Crystallographic and Electroanalytical Analyses of Fexinidazole and Its Major Metabolites

Abstract

Fexinidazole, a drug active against trypanosomiasis and leishmaniasis, is a rare example of a nitroaromatic compound approved under the contemporary drug discovery framework. In an earlier study, we showed that the nitro group is absolutely required for antileishmanial activity. The current study employed X-ray crystallography to unveil the structural intricacies of fexinidazole and its principal metabolites, as well as electroanalytical analyses to characterize the reduction properties of the aromatic nitro group. Fexinidazole showcases a predominantly planar geometry with two distinct conformers. While most metrical parameters were conserved between fexinidazole and its metabolites, differences in the methyl ether bridge and S-methyl tail indicated distinctive preferences in molecular arrangement: conformer I of fexinidazole closely resembles the sulfone metabolite, while conformer II aligns with the sulfoxide metabolite. On the other hand, electroanalytical analysis of fexinidazole revealed a pH-dependent, two-step nitro group reduction mechanism, involving an initial concerted transfer of an electron and a proton, followed by the uptake of three electrons and three protons to likely form a hydroxylamine species. These findings characterize the molecular architecture and reduction mechanism of fexinidazole, providing valuable insights into its structural features and activation mechanism required for anti-infective activity.

Introduction

Fexinidazole (Fex) is a 2-substituted 5-nitroimidazole initially approved by the European Medicine Agency for the treatment of Human African trypanosomiasis (HAT) in 2019. It is the first oral treatment for both the hemo-lymphatic and meningo-encephalitic stages of HAT. Fex also represents a new prototype for leishmaniasis treatment, with in vivo activity comparable to miltefosine.

It has been reported that Fex is rapidly metabolized into two major active metabolites: fexinidazole sulfoxide (Fex–SO) and fexinidazole sulfone (Fex–SO₂). Although only oxidation distinguishes Fex from its metabolites, substantial variations in their activities and toxicities have been reported. Notably, while its metabolites exhibit activity against intracellular amastigotes, Fex itself lacks such efficacy. In addition, Fex–SO₂ is capable of inhibiting the hERG (a human Ether-à-go-go-Related Gene) potassium ion channel current at therapeutic concentrations, a factor implicated for the contraindication of Fex for patients with cardiac rhythm problem. , Conversely, Fex–SO has demonstrated no inhibition of hERG activity up to a 100 μM concentration. In this article, we present for the first time the molecular structures of Fex and its major metabolites, determined through single crystal X-ray diffraction, and discuss the notable differences in their geometrical conformations.

The mechanism responsible for the antiparasitic activity of FEX and metabolites is the reduction of the nitro group by type I nitroreductase (NTR1), which usually carry out stepwise reductions to generate reactive nitroso and hydroxylamine intermediates that lethally damage the parasite’s DNA. , However, the downstream intermediates following Fex reduction remain unidentified. Recognizing the essential role of nitro group reduction in the efficacy of Fex and its metabolites, we performed electroanalytical analyses to precisely define reduction potentials, redox intermediates and electron transfer stoichiometry. These mechanistic insights will also guide NTR1 enzymology studies in confirming the intermediates and reductive activation pathway under physiological conditions.

Results and Discussion

Fexinidazole crystallized with two independent molecules (Figure ) in the asymmetric unit, each representing a distinct conformer. The bond lengths, bond angles, and torsion angles of both Fex conformers, along with its metabolites, align well with typical values in related structures. Their metrical parameters are generally similar, albeit some interesting differences in their molecular structures and crystal packing patterns can be observed.

1.

Molecular structures of conformer I (top) and conformer II (bottom) of fexinidazole. Ellipsoids are shown with 50% probability. The atom numbers used follow patterns common in crystallography which do not necessarily match the numbering in IUPAC names.

Conformer I of Fexinidazole

Bond Lengths

The bond lengths in the imidazole scaffold of conformer I (Fex I) range from 1.33 to 1.38 Å (Table ), closely matching those of unsubstituted imidazole, and typical values for planar and nearly planar benzenoids (1.33–1.48 Å). The C1–C2 bond is shortened, consistent with a C–C bond length adjacent to an aromatic nitro group. Additionally, the nitro nitrogen atom is not pyramidalized, maintaining coplanarity of the nitro group, N ¹-methyl substituent and imidazole ring.

1. Selected Metrical Parameters for the Two Conformers of Fexinidazole and Two Metabolites .

Characteristics	Conformer I	Conformer II	Fex–SO	Fex–SO2
Bond length (Å)
S1–O4	–	–	1.495	1.444
S1–O5	–	–	–	1.444
O1–C5	1.382	1.375	1.368	1.365
O1–C4	1.436	1.439	1.438	1.442
O2–N3	1.229	1.226	1.220	1.228
O3–N3	1.237	1.233	1.235	1.234
N1–C3	1.360	1.356	1.345	1.354
N1–C1	1.378	1.367	1.377	1.380
N2–C3	1.329	1.336	1.338	1.332
N2–C2	1.354	1.359	1.356	1.355
N3–C1	1.422	1.420	1.424	1.420
C1–C2	1.369	1.364	1.365	1.373
Bond angle (°)
O4–S1–O5	–	–	–	118.0
O4–S1–C8	–	–	106.50	108.4
O4–S1–C11	–	–	105.1	109.0
O5–S1–C11	–	–	–	108.1
C8–S1–C11	104.04	103.69	98.6	105.4
C2–C1–N1	107.66	107.98	107.9	107.5
C2–C1–N3	127.63	127.73	127.3	127.9
N1–C1–N3	124.70	124.29	124.8	124.6
O1–C5–C10	116.05	124.46	124.2	115.6
O1–C5–C6	123.76	115.46	115.2	123.8
C7–C8–S1	116.06	116.02	119.3	118.4
C9–C8–S1	125.23	125.20	119.9	120.6
Torsion angles (°)
C3–N1–C1–N3	–179.15	179.41	177.0	–179.66
C12–N1–C3–N2	–177.80	–178.51	179.7	178.98
C1–N1–C3–C4	178.32	177.35	–177.9	–178.93
C5–O1–C4–C3	177.75	–169.32	–173.5	–176.26
N2–C3–C4–O1	–97.08	105.56	102.9	104.8
N1–C3–C4–O1	85.03	–71.74	–78.9	–76.4
C4–O1–C5–C10	–179.60	–9.2	1.0	–176.0
C4–O1–C5–C6	1.0	170.84	–180.0	5.3
O4–S1–C8–C7	–	-	145.3	–157.45
O4–S1–C8–C9	–	–	–30.2	20.98
O5–S1–C8–C9	–	–	–	149.39
C11–S1–C8–C7	166.33	–178.97	–106.1	85.99
C11–S1–C8–C9	–13.62	0.52	78.5	–95.58

^aComplete data are provided in Table S2.

^bFor the Fex conformers, identical atoms are labeled correspondingly with the addition of 20 to the numbers for the second conformer; i.e., C1 correlates with C21, C11 correlates with C31 etc.

A search of the CSD (Cambridge Structural Database) for closely related structures using Mogul (N.B.: very clear outlier values were not considered for most of the following analyses) reveals that the C1–N3 bond length with 1.422 Å almost exactly matches the mean value of 1.421 Å as calculated from 204 deposited structures with nitro substituents on a pyrrole or an imidazole ring. The median value in the known structures is somehow shorter with 1.418 Å while the bond lengths range from 1.390 to 1.486 Å.

Differences in the N–O bond lengths within a nitro group, which range from 1.131 to 1.334 Å in almost 9k related structural motifs in the CSD (mean: 1.227 Å; median: 1.226 Å), are common and attributed to intermolecular interactions involving this group. The N3–O2 and N3–O3 bonds, which are largely involved in the hydrogen bonding network detailed below, have lengths of 1.229 and 1.237 Å, respectively.

Bond lengths related to C4–O1 (1.436 Å) in nearly 5k structures in the CSD range from 1.264 to 1.580 Å (mean = median: 1.424 Å), while those related to C5–O1 (1.382 Å) range from 1.282 to 1.471 Å (mean: 1.372 Å; median: 1.371 Å). The shorter distance of O1 to the phenyl ring compared to the methylene group is likely due to partial conjugation between its lone pair and the aromatic ring and appears to be common. This feature might explain the preferential cleavage of the weaker C4–O1 during Pd/C catalyzed hydrogenation, generating a phenolic intermediate prone to oxidation into an elusive quinone intermediate, potentially contributing to Fex’s liver toxicity.

Bond Angles

The C2–C1–N1 angle in Fex I is 107.66°. An angle with the nitro bearing carbon atom in the center was previously discussed as reflecting a stronger inductive effect of this group, an increased dipole moment and better stability when wider than usual. , In 28 closely related structures in the CSD this angle ranges from 106.62 to 109.07° (mean: 107.74°, median: 107.72°). The angle in Fex I is hence slightly more acute than the average while certainly not indicative of drastically reduced stability.

Torsion Angles

The imidazole ring and its substituents – the nitro group, N1-methyl and C3-substituents – are coplanar, as evident in torsion angles of 179.15° (C3–N1–C1–N3), 177.8° (C12–N1–C3–N2) and 178.3° (C1–N1–C3–C4), respectively. The methylene ether bridge is also nearly coplanar with the imidazole and phenyl rings, as indicated by dihedral angles of 177.75° (C5_phenyl–O1–C4–C3_imi) and 1° (C4–O1–C5_phenyl–C6_phenyl). In contrast, the imidazole and phenyl rings are nearly perpendicular to each other, with an interplanar angle of 83.58°.

The S1-methyl group is slightly out of the phenyl ring plane, with a C11–S1–C8–C7 angle of 166.3° that is more acute than the 0° or 180° expected for coplanarity. This offset suggests some rotational flexibility around the C8_phenyl–S1 bond, and may increase its accessibility for reactions. Consistently, in vitro and in vivo assays demonstrated a rapid oxidation of Fex to Fex–SO and Fex–SO₂ metabolites.

Alternative Conformer of Fexinidazole

A second conformer, conformer II (Fex II), is cocrystallized with Fex I in the same crystal structure. Their bond lengths are rather similar, differing by a maximum of 0.011 Å (for C1–N1/C21–N21) or much less (Table ). However, in Fex II there is the single metrical parameter present (considering all four molecular structures discussed here) that comprises a significant deviation from the commonly observed data in the CSD. The C21–N21 bond length between the two substituted pivot atoms in the imidazole ring is notably short with 1.367 Å (1.378 Å in Fex I). It is in fact the second shortest bond observed, only surpassed by the one in sodium 4-(2-((1-methyl-5-nitro-2-imidazolyl)-thio)­ethoxy)-benzoate with 1.364 Å. In the 31 related structural motifs in the CSD this value ranges from 1.364 to 1.393 Å (mean: 1.379 Å, median: 1.378 Å).

An overlay of Fex I and Fex II with RMSD of 0.568 (Figure ) confirms their overall similarity but with three interesting variations. The first difference lies in the S-CH₃ orientation: In conformer I, it extends into the opposite direction relative to the methoxy bridge arrangement (anti), whereas in conformer II, it points into the same direction as the bridge atoms (syn). Second, the torsion angles for the S-CH₃ group are 166.33° for Fex I, and 178.97° for Fex II, rendering it essentially coplanar with the phenyl ring in the latter. Third, a slight slide of the phenyl ring is observed, which is particularly evident in C5_phenyl–O1–C4–C3_imi angles of 177.75° (Fex I) and −169.32° (Fex II), subtly affecting the methoxy bridge geometry.

2.

Alignment of conformer I (green) and conformer II (blue) of fexinidazole.

Intermolecular Hydrogen Bonding Interactions

A strong intermolecular nonclassical hydrogen bonding network exists between the Fex conformers (Figure ). The most prominent interaction involves the N1 bound C12 methyl group of Fex I, which donates a hydrogen bond to a nitro oxygen of Fex II (Table , C12–H12A···O23; symmetry operator: −x, y + 1/2, −z + 3/2; H···A 2.48 Å) and vice versa (C32–H32A···O3; symmetry operator: −x + 1, −y + 1, −z + 2; H···A 2.49 Å). The nitro oxygen atoms form the core of this network, participating in six of the eight hydrogen bonds (Table ). Hydrogen bonds involving the sulfur atom, and between the phenyl C–H and nitro oxygen atoms provide additional stabilization.

3.

Hydrogen bonding network between conformers of fexinidazole in the crystal packing viewed along the crystallographic b axis.

2. Metrical Parameters of Hydrogen Bonding Interactions in the Two Fex Conformers and Its Two Metabolites .

D–H···A	d(D–H)	d(H···A)	d(D···A)	<(DHA)
Fex conformers
C12–H12A···O23#1	0.98	2.48	3.403	156.3
C12–H12B···S1#21	0.98	2.99	3.803	141.1
C24–H24B···O3#3	0.99	2.55	3.462	152.5
C26–H26···S1#4	0.95	2.90	3.794	158.1
C31–H31A···O23#4	0.98	2.59	3.366	136.1
C31–H31B···O22 #5	0.98	2.56	3.371	139.9
C32–H32A··· O3 #6	0.98	2.49	3.358	148.1
Fex–SO
C2–H2···O3#7	0.95	2.41	3.319	160.1
C6–H6···O4#8	0.95	2.46	3.409	175.0
C11–H11B···N2#9	0.98	2.67	3.315	124.0
C12–H12A···O2#10	0.98	2.42	3.099	125.9
C12–H12B···N2#11	0.98	2.66	3.493	142.6
C12–H12C···O4#12	0.98	2.26	3.155	151.2
Fex–SO2
C6–H6···S1#13	0.95	2.97	3.761	141.2
C6–H6···O5#13	0.95	2.44	3.381	168.9
C11–H11A···N2#14	0.98	2.48	3.306	142.4
C11–H11B···O4#15	0.98	2.40	3.288	151.1
C11–H11C···O2#16	0.98	2.58	3.448	147.5
C12–H12A···O3#3	0.98	2.65	3.216	116.8
C12–H12B··· O3#17	0.98	2.66	3.258	120.0
C12–H12B···N2#5	0.98	2.67	3.541	147.9
C12–H12C···O5#18	0.98	2.27	3.183	154.4

^aSymmetry transformations used to generate equivalent atoms: #1: −x, y + 1/2, −z + 3/2; #2: −x + 1, y + 1/2, −z + 3/2; #3: −x + 1, y – 1/2, −z + 3/2; #4: x + 1, −y + 1/2, z + 1/2; #5: x + 1, y, z; #6: −x + 1, −y + 1, −z + 2; #7: −x + 2, y + 1/2, −z + 2; #8: −x, y + 1/2, −z + 1; #9: −x + 1, y – 1/2, −z + 1; #10: −x + 1, y + 1/2, −z + 2; #11: x, y – 1, z; #12: −x, y – 1/2, −z + 1; #13: x – 1/2, −y + 1/2, −z + 1; #14: x + 1/2, −y + 3/2, −z + 1; #15: x – 1, y, z; #16: −x + 3/2, −y + 1, z −1/2; #17: −x + 2, y – 1/2, −z + 3/2; #18: x + 1/2, −y + 1/2, −z + 1.

Fexinidazole Sulfoxide

The bond lengths in Fex–SO are comparable to those in Fex (Table ). A notable difference is the S1–C8 distance with 1.795 Å. This is a 0.025 Å increase compared to Fex, yet a perfect match with the median value of 1.795 Å reported for closely related moieties in the CSD (mean: 1.792 Å) across 41 deposited structures. This elongation is fully consistent with sulfoxide formation (Figure , top). The sulfoxide S1–O4 bond of 1.495 Å is also rather usual (mean = median: 1.496 Å; range: 1.464–1.590 Å) compared to 107 analogous moieties in the database.

4.

Molecular structures of Fex–SO (top) and Fex–SO₂ (below). Ellipsoids are shown at the 50% probability level.

Although most bond angles are similar, one interesting observation was made when comparing Fex and Fex–SO: While the C7–C8–S1 and C9–C8–S1 angles in Fex I and Fex II are relatively distinct, they are almost the same in Fex–SO. The reason is that in Fex (I and II) the methyl group is more or less in-plane with the aromatic ring and the angle on the side of the methyl group is wider (roughly 125°) while the other is more acute (roughly 116°); in Fex–SO the methyl resides way out-of-plane, removing any steric strain and allowing the angles to relax to be more or less the same (roughly 119.5°).

The configurations of the bridge, phenyl ring and sulfur bound methyl group in Fex II are essentially retained in Fex–SO. However, the sulfoxide oxygen atom moves toward the plane of the aromatic ring without really reaching it (evident in torsion angles for O4–S1–C8–C9 of 145.3° and for O4–S1–C8–C7 of −30.2°), while, as mentioned above, the methyl group points decidedly away from the phenyl ring plane (torsion angles: C11–S1–C8–C9 −106.1° and C11–S1–C8–C7 78.5°) (Figure ). The sulfoxide oxygen and methoxy bridge atoms align in the same direction (syn) of the phenyl ring. The antiperiplanar C11–S1–C8–C7 configuration in Fex I which is syn-periplanar in Fex II changes to syn-clinal in Fex–SO, while the C11–S1–C8–C9 torsion becomes anticlinal. Fex–SO, hence, more closely resemblances Fex II of its parent compound, while retaining general similarity to Fex I. For a better visualization, Fex–SO was overlaid with each conformer of Fex (Figure ). As anticipated, Fex–SO aligns better with conformer II (RMSD 0.238) than with conformer I (RMSD 0.568).

5.

Overlay of Fex–SO with fexinidazole conformer II (A) and conformer I (B), with RMSD of 0.238 and 0.568 respectively. The overlay of the Fex–SO₂ on fexinidazole conformer I (C) and conformer II (D) has RMSD of 0.165 and 0.593 respectively. Color representations: blue for conformer I, green for conformer II, yellow for Fex–SO and orange for Fex–SO₂.

Three moieties not directly involved in the Fex hydrogen bonding network contribute to stabilizing the packing pattern of Fex–SO (Figure S1): 1) the imidazole N2 atom accepts hydrogen bonds from the S–C(11)­H₃ and N1–C(12)­H₃ groups; 2) the sulfoxide oxygen (O4) atom engages in two interactions, including a relatively strong nonclassical hydrogen bond (H···A 2.26 Å) with the N1–C(12)­H₃ group (Table ); 3) a hydrogen atom at imidazole C2 (interacting with O3 of the nitro group) participates in the hydrogen bonding network only in Fex–SO.

Fexinidazole Sulfone

Similar to Fex–SO, most bond lengths, bond angles and torsion angles in Fex–SO₂ closely match those of the two Fex conformers (Table ). The sulfone S1–O4 and S1–O5 bonds are essentially identical in length at 1.444 Å (Figure , bottom). In related structures in the CSD these values do not differ by more than 0.05 Å, while 990 related structures are very similar as they are here. The mean value for the S–O distances is 1.436 Å, with a median value of 1.437 Å and a range of 1.354 to 1.485 Å. The respective bond lengths observed here are therefore somehow longer than average.

The S1–O4 and S1–O5 bonds are shorter than the sulfoxide S1–O4 bond (Table ). This points to a more significant double bond character between sulfur and oxygen in Fex–SO₂ than in Fex–SO. The S–O bond in sulfoxides is considered to have more partial ionic character, with a dipole moment directed toward the oxygen. The precise nature of S–O bonding in sulfoxides, considered to be in-between single and double bonds, is still under investigation and subject to active debate within the scientific community.

The S1–O–C8 bond (1.765 Å) connecting the sulfone with the phenyl ring is more similar in Fex than in Fex–SO. The S–C11 bond to the methyl group (1.763 Å), which is comparable in Fex and Fex–SO, is relatively shorter in Fex–SO₂, indicating a stronger bond. Both bonds are well within the typically observed ranges in the deposited structures in the CSD. These are 1.742 to 1.811 Å for the bond to the aromatic ring (mean: 1.766 Å, median: 1.767 Å, 374 structures) and 1.675 to 1.790 Å to the methyl group (mean: 1.752 Å, median: 1.753 Å, 491 structures).

Typical sulfones adopt a distorted tetrahedral geometry, featuring a larger O–S–O angle and a smaller C–S–C angle. The deviation from the 109.5° angle in the perfect tetrahedron is primarily attributed to the repulsion between the oxygen atoms’ lone pairs. The sulfone group in Fex–SO₂ is no exception in this regard. The O4–S1–O5 bond angle is wide with 118.0°, whereas the C8–S1–C11 angle of 105.4° is the most acute in this moiety, while slightly wider than the respective mean value in the CSD data of 104.5°.

The bond angles O1–C5–C6 and O1–C5–C10 along with the corresponding torsion angles C4–O1–C5–C10 and C4–O1–C5–C6, indicate that Fex–SO₂ more closely resembles Fex I than Fex II, in contrast to Fex–SO, for which the opposite is true. For example, the difference in the torsion angles above between Fex I and Fex–SO₂ are 3.9° and 4.3°, respectively, while those between Fex–SO₂ and Fex II are larger at 166.8° (translates to −13.2°) and 165.4° (translates to −14.6°), respectively (Table ). Furthermore, the overlay of Fex–SO₂ and Fex conformers revealed a remarkably close alignment with Fex I with an RMSD of only 0.165 (Figure ). The similarities between Fex II and Fex–SO, as well as between Fex I and Fex–SO₂ could have important implications for differences in the biological properties between Fex and its major metabolites. ,

Sulfonation, similar to sulfoxidation, induces changes in the torsion angle between the methyl-sulfonyl tail and the phenyl ring compared to Fex. In Fex–SO₂, the dihedral angles C11–S1–C8–C7 and C11–S1–C8–C9 adopt synclinal and anticlinal conformations, respectively. The oxygen O5 in Fex–SO₂ resides in essentially the same position as O4 in Fex–SO. O5 in Fex–SO₂ is synclinal to C7 and anticlinal to C9 and for O4 it is the opposite.

Sulfone containing compounds typically display strong hydrogen bonding networks. Consistent with this, Fex–SO₂ is stabilized by intermolecular hydrogen bonds (Figure S2), with the strongest (H···A 2.27 Å) occurring between the N1-methyl group and a sulfonyl oxygen atom (Table ). The nitro and the sulfonyl oxygen atoms are the major acceptors in this hydrogen bond network. The imidazole nitrogen atom N2 in Fex–SO₂ also acts as a hydrogen bond acceptor, a role it shares in Fex–SO but not in the parent compound.

Electroanalytical Analysis

The mechanism of action of Fex is believed to be associated with a disruption of the replication of protozoal DNA, but how its chemistry impairs the DNA synthesis remains unclear. The key transition for its activity is the in vivo reductive activation of the nitro group by Leishmania or Trypanosoma nitroreductase. In this article, we present the differential pulse voltammetry (DPV) and cyclic voltammetry (CV) analysis to provide insight into the reduction processes of Fex and its metabolites.

Each DPV of Fex reveals a strong cathodic signal at negative potentials (E _pc,2), most likely assignable to the nitro group (Figure , left). Each cathodic peak has an anodic counterpart, which is substantially smaller in its maximal Faradaic current and integral. This event indicates that the reduction is partially quasi-reversible, a case where electron transfer is not instantaneous as in a reversible process and not slow enough that the reaction becomes fully irreversible.

6.

pH-dependent DPV of Fex (left) and CV at 50 mV/s (right) in acidic (3.3 mg), neutral (3.1 mg) and alkaline (3.0 mg) in buffer (5 mL)/DMF (5 mL) medium.

It can be assumed that the reduction process involves an initial electron transfer step followed by a nonelectrochemical reaction (EC _n -mechanism), producing species with an oxidation potential outside of the investigated range. The reduction was observed to be pH-dependent at potentials −0.54 V, −0.65 V and −0.71 V (pH = 3.6, 7.0 and 10.2, respectively), reflecting the influence of the protonation status on the reduction process.

The CV analysis of Fex provided additional insights into the reduction process (Figure , right). It is well established that CV measurements can reveal transient species, including less stable and reversible intermediates, which are often not observed in DPV voltammograms. Similar to those in the DPV, cathodic pH-dependent signals occur in the range of −0.75 V to −0.5 V (E _pc,2). At alkaline conditions, E _pc,2 is less pronounced compared to lower pH values, displaying a slight split with a small shoulder. This feature can be attributed to pH-dependent protonation kinetics. Under acidic conditions at pH 3.6, the high proton concentration enables rapid protonation, thus promoting efficient electron transfer and producing sharp signals. The lower proton concentration at pH = 10.2 may lead to the accumulation of intermediates, such as nitroso species, which can give rise to multiple peak currents. For example, a shoulder-like peak is observed at −0.94 V at pH 10.2. Additionally, the sluggish protonation kinetics under basic conditions contribute to peak broadening.

Aside from E _pc,2, a presignal E _pc,1 is present. This signal also depends on the pH, which is smaller in the observed peak current, but becomes more pronounced at higher scan rates (Figure S3). Therefore, a proposed mechanism for E _pc,1 must include a first step in which a proton and an electron are jointly transferred to Fex (proton-coupled electron transfer or PCET). The second cathodic wave (E _pc,2) then likely facilitates the transfer of the remaining three protons and three electrons, finally leading to a hydroxylamine substituent (Scheme ) as previously reported for related structures. ,

1. Proposed Mechanism for the PCET to the Nitro Group of Fexinidazole.

This is in stark contrast to the results observed for the structurally related antiprotozoal drug metronidazole (MTZ) where such a pH-shift was not observed. Metronidazole is described to be reduced in two main steps: a one electron transfer to MTZ is followed by a concerted three electron–four proton reduction to eventually form a hydroxylamine group.

Electrochemically reversible reoxidation of Fex’s hydroxylamine group is hardly observed in the CV. One broad feature (E _pa,1) occurs in dependence of the applied pH at a potential in between −0.2 and 0.7 V. In acidic conditions it is split into two separate signals, indicating again a two-step mechanism. However, at the current state no reliable reoxidation mechanism can be proposed here.

Fex–SO and Fex–SO₂ essentially provide the same signal set associated with the nitro group as described and discussed for Fex (Figures and S3). One main difference is that Fex shows an anodic feature (E _pa,3) at 1.04 V, while Fex–SO and Fex–SO₂ have no signal in this potential range. As Fex does have a thioether function, E _pa,3 is most likely attributed to its electrochemical oxidation and a subsequent reaction with water eventually leading to a sulfoxide (Fex–SO) or a sulfone (Fex–SO₂). No corresponding reduction peak can be observed in the CV, not even at very high scan rates (Figure S4) or low pH values. Thus, once formed, the sulfoxide or sulfone cannot be rereduced within the observed potential range. Accordingly, the already oxidized metabolites Fex–SO and Fex–SO₂ do not show any cathodic signal here.

7.

CV at 50 mV/s of Fex–SO (left) and Fex–SO₂ (right) in acidic (2.7 mg), neutral (3.0 mg) and alkaline (3.0 mg) buffer (5 mL)/DMF (5 mL) solvent.

This analysis established the fundamental redox behavior of Fex and its metabolites under controlled electrochemical conditions. Similarity is assumed to the biological redox mechanism due to their shared principles, as supported by successful electroanalytical simulations of phase I and phase II redox metabolites of various clinical agents. However, it is important to recognize that the observed reduction mechanisms may not fully reflect the NTR1 bioactivation process within protozoan parasites. This may be due to the complex cellular environment influencing redox mechanisms and intermediate stability, combined with the redox enzymes’ concerted site-specific activity, precise catalytic rates, and resistance to inactivation. Therefore, complementary enzymology or direct enzyme electrochemistry studies are needed to verify that Fex reduction proceeds through the identified intermediates and pathway.

Molecular Orbital Analysis

The frontier orbital energies of Fex were calculated using the B3LYP density functional with the def2-QZVP basis set under tight SCF and geometry optimization criteria. A HOMO–LUMO energy gap of 2.9996 eV suggests moderate reactivity of Fex toward electron transfer reactions. Localized orbitals that allow better molecular interpretations were achieved via ROHF/ROKS wave function calculation. The LUMO is centered on the nitro group, while the HOMO is localized over the sulfide tail (Figure ). This is consistent with the nitro group being the most easily reduced group while the sulfur is the most easily oxidized.

8.

HOMO (green–yellow) and LUMO (pink–purple) orbitals of fexinidazole.

Method

Synthesis and Characterization

Fexinidazole and metabolites were synthesized and characterized according to established procedures. Pure compounds (1 mmol) were then dissolved in small amounts of methanol and allowed to dry under a stream of air for at least a week. This method yielded larger crystals compared to cooling the methanolic solution in a refrigerator.

X-Ray Crystallography and Structural Refinements

The crystallographic data of Fex and metabolites were collected on a Rigaku XtaLAB Synergy HyPix system using Cu-Kα radiation (λ = 1.54184 Å). Numerical face indexed absorption corrected data were processed with the SHELX software-package including programmes SHELXT 2018 for solving the structure and SHELXL2019/2 for refinement employing the WingX GUI. All non-hydrogen-atoms were refined with anisotropic displacement parameters. All hydrogen atoms were refined isotropically at calculated positions using a riding model with their U _iso values constrained to the U _eq of their pivot atoms; 1.5 × U _eq for methyl groups and 1.2 × U _eq for all others. In the refinement of Fex, ten reflexes were omitted as clear outliers.

Crystallographic data for the structures reported herein were deposited with the Cambridge Crystallographic Data Centre (CCDC) and are available as Supporting Information with the reference CCDC numbers 2454038 (Fex), 2454036 (Fex–SO), and 2454037 (Fex–SO₂). Copies of the data can be obtained from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. (FAX: + 44 1223 336033, e-mail: deposit@ccdc.cam.ac.uk, or at http://www.ccdc.cam.ac.uk).

Electroanalytical Methods

Fexinidazole and its metabolites were analyzed by DPV and scan rate dependent CV at three pH-values 3.6, 7.0, and 10.2 by using a 797 CA Computrace instrument from Metrohm (Herisau, Switzerland). The electrode setup consisted of a glassy carbon tip working (WE) and platinum rod counter electrode (CE). The potential at the WE was controlled with a second order type Ag(s)|AgCl(s)|KCl reference electrode (RE). Britton-Robinson Buffer was used for the aqueous phase with an additional 1 M KCl solution to maintain sufficient conductivity. To avoid any solubility issues each experiment was started in a freshly prepared mixture of 50% buffer solution and 50% dimethylformamide (DMF). As solvent mixtures can produce unexpected results in terms of potential stability, the RE potential was cross-checked with additional experiments using the same pH-values as in the main experiments. The well-established redox couple of ferricyanide and ferrocyanide was applied and remained stable under all conditions (Figure S5).

Computational Orbital Analysis

The geometry of Fex was initially optimized with the ORCA package. The optimized geometry was then subject to ROHF/ROKS wave function computation in Ibo-view, which was also utilized to visualize the localized orbitals.

Conclusion and Outlook

The metrical parameters for bond lengths, bond angles, and torsion angles in Fex and its metabolites are generally comparable and fall within the typical ranges. Variations are limited to the sulfur-containing tail and the bridge-phenyl junction, which influence molecular packing and hydrogen bonding networks. These subtle structural differences may contribute to the distinct biological and toxicological profiles of these compounds given that they are retained in solution, but further investigations are required to establish a definitive correlation. The nitro group reduction in fexinidazole is pH-dependent and occurs at negative potentials via a stepwise electron and proton transfer, generating a protonated nitro radical anion intermediate, followed by the formation of a hydroxylamine derivative. These reduction species are presumed to play a role in the biological activity of fexinidazole. The observed redox activity aligned well with the computationally derived frontier orbitals of fexinidazole with the LUMO residing on the nitro group and the HOMO on the thioether tail.

Glossary

Abbreviations

Fex

fexinidazole

Fex–SO

fexinidazole sulfoxide

Fex–SO₂

fexinidazole sulfone

CV

cyclic voltammetry

DPV

differential pulse voltammetry

HAT

Human African trypanosomiasis

PCET

proton-coupled electron transfer.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05115.

• Detailed crystallographic data and additional selected electrochemistry data of Fex and its major metabolites (PDF)

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CDT-Africa, Addis Ababa University, 2Q92+V77 Addis Ababa, Ethiopia

Abdrrahman S. Surur has received the generous Alexander von Humboldt postdoctoral fellowship to perform this project.

The authors declare no competing financial interest.