Methemoglobin Generation by 8-Aminoquinolines - American

Oct 13, 2013 - PQ administration can cause life-threatening hemolytic anemia in some patients. In our efforts to understand the connection between PQ ...
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Methemoglobin Generation by 8‑Aminoquinolines: Effect of Substitution at 5‑Position of Primaquine Haining Liu,† Babu L. Tekwani,‡,§ N. P. Dhammika Nanayakkara,‡ Larry A. Walker,‡,§ and Robert J. Doerksen*,† †

Department of Medicinal Chemistry, ‡National Center for Natural Products Research, and §Department of Pharmacology, University of Mississippi, University, Mississippi 38677, United States S Supporting Information *

ABSTRACT: Currently, the only clinically approved antimalarial drug to treat relapsing malaria is primaquine (PQ), yet PQ administration can cause life-threatening hemolytic anemia in some patients. In our efforts to understand the connection between PQ and methemoglobin formation, the effect of 5substituted primaquine derivatives on the basicity of hemoglobin-bound O 2 was investigated using various computational methods, including quantum mechanics/ molecular mechanics (QM/MM) calculations, molecular dynamics simulations and density functional theory calculations, to determine the geometries, relative energies, spin densities, proton affinities and ionization potentials of various PQ derivatives and PQ···hemoglobin complexes. We found that the protein environment and solvent do not change our previously proposed methemoglobin formation mechanism that 5-hydroxyprimaquine donates an electron to O2, facilitating its conversion to H2O2 and generating methemoglobin. Because of 5-hydroxyprimaquine’s ability to lose an electron by this mechanism, we then used different substituents at primaquine’s 5-position and found that an electron-withdrawing group (EWG) increases the ionization potential of the corresponding derivative. As a result, the EWG-substituted derivatives make the hemoglobin-bound O2 less basic, because of their weaker electron-donating ability. These derivatives hence are predicted to have a lower propensity to generate methemoglobin, which can inform future design of less hemotoxic antimalarial drugs. We also carried out experimental measurement of methemoglobin formation for some of the 5-substituted derivatives.

1. INTRODUCTION Malaria is an infectious disease caused by Plasmodium parasites which has wreaked extensive mortality and morbidity throughout recorded history. Currently, the only clinically approved antimalarial drug to treat relapsing malaria is primaquine (PQ, Figure 1),1,2 the prototype of the 8aminoquinoline (8-AQ) class. This molecule was first synthesized more than sixty years ago.3 However, a serious concern for PQ is that its administration to glucose-6phosphate dehydrogenase (G6PD)-deficient patients can cause life-threatening hemolytic anemia, due to oxidative damage to red cells and their subsequent removal from the circulation.4 A hallmark response to the 8-AQs is the oxidation of hemoglobin to methemoglobin,5−7 which is assumed to be related to the oxidative stress as follows: methemoglobin contains a ferric iron (Fe3+) that is unable to carry oxygen, and methemoglobin formation also gives rise to reactive oxygen species. From various experimental studies, it is known that the extent of hemotoxicity in G6PD-deficient patients varies for different PQ metabolites (Figure 1). For example, it has been found that the hydroxylated metabolites, such as 5-hydroxyprimaquine (5-HPQ),8,9 5,6-dihydroxyprimaquine10 and 6methoxy-8-(N-hydroxy)aminoquinoline,11,12 are more toxic © 2013 American Chemical Society

Figure 1. The structures of primaquine and several of its metabolites carboxyprimaquine, 5-hydroxyprimaquine (5-HPQ), 5,6-dihydroxyprimaquine (5,6-HPQ), 6-methoxy-8-aminoquinoline (MAQ) and 6methoxy-8-(N-hydroxy)aminoquinoline (MAQ-NOH).

than the parent PQ molecule.10,13 In contrast, some other metabolites, such as carboxyprimaquine14,15 and 6-methoxy-8Received: February 14, 2013 Published: October 13, 2013 1801

