Design, synthesis and evaluation of 3-hydroxypyridin-4-ones as inhibitors of catechol-O-methyltransferase

Article abstract of DOI:10.1007/s11030-020-10053-x

Abstract: The most effective treatment of Parkinson’s disease is restoring central dopamine levels with levodopa, the metabolic precursor of dopamine. However, due to extensive peripheral metabolism by aromatic l-amino acid decarboxylase and catechol-O-me

Full text of DOI:10.1007/s11030-020-10053-x

Molecular Diversity
https://doi.org/10.1007/s11030-020-10053-x
ORIGINAL ARTICLE
Design, synthesis and evaluation of 3‑hydroxypyridin‑4‑ones
as inhibitors of catechol‑O‑methyltransferase
Johannie de Beer1 · Jacobus P. Petzer1,2 · Anna C. U. Lourens1,2 · Anél Petzer1,2
Received: 27 December 2019 / Accepted: 13 February 2020
© Springer Nature Switzerland AG 2020
Abstract
The most efective treatment of Parkinson’s disease is restoring central dopamine levels with levodopa, the metabolic
precursor of dopamine. However, due to extensive peripheral metabolism by aromatic l-amino acid decarboxylase and
catechol-O-methyltransferase (COMT), only a fraction of the levodopa dose reaches the brain unchanged. Thus, by preventing
levodopa metabolism and increasing the availability of levodopa for uptake into the brain, the inhibition of COMT would be
benefcial in Parkinson’s disease. Although nitrocatechol COMT inhibitors have been used in the treatment of Parkinson’s
disease, eforts have been made to discover non-nitrocatechol inhibitors. In the present study, the 3-hydroxypyridin-4-one
scafold was selected for the design and synthesis of non-nitrocatechol COMT inhibitors since the COMT inhibitory poten-
tial of this class has been illustrated. Using COMT obtained from porcine liver, it was shown that a synthetic series of ten
3-hydroxypyridin-4-ones are in vitro inhibitors with IC50 values ranging from 4.55 to 19.8 µM. Although these compounds
are not highly potent inhibitors, they may act as leads for the development of non-nitrocatechol COMT inhibitors. Such
compounds would be appropriate for the treatment of Parkinson’s disease.
Graphic abstract
3-Hydroxypyridin-4-ones have been synthesised and evaluated as non-nitrocatechol COMT inhibitors. In vitro, the IC50
values ranged from 4.55 to 19.8 μM.
Keywords 3-Hydroxypyridin-4-one · Catechol-O-methyltransferase · COMT · Inhibition · Parkinson’s disease
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10053-x) contains
supplementary material, which is available to authorized users.
Parkinson’s disease is a neurodegenerative disorder with
bradykinesia, resting tremor and rigidity as the core clinical
features. The motor symptoms of Parkinson’s disease are the
result of the functional loss of dopamine in the striatum due
* Anél Petzer
12264954@nwu.ac.za
Extended author information available on the last page of the article
Vol.:(0123456789)
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Molecular Diversity
to the degeneration of the nigrostriatal neuronal pathway
[1, 2]. The exact cause of Parkinson’s disease is unknown,
and symptoms worsen over time as the disease progresses.
Although current pharmacotherapy provides symptomatic
relief, no treatment has been proven to slow or halt the pro-
gression of the disease [3]. Levodopa, the metabolic precur-
sor of dopamine, remains the most efective treatment for
restoring dopamine levels in the parkinsonian brain [4, 5].
However, due to extensive peripheral enzymatic metabo-
lism by aromatic l-amino acid decarboxylase (AADC) and
catechol-O-methyltransferase (COMT), only a fraction of
the levodopa dose reaches the brain [6–8].
The COMT enzyme was frst described in 1958 by Julius
Axelrod and is responsible for the metabolism of com-
pounds that contain a catechol structure, such as levodopa
and dopamine [8, 9]. It also methylates other hydroxylated
metabolites and catechol compounds in the diet [8]. COMT
catalyses the transmission of a methyl group from S-adeno-
syl-l-methionine (SAMe) to a hydroxyl group of a catechol
substrate in the presence of magnesium. The enzyme is
widely distributed throughout the body with the liver con-
taining the highest COMT activity [9]. Membrane-bound
COMT (MB-COMT) and the soluble isoform of COMT,
S-COMT, are the two major isoforms of the enzyme [10].
S-COMT is dominant in the peripheral tissue, and MB-
COMT is mainly found in the brain.
Fig. 1 The structures of catechol, the nitrocatechol COMT inhibitors,
tolcapone and entacapone, and 3-hydroxypyridin-4-one
aetiology of Parkinson’s disease [16], it may be of clinical
beneft that 3-hydroxypyridin-4-ones display iron chelat-
ing activity [15, 17], which may reduce oxidative damage
to susceptible neurons; and (3) analgesic efects of certain
3-hydroxypyridin-4-ones have further been illustrated in
previous studies, indicating similar potency compared to
aspirin and similar anti-infammatory activity compared to
indomethacin [18]. This may be benefcial for neuroinfam-
mation, which has been implicated in neurodegeneration in
Parkinson’s disease [19].
COMT inhibitors are used in the symptomatic treatment
of Parkinson’s disease where the peripheral inhibition of
the O-methylation of levodopa increases the availability of
levodopa for uptake into the brain [9, 11]. New-generation
COMT inhibitors, such as the nitrocatechol COMT inhibi-
tors (e.g. entacapone or tolcapone), are thus frequently used
as adjunct to levodopa in Parkinson’s disease [12]. The clini-
cal value of nitrocatechol COMT inhibitors is sometimes
limited by adverse efects such as diarrhoea, hepatitis and
neurological reactions, particularly for tolcapone [8].
