Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol O-Methyltransferase

Article abstract of DOI:10.1021/acsmedchemlett.9b00345

A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies, levels of unbound drug exposure are seen in the brain, which exceed their respective IC₅₀s, leading to changes in the levels of dopamine metabolites in a manner consistent with COMT inhibition.

Full text of DOI:10.1021/acsmedchemlett.9b00345

Subscriber access provided by Syracuse University Libraries
Letter
Synthesis and Evaluation of Bicyclic Hydroxypyridones
as Inhibitors of Catechol O-methyltransferase
Glen Ernst, Daniel Akuma, Vinh Au, Ingrid P. Buchler, Spencer Byers, Gregory V Carr, Sabine Defays, Pablo
de Leon, Thierry Demaude, Michael DePasquale, Veronique Durieu, Yifang Huang, Emilie Jigorel, Martha
Kimos, Anna Kolobova, Florian Montel, Florence Moureau, Michael Poslusney, Dominique Swinnen, Marie-
Christine Vandergeten, Nathalie Van houtvin, Huijun Wei, Noelle White, Martyn Wood, and James C Barrow
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00345 • Publication Date (Web): 22 Oct 2019
Downloaded from pubs.acs.org on October 24, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of Catechol O-methyltransferase
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
†
†
†
†
†,‡
Glen Ernst, Daniel Akuma, Vinh Au, Ingrid P. Buchler, Spencer Byers, Gregory V. Carr,
§
†
§
†
§
Sabine Defays, Pablo de León, Thierry Demaude, Michael DePasquale, Véronique Durieu,
Yifang Huang, Emilie Jigorel, Martha Kimos, Anna Kolobova, Florian Montel, Florence
Moureau, Michael Poslusney, Dominique Swinnen, Marie-Christine Vandergeten, Nathalie
Van houtvin, Huijun Wei, Noelle White, Martyn Wood, and James C. Barrow
†
§
†
†
§
§
†
§
§
§
†,‡
†
§
*,†,‡
†Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, Maryland 21205, United States
‡Department of Pharmacology, Johns Hopkins University School of Medicine, 855 North Wolfe Street, Baltimore,
Maryland 21287, United States
§UCB Biopharma, SPRL, B-1420 Braine-L’Alleud, Belgium
KEYWORDS: Catechol O-methyl transferase, catechol mimic, enzyme inhibitors, dopamine metabolism
ABSTRACT: A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase
(
COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent
inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series,
but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies,
levels of unbound drug exposure are seen in the brain which exceed their respective IC50s, leading to changes in
the levels of dopamine metabolites in a manner consistent with COMT inhibition.
Impaired dopamine activity in the prefrontal cortex (PFC)
is thought to contribute to the cognitive deficits in several
conditions such as obsessive-compulsive disorder, traumatic
COMT inhibitors have demonstrated utility and are widely
prescribed for treatment of Parkinson’s disease to increase
levels of exogenously administered L-DOPA through their
1
8
brain injury, ADHD and schizophrenia. An approach toward
ability to inhibit peripheral metabolism. Among known
selectively modulating dopamine signaling in the PFC is by
inhibiting the activity of catechol O-methyltransferase
COMT inhibitor scaffolds, nitrocatechols have distinguished
themselves by providing clinically used drugs tolcapone 1 and
entacapone 2, as well as the more recently approved
opicapone 3 (Figure 1). Although the nitrocatechols show
efficacy for inhibition of COMT, entacapone and opicapone
have negligible CNS penetration and tolcapone gives low but
measurable brain exposure.
symptoms are more effectively treated by COMT inhibitors
with greater brain penetration, and these compounds are
useful for additional neurological and psychiatric conditions
such as cognitive impairment associated with schizophrenia.
Tolcapone has achieved some early clinical success, but its
(
COMT). COMT is a magnesium-containing metalloenzyme
9
that transfers methyl group from the cofactor S-
a
adenosylmethionine (SAM) to dopamine, as well as a number
of endogenous and exogenous catechols. Due to the lack of
expression of synaptic dopamine transporters in the PFC as
1
0
Parkinson’s nonmotor
2,3
compared to the striatum, COMT is the predominant means
of dopamine processing in the PFC.
