malaria 
 is a mosquito - borne infectious disease that is widespread 
 in the tropical and subtropical regions , causing extensive mortality 
 and morbidity . global malarial deaths increased to a peak of 1  817  000 
 in 2004 , decreasing to 1  238  000 in 2010 , mostly affecting 
 those under the age of 5 years . primaquine 
 ( pq , chart 1 ) , an 8-aminoquinoline ( 8-aq ) first 
 synthesized in 1946 , is the only drug 
 approved by the u.s . food and drug administration ( fda ) for the radical 
 cure of relapsing malaria . it is also 
 being used as a prophylactic against all major forms of human malaria and was found to have significant sporontocidal 
 and gametocytocidal activity . the greatest 
 life - threatening problem for pq and other 8-aq antimalarials is the 
 aggravated methemoglobinemia ( in which 
 hemoglobin is oxidized to form methemoglobin , an fe protein 
 that is unable to carry oxygen ) and hemolysis that occurs in patients deficient in glucose-6-phosphate dehydrogenase 
 ( g6pd ) . the hemolysis of 8-aq analogues can be induced by their hydroxylated 
 metabolites , which are formed by the action of cytochrome p450 isoforms . among a number of metabolites of pq shown in chart 1 , it has been found that 5-hydroxyprimaquine ( 5-oh - pq ) , 5,6-dihydroxyprimaquine 
 ( 5,6 - 2oh - pq ) and 6-methoxy-8-(n - hydroxy)aminoquinoline 
 [ 6-meo-8-(n - oh)-aq ] are more toxic than the parent 
 pq molecule . in contrast , carboxyprimaquine 
 ( cpq ) and 6-methoxy-8-aminoquinoline ( 6-meo-8-aq ) are less toxic because 
 they generate less methemoglobin than pq . 8-[(4-amino-1-methylbutyl)amino]-5-[3,4-dichlorophenoxy]-4-methylquinoline 
 ( npc1161 , chart 1 ) , an 8-aq analogue , is a 
 developmental candidate with better antimalarial efficacy and lower 
 toxicity than pq . it is also significantly active 
 against leishmania and pneumocystis carinii infections . in 
 addition , its (  ) enantiomer ( npc1161b ) showed more markedly 
 reduced general toxicity in mice and reduced hematotoxicity in the 
 dog model of methemoglobinemia than its ( + ) enantiomer ( npc1161a ) . however , the properties of the hydroxylated npc1161 metabolites , 
 particularly their potential to cause methemoglobinemia , is not clear . 
 in this work , we evaluated the potential toxicity as well as the feasibility 
 and regioselectivity of formation of hydroxylated metabolites of npc1161 
 using computationally intensive approaches . the properties we studied 
 in this work are identical for the two enantiomers , so the calculations 
 we report were performed on just one enantiomer , npc1161a . the mmff94s 
 force field in schrdinger s macromodel was employed for the conformational search of 
 neutral npc1161a with an energy window of 130 kj / mol . the 25 conformers 
 with the lowest energies were then subjected to geometry optimization 
 and single - point energy calculation at the b3lyp/6 - 31 g * * and 
 b3lyp/6 - 311++g**//b3lyp/6 - 31 g * * levels in the gas phase and at the 
 b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * level in water . twenty conformers 
 were found after all of the b3lyp/6 - 31 g * * geometry optimizations , 
 and their conformational distributions were calculated using the total 
 energies obtained at the above levels , including zero - point vibrational 
 energies ( zpves ) calculated at the b3lyp/6 - 31 g * * level . the ips of 
 the predominant conformers of npc1161a were computed at the above 
 levels . the ips of metabolites singly hydroxylated at all possible 
 positions were also calculated using the above protocols , and the 
 most predominant conformer of neutral npc1161a was selected as a model . potential energy surfaces ( pess ) were scanned at the am1 ( semiempirical 
 molecular model ) or b3lyp/6 - 31 g * * levels to locate the energetic minima 
 of the metabolites of npc1161a hydroxylated at the c2 , c7 , and n1 
 positions . homolytic bond dissociation energies ( hbdes ) of the selected 
 npc1161a conformer were calculated at the b3lyp/6 - 31 g * * and b3lyp/6 - 311++g**//b3lyp/6 - 31 g * * 
 levels in the gas phase . all computations at the semiempirical and 
 quantum mechanical levels were performed using the gaussian 09 software 
 package . if not mentioned elsewhere , 
 the results from the solvation calculations at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level are presented below and discussed , whereas those at all other 
 levels are provided in the supporting information . in our previous studies , we proposed that the ability to lose an electron 
 is correlated in part to the hemotoxicity of antimalarial 8-aq drugs . 
