Structure-metabolism relationships in human-AOX: Chemical insights from a large database of aza-aromatic and amide compounds

Article abstract of DOI:10.1073/pnas.1618881114

Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only "very local" structure-metabolism relationships based on a small number of similar compounds have been developed. This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and themetabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship.

Full text of DOI:10.1073/pnas.1618881114

Structure–metabolism relationships in human-AOX:
Chemical insights from a large database of
aza-aromatic and amide compounds
Susan Lepria, Martina Ceccarellia, Nicolò Milania, Sara Tortorellaa, Andrea Cuccoa, Aurora Valeria, Laura Goraccia,
Andreas Brinkb, and Gabriele Cruciania,1
aDepartment of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy; and bRoche Pharma Research and Early Development,
Drug Disposition and Safety, Roche Innovation Center Basel, F. Hoffmann-La Roche, Ltd., 4070 Basel, Switzerland
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 28, 2017 (received for review November 14, 2016)
Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the
oxidation of aldehyde and aza-aromatic compounds and the hydro-
lysis of amides, moieties frequently shared by the majority of drugs.
Despite its key role in human metabolism, to date only fragmen-
tary information about the chemical features responsible for AOX
susceptibility are reported and only “very local” structure–metabo-
lism relationships based on a small number of similar compounds
have been developed. This study reports a more comprehensive cov-
erage of the chemical space of structures with a high risk of AOX
phase I metabolism in humans. More than 270 compounds were
studied to identify the site of metabolism and the metabolite(s). Both
electronic [supported by density functional theory (DFT) calculations]
and exposure effects were considered when rationalizing the
structure–metabolism relationship.
erature, and general rules for hAOX SMR are missing (17–19). For
instance, Ghafourian and Rashidi (18) developed a quantitative
structure–activity relationship method for phthalazine and quina-
zoline scaffold, based on COSMIC force field, AM1 semiempirical
method, and structural parameters generated by MOPAC and
found a strong dependence of the oxidation rate with the electronic
nature of substituents; Torres et al. (17) reported a qualitative
method based on density functional theory (DFT) to predict the
product of AOX metabolism by examining the energetics of likely
tetrahedral intermediates resulting from nucleophilic attack on
carbon and confirmed the primary role of the electronic effects of
the substituents; later, Jones and Korzekwa (19) developed a
mechanistic computational model to predict intrinsic clearance by
DFT studies, taking into account electronic and steric effect of the
substituents. However, in all papers, the relationships and models
were based on a small number of compounds of similar structure (e.
g., phthalazine and quinazoline), and an attempt made by us to
apply the same strategies in new different chemical entities proved
to be inadequate (for further details, see SI Appendix).
Therefore, we performed in-house AOX-mediated metabolism
experiments covering a diverse chemical domain of commonly
used drugs and drug candidates to obtain a sound base for further
mechanistic investigation and modeling studies.
Regarding AOX oxidation, 198 aza-aromatic compounds were
acquired or synthesized to assess AOX susceptibility, and exam-
ples of the scaffolds investigated are shown in Fig. 1. The full
dataset with more experimental details is reported in SI Appendix,
Tables S1–S5.
aldehyde oxidase amide hydrolysis site of metabolism variously
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decorated heterocycles structure–metabolism relationship
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n recent years, the cytochromes P450 (CYP450)-mediated me-
I
tabolism has been intensively studied. As a consequence, the
development of new and more efficient in silico and in vitro
screening systems contributed to decrease the discontinuation rates
of new drugs in clinical studies by 10% in recent years (1). In
particular, several models for in silico prediction of human
CYP450-mediated metabolism have been reported so far (2–4)
and, more recently, for human FMO3 (5). Nevertheless, a deeper
understanding of non-CYP drug enzyme metabolism is advisable to
further improve screening and models efficacy (6). Among all non-
CYP metabolic enzymes (7), emerging importance has been at-
tributed to human aldehyde oxidase (hAOX), a cytosolic drug-
metabolizing enzyme expressed in human liver that, similarly to
CYPs, contributes to new chemical entities’ oxidation, but acting in
the absence of NADPH cofactor. Drugs that are substrates for
AOX often exhibit high metabolic clearance, resulting in low ex-
posure and hence in decreased drug efficacy in humans. In par-
ticular, AOX catalyzes the oxidation of a wide range of aza-
aromatic scaffolds at the unsubstituted carbon in ortho to the
nitrogen (SI Appendix, Fig. S1 A and B), usually the most electron
deficient (8). Several marketed drugs are well-known substrates of
AOX (e.g., methotrexate, famciclovir, and zaleplon) (9), and sev-
eral compounds have failed due to undetected AOX oxidation (e.
g., BIBX1382, RO-1, FK3453, and carbazeran) (10–14). The role of
AOX in drug development has become more relevant in the last
few years as a result of organic synthesis strategies designed to
reduce cytochromes P450-related metabolism, based on chemical
modifications and decorations that have subsequently increased the
drug reactivity toward AOX (11, 15). In addition, AOX was re-
cently found to be responsible for the rapid hydrolysis of an amide
bond in GDC-0834 (SI Appendix, Fig. S1C), a potent inhibitor of
Bruton’s tyrosine kinase (16). Although the development of struc-
ture–metabolism relationships (SMRs) for hAOX metabolism
prediction would strongly reduce the risk of clinical failure in late
stages of drug discovery, such SMRs are scarcely reported in lit-
Significance
The metabolism of xenobiotics is a critical aspect of drug discov-
ery; nowadays, aldehyde oxidase (AOX) has emerged as a key
metabolic enzyme having a pivotal role in the failures of several
clinical candidates. The lack of homogenous data on possible
substrates and not substrates of this enzyme represents a serious
limit for the development of an in silico model for metabolism
prediction. Here, we present a database of 270 chemically diverse
compounds containing aza-aromatic and/or amide moieties (sus-
ceptible to human AOX), experimentally tested in vitro. The re-
sults herein reported should be useful in the development of a
reliable prediction model, which should be of wide interest in
chemistry, biology, biotechnology, and medicine.
Author contributions: L.G. and G.C. designed research; S.L., M.C., N.M., S.T., A.C., and A.V.
performed research; S.L., N.M., and A.C. contributed new reagents/analytic tools; S.L.,
M.C., N.M., S.T., A.V., A.B., and G.C. analyzed data; S.L., M.C., N.M., S.T., L.G., A.B., and
G.C. wrote the paper; and L.G. and G.C. coordinated and supported the entire project.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. Email: gabriele.cruciani@unipg.it.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1618881114/-/DCSupplemental.
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content of AOX. After 0- and 60-min exposure, samples were
analyzed by liquid chromatography–tandem mass spectrometry
(LC-MS/MS), and then processed with MetaSite and WebMetabase
(Molecular Discovery, Ltd.; www.moldiscovery.com/) (31). In ad-
dition, carbazeran, cinchonine, and famciclovir, well-known AOX
substrates, together with a representative example of each scaffold,
were also tested in the presence of 2,6-dichlorophenolindophenol
(DCPIP), a selective inhibitor of AOX to ensure the reliability of
our method (SI Appendix, Figs. S3 and S4, respectively). The
AOX content of several cytosolic batches was quantified (32)
and the average value was 0.9 μM 0.1 μM. Further details can
be found in Materials and Methods.
DFT Calculation
As mentioned above, the electronic effect is reported to play a
major role in AOX substrate susceptibility; therefore, partial
atomic charges (ACs) were calculated to support the experimental
trend observed and to determine the site of metabolism (SoM).
DFT calculations were run with Gaussian 09 (33) and consisted
of three steps: (i) creation of 3D structures of the substrates,
(ii) ground-state geometry optimization by DFT at a B3LYP level
of theory with 6-31G basis sets, (iii) AC calculations according to
the Merz–Singh–Kollman scheme (34). Interestingly, for the in-
vestigated substrates, the AOX SoM (highlighted by the * symbol in
Tables 1–4) usually corresponds to the most positive unsubstituted
aromatic carbon atom (for more details, see SI Appendix, Fig. S5),
thus suggesting that MK charges can be conveniently used for es-
timating the SoM also where multiple site of reactions are possible.
This computational workflow has been chosen after evaluating the
performance of alternative computational approaches that com-
pare reaction energies to account for the multiple possible reaction
sites (35) (SI Appendix, Table S6). However, this last approach is
less versatile because strictly dependent on the mechanism of re-
action/nature of the intermediate (not fully elucidated yet), more
computational demanding, and slower compared with AC calcu-
lations. For all of these reasons and bearing in mind the goal of a
comprehensive analysis on a large number of compounds, we chose
to proceed with AC calculations (the most relevant results are
reported in SI Appendix, Table S7).
Fig. 1. Examples of the studied aza-aromatic scaffolds: the number of
tested compounds is reported in brackets. The complete dataset is pro-
vided in SI Appendix.
Chemistry
The derivatives of phthalazine were prepared starting from
phthalazine and phthalazone, as summarized in SI Appendix, Fig.
S2 A and B, respectively. 1-Phenylphthalazine (1) was prepared by
direct arylation of phthalazine with phenylboronic acid in very
mild condition (20). Nitration of phthalazine with NaNO3/H2SO4
solution gave 5-nitrophthalazine (2), which was reduced to amino-
phthalazine 3, in turn converted to amide 4 upon 1-[bis(dimethy-
lamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate (HATU)-mediated coupling with 2-pico-
linic acid. The phthalazine ammonium salts 5 and 6 were obtained
from commercial phthalazine after alkylation with methyl and
benzyl halide. Phthalazine 2-oxide (7) was obtained by phthalazine
oxidation with meta-chloroperoxybenzoic acid (mCPBA) (21).
1-Chlorophthalazine hydrochloride 8 was obtained by POCl3-
mediated chlorination of phthalazone (22). 1-Methoxyphthalazine
9 was produced during an attempt of chromatographic purification
on SiO2 (with a methanol-containing eluent) of chloro derivative 8.
1-Chlorophthalazine produced 4-(phthalazin-1-yl)morpholine (10)
and 1-phenoxyphthalazine (11) by nucleophilic substitution in al-
kaline condition. Purine derivatives 12–15 were obtained from
6-chloropurine (2-amino,6-chloropurine for 13d) as described in SI
Appendix, Fig. S2C. Chloro compounds 12a–b were obtained upon
alkylation of purine with benzyl bromide and propargyl bromide in
the presence of K2CO3. The nucleophilic substitution of 6-chlor-
oderivative with phenol, aniline, or cyclohexylamine according to
literature procedures (23, 24) afforded compounds 13a–d. In turn,
bromination of 13b and 13c with Br2 saturated aqueous Na2HPO4
solution gave 14 and 15 (25). Moreover, hypoxanthine and allopu-
rinol reaction with benzyl bromide and K2CO3 in DMF (26) gave
compounds 16–17 and 19–20 (SI Appendix, Fig. S2 D and E).
Furthermore, chlorination of allopurinol with POCl3 gave
4-chloro-1H-pyrazolo[3,4-d]pyrimidine (18) (SI Appendix, Fig. S2E).
Finally, pyrazine based compounds 21–23 were obtained according
to SI Appendix, Fig. S2F. Chloropyrazine 21a was prepared
according to literature procedure (27). Amino derivative 22 was
obtained from chloro 21a by nucleophilic substitution with aniline
(28). Finally, pyrazine 23 was obtained by Suzuki coupling from
bromo derivative 21b, in turn prepared by chloro derivative 21a and
trimethylsilyl bromide (29).
Table 1. AOX susceptibility for pyrimidine-based compounds
Evaluation of AOX-Mediated Metabolism by in Vitro Assays
on Human Liver Cytosol
Compounds 24–26 were analyzed according to LC-MS method B (Materials
and Methods); for 27, method A was used instead.
Metabolism of the 273 selected compounds was evaluated in vitro
according to a modification of a literature procedure (30), using
human liver cytosol (HLC) instead of S9 fraction, due to its higher
*The predicted SoM by DFT calculations is highlighted by a star.
†N, not substrate; Y, substrate (the percentage of substrate consumed after
60-min incubation is reported between brackets).
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protein H-bond donor interactions hot spots (Fig. 2). Compounds
were docked in the catalytic active site, allowing flexibility on both
ligands and cavity amino acids.
Results and Discussion
Oxidation of Aza-Heterocycles by hAOX. All data concerning the
oxidation by hAOX are collected in SI Appendix, Tables S1–S5.
Among the scaffolds investigated, pyridines were the only class
that resulted always in not being susceptible to AOX metabolism
(SI Appendix, Table S1). In this section, a number of scaffolds that
provided interesting insights on the structure–metabolism re-
lationship in hAOX enzyme are discussed.
Fig. 2. (A) Hydrophobic molecular interaction fields generated using the
GRID (41) DRY probe in 4UHX AOX protein catalytic cavity; (B) H-bond donor
molecular interaction fields generated using the GRID N1 probe. Phthalazine
(in green) is added for reference.
For example, the study of the 15 pyrimidines and 4 pyrazines (SI
Appendix, Table S2) suggested that compounds bearing these
scaffolds are barely substrates. When metabolism occurs, the
C2 position seems to be the preferred SoM for oxidation, with all
2-substituted compounds being stable. A comparison of the four
pyrimidine-based compounds in Table 1 exemplifies a general
trend observed for this class of compounds. Indeed, compounds
having EWG group at C4 and a chlorine at C5 (24 and 25)
resulted as substrates, whereas pyrimidines with EDG substituents
at C4 and C6 (26 and 27) were not substrates. This finding high-
lights the importance of electronic effect in AOX susceptibility.
Concerning the 13 quinolines and 15 isoquinolines tested (SI
Appendix, Table S3), they were easily metabolized by AOX at
C2 and C1/C3, respectively, regardless of the substituents’ nature.
Additional insights were extracted from compounds in Table 2.
About quinolines, similar structures were compared. Quinine ester
32 was more stable than quinine 30; a rationale for this behavior is
provided by the correspondent calculated AC at the SoM: the
higher the charge, the more reactive the SoM. Moreover, comparing
Docking Analysis
Compounds identified as substrates by in vitro assay were analyzed
by docking approach with FLAP software (36, 37) using the 4UHX
hAOX 3D structure (4UHX) (38). The protein structure 4UHX
lacks two important polar amino acids (Asp881, Glu882) located in
the loop between the residues Leu880–Ser883. However, these
amino acids are 4.5 Å from the phthalazine substrate; thus, they
may play a relevant role in molecular interactions with substrates.
To avoid misleading results, the two missing amino acids were
added to the 4UHX X-ray structure loop in between Leu880–
Ser883 residues and gently minimized before any computation.
The resulting AOX catalytic cavity encompassed 30 aa that, despite
their different chemophysical nature and polarity, generated large
hydrophobic interaction pathways, with minor occupancy for pro-
tein H-bonding acceptor interaction pathways and almost absent
Table 2. hAOX susceptibility for quinoline- and isoquinoline-based compounds
All compounds were analyzed according to LC-MS method B (Materials and Methods).
*The predicted SoM by DFT calculations is highlighted by a star.
†N, not substrate; Y, substrate (the percentage of substrate consumed after 60-min incubation is reported
between brackets).
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Table 3. hAOX-mediated metabolism for quinoxaline and its isomers
Phthalazone
All compounds were analyzed according to LC-MS method B, with the exception of compounds 1–3, phthala-
zone, and 9, for which method A was used instead (Materials and Methods).
*The predicted SoM by DFT calculations is highlighted by a star.
†N, not substrate; Y, substrate (the percentage of substrate consumed after 60-min incubation is reported
between brackets).
sulfonamides 31 and 33, the presence of the benzotriazole moiety
substrates (as exemplified by compounds 39 and 40 in Table 3),
with the exception of compounds 41 and 42 whose substituents
orient the compounds in an unreactive pose in the catalytic cavity
seems to be detrimental for metabolic susceptibility, suggesting
that this chemical group has an impact on both electronic and
exposure effects. This hypothesis was supported by the calcu- according to our docking studies (SI Appendix, Fig. S6). The steric
lated charges (the AC of 31-SoM is higher than that of 33-SoM;
SI Appendix, Table S7) and by docking analysis. Indeed, ben-
hindrance may in this case overpower the electronic contribution,
because the calculated AC values (SI Appendix, Table S7) suggest
zotriazole was found to compete with quinoline to give a π–π the opposite behavior. Similarly, quinazoline (e.g., 43 and 44) and
stacking interaction with Phe923 and Phe885 (SI Appendix, Fig. phthalazine (e.g., 1, 3, and 9) were highly reactive toward AOX,
S6), previously identified as important residues for recognition with the exception of the nitro-containing compound 2. Finally, 1,6-
in the active site (38). Concerning isoquinolines, we observed
and 1,8-naphthyridines (e.g., 45 and 46) were substrates, whereas
that compounds with EDG at C4, C5, and C7 were substrates, 1,7-naphthyridines like 47 and 48 were not.
A number of other bicyclic scaffolds were investigated (SI
as exemplified by compounds 34 and 35 in Table 2, whereas
EWG-substituted compounds were stable (e.g., 36 and 38,
Table 2).
Comparing 34 to 37, both EDG-substituted compounds, only
the second was stable: as suggested above, the phenyl in 37 can
play the same role of benzotriazole in 33 (and again the AC of
the putative 37-SoM is lower than the one for 34).
Quinoxaline and related regioisomers represent another class of
compounds deeply investigated, with more than 45 compounds
tested (SI Appendix, Table S4). These compounds were mostly
good AOX substrates, with 17 compounds leading to more than
50% of the parent compound consumed after 60-min incubation.
Both EDG- and EWG-substituted quinoxaline were good AOX
Appendix, Table S5). For the eight pyridopyrazines tested, the
SoM could be C2 and C3: when at least one of these positions is
occupied by bulky groups the oxidation does not occur at all (50
and 51). About pyridophthalazines, only two compounds were
tested, resulting as not substrates; however, 53 is already oxi-
dized (similar to phthalazone), whereas 52 is very hindered by
the presence of three phenyl groups (Table 4). The pteridine
scaffold was tested in seven compounds, represented in Table 4
by 54–56. The different oxidation susceptibility of 54 and 56,
only differing in the methyl substituent position, was rational-
ized by considering that the calculated charge on 54-C7 (the
exposed site according to docking analysis; SI Appendix, Fig. S6) is
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Table 4. hAOX susceptibility of miscellaneous bicyclic compounds
All compounds were analyzed according to LC-MS method B, with the exception of compound 19, for which
method A was used instead (Materials and Methods).
*The predicted SoM by DFT calculations is highlighted by a star.
†N, not substrate; Y, substrate (the percentage of substrate consumed after 60-min incubation is reported between
brackets).
‡Hydrolysis reaction contributes to substrate consumption.
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Fig. 3. Inhibition study on 66 with DCPIP (50 μM). The chromatograms report the substrate consumption after 0- (Top) and 60-min (Bottom) HLC incubation
in the absence (Left) or presence (Right) of AOX selective inhibitor DCPIP. For each peak, the corresponding compound is also reported.
Hydrolysis of Amides by hAOX. Among the five pyridazoindoles
tested for AOX susceptibility, all bearing an amide moiety, two
of them (65 and 66), in addition to the oxidated metabolites,
lead to the formation of the corresponding anilines, consistent
with the amide bond hydrolysis. This finding was supported by a
single recently published example of the amidic compound
GDC-0834 (16). To confirm AOX contribution to both hy-
drolysis and oxidation, compound 66 was also tested in the
presence of DCPIP inhibitor. As expected, both reactions were
inhibited (Fig. 3; MS/MS spectra are reported in SI Appendix,
Fig. S7).
Thus, we further investigated the hydrolysis reaction, because
it can cause not only substrate inactivation but also toxification
(liberated anilines are potentially hemato-, nephro-, hepato-
toxic, and carcinogenic) (40).
Therefore, we synthesized (SI Appendix, Fig. S2G) and ac-
quired 75 amides to perform a SMR study (Fig. 4 and Tables 5
and 6; additional information can be found in SI Appendix,
Tables S8 and S9).
higher than the 56-C6 charge (unexposed site according to
docking analysis; SI Appendix, Fig. S6). Indeed, C7 is the
preferred SoM for exposition reasons along with C2, with 55
being the only substrate with C7 occupied but still oxidized.
Similarly, according to AC charges, C6 is the SoM in 2 out of
10 imidazopyrazines, (SI Appendix, Table S7). For example,
concerning representative compounds in Table 4, 57-C6 (sub-
strate) is more electropositive than 58-C6 (not substrate).
Additionally, stability of 59 and 60, with AC charges similar to
57, can be rationalized by the presence of aromatic groups at
C2 (i.e., indole and phenol, respectively). Purine scaffolds were
also tested (SI Appendix, Table S5). Based on our data, 2- or
6-EDG–substituted purines were substrates of AOX (with
unsubstituted N7-H or N9-H), as in compounds 12a and 12b
(Table 4). Again, as shown in SI Appendix, Table S7, the cal-
culated AC on the putative SoM for 12a and 12b (substrates) is
higher than the one calculated for 15 and 16 (not substrates,
Table 4), confirming that the electronic effect may dominate
the exposition effects in determining the AOX oxidation sus-
ceptibility in this scaffold. On the contrary, allopurines were
not substrates (e.g., 18 and 19). The three pyrazolopyridazines
might be assimilated with the phthalazine scaffold: consequently,
compounds 62 and 63 were substrates, whereas 64, similar to
phthalazone, was not a substrate. Other miscellaneous scaffolds
were investigated as reported in SI Appendix, Table S5. In sum-
mary, the presence of indole, pyrazole, and pyridine were detri-
mental for AOX susceptibility. Mostly, EDG substituents favor
the susceptibility of compounds to oxidation. The most evident
exception to this statement is represented by pyrimidines, where
the opposite trend might be due to the higher pKa (>8) of EDG-
substituted compounds, as the protonated species might interact
differently with the catalytic site and through a different mecha-
nism. According to our data, another important trend observed is
that electrophilicity and reactivity seem strictly correlated, al-
though in some cases highly positive-charged carbon atoms are not
oxidized (e.g., 36-C3, SI Appendix, Table S7): electrophilicity alone
cannot discriminate the susceptibility to AOX. This last observa-
tion can be rationalized considering that the initial nucleophilic
attack by molybdenum cofactor (MoCo) requires a moderate
electrophilic center, although the widely accepted mechanisms
and recent experiments with kinetic isotopic effects point to hy-
dride displacement as the critical step (39). Therefore, we suggest
that there should be a trade-off between the SoM nucleophilic and
electrophilic nature, requiring an in-depth modeling study, which
is beyond the aim of this work.
First, amides 67–90 sharing the same acyl thiophene moiety
of GDC-0834 were tested (Table 5). Amides 67–69 carrying
only an EDG in the aniline ortho position resulted as substrates
of AOX, with the parent compound being consumed by 81–
90% after 60 min of incubation. Conversely, 70–71 with EWG
Fig. 4. Design of differently decorated amides inspired to GDC-0834 to
investigate hAOX-mediated amide hydrolysis.
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Table 5. Thiophene-containing anilides tested for AOX-mediated metabolism
All compounds were analyzed according to LC-MS method B, with the exception of compounds 69, 70,
81, and 82, for which method A was used instead (Materials and Methods).
*N, not substrate; Y, substrate (the percentage of substrate consumed after 60-min incubation is
reported between brackets).
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Table 6. Thiophene-free anilides tested for hAOX-mediated metabolism
All compounds were analyzed according to LC-MS method B, with the exception of compounds 91–93, for
which method A was used instead (Materials and Methods).
*N, not substrate; Y, substrate (the percentage of substrate consumed after 60-min incubation is reported
between brackets).
†Substrate consumption is also due to oxidation reaction.
(i.e., carbonyl) were stable to AOX exposition. On the contrary,
correct orientation). Generally, the most populated poses are
nonproductive poses because the molecules interact with the
AOX cavity far from the reactive MoCo. This behavior is
common for both substrate and nonsubstrate molecules. How-
ever, substrates tend to show a higher number of productive
poses, that is, the poses that expose the reactive carbon moiety
to the activated oxygen atoms of the MoCo, with respect to the
nonproductive poses. Conversely, nonsubstrates show a higher
number of nonproductive poses, that is, the poses that expose
unreactive atom moieties to the MoCo, with respect to the
productive ones. Moreover, often the nonsubstrate molecules
do not adopt any of the potential reactive poses. This behavior is
exemplified in Fig. 5 where the substrate 65 and nonsubstrate
105 are reported.
meta or para substitution with both EDG and EWG led to more
stable compounds (72–83).
Thus, provided the same acidic group, the best metabolism
rate is obtained when the amine counterpart is substituted in
the ortho position with an EDG. Moreover, tertiary amides
seemed more stable (84 and 85). Finally, the introduction of
more sterically hindered and less flexible anilines (86 and 87),
or aliphatic amines (88–90), gave stable compounds. Thus, we
concluded that the nature of acyl moiety is less important to
ensure AOX susceptibility. To confirm our hypothesis, furane,
indole, 2-methylenindole, (3-fluorophenyl)propane, cyclohex-
ane, pyrazine, and pyridine compounds 91–100 were tested,
resulting as substrates (Table 6). Regardless of the presence of
only an EDG ortho substituent, isoquinoline 104 was stable
About 10% of the overall poses of 65 are productive for
(Table 6), possibly as a consequence of an intramolecular H bond carbon ring oxidation because, as reported in Fig. 5, the re-
(8). Acquired aliphatic benzamides were also tested (SI Ap-
pendix, Table S9), but they were not AOX substrates. This
finding correlates well with the more favorable hydrolysis of
aromatic amides with respect to the aliphatic ones, due to res-
onance effects. Para-substituted or heterocyclic anilides (e.g.,
102–103, Table 6) and amide-like moieties (hydrazides, ureas,
and carbamates) were not susceptible to AOX metabolism (see
SI Appendix, Table S9, amide-like bond). Furthermore, the
docking analysis of compounds herein reported confirmed the
experimental trends. Indeed, when substrate and nonsubstrate
molecules are docked into the AOX catalytic site, molecules
tend to interact with the AOX cavity in three different ways (far
from MoCo or close to MoCo in correct orientation or not
active carbon atom of the pyridazino ring is well exposed and
oriented toward the MoCo. Interestingly, a similar number of
poses show a potential interaction of the amide moiety with the
MoCo. Indeed, 65 is also hydrolyzed by hAOX. These poses are
mostly stabilized by proficuous hydrophobic interactions and
to a lesser extent by some substrate H-bond donor–protein
H-bond acceptor interactions. Poses where the cyanophenyl
moiety is exposed to MoCo are nonproductive because the
cyanophenyl ring is not reactive to MoCo. Conversely, for the
nonsubstrate 105 the great majority of poses expose unreactive
moieties toward MoCo (the figure reports the exposure of
the unreactive dimethyl-1H-pyrazol and of poorly oriented
pyrimidine moieties). As an example, we decided to test three
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Fig. 5. Comparison of compounds 65 and 105 by
docking analysis. (A) Productive poses for substrate
65 (not all poses are reported for clarity). The most
reactive carbon atom of the pyridazino ring is well
exposed and oriented toward the MoCo. (B) Non-
productive poses for 65 where the unreactive cya-
nophenyl moiety is exposed to MoCo. (C) Productive
poses for hydrolytic reactions of substrate 65 (not all
poses are reported for clarity). The amide moiety is
well oriented toward the MoCo. (D) Nonproductive
poses for nonsubstrate 105 exposing unreactive di-
methyl-1H-pyrazol toward MoCo. (E) Nonproductive
poses for nonsubstrate 105 showing a suboptimal
orientation of the pyrimidine moiety. The phthala-
zine substrate was added to the picture for refer-
ence. (F) 65 (Top) and 105 (Bottom) structures.
additional compounds (106a–c, Fig. 6) with two possible SoM
according to DFT analysis, but exposing only one site toward
the MoCo in the majority of the poses.
putative site of reaction also allowed rationalizing the AOX
susceptibility outcome (Y/N), but this is true only for very similar
structures for which we can assume that the exposure factor play
a similar role. From all of these experiments, it emerges that it is
not at all simple to predict whether a compound is a substrate of
AOX or not. We noted that, although the role of physico-
chemical molecular descriptors may be relevant in case of con-
generic series of compounds, when different molecules are
compared, their contribution to isoform selectivity resulted in
being relatively modest.
In general, when pharmaceutical compounds bearing dif-
ferent scaffolds are compared, more sophisticated modeling
techniques that combine electrophilicity calculation with
productive spatial interaction with the enzyme catalytic site
are required to rationalize SMRs.
Quinazolines 106a–c and their predicted 4-(3H)-quinazolone
metabolites (107a–c) were synthesized (SI Appendix, Fig. S9).
The EDG-substituted quinazoline resulted as AOX substrates
(percentage substrate consumed, 85, 85, and 68%, respectively), and
their metabolism was confirmed by comparison of the RT and
LC-MS/MS spectra of the obtained metabolite and synthesized
quinazolone. Thus, protein–ligand recognition elements able to
correctly expose the reactive ligand moiety to the MoCo cofactor
appear to be the key factors in predicting the hAOX isoform
selectivity and site of metabolism.
In summary, preliminary SMR guidelines are given in Fig. 7.
Although in the majority of aza-heterocycles the ortho C-H is
often oxidized by hAOX, the metabolism can be tuned not
only by addition of substituents at the predicted most positive
C–H, but also in other position (mostly EWG, excepting py-
rimidine) able to reduce the AC. Furthermore, hindered and
highly hydrophobic substituents are often able to drastically
change the SoM exposition, so that metabolic stability can be
achieved. Regarding amides, the amine counterpart is more
relevant to establish hAOX susceptibility: substituents in the
ortho < meta < para position of the anilide counterpart en-
hance the stability, EWG stabilizing the most the amidic bond.
To conclude, we believe the study herein reported represents
a solid base to construct an in silico model to predict the AOX
susceptibility of lead compounds and drug candidates.
Conclusion
In summary, a drug-like database of variously decorated aza-
aromatic scaffolds has been constructed, and all compounds
subsequently tested using the same experimental conditions.
Moreover, the in-depth study on several amides highlighted the
ability of this enzyme to hydrolyze the amidic bond, especially in
EDG-substituted anilides. As with the cytochromes, the bio-
transformation ability of AOX seems to depend on a combi-
nation of electronic and exposure factors. DFT calculations and
docking procedure have been performed to support this finding.
In some cases, comparison of calculated atomic charges on the
Fig. 6. Metabolism of additional designed and synthesized quinazolines
106a–c. * and ** are the sites with the highest AC (SI Appendix, Table S7).
According to docking analysis, * is most favorable exposed toward the MoCo.
Fig. 7. Stability of compounds toward AOX metabolism oxidation (A) and
hydrolysis (B) may be enhanced following the criteria reported in the arrows.
Lepri et al.
PNAS Early Edition
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separation was carried out according to the methods listed below. The
mobile phase was a mixture of water (solvent A) and acetonitrile (solvent B),
both containing formic acid at 0.1%. Method A was as follows: Aeris
Widepore 3.6-μm (C4, 100 × 4.6-mm) column at 30 °C using a flow rate of
0.850 mL/min in a 10-min gradient elution. Gradient elution was as follows: 100:0
(A/B) to 70:30 (A/B) over 9 min, 5:95 (A/B) for 1 min, and then reversion back to
100:0 (A/B) over 0.1 min. Method B was as follows: Acquity UPLC BEH C18 1.7-μm
(C18, 150 × 2.1-mm) column at 40 °C using a flow rate of 0.650 mL/min in a 10-min
gradient elution. Gradient elution was as follows: 99.5:0.5 (A/B) to 5:95 (A/B) over
8 min, 5:95 (A/B) for 2 min, and then reversion back to 99.5:0.5 (A/B) over 0.1 min.
The MS/MS data were processed using “MetaSite 4.2.2 Mass 3.0.22” and
“WebMetabase release-3.1.9” (Molecular Discovery, Ltd.). LC-MS chromatograms
in the reported figures were extracted by Mass Hunter (MassHunter Workstation
Software B.06.00 Qualitative Analysis; Agilent).
Materials and Methods
Materials. Solvents and pooled mixed-gender cryopreserved HLC (1 mg/mL)
were obtained from Sigma Aldrich. Tested compounds were obtained
through chemical synthesis (see above) or acquired from SPECS and
ENAMINE. All tested compounds were >98% pure as determined by
ultrahigh-performance liquid chromatography on Agilent Technologies
6540 UHD accurate-mass quadrupole time-of-flight (QTOF) LC/MS system.
Metabolism Assays on HLC. Metabolism of selected compounds was evaluated
upon incubation with HLC according to a modified Dalvie et al. (30) pro-
cedure. Test compound (10 μM) was incubated at pH 7.4 and 37 °C in the
presence of magnesium chloride hexahydrate (MgCl2·6H2O, 1 mM) and HLC.
After 0 and 60 min of incubation, the reaction was stopped by addition of
250 μL of ice-cold acetonitrile (containing 0.6 μM labetalol as internal stan-
dard). Blank was prepared similarly, but in absence of the investigated
compounds. Similarly, in the inhibition study, test compound (10 μM) was
incubated in the presence (50 μM) or absence of selective inhibitor DCPIP.
Proteins were precipitated by centrifugation at 5,000 rpm for 10 min
(Eppendorf, Italy; centrifuge 5810 R; rotor F-45-30-11) at room temperature
(RT), and an aliquot of supernatant (1 μL) was analyzed by LC-MS/MS on an
Agilent 1200 series HPLC coupled to an Agilent 6540 UHD accurate-mass
QTOF with a dual Jet Stream electron spray ionization source. Analytical
ACKNOWLEDGMENTS. We gratefully acknowledge Molecular Discovery,
Ltd., for Mass-MetaSite and WebMetabase software and financial support;
Nahong Qiu (F. Hoffmann-La Roche) for technical assistance; Istituto di
Scienze e Tecnologie Molecolari–Consiglio Nazionale delle Ricerche (Perugia)
for DFT calculation support; and Istituto di Biostrutture e Bioimmagini–
Consiglio Nazionale delle Ricerche (Naples) for providing the YIQDI-
VASTLK peptide.
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