1
1
COMT exists in two isoforms, designated MB-COMT for
the membrane-bound form and S-COMT for the soluble
1
2
4
form. The isoforms have identical enzymatic domains, with
1
3
impact is limited by hepatic toxicity associated with the drug.
MB-COMT having an extra 50 amino acids at the N-terminus
for membrane anchoring that also influences enzymatic
The risk-benefit profile of tolcapone has severely limited its
more general use. As a result, a need for new COMT inhibitors
exists, particularly those that are CNS penetrant as well as
those arising from alternative pharmacophores to help
mitigate the possible toxicity risk related to the nitrocatechol
moiety.
5
activity. The two COMT isoforms also differ in their
expression pattern, with MB-COMT being more prevalent in
the brain while S-COMT is predominant in the periphery,
although this differential expression is more significant in
6,7
humans as compared to rodents. Selectivity for MB-COMT
over S-COMT may then be theoretically desirable to achieve
the goal of central COMT inhibition.
1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 13
OH
OH
OH
The imine was then reduced with sodium borohydride to give
the O-protected parent 14. Addition of various N-substituents
was achieved through reductive amination, alkylation,
acylation or sulfonylation to enable rapid analogue synthesis
and give penultimate material with the general structure 15.
Deprotection under acidic conditions then afforded the final
hydroxypyridone products 16-41.
OH
OH
OH
Cl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O-
N
O-
O-
N+
O
N+
O
N
N+
O
N+
O
O
O
N
-O
N
Cl
1
2
3
tolcapone
entacapone
opicapone
Figure 1. COMT inhibitors used clinically
MB-COMT inhibition data21 for an initial set of N-
substituted hydroxypyridones are shown in Table 1. The
parent unsubstituted compound 16 shows no measurable
inhibition of the enzyme under assay conditions. Introduction
of aliphatic N-substituents, as with 17 and 18, show
The nitrocatechols are among COMT inhibitors which
have been shown to bind in the catechol binding site of the
enzyme, rather than the site to which the cofactor S-
adenosylmethionine (SAM) occupies. The catechols and their
mimetics are typically bidentate chelators to the magnesium
in the catalytic domain, providing two of the six coordinates
in the octahedral geometry of the dication. Besides
nitrocatechols, other scaffolds which have given rise to COMT
inhibitors are generically shown in Figure 2 including 8-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
incremental improvements in potency, giving
a sub-
micromolar IC50 in the case of 18. This could be viewed as due
to nonspecific binding from the addition of lipophilicity, but
an analysis of subsequent compounds show this to be more
nuanced. The N-benzyl analogue 19 displays an additional
potency improvement with an IC50 of 220 nM. Incorporation
of an additional methylene in the N-phenethyl compound 20
shows a 10-fold loss in potency compared to 19, suggesting a
limit to potency increases that can be attributed merely to
lipophilic bulk. Modification of the phenyl of 19 to the 3-
pyridyl of 21 results in a greater than 20-fold loss in potency.
A change from the methylene linker to a carbonyl, 22, or
sulfonyl, 23 completely eroded MB-COMT inhibition,
highlighting the importance of maintaining the basicity of
that nitrogen for inhibitory activity.
1
4
15
hydroxyquinazolinones 4, 8-hydroxyquinolines 5 and 3-
1
6
hydroxy-4-pyrimidinones 6.
OH
N
OH
O
N
HO
HN
R
R
N
R
O
5
6
4
Figure 2. Scaffolds of additional known COMT inhibitors
Scheme 1. Synthesis of 1,2,3,4-tetrahydro-8H-pyrido[1,2-
a]pyrazin-8-ones 16-41a
16
As reported by Harrison et. al, a particular series of N-aryl
4
-pyridones arose from
a
formal removal of the
X
O
O
O
thiomorpholine ring from the initial screening hit 7. Potency
of these analogues was further enhanced by building off the
N-aryl ring with additional hydrophobic substituents as
shown with 8, which is 10-fold more potent than the HTS hit.
