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. Author manuscript; available in PMC: 2025 Sep 23.
Published in final edited form as: Org Lett. 2025 Jun 26;27(27):7355–7360. doi: 10.1021/acs.orglett.5c02063

General Approach to Access Aldehyde Oxidase Metabolites of Drug Molecules via N-Oxidation/Reissert-Henze-Type Reaction

Christopher M Hanneman a,†, Sanghoon Song a,†, Nicholas P R Onuska b, J Craig Ruble b, Shannon S Stahl a,*
PMCID: PMC12451861  NIHMSID: NIHMS2106854  PMID: 40569179

Abstract

Aldehyde oxidase (AO) contributes significantly to metabolism of drug molecules containing heteroaromatics, and the lack of synthetic mimics for AO-mediated reactivity is a barrier to drug development. Herein, we use tandem N-oxidation and Reissert-Henze-type reaction of quinoline and related heteroaromatics form quinolone derivatives. This reactivity is showcased with small molecule building blocks and active pharmaceutical ingredients with known AO metabolism. The products of the latter substrates map directly onto established AO metabolites.

Graphical Abstract

graphic file with name nihms-2106854-f0001.jpg

Understanding and controlling drug metabolism is crucial to the development of new pharmaceuticals.1,2 Oxidative enzymes play a prominent role in drug metabolism, introducing functional groups that can influence the therapeutic efficacy, increase clearance, and/or form toxic metabolites.3 Regulatory standards by the US Food and Drug Administration often require the identification of drug metabolites to assess toxicity, optimize dosage, and predict metabolite interactions,4 and the synthesis of metabolites can be a major bottleneck during the development process.3 As most oxidative metabolism in humans is attributed to cytochrome P450 (CYP) enzymes,5 significant efforts have been devoted to mimicking the reactivity of CYPs with a metalloporphyrin or other catalysts with an oxo-transfer reagent to synthesize and study potential metabolites.6–13 Aldehyde oxidases (AO) represent another prominent class of enzymes involved in the oxidative metabolism of pharmaceuticals.14–16 Numerous late-stage drug development campaigns have been halted or terminated due to unacceptable metabolite formation via AO pathways that were not predicted by preclinical studies of non-human species.17 CYPs and most chemical and catalytic oxidation methods promote electrophilic reactivity and react more rapidly at electron-rich sites. In contrast, AO features a molybdopterin cofactor that promotes nucleophilic reactivity,18,19 leading to higher reactivity at electron-deficient sites, such as those in heteroarenes (Figure 1A and 1B).20 Existing enzymatic and microbial routes to AO metabolites are only effective for analytical purposes and are often unable to support a full suite of drug metabolism and pharmacokinetic studies.21 Chemical methods that mimic AO reactivity are limited by the dearth of nucleophilic oxidation methods.22 Here, we show how tandem N-oxidation/Reissert-Henze-type reaction of quinolines and other heteroaromatics provides a general approach to access to AO metabolites on preparative scale (Figure 1C).

Figure 1.

Figure 1.

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(A) Comparison of cytochrome P450 (CYP) and aldehyde oxidase (AO) metabolism of ripasudil. (B) Examples of pharmaceutical agents that undergo AO metabolism (C) N-Oxidation/Reissert-Henze sequence to access AO metabolites.

The inherent difficulties posed by AO-mediated metabolism stem from a confluence of factors,17 including the complex biology of AO enzymes and the prevalence of azaheteroaromatics in pharmaceutical agents. The mechanism of AO-catalyzed oxidations has been proposed to proceed via nucleophilic attack of a Mo–OH group coupled to hydride transfer to Mo=S unit.14,23 This pathway raises the possibility that similar reactivity could be achieved with nucleophilic oxidants, such as hypochlorite, peroxyanions (oxone, tBuOO−, ArC(O)O2−), and/or pyridine N-oxides. Screening of such conditions with quinoline as the substrate led to little successful reactivity, however. Sodium hypochlorite afforded the desired 2-quinolone product, but only low yields could be accessed (< 15%; see section 3e of the Supporting Information). The poor results with direct oxidation approaches prompted us to consider other strategies. Previous reports suggested a two-step protocol, involving N-oxidation followed by a Reissert-Henze-type reaction24 of the N-oxide,25–29 could access AO metabolites (Figure 1C). A three-step variation of this method was used to convert quinine and quinidine to their corresponding 2-hydroxy derivatives.25,26 The harsh reaction conditions used to convert an N-acetoxy intermediate (120 °C in Ac2O) into the corresponding 2-acetoxy derivative, prompted us to consider whether milder conditions that could offer a more general, streamlined method to oxidize heteroaromatics.