Based on these observations, a series of 3-hydroxypyri-
din-4-ones were selected for investigation as potential
COMT inhibitors. The 3-hydroxypyridin-4-one scafold
was chosen since the COMT inhibitory potential of several
compounds from this class has been illustrated [13, 14].
3-Hydroxypyridin-4-one is an isostere of the catechol ring,
but is normally not O-methylated by the enzyme (Fig. 1).
Further, it has been illustrated that non-nitrocatechol COMT
inhibitors can be MB-COMT specifc, which may be ben-
efcial when considering that the peripheral side efects of
COMT inhibitors are mostly related to their dopaminer-
gic efects [13]. 3-Hydroxypyridin-4-ones may have three
advantages over traditional nitrocatechol inhibitors: (1)
Several derivatives have been shown to be orally active and
are efciently absorbed from the gastrointestinal tract [15];
(2) due to the presence of excessive iron deposits in the
basal ganglia of the brain that have been associated with the
We thus report the synthesis of ten 3-hydroxypyridin-
4-ones (1–10) and their evaluation as potential inhibitors
of COMT (Table 1) [20]. Such compounds may represent
useful agents for the treatment of Parkinson’s disease with
improved safety profles compared to nitrocatechol COMT
inhibitors. This study explored diferent substituents on the
pyridine nitrogen with simple aromatic and aliphatic substi-
tution (1, 6 and 7), chain elongation and increasing fexibil-
ity (2, 3, 4 and 5), as well as halogen and methyl substitution
on the side chain phenyl ring (8, 9 and 10).
Results and discussion
Chemistry
The 3-hydroxypyridin-4-ones were synthesised by a method
described in the literature, whereby maltol was refuxed with
a suitable primary amine in an acidic environment with etha-
nol as co-solvent (Fig. 2) [21]. After removal of an oily resi-
due, the product precipitated from the reaction solution and
was collected by fltration and purifed by recrystallisation
from hot methanol. Compounds 6 and 7 difered in work-
up and were collected by extraction with ethyl acetate and
purifed by column chromatography. The synthesised com-
pounds were obtained in yields of 4–12%. Characterisation
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Molecular Diversity
Table 1 3-Hydroxypyridin-4-one derivatives and their IC50 values for
the inhibition of porcine liver COMT
COMT inhibition
To determine whether the synthesised 3-hydroxypyridin-
4-ones are in vitro inhibitors of COMT, commercially
available COMT from porcine liver was used as enzyme
source. A modified procedure reported in the literature
was validated and used to measure COMT activity [22].
For this purpose, (−)-norepinephrine served as enzyme
substrate, and the formation of the O-methylated product,
normetanephrine, was quantifed by reversed-phase high-
performance liquid chromatography (HPLC) with fuores-
cence detection. A Km value of 379 µM was estimated for
the methylation of (−)-norepinephrine by COMT, which is
similar to that reported in the literature of 488 µM [22]. The
determination of IC50 values should be carried out with the
substrate concentration approximately equal to Km, and a
typical enzyme reaction thus contained (−)-norepinephrine
(250 µM), SAMe, MgCl2 and the test inhibitors. The reac-
tions were initiated with the addition of the COMT enzyme,
and after 60-min incubation, the reactions were terminated
with perchloric acid. Following centrifugation, the superna-
tant fractions were analysed by HPLC for normetanephrine
content. By measuring COMT activity in the presence of
various concentrations of the test inhibitors, sigmoidal plots
of enzyme catalytic rate versus the logarithm of inhibitor
concentration were constructed from which the IC50 values
were determined (Fig. 3).
Compound
R
COMT IC50 (μM)a
1
–C6H5
–CH2C6H5
–(CH2)2C6H5
–(CH2)3C6H5
–(CH2)4C6H5
–C6H11
10.9 1.06
4.55 0.545
15.0 2.10
18.4 0.974
9.05 1.25
12.2 3.06
19.8 5.86
11.2 1.01
9.31 2.94
5.76 0.664
0.0068
2
3
4
5
6
7
–C5H9
8
–(C6H4)-3′-Cl
–(C6H4)-4′-Cl
–CH2(C6H4)-4′-CH3
9
10
Tolcapone
Entacapone
0.00047
aValues are expressed as the mean SD of triplicate determinations
The 3-hydroxypyridin-4-one derivatives exhibited COMT
inhibitory activity with 2 and 10 as the most potent COMT
inhibitors of the series with IC50 values of 4.55 μM and
5.76 μM, respectively (Table 1). Although active, these
3-hydroxypyridin-4-one derivatives are not as potent as the
reference inhibitors, entacapone (IC50 = 0.00047 µM) and
tolcapone (IC50 = 0.0068 µM). They may, however, serve
Fig. 2 General synthetic route for the synthesis of 3-hydroxypyridin-
4-one derivatives 1–10 by a single-step synthetic pathway. Key: (a)
HCl, H2O, ethanol, 72 h, 110 °C [21]
100
75
50
25
0
of the compounds was carried out by NMR spectroscopy,
IR spectroscopy and mass spectrometry. The purities of the
compounds were estimated by HPLC analyses. It was con-
frmed that the purities (90–99%) of the 3-hydroxypyridin-
4-one derivatives were acceptable for these compounds to
be subjected to biological evaluation. Structures were con-
frmed using 1H NMR, 13C NMR, DEPT90 and DEPT135
spectra. Two-dimensional (2D) spectroscopic data were
1
obtained by analysis of H–1H correlation spectroscopy
(COSY), heteronuclear single-quantum correlation spec-
troscopy (HSQC) and heteronuclear multi-bond correlation
spectroscopy (HMBC) data. Assignments were based on
analysis of 1D spectra in conjunction with the 2D spectra
(COSY, HSQC and HMBC) of these compounds (supple-
mentary material).