We began by examining the same tricyclic scaffold of 7 and
envisioned removal of the N-aryl ring and replacement of the
sulfur atom with nitrogen to give a bicyclic hydroxypyridone
HO
a
O
b
OH
OH
O
11
1
0
X
X
O
O
N
O
O
c
d
OH
N
N
9
(formally 1,2,3,4-tetrahydro-8H-pyrido[1,2-a]pyrazin-8-one).
12
13
NH2
The nitrogen would then serve as a useful position on which
substituents could be introduced, enabling rapid synthesis of
analogues.
X
X
O
O
N
O
N
O
O
HO
e
f
N
NH
N
N
14
15
R
16-41
R
O
N
O
N
O
N
aReagents and conditions: (a) BnCl, NaOH, EtOH/H
5%; or PMB-Cl, CO DMF, 80 °C, 90%; (b)
ethylenediamine, EtOH, 90 °C; (c) MnO , CHCl , 60 °C; (d)
NaBH , MeOH, rt, 48 h, 50%, 3 steps; (e) RX, DIPEA, CHCl
or where R = R’CH , R’CHO, Na(OAc) BH, CHCl ; (f) 6N HCl,
2
O, 60-
HO
HO
HO
7
K
2
3
,
2
3
S
N
R
4
3
,
9
2
3
3
7
EtOH, reflux. Experimental details and characterization data may be
found in Reference 17.
Table 1. SAR of 2-substitutiona
8
MB-COMT IC50
00 nM
1
MB-COMT IC50
.9 nM
8
Figure 3. Origins of existing and inspiration for new
hydroxypyridone COMT inhibitors
O
HO
Compounds were prepared using the synthetic sequence
N
1
7
shown in Scheme 1. Kojic acid, 10, was O-alkylated with
either benzyl chloride or 4-methoxybenzyl chloride to give the
benzyl- or PMB-protected alcohol 11. This material was
reacted with ethylenediamine to give the protected
alcohol/amine 12. A one-pot oxidation/cyclocondensation was
N
R2
2
achieved by reaction with MnO to give the cyclic imine 13.
2
ACS Paragon Plus Environment

Page 3 of 13
ACS Medicinal Chemistry Letters
First, a more focused look at the effect of dimethyl
1
2
3
4
5
6
7
8
9
substitution while maintaining a 2-methyl substituent is
shown in Table 3. A preference for 2,4- and 2,6- disubstitution
(33 and 35, respectively) is exhibited over 2,3- and 2,5-
disubstitution (32 and 34). This trend, while modest in
magnitude, was used to guide subsequent probes for the effect
of pendant phenyl ring substituent patterns on MB-COMT
inhibitory activity in this series.
Table 3. SAR of dimethyl substitution on phenyl of 2-benzyl
derivatives
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
X5
X6
X4
X3
N
N
X2
Human MB-
COMT IC50
Cmpd
X
2
X
3
X
4
X
5
X
6
(
nM)
3
0
CH
CH
CH
CH
CH
3
3
3
3
3
H
CH
H
H
H
H
H
50
a
For assay protocol, see Reference 21
3
2
3
3
H
H
H
H
40
13
All data are the mean values of at least three independent measurements.
3
CH
H
3
An exploration of substitution in the 2- and 4-positions of
the phenyl ring of 19 is shown in Table 2. An overall preference
for 2- versus 4-substitution is seen for some examples (cyano,
24 and 25; and trifluoromethyl, 28 and 29) within a limited set
of matched pairs across both electron withdrawing and
donating substituents, while no clear difference is seen for the
methoxy-substituted pair, 26 and 27. A pair of compounds
with mono 2-substituents but no corresponding 4-substituted
matched pair, 2-methyl 30 and 2-chloro 31 each serve as potent
and intriguing starting points for the effect of disubstitution
on the pendant phenyl ring.
34
H
CH
H
3
H
63
3
5
H
H
CH
3
6.3
All data are the mean values of at least three independent measurements.
The effect of 2,4- and 2,6-dihalo substitution is explored in
Table 4. The 2-fluoro analogue, 36, shows a similar inhibitory
potency as seen with the 2-chloro, 31. Addition of a second
chloro or fluoro substituent to the 4- or 6-positions gives
compounds with either a modest improvement in potency (as
with 38, 40 and 41) or ones that are equipotent with their
respective 2-halo parents (37 and 39), but no loss in potency
was observed.
