Synthesis and evaluation of heterocyclic catechol mimics as inhibitors of catechol-o-methyltransferase (COMT)

Article abstract of DOI:10.1021/ml500502d

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg²⁺. The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

Full text of DOI:10.1021/ml500502d

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Evaluation of Heterocyclic Catechol Mimics as
Inhibitors of Catechol-O-methyltransferase (COMT)
Scott T. Harrison,† Michael S. Poslusney,†,⊥ James J. Mulhearn,† Zhijian Zhao,† Nathan R. Kett,†
Jeffrey W. Schubert,† Jeffrey Y. Melamed,† Timothy J. Allison,‡ Sangita B. Patel,‡ John M. Sanders,§
Sujata Sharma,‡ Robert F. Smith,‡ Dawn L. Hall,‡ Ronald G. Robinson,∥ Nancy A. Sachs,∥
Pete H. Hutson,∥ Scott E. Wolkenberg,*,† and James C. Barrow†,⊥
‡
§
∥
†Department of Medicinal Chemistry, Global Structural Biology, Chemical Modeling and Informatics, and Department of
Neuroscience Research, Merck Research Laboratories, Sumneytown Pike, West Point, Pennsylvania 19486, United States
S
* Supporting Information
ABSTRACT: 3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-
methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the
enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of
the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg2+.
The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed
COMT inhibitors.
KEYWORDS: Catechol O-methyl transferase, schizophrenia, cognition, catechol mimic
chizophrenia is a devastating neurodevelopmental disorder
amine oxidase (MAO) and catechol-O-methyltransferase
(COMT).
S
that affects approximately 1% of the population.1
Symptoms of schizophrenia are categorized into positive,
negative, and cognitive. Negative symptoms and cognitive
impairment are not well-treated with current drugs2 and
account for much of the long-term disability associated with the
disease.3 Therefore, new treatment options are needed,
especially ones that target the negative and cognitive symptoms
of schizophrenia.4
In contrast to the increase in midbrain dopaminergic
signaling thought to be important in the etiology of positive
symptoms,5 impaired dopamine activity in the prefrontal cortex
is thought to contribute to the cognitive deficits in
schizophrenia.6 One strategy to selectively modulate prefrontal
cortex (PFC) dopamine is to take advantage of the differential
modes of clearance of dopamine from different brain regions. In
the midbrain, there is extensive expression of the dopamine
transporter (DAT), which is thought to be primarily
responsible for dopamine clearance from the synapse.7 In
contrast, cortical regions exhibit only low levels of DAT
expression, and dopamine is cleared primarily by enzymatic
catabolism of dopamine, with a contribution from the
norepinephrine transporter (NET).8,9 The primary enzymes
responsible for dopamine catabolism in the PFC are mono-
While MAO A/B inhibitors are clinically used to treat
depression, they show only modest and variable effects on
cognition.10 In contrast, COMT has been extensively
characterized in terms of its role in modulating cognitive
function. The enzyme exists in a membrane-bound (MB-
COMT) form and a soluble form (S-COMT), with the MB
form predominating in the brain.11 Genetic and pharmaco-
logical manipulation of COMT activity has demonstrated
positive effects on cognition in rodent12 as well as human
studies.13,14 These studies provide validation for COMT
inhibition as a promising avenue for treatment of cognitive
deficits in schizophrenia, although no distinctions have yet been
made with regards to selective MB- or S-COMT inhibition.
Both forms of COMT are Mg-dependent and use S-
adenosylmethionine (SAM) as the methyl donor, and both
forms exhibit selectivity for catechol-containing substrates.
Received: December 5, 2014
Accepted: January 16, 2015
© XXXX American Chemical Society
A
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Small molecule inhibition of COMT has been extensively
studied in the context of Parkinson’s disease.15,16 The clinically
used nitrocatechol agents tolcapone 1 and entacapone 2
(Figure 1) were originally developed to improve the
a series of X-ray cocrystal structures of the human enzyme in
complex with novel inhibitors.
