well as between the second carbonyl carbon C(24)of the carboxylic acid and N(13). These differences may be par-ticularly related to the chemical nature of the spacer group. Theunsaturated side chain of the succinyl derivative allows moreapproximation of the side chain to the quinoline ring while thecarbon double bond present in the maleyl derivative leads to amore rigid side chain conformation maintaining larger distance.These features are mostly observed in the monoprotonated formsof the prodrugs when decreased interatomic distances appear,in specially when comparing SPQH+ to its neutral form, SPQ(Table 4). These observations can be seen in Fig. 5.

The conformation changes of the side chain of primaquineand derivatives may be attributed to steric and, specially, toelectronic interactions due to pH medium. The pH variations per-formed in the electrochemical study could explain the increaseof the electrostatic potential, particularly of the quinoline ring,when the pH is elevated from 2 to higher than 5 and in alkalinemedium (pH 10). The analysis of the MEPs of the respectiveft

N

TP

C

(20)

PPP

S .57S .67

M .69M .70

he quinoline ring is registered for PQH+. This analysis cane verified by comparing the interatomic distances, mostlyhose involving the N(1), N(13) and N(19) atoms, presented inable 4 and visualized in Fig. 4. Experimentally, this behavior is

able 5artial electrostatic charges and HOMO eigenvalues calculated by AM1

ompound Atomic charge (eV)

N(1) N(13) N(19) H(N13) C

Q −0.59 −0.62 −0.85 0.31 –QH+ −0.50 −0.35 0.08 0.24 –QH2

2+ 0.04 −0.78 −0.03 0.35 –

PQ −0.59 −0.57 −0.64 0.29 0QPH+ 0.09 −0.76 −0.52 0.43 0

PQ −0.60 −0.69 −0.64 0.32 0PQH+ 0.09 −0.78 −0.59 0.35 0

orms is shown in Fig. 4 to PQ, PQH+ and PQH22+ and at Fig. 5

o SPQ and MPQ and to the correspondent SPQH+ and MPQH+.The pH increasing promoted electronic alterations in the

(19) and N(1) atoms of PQ. When both were protonated (pH

HOMO (eV)

O(21) C(24) O(25) O(26)

– – – – −8.132– – – – −11.565– – – – −14.358

−0.57 0.82 −0.58 −0.63 −8.329−0.63 0.80 −0.61 −0.59 −12.326

−0.54 0.84 −0.55 −0.63 −8.436−0.61 0.83 −0.54 −0.63 −12.464

5110 M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111

Fig. 5. AM1 minimal energy conformers in the aqueous system of the pri-maquine prodrugs SPQ and MPQ and the monoprotonated forms SPQH+ andMPQH+ in the tube model (A). Atoms color: carbon (gray), nitrogen (blue),oxygen (red) and hydrogen (white); molecular electrostatic potential maps (B),corresponding to the orientations in (A), in the ranges of −55 to 23 kcal/mol(SPQ and MPQ) and 15 to 100 kcal/mol (SPQH+ and MPQH+), superimposedonto total electron density of 0.002 eV/au3.

2.15), the extended conformation of the side chain as well as thehighest distance between these protonated nitrogen (Table 4)were justified by the electronic repulsion among their positivecharges. These observations can be also related to the atomicelectrostatic charges, specially of the nitrogen atoms, whichshow an increase to negative values for the N(1) and N(19)atoms (Table 5) following the deprotonation process of PQH2

2+

species to originate PQ. Moreover, in the first deprotonation stepto PQH+ formation, it was also possible to observe a decreaseof the atomic electrostatic charges of that nitrogen atom bondedto the quinoline ring N(13) and its hydrogen atom. This maybe related to electrostatic attraction and consequent approxima-tion of the protonated N(19) to those atoms as well as to thequinoline ring conducting to the contraction of the side chainobserved in PQH+, as previously commented. These electronicchanges can be seen in the MEPs shown in Fig. 4. In PQH2

2+, the

low electronic density of the protonated N(1) and N(19) atomsare represented by deep blue regions. In the PQH+, the electrondonor feature of the quinoline ring is represented by a red-orangecolor (especially in the proximity of the methoxyl oxygen atombonded to the ring). The electronic density decrease in N(13)and its hydrogen atom could be accomplished by the decreasingin the electron density from red to orange color in the corre-spondent region of the map. In this, the protonated N(19) atomis shown in a deep blue color, indicative of a region of low elec-tronic density. At pH higher than 9, where neutral primaquineis the predominant form (PQ), the extended side chain is jus-tified by the repulsion between two electron-rich centers: thequinoline ring (represented by red-orange color) and the nitro-gen N(19), in red color on its region as well, in agreement withthe atomic electrostatic charges distribution, as previously men-tioned. In summary, the atomic electrostatic charges and MEPsshow that the electronic density increases on the quinoline ringas pH increases, providing the decrease in Ep,a values, as ver-ified in the voltammetric study. It is important to mention thatdue to the significant variation of the electronic features of theprimaquine and its protonated forms, the MEPs in Fig. 4 arerepresented in different ranges of energy.