Table 2. SAR of mono-substitution on phenyl of 2-benzyl
derivatives
O
HO
Table 4. SAR of dihalo-substitution on phenyl of 2-benzyl
derivatives
X4
N
O
N
HO
X2
X6
X4
N
N
Human MB-COMT
Cmpd
X
2
X
4
IC50 (nM)
X2
1
9
H
CN
H
H
H
220
100
2000
250
400
60
Human MB-
Cmpd
X
2
X
4
X
6
COMT IC50 (nM)
2
4
1
9
H
F
H
H
H
H
H
H
F
220
50
40
40
10
25
CN
H
36
H
H
F
2
6
OCH
3
3
3
1
Cl
Cl
Cl
F
2
7
H
OCH
3
7
2
8
CF
H
3
H
29
30
CF
H
3
450
50
38
39
40
Cl
H
H
H
CH
Cl
3
38
16
3
1
H
40
Cl
Cl
F
All data are the mean values of at least three independent
measurements.
4
1
Cl
10
All data are the mean values of at least three independent
measurements.
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 13
good permeability and none are P-gp substrates, giving these
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds a good chance of being CNS-penetrant. In rat PK
studies, clearance was quite high, suggesting that rat
hepatocyte stability is not predictive of in vivo clearance for
this scaffold, although reasonable oral bioavailability was
observed. Rat plasma protein binding and rat brain tissue
binding assays indicated that this scaffold has strikingly low
levels of plasma protein binding, with amount of free drug
exceeding 25% for all five compounds tested (Table 5). The
percent free in the brain tissue binding studies was also fairly
high, but not to the degree seen with plasma protein binding.
While having a large free fraction is a boon with regard to
target engagement, it may leave more unbound drug exposed
to metabolism and other clearance mechanisms, which could
Having several compounds that exhibited potent MB-
COMT inhibition in the enzyme assay, we wished to measure
the effects of COMT activity in vivo in the rat. Representative
compounds in this series displayed similar potencies using rat
MB-COMT as with human enzyme (data not shown). As a
result, to simplify the testing funnel, inhibitory activity against
human MB-COMT alone was used as the primary screening
tool.
To determine whether compounds in this series possessed
properties suitable for in vivo studies, we first measured a set
of physicochemical and DMPK properties, both in vitro and in
vivo, which are shown in Table 5 for five compounds which
span a range of potencies. Most of the selected compounds
have reasonable aqueous solubilities, but 35 is less soluble. In
rat hepatocytes, the five compounds tested show moderate to
good stability. In MDCK-MDR1 cells, the compounds display
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
partly explain the high clearance of these compounds in vivo.
Table 5. Potency and ADME properties of 7-hydroxy-3,4-dihydro-1H-pyrido[1,2-a]pyrazin-8-ones
O
HO
X6
X4
N
N
X2
Rat PKb
Kinetic
solubility Stability (Rat)
in water
Hepatocyte
Human
MB-COMT
IC50 (nM)
P
(x 10
cm/s)
app
Rat Plasma Rat Brain
Efflux ratioa
AB/BA
-6
#
X
2
X
4
X
6
%F
T
1/2 (h) -
Cl
(ml/min/kg) Binding
p
Protein
Tissue
6
(μL/min/10
c
d
iv
Binding
(
µg/mL)
60.3
58.0
115
cells)
1
9
H
H
H
H
H
H
220
40
16
< 4
11
25.5
26.1
25.1
11.5
1.0
1.0
1.1
51
57
64
68
19
0.38
0.33
0.80
0.42
0.42
32
49
87
61.4
27.5
52.8
28.0
31.1
12.9
5.9
25.8
11
3
1
Cl
40 Cl
F
11
3
8
Cl Cl
CH
H
10
34.9
9.9
17
1.8
1.3
138
145
3
5
3
H
CH
3
6.3
< 4
21.0
2.8
a
b
c
A:B/B:A efflux ratio from P-glycoprotein expressing MDCK cells. Rat pharmacokinetics after a 1 mpk iv dose and 10 mpk oral dose. Percent
d
free from rat plasma as measured by equilibrium dialysis. Percent free from rat brain homogenate.
