Methemoglobinemia Caused by 8-Aminoquinoline Drugs: DFT Calculations Suggest an Analogy to H₄B’s Role in Nitric Oxide Synthase

Abstract

We suggest a possible mechanism of how 8-aminoquinolines (8-AQ’s) causes hemotoxicity by oxidizing hemoglobin to methemoglobin. In our DFT calculations, we found that 5-hydroxyprimaquine is able to donate an electron to O₂ to facilitate its conversion to H₂O₂. Meanwhile, Fe(II) is oxidized to Fe(III) and methemoglobin is formed. In this mechanism, the 8-AQ drug plays a similar role as that of H₄B in nitric oxide synthase. Furthermore, our study offers an approach to inform the design of less toxic antimalarial drugs.

There is a great need for new antimalarial drugs that can radically cure the liver stage of malaria.^1,2 At present the only FDA-approved drug which can kill liver hypnozoites is primaquine³ (1) (Figure 1), a member of the 8-aminoquinoline (8-AQ) family. In vivo, primaquine is believed to form a number of metabolites, such as carboxyprimaquine⁴ (2), 5-hydroxyprimaquine⁵ (5-HPQ, 3), and 6-methoxy-8-aminoquinoline⁶ (4) (Figure 1). A major concern regarding primaquine and its metabolites is that they can cause life-threatening hemolysis in G6PD-deficient patients.⁷ The hemolysis may be related to the propensity of 8-AQ metabolites to oxidize hemoglobin to methemoglobin, an Fe(III) protein which is unable to carry oxygen.⁸ The reaction also results in the formation of reactive oxygen species (ROS), such as hydrogen peroxide. Since primaquine was first synthesized in 1946,⁹ numerous efforts have been made to reduce the toxicity of 8-AQ drugs.¹⁰ Unfortunately, our knowledge of such toxicity has been limited by a lack of understanding of its chemical mechanism.^4,11

Figure 1.

A schematic illustration of primaquine and metabolites.

In this communication, we suggest a possible mechanism for the methemoglobinemia of 8-AQ drugs. Considering the process of converting O₂ to H₂O₂, it is apparent that in addition to two protons, two electrons must be supplied to the π* orbital of O₂. The question is: where do the two electrons come from? Obviously, iron is able to provide one electron with Fe(II) being oxidized to Fe(III), which results in the conversion of hemoglobin to methemoglobin. The source of the second electron, however, is not clear. In this work we hypothesize that an 8-AQ metabolite may provide this second electron, itself being converted into a radical cation. Interestingly, if this were true, the 8-AQ metabolite then plays a similar role to that of tetrahydrobiopterin (H₄B) in nitric oxide synthase (NOS). NOS is an iron-heme containing enzyme that catalyzes the formation of nitric oxide from L-arginine. It has been found that NOS needs the cofactor H₄B during catalysis to transfer an electron to the Fe–O₂ moiety.¹² In particular, Shaik and coworkers¹³ studied the active site protonation states of NOS using QM/MM methods and found that when O₂ is doubly protonated to form the Fe–H₂O₂ complex, H₄B is converted to a radical cation and transfers an electron to aid the formation of H₂O₂. Similarly, when complexed with hemoglobin, an 8-AQ or an 8-AQ metabolite could assist the formation of H₂O₂ analogously as does H₄B in NOS.

To test this hypothesis, we performed a density functional theory (DFT) study on unprotonated, singly protonated and doubly protonated hemoglobin–8-AQ complexes. 5-HPQ (3) was chosen for this study because it is known to cause methemoglobinemia directly and is able to form H₂O₂.¹⁴ By contrast, primaquine itself appears to require metabolic activation to elicit methemoglobin formation.¹⁰ The terminal amine of the 5-HPQ’s 8-amino alkyl chain was assumed to be protonated to match physiological pH and the asymmetric carbon alpha to the 8-amine (see Figure 1) was chosen in the S form. The initial 5-HPQ–hemoglobin complex was obtained by docking 5-HPQ into hemoglobin using the Glide program.¹⁵ The crystal structure (PDB: 2D5X),¹⁶ in which a ligand 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid makes a hydrogen bond with the propionate group of heme, was used for the docking. Detailed docking procedures are provided in the Supporting Information. The docked structure showed an interaction of the terminal 8-amino –NH₃⁺ group of 5-HPQ with the carboxylic side chain of heme (Figure 2a), most likely due to the formation of a strong electrostatic interaction. On the basis of this result, a chemical model to be used for DFT calculations was derived (Figure 2b). All DFT calculations were performed using the spin unrestricted approach. Geometries were optimized using the Jaguar program¹⁷ at the B3LYP¹⁸ level with the LACVP* basis set. Relative energies were obtained at those geometries using Jaguar’s Poisson-Boltzmann self-consistent reaction field method with a dielectric constant (ε) of 4.0 at the B3LYP/LACV3P** level of theory. The above computational approach to study metalloenzymes has recently been applied to similar systems and reviewed in depth.¹⁹