Ring-opening of the thiomorpholine ring embedded in 3
provides N-aryl 4-pyridinone analogues, which retain COMT
inhibition, and rapid analogue synthesis in this series was
enabled via the condensation reactions shown in Supporting
Information Scheme S1. The condensation of amines with a
variety of known kojic acid derivatives28,29 proceeded in good
yields and enabled the exploration of both the N-substituent as
well as the 4-pyridinone ring substituent structure−activity
relationship (SAR) in parallel. N-Alkyl and N-aryl derivatives
were prepared in the 6-hydroxymethyl-4-pyridinone series
(Table 1).
The N-alkyl analogues 6 and 7 lose most or all potency when
compared with 3 (Table 1). Among the N-aryl analogues, p-
methyl substitution provided an inhibitor approximately
a
Table 1. Profiles of Compounds 6−18
Figure 1. Marketed COMT inhibitors and hits from high-throughput
screening. For assay protocol, see refs 23 and 26.
pharmacokinetics of exogenously administered L-DOPA.17
While they are effective in blocking peripheral COMT activity,
entacapone has negligible brain penetration, and tolcapone has
low but measurable levels in the brain.18 Despite low brain
penetration, tolcapone improved cognition in schizophrenic
patients in an early phase clinical study.14 However, its
association with serious liver injury, including three deaths,
and requirement for liver function monitoring during dosing
limit its potential as a widespread treatment for schizophrenia.19
Since MB-COMT is the most prevalent isoform in human
brain, selective inhibition of MB-COMT has potential for
reducing the peripheral side effects of S-COMT inhibition.20−22
Further, MB-COMT is also reported to be overexpressed in
schizophrenia patients.21 Therefore, a new potent, selective,
and brain-penetrant COMT inhibitor has the potential for
widespread usage to treat cognitive deficits in schizophrenia.
This potential prompted a high-throughput screen of the
Merck compound collection using recombinantly expressed
MB-COMT.23 This screen identified several classes of
compounds related to the Mg2+-binding pharmacophore in
tolcapone, including catechols and heterocyclic catechol mimics
including 3-hydroxy-4-pyridinones (3), 5-hydroxy-4-pyrimidi-
nones (4), and 3-hydroxy-2-pyridinones (5, see Figure 1). Hits
3−5 are related to weak heterocyclic COMT inhibitors first
described in 1973.24 Because of the toxicity risks associated
with catechols (potential to form reactive o-quinones)25 and
the clinically observed hepatotoxicity of tolcapone, efforts were
directed toward optimization of these noncatechol lead
structures 3−5, which exhibit improved toxicity profiles versus
1 and 2 (tolcapone and entacapone).26 Specifically, 3−5 exhibit
>1000-fold lower potency than tolcapone and entacapone in an
assay measuring mitochondrial membrane potential disruption,
which has been associated with nitrocatechol hepatotoxicity.26
S-COMT can be crystallized,27 and optimization was guided by
a
For assay protocol, see refs 23 and 26.
B
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equipotent with the parent phenyl (compare 8 and 11), while
o-substitution diminished potency (e.g., 12).
Crystallographic studies with human S-COMT, which
confirm the Mg2+-binding properties of the HTS hits and
thus their similarity to tolcapone27,30 (Figure 2A), may explain
groups investigated. The effect on MB-COMT potency varied
from minimal (compare 8 and 10) to approximately 8-fold
(compare 8 or 10 versus 9 as well as 13 versus 14), and
scaffolds A and C were preferred over B. This small data set
suggested substitution may not be required for maximal
potency, and a larger study of the 4-pyridinone 6-position
substituent was undertaken to explore this further.