Table 6. In vivo exposures of COMT inhibitors and their effects on biomarkers of dopamine metabolism
Human
Total
Plasma
Total
Brain
(nM)
Free
Plasma Brain
(nM)
Free
MB-
COMT
IC50
Free
Dose
Time
(h)
CSF
(nM)
Total
B/P
#
K
p,uu
Brain HVAc DOPACc
/ IC50
(
mpk)
a
a
(
nM)
(nM)
(
nM)
< 1
220
40
1
15
4
4
4
4
4
4
3805
8884
2732
3579
1037
801
< 732
4373
4795
1912
< 183
4902
989
355
< 0.19
0.49
1.75
35
BLQ
564
285
493
430
16
N/A
0.10
0.38
0.26
1.48
0.06
N/A
2.6
7.1
28%
13%
304%
126%
197%
217%
209%
203%
19
100
100
100
100
100
5454
751
3
1
29%
25%
22%
29%
4
0
8
0.53
3.77
0.70
1890
290
249
16
31
3
3911
78
10
43
35
564
214
6.3
2.5
a
Free plasma and brain levels calculated using ratios listed in Table 5 on plasma and brain samples taken immediately after CSF sampling.
p,uu is the ratio of free brain to free plasma. CSF HVA and DOPAC changes (% of vehicle) 4 hours after 100 mpk oral dose compared to
vehicle (n=7-8 rats per group), except 1 (15 mpk ip). BLQ = Below the limit of quantitation.
b
c
K
4
ACS Paragon Plus Environment

Page 5 of 13
ACS Medicinal Chemistry Letters
We then proceeded to test the in vivo efficacy of these
remarkably low levels of protein and tissue binding, increasing
the availability of circulating compounds to interact with the
target of interest. In rat biomarker studies, levels of unbound
drug exposure are seen in the brain which exceed their
respective IC50s. This target inhibitory coverage manifests
itself in altering the levels of dopamine metabolites in a
manner consistent with COMT inhibition, although not
reflecting the magnitude of change in DOPAC concentration
as seen with tolcapone.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds in rats, while mindful of the pharmacokinetic
data. Inhibition of COMT in the CNS should produce
measurable changes in dopamine metabolites in the central
nervous system: an increase in the concentration of
dihydroxyphenyl acetic acid (DOPAC) and a decrease in that
of homovanillic acid (HVA). These dopamine metabolites
were measured in rat CSF, while concentrations of COMT
inhibitors were assessed in CSF, brain homogenate and
plasma concurrently. Taken as a whole, these measurements
provide an assessment of both CNS penetration and activity
for the dosed compounds. It has been previously shown that
CSF levels of HVA and DOPAC are responsive to brain-
ASSOCIATED CONTENT
Supporting Information
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
penetrant COMT inhibitors, and that these levels mirror
(Procedures for synthesis and characterization of compounds,
details of COMT enzymatic assays, and a table of S-COMT
activity for key compounds)
20
those seen in total brain. All compounds in Table 5 have IC50
s
for S-COMT that are greater than 5 µM (supplementary table
1
5
S1), giving confidence that any changes in biomarker level
would be driven by MB-COMT inhibition.
Table 6 shows the results from a single oral administration
100 mpk) of COMT inhibitors to rats (n = 7-8). The animals
AUTHOR INFORMATION
Corresponding Author
(
were sacrificed 4 h post-dose, then total drug was measured in
plasma, brain and CSF while dopamine metabolite levels were
determined in CSF. The free plasma and brain concentrations
were calculated using the plasma protein and brain tissue
binding data (Table 5). LC-MS was used to measure the levels
of HVA and DOPAC in CSF; the percent change in these levels
versus vehicle treated control animals is shown in Table 6.
Tolcapone, 1, was used as a positive control (15 mpk, ip
injection) and gave expected movement of biomarkers, where
HVA was decreased and DOPAC increased relative to control.
All five compounds tested decreased HVA levels to the same
degree as tolcapone. Increases in DOPAC were below the
levels seen with tolcapone, and the change seen with 19 was
only slightly increased versus control. The other four
compounds roughly doubled the levels of DOPAC as
compared to control, but below that seen with tolcapone.