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aminoquinoline,11,12 result in less methemoglobin formation than PQ,5,13,16 and hence are less toxic. Although this limited information was available, the mechanism of methemoglobin formation remained unknown for many years. Despite tremendous efforts to discover less toxic antimalarial drugs in recent years,4,17−24 there has been only limited progress. We feel that it is important to understand the chemical mechanism of the methemoglobinemia caused by the PQ derivatives to better aid this discovery process. Recently, we have considered the possible methemoglobinemia mechanism from three angles. First, our density functional theory (DFT) calculations25 excluded the possibility that PQ and its metabolites accept an electron, donated from the ferrous iron in hemoglobin, because PQ and 5-HPQ have essentially the same adiabatic electron affinities, which is inconsistent with the fact that 5-HPQ is much more toxic than PQ. Second, we considered and disproved the hypothesis that 8-AQs enhance methemoglobinemia by lowering the ionization potential of hemoglobin when they bind to it.26 Third, the binding of 5HPQ to hemoglobin and the subsequent process of protonation of O2 were studied using docking and DFT methods.27 Based on the binding mode of 5-HPQ with hemoglobin obtained from docking calculations,27 when O2 is singly and doubly protonated, 5-HPQ donates ∼0.6 and 1 electron to the O2 moiety, respectively, hence facilitating its conversion to H2O2 and simultaneously resulting in the formation of methemoglobin. Interestingly, in this mechanism 5-HPQ plays a similar role to that of the cofactor tetrahydrobiopterin (H4B) in the enzyme nitric oxide synthase (NOS), which was found to be able to transfer an electron to the Fe−O2 moiety via the carboxylate group of heme during catalysis.28−34 Notably, the ability of the heme carboxylate group to transfer an electron was found in other enzymes as well.35 In this work, the effect of 5-substituted primaquine derivatives on the basicity of hemoglobin-bound O2 was investigated using quantum mechanics/molecular mechanics (QM/MM) calculations, molecular dynamics simulations and density functional theory calculations, to determine the geometries, relative energies, spin densities, proton affinities and ionization potentials of PQ and various PQ derivatives as well as of their complexes with hemoglobin. Our previous study 27 suggested a rational approach to design less methemoglobinemia-toxic PQ-related antimalarial drugs by altering the electron-donating ability of PQ using different exocyclic substituents. In this paper, we focused on PQ derivatives with different substituents at the 5-position, since the metabolite which is hydroxylated at this position, 5-HPQ, has long been known to cause methemoglobinemia and oxidative stress.8,9 We calculated the effect of these derivatives on the basicity of hemoglobin-bound O2 in order to derive a trend that could be used to inform the future design of less toxic antimalarial drugs. We also carried out experimental measurement of methemoglobin formation for some of the 5substituted derivatives.