Primaquine modifications into its prodrugs (SPQ and MPQ)promoted little significant alterations in the partial electrostaticcharge of the nitrogens N(1) and N(13). The decrease on thenegative charge in N(19) due to the amide bond formation wastdtmaMtp

tqadbTegaecbqctorOvetc

he major finding (Table 5). A discrete increase of the electronicensity on the quinoline ring can be observed, especially in SPQ,hrough the larger extension of the orange-green color in detri-

ent of the light blue color, which stayed more restricted to theromatic hydrogens, as observed to PQ in Fig. 4 and to SPQ andPQ in Fig. 5. This increase promoted little significant varia-

ions in the oxidative potentials, as can it be seen in Table 1 andrevious commented.

For the prodrugs, the N(1) assumed a positive charge charac-er with its protonation (Table 5), condition that transformed theuinoline ring in an electron acceptor center and the consequentpproximation of the side chain, especially that of the succinylerivative, SPQH+. The side chain of the maleyl derivative alsoecame closer, in opposition to the verified in its neutral form.he observed higher approximation in the SPQH+ can be due tolectronic interactions between electron donor centers: the oxy-en atoms of the O(21) amide bonds and the O(25) of carboxyliccid group. For this reason, these oxygen atoms have their partiallectrostatic charges become more negative and its respectivearbon atoms C(20) and C(24), more positive (Table 5). Thisehavior would explain the higher electronic density on theuinoline ring of SPQ when compared to MPQ. In the latter, thearbon double bond seems to allow a resonance effect betweenhe carboxyl and the amide groups. As a consequence, it wouldccur an electron displacement in the direction of the acid group,educing the electronic availability of the side chain (especially(25)) to stabilize the protonated quinoline ring. These obser-ations could be verified throughout the analysis of the partiallectrostatic charges of the C(24), O(25) and O(26) atoms ofhe referred species. Higher similarity was observed among theharges of the MPQ and MPQH+ forms, contrarily to SPQH+.

M.A. La-Scalea et al. / Electrochimica Acta 51 (2006) 5103–5111 5111

The analysis of the correspondent minimal energy conformers(Fig. 5) strongly contributed to this discussion and assists inthe comprehension of the differences in interatomic distancesand orientations adopted for each derivative. The correspondentMEPs showed a higher electronic density on the side chain ofMPQH+ (deeper green color), especially near O(25) and O(26),and a minor electronic density on the alcoholic-hydrogen atomregion when compared to that of SPQH+.

In addition, the energy of the highest occupied molecularorbital (HOMO) was also calculated (Table 5). The increase ofHOMO eigenvalues occurred in parallel to the deprotonationprocess as a consequence of pH elevation, a common behaviorfor all species. Closer eigenvalues were observed for PQ, SPQand MPQ in accordance to the discrete differences in Ep,a values.It is important to observe that in acidic medium, the SPQH+ andMPQH+ species had more oxidative character than PQH2

2+,as shown in Table 1, and confirmed by the HOMO eigenval-ues (Table 5). In the correspondent MEPs, these comparisonscould be accomplished by the higher electronic density in thequinoline ring of SPQH+ and MPQH+, represented in orange togreen (Fig. 5), while in the same range, the quinoline ring ofPQH2

2+ was totally deficient of electronic density, representedin deep blue (data not shown). Considering the predominantspecies PQH+ and the SPQ and MPQ at pH 7.58, the physio-logical condition, it might be supposed that these prodrugs weremore active than their prototype.

trwt

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to M.A. La-Scalea (process no. 01/09418-0), Capes-Prodoc forthe fellowship to C.M.S. Menezes (process no. 00019-03-8), andCNPq, for research fellowship to E.I. Ferreira.

References

[1] M.A. La-Scalea, M.C. Chung, S.H.P. Serrano, M.L. Cruz, E.I. Ferreira,Bioelectrochemistry 53 (2001) 55.

[2] D.L.A. Faria, P.S. Santos, Spectrochim. Acta 47A (1991) 1653.[3] Y.G. Chyan, M.W. Hsiao, C.M. Catuara, C.T. Lin, J. Phys. Chem. 98

(1994) 10352.[4] J. Vasquez-Vivar, O. Augusto, Biochem. Pharmacol. 47 (1994)

309.[5] A. Korolkovas, Essentials of Medicinal Chemistry, 2nd ed., John Wiley

& Sons, 1988, p. 579.[6] K.A. Fletcher, P.F. Barton, J.A. Kelly, Biochem. Pharmacol. 37 (1988)

2683.[7] K.N. Bangchang, J. Karbawang, D.J. Back, Biochem. Pharmacol. 44

(1992) 587.[8] J.L. Johnson, D.F. Worth, N.L. Colbry, L.M. Werbel, J. Heterocyc.

Chem. 21 (1984) 1093.[9] J. Vasquez-Vivar, O. Augusto, J. Biol. Chem. 267 (1992) 6848.

[10] S.N. Kelman, S.G. Sullivan, A. Stern, Biochem. Pharmacol. 31 (1982)2409.

[11] P.J. Thornaley, A. Stern, J.V. Bannister, Biochem. Pharmacol. 32 (1983)3571.

[12] R.H. Bisby, Free Rad. Res. Commun. 5 (1988) 117.[13] O. Augusto, C.L.V. Weingrill, S. Schreier, H. Amemiya, Arch. Biochem.

Biophys. 244 (1986) 147.[14] M.C. Chung, M.F. Goncalves, W. Colli, E.I. Ferreira, M.T.M. Miranda,

[

[

[

[

[[

[

[

[

[

[

[

[

[

To inform, HOMO is, by its definition, related to the elec-ron donor capacity of a substance, feature correlated with theedox potential. Therefore, for the studied compounds in thisork, HOMO could be adopted as an indicative parameter of

he voltammetric behavior of primaquine and its derivatives.

. Conclusions

Although SPQ and MPQ prodrugs display the same volt-metric behavior of primaquine, the molecular modification

ontributed to facilitate the drug electrooxidation. Moreover, thexidation is strongly pH-dependent in acidic medium, indicatinglearly that the deprotonation of nitrogen quinoline is a determi-ant step showing that the neutral forms are more electroactive.inally, in order to relate the obtained results, in this work, to

he proposed mechanism of action to primaquine [4,6,9] and itserivatives, and taking into account the voltammetric behavior,t may be predicted that in physiological pH, the SPQ and MPQpecies would be more active than the corresponding PQH+.iological assays of the prodrugs in experimental malaria orhagas’ diseases might prove the hypothesis herein advanced.

cknowledgements

We thank FAPESP for the financial support (process nos.1/01192-3 and 03/10763-0) and for the postdoctor fellowship

J. Pharm. Sci. 86 (1997) 1127.15] M.C. Chung, Planejamento e sıntese de pro-farmacos recıprocos de

nitrofural e primaquina potencialmente antichagasicos, Ph.D. Thesis,FCF,USP, 1996, 196 pp.

16] F.C. de Abreu, P.A.D. Ferraz, M.O.F. Goulart, J. Braz. Chem. Soc. 13(2002) 19.

17] H.-D. Holtje, G. Folkers, in: R. Manhold, H. Kubinyi, H. Timmerman(Eds.), Methods and Principles in Medicinal Chemistry, vol. 5, VCH,Weinheim, 1996, p. 23.

18] J. Lurie, Handbook of Analytical Chemistry, Mir Publishers, Moscow,1978, p. 263.

19] T.A. Halgren, J. Comput. Chem. 17 (1996) 490.20] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, J. Am. Chem.

Soc. 107 (1985) 3902.21] C.C. Chambers, G.D. Hawkins, C.J. Cramer, D.G. Truhlar, J. Phys.

Chem. 100 (1996) 16385.22] J.R. Rubin, P. Swaminathan, M. Sundaralingam, Acta Cryst. C 48 (1992)

379.23] E.R. Brown, J.R. Sandifer, in: B.W. Rossiter, J.F. Hamilton (Eds.), Phys-

ical Methods of Chemistry, John Wiley and Sons, New York, 1986, p.273.

24] C.D. Hufford, J.D. McChesney, J.K. Baker, J. Heterocycl. Chem. 20(1983) 273.

25] J.R. Perussi, V.E. Yushmanov, S.C. Monte, H. Imasato, M. Tabak, Phys-iol. Chem. Phys. Med. NMR 27 (1995) 1.

26] E. Mutschler, H. Derendorf, Drug Actions. Basic Principlesand Therapeutic Aspects, Medpharm, CRC Press, Stuttgart, 1995,p. 5.

27] A.R. Tarlov, A.S. Alvin, P.E. Carson, P.E. Brewer, Arch. Intern. Med.109 (1962) 209.

28] A.K. Bhattacharjee, J.M. Karle, J. Med. Chem. 39 (1996) 4622.