-2
-1
0
1
2
Log [I]
Fig. 3 Sigmoidal plot for the inhibition of COMT by compound 2.
Each data point represents the mean standard deviation (SD) of trip-
licate measurements
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Molecular Diversity
as leads for the development of non-nitrocatechol COMT
inhibitors with better side efect profles [13]. Further inves-
tigation in this regard is required. Based on the IC50 values,
preliminary structure–activity relationships (SARs) were
derived for the inhibition of COMT by the 3-hydroxypyri-
din-4-ones: (1) Phenyl substitution of the 3-hydroxypyridin-
4-one moiety (1) resulted in a higher IC50 value (and thus
lower inhibition potency) compared to benzyl substitution
(2). Benzyl substitution of the 3-hydroxypyridin-4-one moi-
ety also resulted in improved COMT inhibition compared to
phenylethyl (3), phenylpropyl (4) and phenylbutyl (5) substi-
tution. This showed that chain elongation of the substituent
reduces COMT inhibition potency and that the methylene
(CH2) linker between the 3-hydroxypyridin-4-one moiety
and phenyl ring was more optimal than the other linkers con-
sidered [(CH2)2, (CH2)3 and (CH2)4]. When comparing phe-
nyl (1) substitution of the 3-hydroxypyridin-4-one moiety to
cyclohexyl substitution (6), the aromatic substituent yielded
slightly more favourable inhibition than the aliphatic substit-
uent. Compared to cyclohexyl substitution (6), a reduction
in COMT inhibition potency was observed for cyclopentyl
substitution (7). In fact, 7 was the weakest COMT inhibitor
of the series of 3-hydroxypyridin-4-one derivatives. When
comparing the phenyl-substituted derivative (1) with the
derivatives containing chlorine on the phenyl ring, com-
pounds 8 and 9, it was apparent that chlorine substitution
on the meta and para positions of the phenyl ring did not sig-
nifcantly afect COMT inhibition potency. Similarly, when
the benzyl-substituted derivative 2 was substituted with a
methyl substituent on the para position of the phenyl ring
(to yield 10), COMT inhibitory activity remained relatively
unchanged. Benzyl substitution of the 3-hydroxypyridin-
4-one moiety (2 and 10) thus yielded the most potent COMT
inhibitory activity among the compounds evaluated. The
reference inhibitors, entacapone (IC50 = 0.00047 µM) and
tolcapone (IC50 = 0.0068 µM), proved to be highly potent
COMT inhibitors.
Molecular modelling
In an attempt to determine potential binding orientations and
interactions of the 3-hydroxypyridin-4-ones with the COMT
active site, molecular docking was carried out. The protein
model of human COMT complexed with 3,5-dinitrocat-
echol was obtained from the Protein Data Bank (PDB code:
3BWM) [23]. The catechol binding site of COMT presents
as a shallow cleft on the protein surface. It is defned by
Met40, Leu198, Tyr200 as well as the “gatekeeper” residues
Trp38 and Pro174, which ensures the correct orientation of
the substrate for methylation [23]. Mg2+, which is a co-factor
for the methylation reaction, is octahedrally coordinated to
the side chains of Asp141 and Asp169, Asn170, the two
hydroxy groups of the catechol substrate, and a water mol-
ecule [23]. Although not shown, the 1-hydroxy group of
3,5-dinitrocatechol is within hydrogen bonding distance to
Glu199 and Asn170 in the shallow catechol binding site,
while the 2-hydroxy and 3-nitro groups are within hydrogen
bond distance to Lys144 (Fig. 4). The potential for hydrogen
bonding and coordination with the Mg2+ ion underscores the
importance of the catechol structure for binding to COMT.
Molecular docking was carried out according to the pre-
viously reported protocol with the CDOCKER application
of the Discovery Studio [24]. The protein models were
prepared by frst calculating the pKa values and protona-
tion states of the amino acid residues, which was followed
by an energy minimisation with the protein backbone con-
strained. The structures of the ligands (3,5-dinitrocatechol
and compound 2) were drawn in Discovery Studio, and
after docking with CDOCKER, the docked orientations
were refned using in situ ligand minimisation. Among the
Fig. 4 The interactions of
3,5-dinitrocatechol (magenta)
with the active site of human
COMT (PDB code: 3BWM).
The docked orientation (teal)
of 3,5-dinitrocatechol is also
shown. (Color fgure online)
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Molecular Diversity
ten solutions generated, solutions existed for each ligand
where the catechol hydroxyl groups of 3,5-dinitrocatechol
or the 3-hydroxypyridin-4-one system of 2 are correctly
placed for coordination with the Mg2+. As these are most
probable, these solutions were selected and are presented
here. The accuracy of the docking procedure was evaluated
by redocking the co-crystallized ligand, 3,5-dinitrocat-
echol, into the active site of COMT. The root mean square
deviation (RMSD) of the selected docked orientation from
the position of the co-crystallised ligand was calculated to
be 0.418 Å. RMSD values < 1.5 Å are considered to be
successful, and this protocol was thus deemed appropriate
for docking experiments with COMT [25].
Conclusion
This study investigated a series of 3-hydroxypyridin-4-one
derivatives as potential inhibitors of COMT. Benzyl sub-
stitution of the 3-hydroxypyridin-4-one moiety (2 and 10)
yielded the most potent COMT inhibition of the series,
with the most potent inhibitor exhibiting an IC50 value of
4.55 µM. Although the 3-hydroxypyridin-4-ones are rela-
tively weak COMT inhibitors compared to the nitrocat-
echol compounds currently used, they may act as leads for
the development of non-nitrocatechol COMT inhibitors
for the treatment of Parkinson’s disease. In this respect,
3-hydroxypyridin-4-ones may exhibit other pharmacologi-
cal actions that are relevant to neurodegenerative disorders
(e.g. iron chelating, antioxidative and anti-infammatory
activities).
The result of the docking study shows that the
3-hydroxypyridin-4-one system of 2 is correctly placed for
coordination with the Mg2+ (Fig. 5). Although not shown,
the 3-hydroxy group is within hydrogen bond distance
from Asp141 and Lys144. The 2-methyl group overlaps
with the 3-nitro group of 3,5-dinitrocatechol. The 1-ben-
zyl side chain projects towards the exterior of the enzyme
and mainly undergoes van der Waals interactions with
Trp38 and Trp143. It may be concluded that compound 2
has the ability to adopt a similar orientation to 3,5-dini-
trocatechol within the COMT active site and forms the
appropriate polar interactions. The molecular reason for
its lower potency compared to the nitrocatechol inhibi-
tors, however, is not apparent. Based on the similarity of
binding and interaction network between 3,5-dinitrocat-
echol and 2, it may be postulated that with appropriate
structure modifcation, the COMT inhibition potencies of
3-hydroxypyridin-4-one may be signifcantly improved.
Experimental section
Chemicals and instrumentation
Reagents and solvents were obtained from Sigma-Aldrich
and were used without any further purifcation. Deuter-
ated dimethylsulfoxide (DMSO-d6) was used for nuclear
magnetic resonance (NMR) spectroscopy and was pur-
chased from Merck. A Bruker Avance III 600 spectrom-
eter was used to record proton (1H) and carbon (13C) NMR
spectra at 600 MHz and 150 MHz, respectively. To pro-
cess and analyse the NMR data, MestReNova2 was used.
Data reported included integration (e.g. 1H), multiplicity
and coupling constants (J) given in hertz (Hz). Chemical
shifts (δ), in parts per million (ppm), were referenced to
Fig. 5 The docked orientation
and predicted interactions of
compound 2 (orange) with the
active site of human COMT.
The orientation of 3,5-dini-
trocatechol (magenta) is also
shown. (Color fgure online)
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Molecular Diversity
1
the residual solvent (DMSO-d6) signal at 2.5 ppm for H
the reaction progress indicated that it was necessary to add
an additional 10 mmol (0.5 equiv.) of the primary amine to
complete the reaction. After refux was completed, a dark
oily residue was allowed to separate to the bottom of the
round-bottom fask. While still hot, the light yellow clear top
layer was decanted from the oily residue. The clear solution
was covered and incubated overnight at room temperature
during which a white solid precipitated from the solution.
The precipitate was collected by fltration the following
morning and allowed to air-dry overnight. Recrystallisation
from hot methanol gave a white crystalline solid.
NMR and 39.5 ppm for 13C NMR. Spin multiplicities were
presented as s (singlet), br s (broad singlet), d (doublet),
br d (broad doublet), ddd (doublet of doublet of doublets),
t (triplet), br t (broad triplet), p (pentet) or m (multiplet).
Note that the OH proton of the 3-hydroxy group was not
1
observed in the H NMR spectra. A Bruker micrOTOF-Q
II mass spectrometer functioning in atmospheric-pressure
chemical ionisation (APCI) mode was used to record high-
resolution mass spectra (HRMS). Infrared spectra were
obtained using an Alpha Bruker Fourier Transform Infra-
red (FTIR) spectrometer. Opus 7.0.129 software was used
to process and record IR data, reporting absorption bands in
cm−1. TLC (thin-layer chromatography) was used to monitor
the progress of the reactions and to determine whether the
reactions were complete. The reagents and products were
dissolved in methanol, applied to silica gel 60 aluminium-
coated TLC sheets (Merck) and developed in an appropriate
mobile phase. Ethyl acetate was used as eluent during TLC
of compounds 1, 2, 8 and 9, while a mixture of methanol
and ethyl acetate (9:1) was used as mobile phase for com-
pounds 3–7 and 10. The developed TLC sheets were visu-
alised under an UV lamp at a wavelength of 254 nm. HPLC
(high-performance liquid chromatography) analyses were
performed to estimate the purities of the synthesised com-
pounds. These were conducted with an Agilent 1100 HPLC
system equipped with a quaternary pump and an Agilent
1100 series diode array detector. HPLC-grade acetonitrile
(Merck) and Milli-Q water (Millipore) were used for the
preparation of the mobile phase. A Venusil XBP C18 col-
umn (4.60×150 mm, 5 µm) was used as analytical column.
Initially, the mobile phase consisted of 30% acetonitrile and
70% Milli-Q water and the fow rate was set to 1 mL/min. A
solvent gradient programme was initiated at the beginning
of each HPLC run. This was done by linearly increasing the
composition of the acetonitrile in the mobile phase to 85%
acetonitrile over a 5-min period. HPLC runs lasted 15 min,
and a 5-min period was allowed for equilibration of the sys-
tem. The test compounds were dissolved in acetonitrile to
a concentration of 1 mM, and 20 µL of these solutions was
injected into the HPLC system. The eluent was monitored
at a wavelength of 254 nm.
Synthesis of 3‑hydroxypyridin‑4‑one derivatives 6
and 7
The synthesis of 6 and 7 follows the same general proce-
dure as described for the 3-hydroxypyridin-4-one deriva-
tives above, but difers in the work-up. After refux was
completed, the reactions did not yield dark oily residues,
and no precipitate was obtained after cooling of the reac-
tions. Instead, the resulting reaction mixture remained a
clear red orange solution. After cold distilled water (20 mL)
was added to the reaction, it was extracted to ethyl acetate
(1×20 mL for 6; 2×30 mL for 7). The combined organic
layers were dried over anhydrous magnesium sulphate and
fltered, and the solvent was removed by rotary evaporation.
The crude product was then allowed to air-dry to yield a red
powder that was purifed with silica gel column chromatog-
raphy (methanol/ethyl acetate, 9:1).
3‑Hydroxy‑2‑methyl‑1‑phenylpyridin‑4‑one (1) The title com-
pound was prepared from maltol and aniline in a yield of
6.56% (132 mg): mp 223.3–223.9 °C (methanol) (lit. 221–
222 °C [21]), white crystals. 1H NMR (600 MHz, DMSO-d6)
δ 7.60–7.50 (m, 4H, H-6, H-3′/5′, H-4′), 7.48–7.43 (m, 2H,
H-2′/6′), 6.22 (d, J =7.3 Hz, 1H, H-5), 1.96 (s, 3H, CH3).
13C NMR (151 MHz, DMSO-d6) δ 169.7 (C-4), 145.1 (C-3),
141.6 (C-1′), 137.9 (C-6), 129.7 (C-3′/5′), 129.1 (C-4′),
128.8 (C-2), 127.0 (C-2′/6′), 111.0 (C-5), 13.4 (CH3). APCI-
HRMS m/z: calcd for C12H12NO2 (MH+), 202.0863, found
202.0854; purity (HPLC): 99% (254 nm). FTIR v/cm−1
:
3197 (broad OH), 1626 (C=O), 1578 (C=C), 1235 (C–N).
Synthesis of 3‑hydroxypyridin‑4‑one derivatives
1–5, 8–10
1‑Benzyl‑3‑hydroxy‑2‑methylpyridin‑4‑one (2) The title
compound was prepared from maltol and benzylamine in a
yield of 4.88% (105 mg): mp 205.6–207.1 °C (methanol),
1
The synthesis of the 3-hydroxypyridin-4-one derivatives
was achieved in a single-step synthesis and is based on a
modifcation of the procedure reported in the literature [21].
Commercially available maltol (20 mmol, 1 equiv.) was
heated under refux at 110 °C with a suitable primary amine
(30 mmol, 1.5 equiv.) in the presence of HCl (32%, 0.8 mL),
water (36 mL) and ethanol (4 mL) for 72 h. TLC analysis of
white crystals. H NMR (600 MHz, DMSO-d6) δ 7.75 (d,
J = 7.3 Hz, 1H, H-6), 7.37 (br t, J = 7.6 Hz, 2H, H-3′/5′),
7.33–7.27 (m, 1H, H-4′), 7.06 (br d, J=7.0 Hz, 2H, H-2′/6′),
6.21 (d, J = 7.2 Hz, 1H, H-5), 5.25 (s, 2H, H-1″), 2.12 (s,
3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 169.3 (C-4),
145.8 (C-3), 138.6 (C-6), 137.1 (C-1′), 129.1 (C-2), 129.0
(C-3′/5′), 127.7 (C-4′), 126.1 (C-2′/6′), 110.9 (C-5), 56.0
1 3

Molecular Diversity
(C-1″), 11.6 (CH3). APCI-HRMS m/z: calcd for C13H14NO2
(MH+), 216.1019, found 216.1003; purity (HPLC): 99%
(254 nm). FTIR v/cm−1: 3175 (broad OH), 1626 (C=O),
1574 (C=C), 1234 (C–N).
(HPLC): 98% (254 nm). FTIR v/cm−1: 3079 (broad OH),
1626 (C=O), 1567 (C=C), 1223 (C–N).
1‑Cyclohexyl‑3‑hydroxy‑2‑methylpyridin‑4‑one (6) The title
compound was prepared from maltol and cyclohexylamine
in a yield of 6.86% (142 mg): mp 191.6–202.8 °C (metha-
nol), red solid. 1H NMR (600 MHz, DMSO-d6) δ 7.67 (br
s, 1H, H-6), 6.15 (br s, 1H, H-5), 4.02 (br s, 1H, H-1′), 2.32
3‑Hydroxy‑2‑methyl‑1‑(2‑phenylethyl)pyridin‑4‑one (3) The
title compound was prepared from maltol and 2-phenyl-
1-ethylamine in a yield of 6.42% (147 mg): mp 159.4–
160.8 °C (methanol), white crystals. H NMR (600 MHz,
(s, 3H, CH3), 2.09–0.74 (m, 10H, H-2′/6′, H-3′/5′, H-4′). 13
C
1
DMSO-d6) δ 7.45 (d, J = 7.3 Hz, 1H, H-6), 7.29 (br t,
J = 7.3 Hz, 2H, H3′/5′), 7.26–7.17 (m, 3H, H-2′/6′, H-4′),
6.06 (d, J=7.2 Hz, 1H, H-5), 4.14 (t, J=7.5 Hz, 2H, H-1″),
2.95 (t, J=7.5 Hz, 2H, H-2″), 2.25 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ 168.9 (C-4), 145.4 (C-3), 137.52
(C-6), 137.46 (C-1′), 129.0 (C-2′/6′ or C-3′/5′), 128.7 (C-2),
128.5 (C-2′/6′ or C-3′/5′), 126.7 (C-4′), 110.6 (C-5), 53.9
(C-1″), 36.3 (C-2″), 11.3 (CH3). APCI-HRMS m/z: calcd
for C14H16NO2 (MH+), 230.1176, found 230.1186; purity
(HPLC): 99% (254 nm). FTIR v/cm−1: 3188 (broad OH),
1625 (C=O), 1558 (C=C), 1231 (C–N).
NMR (151 MHz, DMSO-d6) δ 168.6 (C-4), 145.3 (C-3),
133.3 (C-6), 128.8 (C-2), 110.8 (C-5), 59.0 (C-1′), 32.5
(C-2′/6′), 25.3 (C-3′/5′), 24.6 (C-4′), 11.3 (CH3). APCI-
HRMS m/z: calcd for C12H18NO2 (MH+), 208.1332, found
208.1344; purity (HPLC): 92% (254 nm). FTIR v/cm−1
:
3081 (broad OH), 1619 (C=O), 1566 (C=C), 1216 (C–N).
1‑Cyclopentyl‑3‑hydroxy‑2‑methylpyridin‑4‑one (7) The title
compound was prepared from maltol and cyclopentylamine
in a yield of 8.39% (162 mg): mp 51.4–51.5 °C (methanol),
red solid. 1H NMR (600 MHz, DMSO-d6) δ 7.62 (br s, 1H,
H-6), 6.17 (br s, 1H, H-5), 4.61 (br s, 1H, H-1′), 2.46–1.28
(m, 11H, H-2′/5′, H-3′/4′, CH3). 13C NMR (151 MHz,
DMSO-d6) δ 168.7 (C-4), 145.4 (C-3), 133.1 (C-6), 129.3
(C-2), 111.1 (C-5), 60.9 (C-1′), 32.5 (C-2′/5′), 23.7 (C-3′/4′),
11.7 (CH3). APCI-HRMS m/z: calcd for C11H16NO2 (MH+),
194.1176, found 194.1174; purity (HPLC): 91% (254 nm).
FTIR v/cm−1: 3078 (broad OH), 1621 (C=O), 1562 (C=C),
1212 (C–N).
3‑Hydroxy‑2‑methyl‑1‑(3‑phenylpropyl)pyridin‑4‑one (4) The
title compound was prepared from maltol and 3-phenyl-
1-propylamine in a yield of 12.92% (294 mg): mp 157.2–
1
159.3 °C (methanol), white crystals. H NMR (600 MHz,
DMSO-d6) δ 7.57 (d, J=7.3 Hz, 1H, H-6), 7.31–7.25 (m,
2H, H-3′/5′), 7.25–7.16 (m, 3H, H-2′/6′, H-4′), 6.13 (d,
J=7.2 Hz, 1H, H-5), 3.94 (t, J=7.4 Hz, 2H, H-1″), 2.60 (t,
J=7.8 Hz, 2H, H-3″), 2.23 (s, 3H, CH3), 1.94 (p, J=7.8 Hz,
2H, H-2″). 13C NMR (151 MHz, DMSO-d6) δ 168.9 (C-4),
145.5 (C-3), 140.8 (C-1′), 137.5 (C-6), 128.6 (C-2), 128.4
(C-2′/6′ or C-3′/5′), 128.2 (C-2′/6′ or C-3′/5′), 126.0 (C-4′),
110.7 (C-5), 52.4 (C-1″), 31.8 (C-2″, C-3″), 11.3 (CH3).
APCI-HRMS m/z: calcd for C15H18NO2 (MH+), 244.1332,
found 244.1331; purity (HPLC): 99% (254 nm). FTIR v/
cm−1: 3118 (broad OH), 1624 (C=O), 1572 (C=C), 1219
(C–N).
1‑(3‑Chlorophenyl)‑3‑hydroxy‑2‑methylpyridin‑4‑one (8) The
title compound was prepared from maltol and 3-chloro-
aniline in a yield of 5.06% (119 mg): mp 185.6–186.2 °C
1
(methanol), white solid. H NMR (600 MHz, DMSO-d6)
δ 7.68 (br t, J=2.0 Hz, 1H, H-2′), 7.64–7.55 (m, 3H, H-6′,
H-5′, H-6), 7.46 (ddd, J=7.7, 2.1, 1.2 Hz, 1H, H-4′), 6.23
(d, J = 7.3 Hz, 1H, H-5), 1.97 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ 169.8 (C-4), 145.1 (C-3), 142.7
(C-1′), 137.9 (C-6), 133.8 (C-3′), 131.2 (C-6′), 129.4 (C-5′),
128.9 (C-2), 127.3 (C-2′), 126.2 (C-4′), 111.2 (C-5), 13.4
(CH3). APCI-HRMS m/z: calcd for C12H11ClNO2 (MH+),
236.0473, found 236.0446; purity (HPLC): 94% (254 nm).
FTIR v/cm−1: 3057 (broad OH), 1627 (C=O), 1561 (C=C),
1245 (C–N).
3‑Hydroxy‑2‑methyl‑1‑(4‑phenylbutyl)pyridin‑4‑one (5) The
title compound was prepared from maltol and 4-phenyl-
1-butylamine in a yield of 4.90% (126 mg): mp 171.6–
1
173.7 °C (methanol), white crystals. H NMR (600 MHz,
DMSO-d6) δ 7.55 (d, J=7.3 Hz, 1H, H-6), 7.30–7.24 (m,
2H, H-3′/5′), 7.21–7.14 (m, 3H, H-2′/H-6′, H-4′), 6.09 (d,
J=7.2 Hz, 1H, H-5), 3.94 (t, J=7.3 Hz, 2H, H-1″), 2.59 (t,
J=7.6 Hz, 2H, H-4″), 2.25 (s, 3H, CH3), 1.68–1.50 (m, 4H,
H-2″, H-3″). 13C NMR (151 MHz, DMSO-d6) δ 168.8 (C-4),
145.5 (C-3), 141.8 (C-1′), 137.6 (C-6), 128.5 (C-2), 128.32
(C-2′/6′ or C-3′/5′), 128.28 (C-2′/6′ or C-3′/5′), 125.8 (C-4′),
110.5 (C-5), 52.7 (C-1″), 34.6 (C-4″), 29.8 (C-2″ or C-3″),
27.7 (C-2″ or C-3″), 11.3 (CH3). APCI-HRMS m/z: calcd
for C16H20NO2 (MH+), 258.1489, found 258.1481; purity
1‑(4‑Chlorophenyl)‑3‑hydroxy‑2‑methylpyridin‑4‑one (9) The
title compound was prepared from maltol and 4-chloroani-
line in a yield of 5.09% (120 mg): mp 298.1–298.4 °C (meth-
1
anol), white solid. H NMR (600 MHz, DMSO-d6) δ 7.63
(d, J=8.7 Hz, 2H, H-2′/6′ or H-3′/5′), 7.55 (d, J =7.3 Hz,
1H, H-6), 7.51 (d, J=8.7 Hz, 2H, H-2′/6′ or H-3′/5′), 6.22
(d, J = 7.3 Hz, 1H, H-5), 1.96 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ 169.8 (C-4), 145.1 (C-3), 140.4
(C-1′), 137.9 (C-6), 133.7 (C-4′), 129.7 (C-2′/6′ or C-3′/5′),
1 3

Molecular Diversity
129.1 (C-2′/6′ or C-3′/5′), 128.7 (C-2), 111.1 (C-5), 13.4
(CH3). APCI-HRMS m/z: calcd for C12H11ClNO2 (MH+),
236.0473, found 236.0465; purity (HPLC): 90% (254 nm).
FTIR v/cm−1: 3047 (broad OH), 1622 (C=O), 1582 (C=C),
1233 (C–N).
For the HPLC analysis of normetanephrine, an Agilent
1100 HPLC system equipped with a Shimadzu RF-551 fuo-
rescence detector was used. A Phenomenex USP L1 Luna
C18 column (250 × 4.6 mm, 5 μm) was used for chroma-
tographic separation. A mixture of an aqueous phase and
an organic phase was used to prepare the mobile phase.
The aqueous phase consisted of sodium phosphate bufer
(10 mM, pH 7.0), boric acid (80 mM) and sodium 1-hex-
anesulfonate (4 mM). The organic phase consisted of ace-
tonitrile, and the aqueous and organic phases were mixed
in a ratio of 85:15. The HPLC system was set to a fow
rate of 1 mL/min with a sample injection volume of 20
μL, and fuorescence detection was carried out at an exci-
tation wavelength of 283 nm and an emission wavelength
of 315 nm. A retention time of 4.7 min was recorded for
normetanephrine [22].
3‑Hydroxy‑2‑methyl‑1‑(4‑methylbenzyl)pyridin‑4‑one
(10) The title compound was prepared from maltol and
4-methylbenzylamine in a yield of 7.68% (176 mg):
1
mp 213.7–226.0 °C (methanol), white solid. H NMR
(600 MHz, DMSO-d6) δ 7.73 (d, J = 7.3 Hz, 1H, H-6),
7.18 (d, J = 7.8 Hz, 2H, H-3′/5′), 6.96 (d, J = 7.9 Hz, 2H,
H-2′/6′), 6.19 (d, J=7.2 Hz, 1H, H-5), 5.19 (s, 2H, H-1″),
2.27 (s, 3H, CH3 benzyl), 2.12 (s, 3H, CH3 pyridone). 13
C
NMR (151 MHz, DMSO-d6) δ 169.2 (C-4), 145.8 (C-3),
138.5 (C-6), 137.0 (C-1′), 134.0 (C-4′), 129.5 (C-3′/5′),
129.0 (C-2), 126.1 (C-2′/6′), 110.8 (C-5), 55.8 (C-1″), 20.7
(CH3 benzyl), 11.6 (CH3 pyridone). APCI-HRMS m/z: calcd
for C14H16NO2 (MH+), 230.1176, found 230.1166; purity
(HPLC): 93% (254 nm). FTIR v/cm−1: 3203 (broad OH),
1627 (C=O), 1576 (C=C), 1239 (C–N).
Molecular docking studies
Discovery Studio 3.1 (Accelrys) was used to carry out
molecular docking. The protein model of human COMT
was obtained from the Protein Data Bank (PDB code:
3BWM) [23]. The default ‘prepare protein’ option was used
to protonate the model, and the pKa values and protona-
tion states of the ionisable amino acids were calculated at
pH 7.4. The valences of the cofactors and co-crystallised
ligand (3,5-dinitrocatechol) were corrected, and the protein
models were automatically typed with the Momany and
Rone CHARMm forcefeld. The Smart Minimizer algo-
rithm was used to minimise the protein structure using a
fxed backbone constraint and the generalised born approxi-
mation with molecular volume (GBMV) as solvent model
(50,000 steps). The crystal water molecules were deleted,
and the binding site was defned from the position of the
co-crystallised ligand. The structures of 2 and 3,5-dinitro-
catechol were constructed in Discovery Studio and prepared
using the ‘prepare ligands’ function. The Momany and Rone
CHARMm forcefeld was used to assigned atom potential
types and partial charges to the ligands. The prepared ligands
were then docked into the active site of the COMT model
using the CDOCKER algorithm, allowing for ten random
ligand conformations and the heating target temperature set
to 700 K in full potential mode. The docking solutions were
fnally refned using in situ ligand minimisation with the
Smart Minimizer algorithm. The illustrations were prepared
with the PyMOL molecular graphics system [26].
COMT inhibition studies
Purifed COMT from porcine liver (≥150 units/mg), nor-
metanephrine hydrochloride and SAMe were obtained from
Sigma-Aldrich. The enzyme stock solution was prepared by
dissolving the purifed COMT (750 units/5 mL) in sodium
phosphate bufer (25 mM, pH 7.8) containing dithiothrei-
tol (0.5 mM). The test inhibitors were prepared in DMSO
and added to the reactions at concentrations of 0.1–100 µM,
while the reference inhibitors, tolcapone and entacapone,
were evaluated at 0.0003–1 µM. The fnal DMSO concen-
tration in the reactions was 4%. The enzyme reactions (125
µL) contained MgCl2 (2 mM; 10 µL), (−)-norepinephrine
(250 µM; 35 µL), SAMe (200 µM, 25 µL) and the test inhibi-
tor (5 µL). After the reactions were pre-incubated at 37 °C
in a water bath for at least 10 min, a 50-µL aliquot of the
enzyme stock solution was added. The reactions were incu-
bated for 60 min and subsequently terminated by adding
12.5 µL perchloric acid (1 M). The samples were centri-
fuged at 16,000×g for 10 min, and the supernatants were
analysed by HPLC. The peak areas of normetanephrine
were recorded, and the corresponding concentrations of nor-
metanephrine formed were calculated using a calibration
curve (1–30 µM). Using the Prism 5 (GraphPad) software
package, the data were ftted to the equation for one-site
competition to yield a sigmoidal curve of concentration of
normetanephrine formed versus the logarithm of inhibitor
concentration. IC50 values were estimated from these plots.
For each test inhibitor, the IC50 values were determined in
triplicate and expressed as mean SD.
Acknowledgements The NMR and MS spectra were recorded by
André Joubert and Johan Jordaan of the Laboratory for Analytical
Services (LAS) in the focus area Chemical Resource Benefciation,
respectively, while HPLC analyses were carried out by Jan du Preez
of the Analytical Technology Laboratory (ATL) at the North-West
University (NWU).
1 3

Molecular Diversity
Walker TL, Kershner JL, Bhandari N, Hutson PH, Sachs NA
(2012) Characterization of non-nitrocatechol pan and isoform
specifc catechol-O-methyltransferase inhibitors and substrates.
ACS Chem Neurosci 3:129–140. https://doi.org/10.1021/cn200
109w
Funding The fnancial assistance of the National Research Founda-
tion (NRF) of South Africa [Grant specifc unique Reference Numbers
(UID) 85642, 96180] towards this research is hereby acknowledged.
The Grantholders acknowledge that opinions, fndings and conclu-
sions or recommendations expressed in any publication generated by
the NRF supported research are that of the authors and that the NRF
accepts no liability whatsoever in this regard.
14. Borchardt RT (1973) Catechol O-methyltransferase. 4. In vitro
inhibition by 3-hydroxy-4-pyrones, 3-hydroxy-2-pyridones, and
3-hydroxy-4-pyridones. J Med Chem 16:581–583. https://doi.
org/10.1021/jm00263a047
Compliance with ethical standards
15. Rai BL, Liu ZD, Liu DY, Lu SL, Hider RC (1999) Synthesis,
physicochemical properties and biological evaluation of ester
prodrugs of 3-hydroxypyridin-4-ones: design of orally active
chelators with clinical potential. Eur J Med Chem 34:475–485.
https://doi.org/10.1016/S0223-5234(99)80097-X
Conflict of interest The authors declare that they have no confict of
interest.
16. Zucca FA, Segura-Aguilar J, Ferrari E, Munoz P, Paris I, Sulzer
D, Sarna T, Casella L, Zecca L (2017) Interactions of iron, dopa-
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Molecular Diversity
Afliations
Johannie de Beer1 · Jacobus P. Petzer1,2 · Anna C. U. Lourens1,2 · Anél Petzer1,2
1
2
Centre of Excellence for Pharmaceutical Sciences, North-
West University, Private Bag X6001, Potchefstroom 2520,
South Africa
Pharmaceutical Chemistry, School of Pharmacy, North-West
University, Private Bag X6001, Potchefstroom 2520,
South Africa
1 3