calculations at the B3LYP/LACV3P+** level. The OPLS2005 force field was used for the MM region in both geometry optimizations and single point calculations, i.e., B3LYP/LACV3P+**/OPLS2005// B3LYP/LACVP/OPLS2005. The QM region is shown in Figure S3 in the Supporting Information. Specifically, it includes the iron, heme, O2, a histidine residue that is coordinated with iron and modeled as an imidazole, and the PQ derivative. The total charge of the QM region for the unprotonated system is 0, while that for the singly and doubly protonated systems is +1 and +2, respectively. The hydrogen cap method as implemented in QSite was used to treat the cutoff of covalent bonds. For the systems taken from molecular dynamics snapshots, the Cl− counterion and the water layer beyond 5 Å from the protein surface were removed. It has been recently found in several studies44−46 that the OLYP functional47 is better than B3LYP in describing the energies and spin distributions for iron containing systems. We also used this functional on the lowest energy spin states of each complex considered in this work. The calculated spin densities and proton affinities are shown in Table S3 in the Supporting Information. It was found that the use of this functional did not change the conclusions obtained with the B3LYP functional. For example, when a 5-EWG (electron-withdrawing group) substituted PQ derivative binds to hemoglobin, the proton affinities of O2 are smaller than when the 5-EDG (electron-donating group) substituted derivatives bind to hemoglobin. 2.2. Molecular Dynamics (MD) Simulations. We first took the 5-hydroxyprimaquine···hemoglobin complex obtained from previous docking calculations,27 in which the protonation states of ionizable residues were set by the Protein Preparation Wizard as implemented in the Schrödinger program. Specifically, all the lysine and histidine residues were protonated while all the glutamic acid and aspartic acid residues were deprotonated. A detailed description of the docking approach and the obtained binding poses is provided in ref 27. The most preferred binding complex showed an interaction between the terminal −NH3+ of the side chain attached at the 8-position of 5-HPQ and the propionate group of heme, i.e., −NH3+···−OOC−. We manually transferred a proton from the −NH3+ to the carboxylic group of heme, thus forcing the formation of the −H2N···HOOC− interaction instead. This system was minimized using the OPLS2005 force field as implemented in the MacroModel48 program before being subjected to the MD simulation. The MD simulations were performed using the Desmond program.49,50 The OPLS2005 force field was used. The system was first solvated using the TIP3P explicit solvation model in a cubic water box with the length of each dimension set to 66.5 Å. The whole system contained 8,961 water molecules. One Cl− ion was added to balance the charge. A detailed description of the relaxation and simulation protocol used is provided in the Supporting Information. After the system was relaxed, a 5 ns production MD simulation in the NPT ensemble at 300 K and 1 atm was performed. The smooth particle mesh Ewald method and a cutoff of 9 Å were used to treat long-range electrostatic interactions. 2.3. DFT Calculations. The Gaussian 09 program51 was used for all DFT calculations. Geometries were optimized in the gas phase using the B3LYP52−54 method with the 6-31G(d,p) basis set. Frequency calculations were also performed at this level to confirm that all the optimized structures correspond to minima on the potential energy surface and to obtain zero-point vibrational energies (ZPVE). Relative energies in the gas phase were obtained by performing single point calculations at the B3LYP/6-311+G(2df,p) level based on the above optimized geometries and corrected with the ZPVE from the smaller basis set, i.e., B3LYP/6-311+G(2df,p)// B3LYP/6-31G(d,p) + ZPVE. 2.4. Methemoglobin Formation Assay. The analogues were evaluated in vitro for methemoglobin formation.55 The reaction mixture for the in vitro methemoglobin formation assay contained 100 μL of washed glucose 6-phosphate dehydrogenase deficient human erythrocytes [suspended in PBSG (phosphate-buffered saline with glucose) with 50% hematocrit], 5 μL of the compound dissolved in distilled water or DMSO and 395 μL of PBSG to make the final

2. EXPERIMENTAL SECTION 2.1. Quantum Mechanics/Molecular Mechanics (QM/MM) Calculations. The QM/MM calculations were performed using the QSite program.43 The B3LYP/LACVP level was used for geometry optimizations in the QM region. This level of theory has been widely used in previous QM/MM studies on similar heme-iron enzymes.36,37 Relative energies were obtained by performing single point 1802

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volume of the reaction mixture to be 500 μL. No liver microsomes were included. The reaction mixtures with appropriate erythrocyte controls without drugs were also set up simultaneously. Each assay was set up at least in duplicate. The reaction mixtures were incubated at 37 °C in a metabolic water bath for one hour with constant shaking at 80 rpm. After one hour the reaction mixtures were chilled on ice and methemoglobin levels were measured with a CO-Oximeter (IL-682).

3. RESULTS AND DISCUSSION 3.1. QM/MM Calculations on 5-HPQ···Hemoglobin Complex. We first started with 5-HPQ, which we previously studied using a simplified model derived from docking calculations,27 including iron, 5-HPQ, heme, an imidazole coordinated to the iron to represent the histidine in hemoglobin and either O2, OOH, or H2O2 in the distal position. In this work, we took the docking model complex and added in the rest of the hemoglobin protein using the quantum mechanics/molecular mechanics (QM/MM) method in order to see if the protein environment affected the properties of the system. The unprotonated, singly protonated and doubly protonated complexes were optimized and are shown in Figure 2. The relative energies and calculated spin densities at all possible multiplicities are shown in Table 1. The previous docking structure27 showed an interaction between the terminal −NH3+ of 5-HPQ and the carboxylic group of the heme, i.e., −NH3+···−OOC−. Upon QM/MM geometry optimization, such an interaction collapsed in all the protonation states, resulting in the formation of a neutral −H2N···HOOC− hydrogen bond, similar to what we found in previous DFT calculations on the model system.27 The singlet spin state was the lowest in energy in both the unprotonated and singly protonated complexes, while the quintet spin state was the lowest in energy in the doubly protonated complexes. Using the ground state energies of the three protonation states, the proton affinities (PAs) of O2 were calculated (Table 1). The first PA (PA1) reflects the process of converting O2 to OOH, while the second PA (PA2) reflects the process of converting OOH to H2O2. PA1 and PA2 were calculated to be 1034.5 and 910.6 kJ mol−1, respectively, for this system with 5-HPQ bound to hemoglobin. Both of the PAs are considerably higher than that of H2O, which is 712.7 kJ mol−1 at the same QM level. Hence, such protonation processes are indeed likely to occur in biological systems. Importantly, the inclusion of the protein environment did not change our previous finding27 that 5-HPQ is able to donate an electron to O2, which facilitates its conversion to H2O2. In the singly and doubly protonated states, 5-HPQ donates ∼0.7 and 1 electron, respectively, as shown from the calculated spin densities in Table 1. This electron and another electron from iron are inserted into the π* orbital of O2, which undergoes a two-electron reduction to form H2O2. In the meantime, 5-HPQ is converted to a radical cation and iron is converted to the ferric state. 3.2. Molecular Dynamics Simulations. Since the −H2N···HOOC− hydrogen bonding interaction, rather than the −NH3+···−OOC− ionic interaction, is favored in the above QM/MM geometry optimization, an MD simulation was performed to characterize the dynamical stability of such an interaction. The construction of the −H2 N···HOOC− interaction from the docking models is detailed in the Experimental Section. Figure 3a shows the root-mean-square deviation (RMSD) of the backbone atoms with respect to simulation time. The RMSD quickly increased during the first

Figure 2. The QM/MM optimized structures of the (a) unprotonated, (b) singly protonated, and (c) doubly protonated 5-HPQ··· hemoglobin complex in the lowest energy spin states.

few picoseconds of the simulation, because of the relaxation of the system, and was well converged to ∼1.3 Å in the last several nanoseconds of the simulation. Hence, the system reached a dynamically stable state. To study the −H2N···HOOC− interaction, the distance between the heavy atoms that are involved in this interaction, 1803

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Table 1. Relative Energies, Spin Densities and Proton Affinities of O2 in the Unprotonated (O2), Singly Protonated (OOH) and Doubly Protonated (HOOH) 5-HPQ···Hemoglobin Complex in All Possible Multiplicities, Using the QM/MM Method structure O2

OOH

HOOH

a

Ma

ΔEb

Fe

Oinner

Oouter

heme

5-HPQ

1 3 5 7 1 3 5 7 1 3 5 7

0.0 22.5 70.0 63.4 0.0 5.4 69.5 47.6 42.5 43.6 0.0 18.8

1.24 1.06 2.14 3.88 0.97 0.97 3.04 4.15 1.04 1.05 2.90 4.28

−0.44 0.32 0.96 0.96 0.11 0.11 −0.15 0.59 0.00 0.00 0.01 0.02

−0.71 0.66 0.97 0.97 −0.01 −0.01 −0.06 0.21 0.00 −0.01 0.01 0.01

−0.08 −0.04 −0.10 0.15 −0.35 0.25 0.50 0.39 −0.02 −0.03 −0.01 0.59

0.00 0.00 0.00 0.00 −0.73 0.70 0.69 0.62 −1.01 1.00 0.99 1.00

PAc

1034.5

910.6

Multiplicity. bRelative energy (kJ mol−1) cProton affinity (kJ mol−1).

Furthermore, the calculated spin densities (Table S1 in the Supporting Information) suggested a similar amount of electron transfer occurred when O2 was singly and doubly protonated. The proton affinities (PAs) of O2 were also calculated using the 2.5 and 4 ns snapshots for the initial geometries, and were found to be larger by 46.3−72.2 kJ mol−1 than those obtained using the docking structure for the initial geometry (Table S1 in the Supporting Information). This is probably due to the stabilization of the positive charges by the water molecules explicitly included in the MD simulation in the singly and doubly protonated complexes. Thus, the inclusion of solvent yielded an increase in the calculated basicity of O2. However, when different PQ derivatives bind to hemoglobin, it is the trend of the change in the PA of O2, rather than the absolute PA values, which sheds light on the design of antimalarial drugs with less potential to generate methemoglobin. Considering that it is computationally time-consuming to use multiple MD snapshots for QM/MM calculations, it was more feasible to use the docking structure as the initial geometry in all subsequent QM/MM calculations with a different PQ derivative bound to hemoglobin. An increase of PA by up to ∼70 kJ mol−1 may be possible when solvent is included. However, we expected such increases to be similar for each PQ derivative considered in this study, since they all bind in the same environment. The structure obtained from docking calculations was also directly taken for a 5 ns MD simulation in which the ionic −NH3+···−OOC− interaction remains during the simulation. It was found that the existence of the −NH3+···−OOC− interaction did not change the fact that 5-HPQ is able to donate an electron to O2, similar to what we have previously identified.27 A detailed analysis is provided in the Supporting Information. 3.3. Ionization Potentials of Other PQ derivatives. The electron-donating abilities of PQ and six derivatives were then considered. Their substituents include electron-donating groups (EDGs) and electron-withdrawing groups (EWGs). We compared their calculated gas phase IPs (Table 2) to obtain the trend of how the change of this exocyclic substituent affects the IP. In the polar enzymatic environment, the IPs of these molecules are expected to decrease. However, it is likely that the trend obtained from the gas-phase calculations will not change because all the derivatives have a similar binding environment in hemoglobin. For comparison, the gas-phase IP

Figure 3. (a) The root-mean-square deviation (RMSD) of the backbone atoms versus simulation time, and (b) the N···O distance in the key −H2N···HOOC− interaction versus simulation time, in the MD simulation of the 5-HPQ···hemoglobin complex that has a neutral −H2N···HOOC− hydrogen bonding interaction between 5-HPQ and heme.

i.e., N···O, was monitored (Figure 3b). If the distance was within 3 Å, we considered it to be a hydrogen bonding interaction. As seen in Figure 3b, at most times this −H2N··· HOOC− interaction existed in the simulation. During the 5 ns production run, this interaction had an occupancy of 91%. Hence, the −H2N···HOOC− hydrogen bond was a dynamically stable interaction in the simulation and is predicted to be important in vivo. From the MD simulation, two snapshots at 2.5 and 4 ns were taken as the initial geometries for QM/MM optimizations for the unprotonated, singly protonated and doubly protonated states. The −H2N···HOOC− interaction was found to remain in all the optimized structures. In addition, the inclusion of the solvent (explicit water molecules from the MD simulations) did not change the lowest energy spin state for any of the three protonation states (Table S1 in the Supporting Information). 1804

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also lower than that of H4B, suggesting that they have a higher electron-donating ability than H4B. Hence, like H4B, they may be able to donate an electron in biological systems and are perhaps even better than H4B for this role. In compounds 5−7, the 5-position is substituted with an EWG, −CF3, −CN or −NO2. Their IPs are higher than that of PQ by 39.7, 46.3, and 49.0 kJ mol−1, respectively, suggesting that they should be less able to donate an electron. According to our proposed mechanism that the methemoglobinemia potential of PQ derivatives is related to their electron-donating ability, these molecules should have lower methemoglobingenerating potential than either PQ or its derivatives with an EDG at the 5-position. 3.4. QM/MM Calculations on PQ Derivatives. In order to investigate their methemoglobinemic potential, PQ (1) and its derivatives (2, 4−7) were then studied bound to hemoglobin. To make a consistent comparison, the above 5HPQ···hemoglobin QM/MM model built based on the docking structure was used as the initial geometry. Considering the fact that there is a lack of experimental information on how PQ and/or its derivatives bind to hemoglobin, the PQ derivatives considered in this study were assumed to bind in a similar manner. It was found that after the QM/MM geometry optimizations the binding of the different molecules did not significantly change the nearby backbone helical structure of the protein (Figure S1 in the Supporting Information). Hence, this is a reasonable binding mode for this type of ligand.