Established oxidation methods to access quinoline-N-oxides30 were evaluated and led to prioritization of two methods for the first step in the sequence. Catalytic methyltrioxorhenium (MTO) with hydrogen peroxide as the oxidant (Figure 2A-i) is efficient and produces minimal side products, facilitating purification of the desired heterocyclic N-oxide intermediates.30 Meta-chloroperbenzoic acid (mCPBA) is also efficient and enables better control over oxidant stoichiometry. The latter method generates a benzoic acid byproduct that can be challenging to separate, however. Many pharmaceutically relevant functional groups are tolerated by these methods, with some exceptions, including amines and thioethers (see section 3b of the Supporting Information for discussion). For substrates with tertiary amines, it was often possible to employ an oxidation/reduction sequence involving oxidation of both the aromatic heterocycle and the amine, followed by selective reduction of the amine N-oxide with sodium bisulfite. This sequence is illustrated in Figure 2A-ii for quinine.25,31,32

Figure 2.

Figure 2.

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(A) Strategies for N-oxide synthesis featuring: i. methyltrioxorhenium/H2O2 oxidation, or direct oxidation by mCPBA, and ii. mCPBA over-oxidation and subsequent reduction of the amine N-oxide. (B) Optimization of the Reissert-Henze-type reaction. Yields determined by 1H NMR spectroscopy (int. std. = maleic acid). Yield of competing 2-chlorination in parentheses.

The quinine N-oxide 27b was then used to evaluate new conditions for the second step in the sequence (Figure 2B). Aqueous conditions have been described previously with small heterocyclic N-oxides,28 but this method is problematic for complex molecules with poor water solubility. Initial reactions of 27b with different activating agents (MsCl, BzCl, PyBroP, Ac2O)27–29 in organic solvent led to poor yields and/or selectivity (Figure 2B, entries 4–7). In the best case, significant 2-chlorination of quinine was observed with MsCl as the activation reagent (entry 4). This complication was bypassed, however, by using p-toluenesulfonic anhydride (Ts2O), affording 27c in 57% yield when the reaction was conducted with 15 equiv of H2O as the nucleophile (entry 1). Only 10% yield of 27c was obtained with no water added, indicating that the tosylate counteranion is not effective as the nucleophile (entry 2), but the yield with water included improved to 71% yield when the reaction was conducted at 50 °C (entry 3). Other anhydride agents were less successful. For example, acetic anhydride was unreactive under mild conditions and triflic anhydride reacted rapidly with water to give low yields of 27c (see Table S1 in the Supporting Information). Use of other oxygen-based nucleophiles, such as NaOAc and Me3SiOK also led to lower yields (Table S3). The role of water as the nucleophile was confirmed by a reaction of quinoline N-oxide 1b with 15 equiv of 18O-enriched water (20% 18O) (eq 1). The resulting quinolone 1c integrated 18O at a level close to that of the 18O-enriched water. The small reduction in 18O content is attributed to reaction of tosylate anion as the nucleophile, followed by hydrolysis.

graphic file with name nihms-2106854-f0002.jpg (1)

The conditions identified in this effort were then evaluated with an array of azaheteroaromatic substrates, including quinoline, isoquinoline, and quinoxaline derivatives (Figure 3A), to assess the generality of the two-step procedure. The N-oxidation/Reissert-Henze sequence shows good reactivity to access the 2-oxo heterocycles with diverse substrates. Useful linchpins, such as aryl halides are tolerated under the reaction conditions (2c–3c, 9c, 11c, 13c–15c, 20c), highlighting the viability of this method for de novo metabolite synthesis for cases in which late-stage oxygenation may be unsuccessful. Labile benzylic C–H (4c, 9c, 18c) bonds remained unreactive, highlighting the orthogonal nature of this oxidation method to conventional electrophilic oxidation methods and resembling the contrast between CYP and AO metabolism. The reaction was relatively insensitive to electronic effects (5c, 8c, 10c–13c, 17c); however, steric effects resulted in low conversion for the N-oxidation reaction (see Table S6 of the Supporting Information for additional discussion). Hydroxyquinolines (8c), which represent important pharmacophores, are compatible with the reaction conditions.33 As noted above, substrates bearing tertiary amine subunits are susceptible to overoxidation. In the reactions of 12c and 13c, using mCPBA (N-oxidation method B) enabled selective oxidation of the quinoline ring in the presence of the appended tertiary amine. For 12c, Boc protection of the secondary amine prior to reaction prevented N-oxidation at that site, and the Boc is removed in situ during the Reissert-Henze reaction, affording 12c as the tosylic acid salt. 5-Nitroquinolines such as 12c have been implicated in both oxidative and reductive transformations of AO.34 Isoquinolines (14c–18c) are also effective substrates. Oxygenation proceeds at the C1 site, with the exception of 17c, which has a cyano substituent already present at this site. Finally, quinoxalines undergo smooth conversion to the 2-oxo derivatives (19c, 20c). During the N-oxidation step, only a single nitrogen atom was oxidized in these substrates, likely reflecting attenuation of nucleophilicity of the second nitrogen following N-oxygenation.35 In the case of 20c, site-selectivity was confirmed through 1H–15N HMBC analysis (specifically, N1 C8–H HMBC correlation) and corroborated by the J-coupling values and chemical shifts of the proximal protons.36

Figure 3.

Figure 3.

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(A) 2-Hydroxylation of quinolines, isoquinolines, and quinoxalines. (B) Oxidation of pharmaceutically relevant substrates; names of the parent drug molecules are noted for context. Isolated yields, reflecting conversion of the N-oxide to the corresponding 2-oxo derivatives; 1H NMR spectroscopic yields (int. std. = maleic acid) shown in parentheses. aisolated as the tosylic acid salt. bBoc protecting group was removed under reaction conditions. cConducted at 60 °C with 3 equiv of potassium trimethylsilanolate as oxygen atom source.

Overall, the results in Figure 3A confirm that the N-oxidation/Reissert-Henze sequence represents an effective synthetic surrogate for AO-like reactivity. Subsequent efforts, therefore, sought to assess this reactivity with active pharmaceutical ingredients (APIs) and related late-stage structures (Figure 3B).17 We began our study with lenvatinib, a kinase inhibitor developed to treat thyroid cancer that undergoes both CYP and AO metabolism.37,38 Lenvatinib exhibits poor solubility in common organic solvents, and this feature was exacerbated upon conversion to the N-oxide 21b,39 complicating chromatographic purification (C18, preparative HPLC). This challenge was overcome by using centrifugal partition chromatography (CPC), a preparative, high-resolution liquid-liquid extraction method,40 which accessed 21b in 78% isolated yield (see section S1b of the Supporting Information for details). Treatment of 21b under the Reissert-Henze-type reaction conditions at 50 °C afforded 21c in 91% in situ yield (55% isolated yield). The challenges encountered here with solubility and purification of the oxidized compounds are not unique to lenvatinib.

Tilbroquinol is an hydroxyquinoline-derived drug approved for use as an antiparasitic and later removed due to hepatotoxicity.41 The N-oxide, 22b, was recalcitrant toward N-oxidation via MTO, possibly due to the steric effect of the the C-8 substituent. Use of mCPBA generated 22b in 32% yield, and subsequent conversion to the quinolone enabled isolation of 22c in 39% yield.

Pfizer has reported a c-MET inhibitor, pf-04217903, that is labile to aldehyde oxidase metabolism and contains multiple heteroaromatic rings that could compete for N-oxidation.42 Once again, mCPBA was used to enable more precise control over the oxidant stoichiometry to avoid over-oxidation and maximize yield. 1H–15N HMBC spectroscopy was used to establish selective oxidation of the quinoline ring, and subsequent conversion to 23c proceeded efficiently in 72% isolated yield.

Cabozantinib43 (24a), a kinase inhibitor developed to treat thyroid cancer, and a gardiquimod44 derivative (25a), a toll-like receptor-7 agonist, were commercially available as the N-oxides (24b, 25b). Subjection of these compounds to the Reissert-Henze-type reaction conditions formed the corresponding quinolones 24c and 25c in 29% and 98% isolated yields respectively. The comparatively poor yield with cabozantinib N-oxide likely reflects the electron-donating substituents on the quinoline ring. The optimal outcome was achieved by modifying the reaction conditions, including using elevated temperature (60 °C) and potassium trimethylsilanolate as a more potent oxygen nucleophile than water. Effective reaction of the N-oxide of gardiquimod is notable because the imidazoquinoline core in is featured in numerous other drug candidates.45

SGC707 (26a)46 is a selective allosteric inhibitor of protein arginine methyltransferase 3 (PRMT3). Successful oxidation of SGC707 to access isoquinolone 26c demonstrates applicability of this in the two-step sequence to metabolite synthesis from isoquinoline-based drugs.

Synthesis of 27c from the anti-malarial drug quinine was described above (Figure 2B). This oxidation method does not encounter any interference with the unprotected secondary alcohol present nor competing epoxidation of the alkene during the reaction with mCPBA.47 SB-277011 (28a) is a dopamine D3 receptor antagonist developed for the treatment of addiction.48 Preparation of the N-oxide was achieved by using the oxidation/reduction strategy C, similar to that use for quinine. The resulting quinolone 28c was then accessed in excellent yield.

Ripasudil (29a) and fasudil (30a) are both Rho-kinase (ROCK) inhibitors that treat numerous ailments and are rapidly converted to their 2-oxygenated metabolites upon ingestion, resulting in unexpectedly high clearance values.19,27,49 These metabolites are readily accessed with our protocol (29c, 30c) from the Boc-substituted drug molecules. To demonstrate the scalability and robustness of the chemistry developed herein, Boc-fasudil was subjected to reaction on 10 mmol scale, and the active metabolite was generated in 99% yield. Development of a liquid-liquid extraction method avoided a need for large-scale reverse-phase column chromatography and accessed the purified product in 88% yield. These results showcase the utility of this chemistry to generate preparative quantities of metabolites.

The results outlined in this study introduce a straightforward sequence to oxidize quinolines and related heterocycles to metabolites derived from aldehyde oxidase. The net reactivity is orthogonal to that typically accessed by CYPs and related chemical oxidation methods. The method begins with creation of an internal oxidant in the form of an N-oxide that can be induced to react at the adjacent C–H site, affording a product arising from formal “nucleophilic” oxidation. This reactivity mimics aldehyde oxidase metabolism and is capable of generating the corresponding drug metabolites in preparative yields. Access to these and similar metabolites should have broad impact on drug development efforts. Efforts are now focused on complementary reactivity for the oxidation of pyridines and related monocyclic heteroaromatic compounds, which are even more prevalent in drug molecules. These structures have higher aromatic stabilization energy relative to quinolines and, thus, are less effective with the methods outlined herein.

Supplementary Material

SI

The Supporting Information is available free of charge on the ACS Publications website.

Experimental details, procedures, characterization data, and NMR spectra (PDF)

ACKNOWLEDGMENT

We thank Dr. McKenna Goetz (UW-Madison) for editorial assistance. This work was funded by Eli Lilly and Company (Lilly Research Award Program), with additional support from the NIH (R35 GM134929). The spectrometers were supported by the NSF (CHE-1048642) and by a generous gift from Paul J. and Margaret M. Bender. The mass spectrometer was supported by the NIH (S10-OD020022)

Footnotes

The authors declare no conflicts of interest.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SI
NIHMS2106854-supplement-SI.pdf (4.9MB, pdf)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

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