Figure 2.

(a) The interaction of 5-HPQ with hemoglobin from docking into 2D5X. For clarity, only 5-HPQ and heme are shown. The sidechain terminal amine was found to be in close proximity to the heme carboxyl moiety, whereas the alkyl group at the 8-position and the quinoline ring are more than 3 Å away from the heme and form hydrophobic interactions with Leu86 and Leu83. (b) The chemical model used for DFT calculations.

Figure 3 shows the optimized structures of unprotonated, singly protonated, and doubly protonated O₂–hemoglobin–5-HPQ complexes. Upon optimization, the terminal –NH₃⁺ group of 5-HPQ in all the protonation states transfers a proton to the carboxylic group of heme, forming a –H₂N···HOOC– hydrogen bond. For the unprotonated complex, the lowest energy structure is in the singlet spin state (Table 1). In this spin state, an electron is transferred from iron to one of the π* orbitals of O₂ to form Fe^III -O₂^•−. The O₂ moiety shows superoxide character (electron configuration σ_x² π_x² π_y² π_x^*2 π_y^*1 σ_x^*0) with the O–O bond length at 1.27 Å. Thus, the O₂ is in the one-electron reduced state. This electronic assignment is in agreement with computational studies on similar systems.^20–24 It should be noted that in all the possible spin states, the spin density on 5-HPQ is 0.00, suggesting that no electron is transferred from 5-HPQ to O₂.

Figure 3.

The optimized structures of (a) unprotonated, (b) singly protonated and (c) doubly protonated O₂ hemoglobin–5-HPQ complexes in the lowest energy spin states.

Table 1.

Relative Energies (kJ mol⁻¹) and Spin Densities for the Unprotonated (O₂), Singly Protonated (OOH) and Doubly Protonated (H₂O₂) Species in all the Possible Spin Multiplicities.

Structure	M	ΔE	Spin Density
			Fe	Oinner	Oouter	heme	5-HPQ
O2	1	0.0	1.37	−0.56	−0.73	−0.08	0.00
	3	16.3	1.10	0.33	0.62	−0.04	0.00
	5	32.3	2.14	0.94	0.97	−0.08	0.00
	7	17.5	3.80	1.00	0.99	0.17	0.00
OOH	1	0.3	0.84	0.19	0.02	−0.42	−0.63
	3	0.0	0.85	0.19	0.03	0.32	0.64
	5	62.1	3.06	−0.16	−0.07	0.62	0.59
	7	31.4	4.13	0.40	0.09	0.75	0.61
HOOH	1	11.8	1.05	0.00	0.00	−0.03	−1.01
	3	0.0	1.06	0.00	0.00	−0.04	1.01
	5	38.7	3.03	0.02	0.00	−0.12	1.01
	7	10.2	4.26	0.04	0.00	0.59	1.01

In the singly protonated complex, the lowest energy structure is in the triplet spin state. The singlet spin state, however, lies only marginally higher in energy by 0.3 kJ mol⁻¹. In the NOS system complexed with OOH, the singlet and triplet states were similarly found to be very close in energy.¹³ The affinity of the unprotonated complex to accept one proton was calculated to be 1133.5 kJ mol⁻¹ using the free energies²⁵ of the unprotonated and singly protonated complexes in the ground state. In the triplet state, iron is still in the Fe(III) state. The O–O bond is further lengthened to 1.44 Å, indicating that it is essentially a single bond. Thus, an electron has been promoted to the other π* orbital of O₂^•− to convert it to O₂²⁻ with electron configuration σ_x²π_x² π_y ²π_x^*2 π_y ^*2σ_x^*0. Hence, the O₂ is now in the two-electron reduced state. From the calculated spin distribution (Table 1), it can be concluded that this electron is partially supplied by each of heme and 5-HPQ, with 5-HPQ contributing ~0.64 electrons. In the triplet, quintet and septet states, the unpaired electron shared between heme and 5-HPQ aligns ferromagnetically with that in iron, while in the single state, they align antiferromagnetically.