*
Phone: +1 410 955 0894. Fax: +1 410 955 1044. E-mail
james.barrow@libd.org.
ORCID
James C Barrow 0000-0001-5115-9300
Author Contributions
I.B., S.D., P.d.L., T.D., V.D., G.E., Y.H., M.P., D.S., F.M., M.-C.
V., N. V., and J.C.B. designed and synthesized compounds.
M.K., H.W., M.W., E.J., and J.C.B. developed the in vitro
assays. D.A., V.A., S.B., G.C., M.D., A.K., E.J., D.S., N.W., and
J.C.B. developed and analyzed the in vivo assays. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
All five hydroxypyridone analogues exhibited good
peripheral exposure and brain levels. Free drug
concentrations in brain homogenate for all compounds
exceeded their respective MB-COMT IC50s by at least twofold,
Funding Sources
Funding of parts of this work from the National Institutes of
Men-tal Health NIMH R01 MH107126 is gratefully
acknowledged.
with 38 displaying a 40-fold level of coverage over its IC50
.
However, the change in the biomarker levels is roughly the
same for all compounds except 19, and does not reflect the
range of free compound concentration coverage above the
IC50s. While these compounds clearly inhibit COMT in vivo,
they do not increase the levels of DOPAC as robustly as
tolcapone. A possible explanation for this is off-target activity
at other enzymes of dopamine processing; however,
compounds 31 and 38 were tested for, and had no activity
against, monoamine oxidases A and B (IC50> 10 uM) and very
weak inhibition of tyrosine hydroxylase (IC50~10 uM).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We gratefully acknowledge Yves Lamberty (UCB) for the
design of in vivo emperiments and for constructive
pharmacology discussions and David Boucaut (UCB) for
assistance with in vivo studies. We also gratefully
acknowledge Eric Gillent (UCB) and Benoît Culot (UCB) for
bioanalysis assistance, and Benoît Mathieu (UCB), Geraldine
Longfils (UCB) and Ariane Descamps (UCB) for analytical
chemistry support.
In summary, a series of bicyclic pyridones were prepared
and evaluated for their ability to inhibit COMT, and several
potent inhibitors of MB-COMT were identified. Incorporation
of a halo or methyl 2-substituent on the benzyl group attached
to the basic nitrogen of the core scaffold gave compounds with
increased potency; further methyl or halo substitution at the
4- or 6-position gave the most potent inhibitors within this
series. Although rat pharmacokinetic studies showed medium
to high levels of clearance for this series, they also have
ABBREVIATIONS
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 13
%
F, bioavailability; ADHD, attention deficit hyperactivity
Catechol-O-Methyltransferase in Cognition and Stress Reactivity
in Mice. J. Neurosci. 2008, 28 (35), 8709–8723.
8) Learmonth, D. A.; Kiss, L. E.; Soares-da-Silva, P. The
Chemistry of Catechol-O-Methyltransferase Inhibitors. Int. Rev.
Neurobiol. 2010, 95, 119–162.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
disorder; ADME, absorption, distribution, metabolism, and
excretion; BLQ, below limit of quantitation; BnCl, benzyl
chloride; B/P, ratio of concentration of drug in brain to
(
3 p
concentration of drug in plasma; CHCl , chloroform; Cl ,
(
9)
Bonifácio, M. J.; Nuno Palma, P.; Almeida, L.; Soares-da-
plasma clearance; COMT, catechol-O-methyltransferase;
Cmpd, compound; CNS, central nervous system; CSF,
cerebrospinal fluid; DIPEA, N,N-diisopropylethylamine; DMF,
dimethylformamide; DMPK, drug metabolism and
pharmacokinetics; DOPAC, dihydroxylphenyl acetic acid;
EtOH, ethanol; F , fraction unbound; H O, water; HCl,
u 2
hydrochloric acid; HTS, high-throughput screening; HVA,
homovanillic acid; IC50, compound/substance concentration
Silva, P. Catechol-O-Methyltransferase and Its Inhibitors in
Parkinson’s Disease. CNS Drug Reviews. 2007, 13 (3), 352–379.