Table 2. Calculated Ionization Potentials of Selected PQ Derivatives and H4B

a

Ionization potential (kJ mol−1).

of H4B, the cofactor in NOS that is known to be able to donate an electron during catalysis, was also calculated. When the 5-position was substituted with an EDG, the IPs of the corresponding molecules were found to be lower than that of PQ. For example, the IPs of compounds 2, 3 and 4, with a −CH3, −OH and −OCH3 group at the 5-position, respectively, are 18.5, 38.0, and 37.8 kJ mol−1 lower than that of PQ, respectively. Furthermore, the IPs of compounds 3 and 4 are

Table 3. Spin Densities and Proton Affinities of O2 in the Complexes with Each PQ Derivative Bound to Hemoglobin in Its Lowest Energy Multiplicity Using the QM/MM Method

1 (R = −H)

2 (R = −CH3)

3 (R = −OH)

4 (R = −OCH3)

5 (R = −CF3)

6 (R = −CN)

7 (R = −NO2)

a

O2 OOH HOOH O2 OOH HOOH O2 OOH HOOH O2 OOH HOOH O2 OOH HOOH O2 OOH HOOH O2 OOH HOOH

Ma

Fe

Oinner

Oouter

heme

ligand

1 3 5 1 3 5 1 1 5 1 1 5 1 3 5 1 3 5 1 1 5

1.22 0.94 2.90 1.24 0.95 2.90 1.24 0.97 2.90 1.20 0.91 2.92 1.25 0.97 2.90 1.24 0.92 2.90 1.29 0.86 2.90

−0.43 0.12 0.01 −0.44 0.12 0.01 −0.44 0.11 0.01 −0.42 0.16 0.01 −0.45 0.54 0.01 −0.44 0.50 0.01 −0.47 0.20 0.01

−0.70 −0.01 0.01 −0.71 −0.01 0.01 −0.71 −0.01 0.01 −0.70 0.00 0.01 −0.72 0.27 0.01 −0.71 0.25 0.01 −0.73 0.02 0.01

−0.08 0.41 −0.01 −0.08 0.36 −0.01 −0.08 −0.35 −0.01 −0.07 −0.25 −0.03 −0.08 0.06 −0.01 −0.08 0.09 −0.01 −0.09 −0.98 −0.01

0.00 0.54 1.00 0.00 0.61 1.00 0.00 −0.73 0.99 0.00 −0.82 1.00 0.00 0.17 1.00 0.00 0.26 1.00 0.00 −0.14 1.00

PAb 993.7 896.5 1020.7 907.7 1034.5 910.6 1047.2 909.9 971.0 904.7 969.8 898.0 973.3 888.0

Multiplicity. bProton affinity (kJ mol−1). 1805

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was found between the IPs and PA1 + PA2. Hence, the ability of one of the PQ derivatives to donate an electron indeed does determine, at least partially, the basicity of the hemoglobinbound O2. If this proposed mechanism is responsible for methemoglobin generation by the 8-AQs, it suggests that an approach to decrease the toxicity of primaquine would be the use of an EWG at the 5-position. 3.5. Comparison to Nitric Oxide Synthase and Other Similar Enzymes. As we previously discussed,27 in this proposed methemoglobinemia mechanism the PQ derivatives play a similar role to that of the H4B cofactor in nitric oxide synthase (NOS) in donating an electron to the Fe−O2 moiety. In NOS, H4B similarly hydrogen bonds with the propionate group of heme via its −N3H and exocyclic −NH2 moieties (Figure S2 in the Supporting Information). It has been found35 that the heme propionate is able to act as an electron transfer conduit in several other enzymes, such as manganese peroxidase, ascorbate peroxidase and diheme cytochrome c peroxidase. The current findings add in PQ and its derivatives as possible donors to transfer an electron via the heme propionate, which may account for their methemoglobingenerating potential. During the catalytic functioning of NOS, the protonation state of H4B, however, is not clear. It was proposed38,39 that N3 may be deprotonated by the carboxylic group of heme, hence forming a neutral radical H3B•. Other experimental40−42 and theoretical29,30 studies, however, favor the formation of a radical cation H4B•+. The PQ derivatives differ from H4B in that they bind at the protein surface. Hence, the much more polar hydrophilic environment created by the proximity of bulk solvent has a great stabilizing effect on the radical cation generated in the electron transfer process. Therefore, the PQ derivatives seem to be more adaptable than H4B regarding their protonation state, since both the neutral and protonated 5HPQ were found to be able to donate an electron to O2 when bound to hemoglobin. 3.6. Experimental Measurement of Methemoglobin Formation. In order to test our proposed mechanism, we performed experiments to measure the in vitro methemoglobin formation by 1, 2, 3 and 4. We were unable to synthesize the primaquine derivatives with an EWG at the 5-position (5, 6 and 7), and hence do not have their methemoglobin formation data. As can be seen from Table 4, 5-HPQ (3) and 5methoxyprimaquine (4) result in more methemoglobin formation than PQ (1). This is in agreement with the above computational results that EDG substituents cause more methemoglobin generation. However, 5-methylprimaquine

The geometries of the unprotonated, singly protonated and doubly protonated complexes of these six compounds bound to hemoglobin in all the possible multiplicities were optimized. The spin densities of the lowest energy multiplicity for all three protonation states are listed in Table 3. Those for all the other multiplicities are provided in Table S2 in the Supporting Information. In the unprotonated case, the singlet spin state has the lowest energy in all the systems considered. In this spin state, an electron has been promoted from Fe(II) to O2, which is in agreement with previous studies on similar systems.27,36,37 In addition, all the PQ derivatives remain neutral, having spin densities equal to 0.0 for each. For the singly protonated case, the singlet and triplet spin states similarly lie very close in energy, as previously found for 5-HPQ in our earlier calculations.27 Except for the −OH (3), −OCH3 (4) and −NO2 (7) systems, the lowest energy state in the singly protonated complex is the triplet spin state. When the 5position is substituted by an EDG (2, 3 and 4), the resulting PQ derivative donates ∼0.5−0.8 electron to the O2 moiety as suggested by the calculated spin densities. However, when the 5-position is substituted by an EWG (5, 6 and 7), those derivatives donate significantly less of an electron to O2. Specifically, in the ground state the spin densities on the PQ derivatives in 5, 6 and 7 are 0.17, 0.26 and −0.14, respectively. This is likely due to the weaker ability of the EWG-substituted PQs to donate an electron, as suggested by the calculated ionization potentials (cf. Table 2). In the doubly protonated complexes, the quintet spin state was found to be the lowest in energy in all systems considered. In those complexes, the PQ derivative donates one electron to O2, and is itself converted to a radical cation form. This results in the formation of a neutral H2O2 and iron being in the Fe(III) state, which indicates that hemoglobin has been converted to methemoglobin. The calculated first and second proton affinities (PA1 and PA2) of O2 with these PQ derivatives bound to hemoglobin are listed in Table 3. As can be seen, when the EWG-substituted derivatives (5, 6 and 7) are bound, the PAs are much smaller than those of the bound EDG-substituted derivatives (2, 3 and 4), which suggests that in the EWG-substituted derivative complexes O2 should be less basic and less easily protonated to form H2O2. We concluded that this occurs in part because the EWG-substituted derivatives have a lower ability to donate an electron. Hence, it is worth considering if there is a mathematical relationship between the IPs of the PQ derivatives and the PAs of O2. We used the sum of PA1 and PA2 to derive such a relation, since that sum represents the overall basicity of O2. A linear relationship (r2 = 0.94, Figure 4)

Table 4. Experimentally Determined in Vitro Methemoglobin Formation by Selected Primaquine Derivatives

R 1 2 3 4

Figure 4. The sum of the first and second proton affinities of O2 (PA1 + PA2) versus the ionization potential (IP) of the PQ derivatives considered in this study. 1806

−H −CH3 −OH −OCH3

% methemoglobin 1.35 0.85 54.36 2.40

± ± ± ±

0.53 0.51 13.45 0.27

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−NH3+···−OOC− interaction. This material is available free of charge via the Internet at http://pubs.acs.org.

(2) seems to be an exception. Specifically, it has an EDG substituent but generates slightly less methemoglobin than PQ. Perhaps, in addition to the ability to lose an electron, specifics of binding of the ligand to hemoglobin also affect the methemoglobin formation. It should be noted that the binding pocket is located at the protein surface, which is hence in a rather hydrophilic environment. It is clear that 5-methylprimaquine is less polar than 5-HPQ and 5-methoxyprimaquine. As a result, the binding of 5-methylprimaquine with hemoglobin is very likely to be weaker than that of 5-HPQ or 5methoxyprimaquine. This may help explain why 5-methylprimaquine generates less methemoglobin than PQ. Nevertheless, when strong and polar EDGs, such as −OH and −OCH3, are substituted at the 5-position, the resulting derivatives indeed generate more methemoglobin than PQ. Further studies are needed to verify our predictions for 5-EWG substituted PQ derivatives. In addition, other factors that determine the methemoglobin formation should be investigated in the future.



Corresponding Author

*E-mail: [email protected]. Funding

This project was partially supported by a grant to the University of Mississippi, W81-XWH-10-2-0059, awarded and administered by the U.S. Army Medical Research & Material Command (USAMRMC) and the Telemedicine & Advanced Technology Research Center (TATRC), at Fort Detrick, MD. NCNPR is partially supported by a U.S. Department of Agriculture, Agriculture Research Service, Cooperative Agreement # 58-6408-2-0009. This investigation was conducted in part in a facility constructed with support from Research Facilities Improvements Program (C06 RR-14503-01) from the NIH National Center for Research Resources. Notes

4. CONCLUSION In this paper, the effect of several 5-position substituted PQ derivatives on the proton affinities of hemoglobin-bound O2 was studied using a variety of computational methods. We also reported experimental methemoglobin formation results for some of the derivatives. We found that the inclusion of the protein environment and water solvent does not change our previously proposed methemoglobinemia mechanism27 that 5HPQ is able to donate an electron to O2 to facilitate its conversion to H2O2. Following this finding, we then considered the parent PQ molecule and five other derivatives with typical EDGs (−CH3 and −OCH3) or typical EWGs (−CF3, −CN and −NO2) at the 5-position. An EWG at the 5-position was able to increase the IP of the PQ derivative, hence making it more difficult for it to donate an electron. All these PQ derivatives were used to bind to hemoglobin. We found that, in the singly protonated state, the 5-EWG substituted PQ derivatives donate a smaller fraction of an electron than the 5-EDG substituted derivatives. This additionally causes O2 to be less basic and makes it more difficult for it to be protonated to form H 2 O 2 , which should in turn generate less methemoglobin. As a result, a rational approach to design of less toxic antimalarial drugs may be to use an EWG as the exocyclic substituent at the 5-position on primaquine. However, one must also consider the susceptibility of such analogues to metabolic transformation in order to lead to biological activation, which seems to be an important factor in the case of 8-AQs. Furthermore, since the mechanism of the antimalarial action of these drugs is still unknown, the impact of such changes on their desired efficacy would have to be explored.



AUTHOR INFORMATION

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Thanks to Rajnish Sahu for technical support. Computer time and resources from the Department of Medicinal Chemistry, University of Mississippi, and the Mississippi Center for Supercomputer Research are greatly appreciated.



ABBREVIATIONS PQ, primaquine; QM/MM, quantum mechanics/molecular mechanics; EWG, electron-withdrawing group; 8-AQ, 8aminoquinoline; G6PD, glucose-6-phosphate dehydrogenase; 5-HPQ, 5-hydroxyprimaquine; DFT, density functional theory; H4B, tetrahydrobiopterin; PA, proton affinity; RMSD, rootmean-square deviation; MD, molecular dynamics; EDG, electron-donating group; NOS, nitric oxide synthase; PBSG, phosphate-buffered saline with glucose



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ASSOCIATED CONTENT

S Supporting Information *

Superimposition of all the optimized lowest energy spin states of the unprotonated complexes (Figure S1), schematic illustration of the binding of H4B in nitric oxide synthase (Figure S2), schematic illustration of the QM region (Figure S3), the relative energies and spin densities for structures taken from MD snapshots (Table S1) and all the PQ derivatives bound to hemoglobin (Table S2), spin densities and proton affinities of O2 using the OLYP method (Table S3), the detailed protocol for the relaxation step used in Desmond, and discussion of the MD simulation of the system that contains the 1807

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