Analogues varying the 4-pyridinone 6-position substituent
were prepared from three routes including condensation of
kojic acid derivatives (Supporting Information Scheme S1),
nucleophilic addition to the aldehyde (Supporting Information
Scheme S2), and de novo 4-pyridinone ring synthesis31
(Supporting Information Scheme S3). These synthetic routes
provided analogues whose in vitro characterization is described
in Table 2.
a
Table 2. Profiles of Compounds 19−26
Figure 2. (A) Superposition of the 18/human S-COMT structure
(orange, PDB 4XUC) and tolcapone (1)/rat S-COMT structure
(gray, PDB 3S68). Human and rat S-COMT exhibit 74% sequence
identity overall and 94% sequence identity within the catechol binding
site (residues within 6 Å of tolcapone). Inhibitors are shown as sticks,
together with Mg2+ (green spheres), the cofactor S-adenosylmethio-
nine (SAM), and residues completing the octahedral complex around
the Mg2+. The novel inhibitor 18 binds in an orientation very similar
to tolcapone, with the two oxygen atoms of the hydroxypyridinone
chelating the active site Mg2+ atom. (B) Detailed view of 18/human S-
COMT complex (PDB 4XUC). The distal biaryl group of 18 makes
contacts with hydrophobic residues at the edge of the S-COMT active
site (shown as sticks).
this loss in potency as the structure of 18 suggests that there is
little room above and below the plane of the metal-binding
pharmacophore to accommodate the o-substituent in 12 due to
the presence of Trp 88 and Pro 224 (Figure 2B). Among the
methyl-substituted analogues, 3,5-dimethyl 13 was most potent.
Larger hydrophobic groups, both aromatic and aliphatic,
provide an additional potency boost when incorporated at
either meta or para positions (see 15−18). In the case of 18,
this may be explained by the formation of hydrophobic contacts
between the extended side chain of the inhibitor and residues
Trp 88, Cys 223, Leu 248, Arg 251, and Val 253 (Figure 2B).
The nature of these contacts differ from those observed with
tolcapone30 (Figure 2A), which because of the geometry of the
ketone linker between the aryl groups projects into solvent
instead of making additional contacts with the protein.
a
For assay protocol, see refs 23 and 26.
A range of substituents was explored in the 6-position,
including no substitution (18), methyl (19), aryl (20), and 1-
hydroxyalkyls (22−26). The observed SAR among these
analogues is consistent with the shallow and solvent-exposed
nature of the binding site and with the observation that 18
would direct 6-position substituents toward solvent in a
relatively unencumbered manner. This is consistent with the
Among the initial set of analogues prepared, the effect of 4-
pyridinone ring substitution depended on the nature and
position of the substituent and was similar across the N-aryl
C
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observation that stereochemistry in the 1-hydroxyalkyls did not
seem to affect potency, as enantiomers 22a and 22b exhibit
nearly identical IC50 values. The basic amine in 26 was not well
tolerated. Generally, rat MB-COMT activity tracked well with
human MB-COMT activity, and compounds exhibited a 10- to
20-fold selectivity for human MB-COMT versus S-COMT,
something not explained by our crystallographic studies with S-
COMT.
hydroxypyrimidinone scaffold, was observed crystallographi-
cally (Figure 3A). Although we cannot discount the effects of
A possible origin of MB-/S-COMT selectivity, though
speculative, is contact between the transmembrane domain of
MB-COMT and the binding site for these inhibitors as
suggested by modeling (see Figure 9B in ref 32). Such
interactions could be affected by the nature of the aromatic
group projecting from the Mg binder and are consistent with a
preference for hydrophobic groups in what appears, from the S-
COMT structures, to be a solvent-exposed area.
On the basis of the attractive profile of 18, related analogues
27a and 27b were prepared according to the routes in
Supporting Information Scheme S4. Generation of the 4-
pyrimidinone was accomplished via selective nucleophilic
addition of allyl alcohol, followed by allyl deprotection and
methylation (for 27b). Suzuki reaction and methyl ether
deprotection with BBr3 provided 27a and 27b.
These structures incorporate a meta-biaryl with a hydrox-
ypyrimidinone-based metal binder similar to that in the HTS
lead 4 (Table 3). Unsubstituted pyrimidinone 27a is a substrate
a
Table 3. Profiles of Compounds 27a, 27b, and 28
Figure 3. (A) Superposition of the novel heterocyclic inhibitors 18
(orange) and 27b (green) in complex with human S-COMT. As with
other inhibitors, 27b chelates the active site Mg2+ (PDB 4XUE). (B)
Putative hydrogen bonding network between 27b and S-COMT is
shown as black dashed lines (PDB 4XUE). Compound 27b is believed
to donate a hydrogen bond to Glu 249 and accept a hydrogen bond
from Asn 220. This is thought to result in the biaryl substituent being
oriented very differently than in 18 although it affects the active site
residues only slightly. Hydrogen atoms were added and optimized
using the Schrodinger Protein Preparation Wizard (Suite 2012:
̈
Maestro, version 9.3, Schrodinger, LLC, New York, 2012.).
̈
crystal packing, this may suggest that hydrogen bonds involving
the phenolic group of 27b and residues Asn 220 and Glu 249
(Figure 3B) may help orient inhibitors in the COMT binding
site.33
The putative H-bond to Glu249 is significant because it
appears to orient 27a and 27b such that the carbonyl oxygen
atom in these inhibitors is in close proximity to the methyl
group of SAM. This arrangement suggests a rationale for the
difference in COMT-catalyzed methylation susceptibility of 27a
and 27b since the nucleophilicity of the carbonyl oxygen atoms
in 27a and 27b differ. In 27a, the contribution of a
dihydroxypyrimidine tautomer renders the oxygen more
nucleophilic, and methylation by COMT is observed
experimentally. In 27b, the dihydroxypyrimidine tautomer is
not accessible since the pyrimidinone N-methyl group
maintains the carbonyl oxygen atom in the keto form; this
reduces its nucleophilicity and renders the compound a COMT
inhibitor and nonsubstrate. This proposed mechanism is
consistent with the observed differences between 27a and
27b and is distinct from the nitrocatechols such as tolcapone
a
For assay protocol, see refs 23 and 26.
of human COMT; its N-Me analogue 27b is not (vide infra)
and exhibits potency similar to the hydroxy-4-pyridinone 18
even though their substitution patterns differ, i.e., the biaryl is
para to the pyrimidinone hydroxyl group in 27b, while it is
meta in 4-pyridinone 18. A difference in the orientation of the
aryl substituents in 18 and 27b, rather than an alteration in the
orientation of the hydroxyl groups through rotation of the
D
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(1), where O-alkylation does not occur due to the electron-
withdrawing nitro substituent.
AUTHOR INFORMATION
■
Corresponding Author
An additional 4-pyridinone analogue of 18 was prepared
incorporating a boronic acid substituent ortho to the metal
binding keto group. Synthesis of this analogue (28) from O-
benzyl 18 proceeded with selective bromination followed by
Mg−bromine exchange, borylation, and deprotection (Support-
ing Information Scheme S5). The boronic acid in 28 was
designed to mimic the nitro group found in numerous COMT
inhibitors (for a review of recent applications of boronic acids
in medicinal chemistry, see ref 34) and was found to be
tolerated (compare 28 and 18), but not potency-enhancing for
human or rat MB-COMT. Our crystallography studies indeed
confirm that the boronic acid is isosteric with the nitro group
from tolcapone (Figure 4), but we expect its electronic effect to
deviate from nitro significantly (Hammett constants σp for
−B(OH)2 and −NO2 are 0.12 and 0.78, respectively).35
*Phone: 215-652-0821. E-mail: scott_wolkenberg@merck.com.
Present Address
⊥Lieber Institute for Brain Development, Johns Hopkins
School of Medicine, Room 336, John G. Rangos, Sr., Building,
855 North Wolfe Street, Baltimore, Maryland 21205, United
States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Ray McClain, Anna Dudkina, April
Cox, and Semhal Berhane for purifying novel analogues and
Michael Bogusky and Charles Ross, III for NMR and HRMS
determinations. We thank Stephen M. Soisson for assistance
depositing crystal structure coordinates.
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* Supporting Information
X-ray crystallography, syntheses, and characterization data for
key compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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