(10)
Russ, H.; Müller, T.; Woitalla, D.; Rahbar, A.; Hahn, J.;
Kuhn, W. Detection of Tolcapone in the Cerebrospinal Fluid of
Parkinsonian Subjects. Naunyn. Schmiedebergs. Arch. Pharmacol.
1
999, 360 (6), 719–720.
11) Müller, T.; TANIMOS Study Investigators. Tolcapone
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Addition Improves Parkinson’s Disease Associated Nonmotor
Symptoms. Ther. Adv. Neurol. Disord. 2014, 7 (2), 77–82.
required for 50% inhibition; K
2 3
CO , potassium carbonate;
ratio of free concentration of drug in brain to free
K
p,uu
,
(12)
Tunbridge, E. M.; Harrison, P. J.; Weinberger, D. R.
concentration of drug in plasma; LC-MS, liquid
chromatography−mass spectrometry; L-DOPA, L-3,4-
dihydroxyphenylalanine; MB-COMT, membrane bound
catechol-O-methyltransferase; MDCK, Madin−Darby canine
kidney cells; MDR1, multidrug resistance protein 1, MeOH,
Catechol-O-Methyltransferase, Cognition, and Psychosis:
Val158Met and Beyond. Biological Psychiatry. 2006, 60 (2), 141–151.
(13)
and Safety Review (2007). Clin. Neuropharmacol. 2007, 30 (5),
87–294.
14) Bissantz, C.; Bonnafous, R.; Buettelmann, B.; Jakob-
Roetne, R.; Lerner, C.; Rudolph, M. Preparation of 8-
hydroxyquinazolin-4(3H)-one derivatives as catechol-O-
methyltransferase (COMT) inhibitors. WO2014102233, 2014.
15) Buchler, I.; Akuma, D.; Au, V.; Carr, G.; de León, P.;
Olanow, C. W.; Watkins, P. B. Tolcapone: An Efficacy
2
(
methanol; MnO
borohydride; nM, nanomoles per liter of solution;
Na(OAc) BH, sodium triacetoxyborohydride; NaOH, sodium
apparent intrinsic permeability; PFC,
cortex; P-gp, P-glycoprotein 1; PK,
2 4
, magnesium (IV) oxide; NaBH , sodium
3
hydroxide;
prefrontal
app
P ,
(
DePasquale, M.; Ernst, G.; Huang, Y.; Kimos, M.; Kolobova, A.;
Poslusney, M.; Wei, H.; Swinnen, D.; Montel, F.; Moureau, F.;
Wood, M. Optimization of 8-Hydroxyquinolines as Inhibitors of
Catechol O-Methyltransferase. J. Med. Chem. 2018, 61 (21), 9647–
9665.
(16) Harrison, S. T.; Poslusney, M. S.; Mulhearn, J. J.; Zhao,
Z.; Kett, N. R.; Schubert, J. W.; Melamed, J. Y.; Allison, T. J.; Patel,
S. B.; Sanders, J. M.; Sharma, S.; Smith, R. F.; Hall, D. H.; Robinson,
R. G.; Sachs, N. A.; Hutson, P. H.; Wolkenberg, S. E.; Barrow, J. C.
Synthesis and Evaluation of Heterocyclic Catechol Mimics as
Inhibitors of Catechol-O-Methyltransferase (COMT). ACS Med.
Chem. Lett. 2015, 6 (3), 318–323.
pharmacokinetics; PMB, p-methoxybenzyl; PMB-Cl, p-
methoxybenzyl chloride; PPB, plasma protein binding; SAM,
S-adenosyl methionine; SAR, structure−activity relationship;
S-COMT, soluble catechol-O-methyltransferase; T1/2, half-life
of the product
REFERENCES
(
1)
Lachman, H. M.; Papolos, D. F.; Saito, T.; Yu, Y.-M.;
Szumlanski, C. L.; Weinshilboum, R. M. Human Catechol-O-
Methyltransferase Pharmacogenetics: Description of a Functional
Polymorphism and Its Potential Application to Neuropsychiatric
Disorders. Pharmacogenetics. 1996, 6 (3), 243–250.