 in this work , we calculated the ionization potentials ( ips ) of antimalarial 
 candidate npc1161a to evaluate its possible hemotoxicity . to locate 
 the most favorable conformers to be used to calculate the ips for 
 this very flexible compound , a monte carlo random conformational search 
 at the mmff94s level of theory was performed , using schrdinger s 
 macromodel software package , yielding 
 453 conformers within an energy window of 130 kj / mol ( figure s1 , supporting information ) . the 25 conformers with 
 the lowest energies within an energy cutoff of 8.4 kj / mol ( 2 
 kcal / mol ) were submitted to full hybrid density functional theory 
 ( dft ) geometry optimization , leading to 20 conformers located at the 
 b3lyp/6 - 31 g * * level in the gas phase ( figure 1 ) . harmonic vibrational frequencies were calculated at the same level 
 to confirm that they were minima on the potential energy surface , 
 and single - point energies were computed at the b3lyp/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in the gas phase and at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . the rotation of the phenolic group about c5o 
 ( c1 ) is the major geometric difference between conformers 110 and 1120 , in which the dihedral angles of c6c5o  c1 
 are about 77 and 77 , respectively . this rotation 
 is the only significant geometric difference between the conformers 01 and 11 , 02 and 20 , 03 and 12 , 05 and 14 , and 10 and 19 . among the above 
 conformers , weak chn and/or nhn 
 hydrogen bonds were found in conformers 03 , 05 , 10 , 12 , 14 , and 19 . the distances of n6h(c7 ) and n6h(c2 ) 
 in conformers 03 and 12 are 2.67 and 2.54 
  , respectively ; those of n6h(n1 ) , 
 n6h(c3 ) , and n1h(c5 ) 
 in 05 and 14 are 2.23 , 2.65 , and 2.62  , 
 respectively ; and those of n6h(c2 ) 
 in 10 and 19 are each 2.42  . conformational 
 analysis ( table 1 ) indicated that these conformers 
 with hydrogen bonds are negligible in water , as evidenced by the fact 
 that they accounted for only 4% at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . the conformers with the most extended side chain , 
 specifically 02 and 20 , were found to be 
 the major conformers , accounting in total for 45% at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . optimized geometries of conformers of neutral npc1161a 
 at the b3lyp/6 - 31 g * * 
 level in the gas phase . relative energy ( kj / mol ) , including 
 zero - point energy at the b3lyp/6 - 31 g * * level in the gas phase . the geometries of 
 all conformers found in water with a boltzmann 
 contribution greater than 2% were employed as the starting points 
 to locate the ionized conformers of npc1161a at the b3lyp/6 - 31 g * * 
 level in the gas phase . single - point energies and the conformational 
 distribution were computed using the same protocols described above . the ionized conformers 02 and 20 were again 
 found to be the major ones , with contributions of 26% and 28% , respectively , 
 in water at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * level ( table 1 ) . the ionization potentials were calculated at 
 the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * level in water and are 
 listed in table 2 . the greatest difference 
 in ip values was found to be only 3.14 kj / mol at the b3lyp - scrf(pcm , 
 h2o)/6 - 311++g**//b3lyp/6 - 31 g * * levels , indicating that the ips in 
 this case are relatively insensitive to conformation . thus , if not 
 mentioned elsewhere , hereafter we employed conformer 02 ( npc1161a02 ) as a conformer model for the further computations because 
 it was found to be the most predominant one in water . zero - point energy at the b3lyp/6 - 31 g * * 
 level in the gas phase included . because hydroxylated metabolites of 8-aq have been postulated to 
 be responsible for the 8-aq hemotoxicity , ionization potentials of hydroxylated metabolites of the conformer 
 npc1161a02 were calculated at b3lyp - srcf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . potential energy surfaces were scanned at the am1 
 level in the gas phase , and the predominant conformers of metabolites 
 hydroxylated at the c2 , c7 , and n1 positions ( figures s2s4 , supporting information ) were used for the geometry 
 optimization . figure 2 depicts the optimized 
 geometries of metabolites of neutral conformer npc1161a02 hydroxylated 
 at all possible positions at the b3lyp/6 - 31 g * * level in the gas phase . their relative energies ( table 3 ) and ionization 
 potentials ( table 4 ) were computed at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . the metabolite hydroxylated at the c2 position was 
 found to be the most stable , at least 20 kj / mol more stable than any 
 other , whereas those hydroxylated at 2me , 4me , n6 , 
 and n1 were found to be the least stable , for both the neutral 
 and ionized states . the calculated ips indicated that the introduction 
 of a hydroxyl group into npc1161a02 would have little effect on its 
 ability to lose an electron , except if the hydroxylation were at the 
 n1 position . compared to its parent compound , the hydroxylation 
 of npc1161a02 at the n1 position would dramatically lower 
 its energy barrier to donating an electron by 44.37 kj / mol , implying 
 that this hydroxylated metabolite should contribute the most to the 
 potential hemotoxicity of npc1161a . interestingly , the hydrogen atom 
 in the hydroxyl group attached at n1 in the neutral hydroxylated 
 metabolite ( oh@n1 in figure 2 ) automatically 
 shifted to the n1 position while being converted into a radical cation , 
 as shown in the potential energy surface scanned at the b3lyp/6 - 31 g * * 
 level in the gas phase ( figure 3 ) . the n1o 
 bond length decreased from 1.34  in the neutral form to 1.29 
  in the radical , and the ( n1)o  h bond dissociated 
 ( the distance increased from 1.09  in the neutral form to 1.61 
  in the radical ) , whereas the hydrogen shifted to form the ( n1o)h  n1 
 bond ( the distance was decreased from 1.40  in the neutral form 
 to 1.05  in the radical ) . the hemotoxic contribution from the 
 metabolites hydroxylated at the c2 and c7 positions was predicted 
 not to be significant . the hydroxylated metabolites at all other possible 
 positions were found to have calculated ips indistinguishable from 
 those of their parent . optimized geometries of the metabolites of neutral conformer 
 npc1161a02 
 hydroxylated at all possible positions at the b3lyp/6 - 31 g * * level 
 in the gas phase . potential energy surface 
 of the hydrogen shift in the metabolite 
 of npc1161a02 hydroxylated at the n1 position while being 
 converted to a radical cation ( scanned in the gas phase at the b3lyp/6 - 31 g * * 
 level ; key distances are shown in ngstroms ) . including zero - point energy at 
 the b3lyp/6 - 31 g * * level in the gas phase . including zero - point energy at the 
 b3lyp/6 - 31 g * * level in the gas phase . cytochrome p450 isoforms 
 are the major enzymes involved in drug metabolism , accounting for 
 about 75% of the total number of different metabolic reactions , and have been shown to be responsible for causing 
 oxidative stress and hemotoxicity of 8-aqs , specifically by catalyzing the hydroxylation of 8-aqs . both experimental 
 observations and theoretical calculations indicated that the barrier to h - abstraction , 
 the first and rate - controlling step in the hydroxylation carried out 
 in cyp450 , exhibits a linear correlation with the homolytic c  h 
 bond dissociation energy ( hbde ) , implying the possibility and reliability 
 of predicting the feasibility and regioselectivity of hydroxylation 
 of substrates in the protein environment of p450 . therefore , in this 
 work , we also calculated the hbdes of c  h / n  h bonds 
 at all possible positions in npc1161a to evaluate the feasibility 
 and regioselectivity of forming hydroxylated metabolites in npc1161a , 
 at the b3lyp/6 - 311++g**//b3lyp/6 - 31 g * * level in the gas phase . the conformer npc1161a02 with an extended 
 side chain was selected as a model for the hbde computations . the 
 calculated hbdes are listed in table 5 , from 
 which one can see that the methyl group at c4 , the methoxyl group 
 at c6 , and the positions on the side chain are the easiest to be hydroxylated , 
 specifically in the order of ( c4)me , c2 , c5 , n1 , 
 ( c6)ome , n6 , and c4 at the b3lyp/6 - 311++g**//b3lyp/6 - 31 g * * 
 level . the positions in the quinoline and phenolic rings are predicted 
 as relatively difficult to be hydroxylated . the molecular orbital 
 analysis of neutral conformer npc1161a02 ( figure 4 ) indicated that the electronic density in the highest occupied 
 molecular orbital ( homo ) was localized more at n1 ( and at 
 c5 ) whereas the electronic density in the lowest unoccupied molecular 
 orbital ( lumo ) was localized more at n1 ( and at c4 ) , implying that 
 the n1 atom is more likely than all other atomic centers in 
 the molecule to pair with an electron from the oxygen attached to 
 the iron in the active site of cyp450 . the biochemical interaction / reaction 
 between npc1161 enantiomers and cyp450 isoforms should also be taken 
 into account to interpret the enantiomeric differences in hemotoxicity 
 contributions from the hydroxylation of npc1161 . including 
 zero - point energy at the 
 b3lyp/6 - 31 g * * level in the gas phase . calculated frontier molecular 
 orbitals of neutral conformer npc1161a02 
 at the b3lyp/6 - 31 g * * level in the gas phase . conformational analysis 
 of the very flexible antimalarial candidate 
 npc1161a showed that an extended conformation of the side chain was 
 found in 45% and 54% of the boltzmann - weighted structures for the 
 neutral and ionized conformers , respectively , with conformers 02 and 20 predominating , at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . the metabolite hydroxylated at the n1 position 
 was suggested as contributing the most to the potential hemotoxicity 
 of npc1161a , as evidenced by its lowest ip of 430 kj / mol at 
 the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * level in water . the 
 calculated homolytic bond dissociation energies demonstrated the preference 
 for hydroxylation on the side chain . the results in this work should 
 apply equally to both enantiomers npc1161a and npc1161b , whereas differences 
 in their activities and toxicities could arise from interactions with 
 proteins such as transporters or other metabolizing enzymes . further 
 theoretical investigations of biochemical interactions and reactions 
 between npc1161 enantiomers and cyp450 isoforms are currently in progress 
 to elucidate the source of toxicity differences between enantiomers .
for antimalarial 8-aminoquinoline 
 ( 8-aq ) drugs , the ionization 
 potential ( energy required to remove an electron ) of their putative 
 metabolites has been proposed to be correlated in part to their hemotoxicity 
 potential . 
 npc1161 is a developmental candidate as an 8-aq antimalarial 
 drug . in this work 
 , the ionization potentials ( ips ) of the s - npc1161 ( npc1161a ) hydroxylated derivatives , which are 
 possible metabolites derived from action of endogenous cytochrome 
 p450 ( cyp450 ) enzymes , were calculated at the b3lyp - scrf(pcm)/6 - 311++g**//b3lyp/6 - 31 g * * 
 level in water . 
 the derivative hydroxylated at n1 ( 8-amino ) 
 was found to have the smallest ip of 430 kj / mol , predicting 
 that it would be the most hemotoxic . 
 the calculated ips of the derivatives 
 hydroxylated at the c2 and c7 positions were 475 and 478 
 kj / mol , respectively , whereas the calculated ips of those hydroxylated 
 at all other possible positions were between 480 and 490 kj / mol . 
 the 
 homolytic bond dissociation energies ( hbdes ) of all c  h / n  h 
 bonds in npc1161a were also calculated . 
 the smaller hbdes of the c 
 h / n 
 h 
 bonds on the 8-amino side chain suggest that these positions are more 
 easily hydroxylated compared to other sites . 
 molecular orbital analysis 
 implies that the n1 position should be the most reactive center 
 when npc1161 approaches the heme in cyp450 .