In the doubly protonated complex, the lowest energy structure is also in the triplet state. The affinity of the singly protonated complex to accept an additional proton was calculated to be 1126.6 kJ mol⁻¹ using the free energies²⁵ of the singly protonated and doubly protonated complexes in the ground state. The proton affinity of H₂O was calculated to be 997.2 kJ mol⁻¹ at the same level. The affinities of O₂ to accept protons are larger than that of H₂O, suggesting that the hemoglobin ···5-HPQ bound O₂ is more basic than H₂O and should be able to be protonated by H₃O⁺. In the triplet spin state of the doubly protonated complex, iron remains in the Fe(III) state. The O–O bond lengthens slightly further to 1.45 Å and the spin densities on both oxygen atoms are 0.00. Thus, as for the singly protonated complex, O–O is closed-shell and single bonded with electron configuration σ_x²π_x²π_y²π_x^*2 π_y^*2 σ_x^*0. However, in addition to the electron donated by iron, the second electron transferred to the π* orbital of O₂ is now purely provided by 5-HPQ, which is converted to a radical cation, as shown from its calculated spin density of 1.01.

In a separate set of gas phase calculations, we fixed the lengths of the two terminal N–H bonds to prevent the proton transfer to the carboxylic acid side chain of the heme. This restriction to the –H₃N⁺···⁻OOC– interaction, however, did not affect the conclusions obtained (see Supporting Information). In addition, to take into account that the binding site is located at the protein surface, single point calculations were performed on selected structures with ε of 80.37 (representing water), but in this case also similar results were obtained (Supporting Information).

These results show that 5-HPQ indeed plays a similar role for hemoglobin as H₄B does for NOS. It is, in fact, an even better electron donor than H₄B. In Shaik et al.’s study¹³ on NOS, the process of electron transfer from H₄B was only observed when O₂ is doubly protonated. In contrast, when O₂ is singly protonated in the hemoglobin–5-HPQ complex, 5-HPQ donates nearly two thirds of an electron to O₂. This may be explained in part by the fact that 5-HPQ has a lower ionization energy than H₄B. The gas phase calculation at the B3LYP/6-311+G(2df,p)//B3LYP/6-31G(d,p) level shows that H₄B has an ionization energy of 593.9 kJ mol⁻¹. However, that of 5-HPQ is 13.2 kJ mol⁻¹ lower in energy at 580.7 kJ mol⁻¹. Thus, 5-HPQ is more capable of donating an electron than H₄B.

Furthermore, it should be noted that the binding pocket is located at the surface of hemoglobin. In addition to the –H₂N···HOOC– hydrogen bond, the ligand interacts hydrophobically with the amino acid residues of the protein. The relatively non-specific hydrophobic interactions suggest that the proposed chemical mechanism may be general to the class of aromatic compounds that can form a hydrogen bond with the carboxylic arm of heme via an amine or a hydroxyl group. Indeed, certain small compounds such as aniline and its metabolites are also able to catalyze the formation of methemoglobin in vivo.²⁶ These processes may follow a similar mechanism to that discussed in this communication.

In the present work, we suggest a possible explanation of the methemoglobinemia caused by 8-AQ drugs. Both iron and 8-AQ donate an electron to the π* orbital of O₂. This thus facilitates the formation of H₂O₂. In the meantime, Fe(II) is converted to Fe(III). Therefore, methemoglobin, which cannot bind O₂, is formed. Notably, in this mechanism 8-AQ plays a similar role as that of H₄B in NOS. In principle, modification of the exocyclic substituents of 8-AQ’s will affect their electron donating ability, which suggests a rational plan for discovery of new, less toxic 8-AQ drugs.