(17)
-Hydroxytetrahydropyrido[1,2-a]pyrazin-8-ones
inhibitors and their preparation. WO2017091818, 2017.
18) Baker, M; Parton, T. Kinetic determinants of hepatic
Barrow, J.; Ernst, G; Swinnen, D.; Montel, F.; Defays, S.
7
as COMT
(2)
Yavich, L.; Forsberg, M. M.; Karayiorgou, M.; Gogos, J.
A.; Männistö, P. T. Site-Specific Role of Catechol-O-
Methyltransferase in Dopamine Overflow within Prefrontal
Cortex and Dorsal Striatum. J. Neurosci. 2007, 27 (38), 10196–
(
clearance: Plasma protein binding and hepatic uptake.
Xenobiotica, 2007, 37 (10–11), 1110–1134.
1
0209.
3)
(
19) Hutson, P. H.; Curzon, G. Dopamine metabolites in rat
cisternal cerebrospinal-fluid major contribution from
extrastriatal dopamine neurons. J. Neurochem. 1986, 46, 186−190.
20) Robinson, R. G.; Smith, S. M.; Wolkenberg, S. E.; Kandebo,
(
Käenmäki, M.; Tammimäki, A.; Myöhänen, T.;
-
Pakarinen, K.; Amberg, C.; Karayiorgou, M.; Gogos, J. A.;
Männistö, P. T. Quantitative Role of COMT in Dopamine
Clearance in the Prefrontal Cortex of Freely Moving Mice. J.
Neurochem. 2010, 114 (6), 1745–1755.
(
M.; Yao, L. H.; Gibson, C. R.; Harrison, S. T.; Polsky-Fisher, S.;
Barrow, J. C.; Manley, P. J.; Mulhearn, J. J.; Nanda, K. K.; Schubert,
J. W.; Trotter, B. W.; Zhao, Z. J.; Sanders, J. M.; Smith, R. F.;
McLoughlin, D.; Sharma, S.; Hall, D. L.; Walker, T. L.; Kershner, J.
L.; Bhandari, N.; Hutson, P. H.; Sachs, N. A. Characterization of
non-nitrocatechol pan and isoform specific catechol-O-
methyltransferase inhibitors and substrates. ACS Chem. Neurosci.
(
4)
Männistö, P. T.; Kaakkola, S. Catechol-O-
Methyltransferase (COMT): Biochemistry, Molecular Biology,
Pharmacology, and Clinical Efficacy of the New Selective COMT
Inhibitors. Pharmacol. Rev. 1999, 51 (4), 593–628.
5)
K.; Julkunen, I.; Taskinen, J. Kinetics of Human Soluble and
Membrane-Bound Catechol O-Methyltransferase: Revised
(
Lotta, T.; Vidgren, J.; Tilgmann, C.; Ulmanen, I.; Melen,
2
012,
3
(2),
129−140
A
(
21) Kimos M, Burton M, Urbain D, Caudron D, Martini M,
Mechanism and Description of the Thermolabile Variant of the
Enzyme. Biochemistry. 1995, 34 (13) 4202–4210.
Famelart M, Gillard M, Barrow J, Wood M. Development of an
HTRF Assay for the Detection and Characterization of Inhibitors
of Catechol-O-Methyltransferase. J. Biomol. Screen. 2016, 21, 490.
(
6)
Reenilä, I.; Männistö, P. T. Catecholamine Metabolism
in the Brain by Membrane-Bound and Soluble Catechol-O-
Methyltransferase (COMT) Estimated by Enzyme Kinetic Values.
Med. Hypotheses 2001, 57 (5), 628–632.
(
7)
Papaleo, F.; Crawley, J. N.; Song, J.; Lipska, B. K.; Pickel,
J.; Weinberger, D. R.; Chen, J. Genetic Dissection of the Role of
6
ACS Paragon Plus Environment

Page 7 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TABLE OF CONTENTS GRAPHIC:
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 13
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
0x26mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 9 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
61x39mm (300 x 300 DPI)
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 10 of 13
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
88x31mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 11 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
4x58mm (300 x 300 DPI)
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 12 of 13
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
63x95mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 13 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment