Synthesis and biological evaluation of a novel series of "ortho-nitrated" inhibitors of catechol-O-methyltransferase

Article abstract of DOI:10.1021/jm0580454

Novel regioisomeric "ortho-nitrated" catechols related to the catechol-O-methyltransferase (COMT) inhibitors BIA 3-202 3 and BIA 3-335 4 were synthesized and biologically evaluated. Changing the position of the nitro group from the "classical" meta- to th

Full text of DOI:10.1021/jm0580454

8070
J. Med. Chem. 2005, 48, 8070-8078
Synthesis and Biological Evaluation of a Novel Series of “Ortho-Nitrated”
Inhibitors of Catechol-O-methyltransferase
David A. Learmonth,† Maria Joa˜o Bonifa´cio,‡ and Patr´ıcio Soares-da-Silva*,‡
Laboratory of Chemistry and Laboratory of Pharmacology, Department of Research & Development, BIAL,
4745-457 S. Mamede do Coronado, Portugal
Received August 4, 2005
Novel regioisomeric “ortho-nitrated” catechols related to the catechol-O-methyltransferase
(COMT) inhibitors BIA 3-202 3 and BIA 3-335 4 were synthesized and biologically evaluated.
Changing the position of the nitro group from the “classical” meta- to the ortho-position relative
to the side-chain substituent of the nitrocatechol pharmacophore exerted profound effects on
selectivity and duration of COMT inhibition. Alkylaryl compounds 7a-d possessed shorter
duration of action than their regioisomers, but 7b displayed reversed selectivity over 3 at 3
and 6 h, exhibiting preferential central inhibition. In the amino-substituted series, ortho-nitrated
regioisomer 14k was less peripherally selective than 4 and short-acting, whereas decahydro-
quinoline 14g displayed an unprecedented combination of long-acting and selective peripheral
inhibition. 7b could provide a useful tool to probe the pharmacological utility of short-acting,
centrally selective COMT inhibitors in the treatment of depression in Parkinsonian patients,
and 14g represents a promising candidate for clinical evaluation as an adjunct to L-Dopa
therapy.
Introduction
Despite the fact that levodopa (3-(3,4-dihydroxyphe-
nyl)-L-alanine, L-Dopa) was introduced into clinical
practice as long ago as the early 1960s, it still continues
to be the gold standard drug for the symptomatic
treatment of Parkinson’s disease (PD).1-3 PD is a
dopamine-deficiency disorder resulting from the degen-
eration of striatal dopaminergic neurons. Because dopam-
ine itself is unable to permeate across the blood-brain
barrier (BBB), Parkinsonian patients are treated with
the dopamine precursor L-Dopa in combination with an
aromatic amino acid decarboxylase (AADC) inhibitor
such as carbidopa or benserazide,4,5 which prevent
premature decarboxylation of L-Dopa in the periphery.
However, when this decarboxylation degradation path-
way is blocked, catechol-O-methyltransferase (COMT),
catalyzed O-methylation of L-Dopa to give 3-O-methyl-
L-Dopa (3-OMD), becomes the predominant degradation
pathway in the periphery6-8 such that only a very
limited percentage of the orally administered L-Dopa
dose actually reaches the site of action intact.
Accordingly, keen interest has been maintained in the
development of novel, more clinically effective inhibitors
of COMT.9,10 COMT11 is a magnesium-dependent en-
zyme which catalyses the transfer of a methyl group
from its cofactor S-adenosyl-L-methionine (SAM) to
substrates containing a catechol motif. COMT is now
known to play a key role in the inactivation of endog-
enous catecholamines,12 catechol estrogens,13 and the
detoxification of several xenobiotic catechols.14,15
It has been postulated that it should be possible to
improve the bioavailability of L-Dopa by reducing meta-
Figure 1. Chemical structures of tolcapone (1), entacapone
(2), BIA 3-202 (3), BIA 3-335 (4), [2-(3,4-dihydroxy-2-nitro-
phenyl)vinyl]phenyl ketone (5), and nitecapone (6).
bolic O-methylation in the periphery. On one hand,
3-OMD is not known to provide any beneficial thera-
peutic effect and has a long elimination half-life com-
pared to L-Dopa, which means that it accumulates in
plasma and tissues.16 On the other hand, 3-OMD may
compete with L-Dopa for the same active transport
system17,18 that permits permeation across the BBB, and
a close relationship between the accumulation of 3-OMD
and the “end-of-dose” or ‘wearing-off’ syndrome has been
described.16 In principle therefore, COMT inhibition
should protect L-Dopa from undesirable metabolic deg-
radation in the periphery and facilitate passage into the
brain, thereby extending the duration of antiparkinso-
nian action and permitting a reduction in the dose and/
or number of daily doses of L-Dopa.
Of the second-generation COMT inhibitors, the most
commonly known are tolcapone (1)19and entacapone
(2)20 (Figure 1). Both of these molecules contain a
catecholic pharmacophore to which the highly electro-
negative nitro group has been added to occupy the meta
position relative to the side-chain substituent. Both of
these molecules are potent, tight-binding inhibitors of
* Author to whom correspondence should be addressed. Tel: 351-
22-9866100. Fax: 351-22-9866192. E-mail: psoares.silva@bial.com.
† Laboratory of Chemistry.
‡ Laboratory of Pharmacology.
10.1021/jm0580454 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/17/2005

Inhibitors of Catechol-O-methyltransferase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8071
COMT, which themselves are very poor substrates for
the enzyme21,22 unlike earlier, nonfunctionalized cat-
echolic inhibitors.23 Pharmacologically, 1 can be distin-
guished from 2 in that it is an entirely indiscriminate
inhibitor of both central and peripheral COMT, whereas
2 is a peripherally selective inhibitor. The nitro group
is thought to have the twofold function of stabilizing the
ionized inhibitor-enzyme complex and to reduce the
nucleophilicity of the hydroxyl group of the catechol
usually susceptible to methylation. Although both of
these molecules were approved and reached the mar-
ketplace as adjuncts to L-Dopa therapy, 1 was subse-
quently withdrawn due to hepatotoxicity concerns.24,25
Presently, 1 can only be administered to Parkinsonian
patients unresponsive to other PD treatments and
strictly only with a regular liver function testing regime,
which is both expensive and inconvenient for the
patient. The actual mechanism(s) by which 1 induces
liver toxicity remain controversial, but hepatotoxicity
has not thus far been associated with 2, which is
generally regarded as a safe drug. This may be signifi-
cant, given that it shares with 1 the same nitrocatecholic
pharmacophore. Studies into the metabolism of 1 in
humans26 have shown that among extensive phase I
transformations (methylation, oxidation, and reduction),
significant reduction of the nitro group occurs to form
the corresponding amino metabolite that then under-
goes a number of phase II conjugation reactions includ-
ing acetylation of the amino group. On the other hand,
analogous metabolic reduction of the nitro group of 2 is
only observed in rats, not humans.27 Recent in vitro
studies28 showed that these metabolites of 1 could be
further oxidized to reactive intermediates capable of
forming covalent adducts with hepatic proteins, thereby
explaining the potential of 1, but not 2, to cause
hepatocellular injury. Other workers have found that 1
but not 2 is a potent uncoupler of oxidative phosphory-
lation in mitochondria29,30 and is thereby able to reduce
the cell’s capacity to generate ATP, and they have
proposed this aspect as the source of toxicity for 1.
Furthermore, it was recently reported31 that tolcapone
has a significantly higher lipophilicity compared to that
of 2 at physiological pH (1, log Papp 1.03 vs 2, log Papp
0.174); thus, it would be expected that 1 would be able
to permeate across cell membranes more efficiently than
2 and, once therein, could exert the above-mentioned
uncoupling effects.
onstrated through structure-activity relationships and
molecular modeling studies that variation of the side-
chain substituent was found to exert profound influence
on both the peripheral selectivity and duration of COMT
inhibition. Thus, although the nitrocatechol moiety may
be regarded as responsible for “anchoring” the inhibitor
molecule to the magnesium ion at the active site of the
enzyme, due consideration must be given to appropriate
side-chain substitution of the pharmacophore, because
this side chain can modulate the overall physicochemical
properties and, thus, performance of the compounds.
Animal and human studies indicate that 3 and 4 are
endowed with significantly improved pharmacokinetic
and pharmacodynamic properties over both 1 and 2 and
are currently in advanced clinical investigation as
potential adjuncts to L-Dopa/AADC therapy in human
studies.36,37
During this work, we became increasingly curious as
to what pharmacological effects might be observed by
changing the position of the nitro group from the meta
position (i.e., 3,4-dihydroxy-5-nitro substitution pattern
as in 3, 4, and their analogues) to the ortho position (i.e.,
3,4-dihydroxy-2-nitrophenyl substitution pattern) in
relation to the side-chain substituent. To the very best
of our knowledge, no systematic comparative study of
the pharmacology of regioisomerically nitrated deriva-
tives of COMT inhibitors currently in clinical use or
development has thus far been published. However, over
a decade ago, Perez et al.38 reported the synthesis and
inhibitory activity of a series of regioisomeric dihydroxy
nitrobenzaldehydes against COMT isolated from pig
liver. These authors concluded that the substitution of
the catechol with a nitro group ortho to both one
hydroxyl group and the carbonyl group (3,4-dihydroxy-
2-nitro) gave the most potent COMT inhibitor in this
series, on the basis of the evidence of a lower IC50 than
that of the corresponding 3,4-dihydroxy-5-nitro regioi-
somer. The same authors went on to synthesize and
evaluate the kinetics of COMT inhibition in vivo by a
series of ortho- and meta-nitrated vinyl-substituted
catechols39-41 derived from these benzaldehydes and
concluded that the ortho-nitrated vinylphenyl ketone (5)
(Figure 1) was a particularly potent and selective COMT
inhibitor, with an apparent Ki (Kapp) 3.5-fold lower than
nitecapone (6) (Figure 1). Despite this promise, it
appears that 5 did not progress further in terms of
clinical development, and indeed we are unaware of any
ortho-nitrated COMT inhibitor currently in develop-
ment.
Notwithstanding the reduced toxicity risk, however,
2 is endowed with a very short in vivo half-life32 which
means that the dosages are high and must be repeatedly
administered throughout the day. Indeed, the clinical
efficacy of 2 has been recently questioned,33 and this
aspect is often cited by patients as the principal motive
for cessation of treatment.
Recent efforts from this group sought to address these
problems, and this work led to the discovery of BIA
3-202 (3)34 and BIA 3-335 (4)35 (Figure 1). Immediately,
one can see that both of these structures also display
what can now be regarded as the “classical” 3,4-
dihydroxy-5-nitrophenyl pharmacophore, wherein the
nitro group is located at the meta position relative to
the carbonyl substituent. Both 3 and 4 are potent, tight-
binding, long-acting, and highly selective inhibitors of
peripheral COMT. In either case, it was clearly dem-
Accordingly, we proceeded to synthesize a restricted
series of ortho-nitrated catechols including several
analogues of 3 and 4 to ascertain the effect of changing
the position of the nitro substituent on pharmacological
parameters such as potency, duration, and selectivity
of COMT inhibition.
Chemistry
The ortho-nitrated analogues of the previously re-
ported34 BIA 3-202 homologous series (7a-d, Table 1)
were prepared according to Scheme 1. The hydroxyl
group of vanillin (8, R ) H) was temporarily protected
as the benzyl ether (8, R ) Bn) by the Williamson
reaction and was reacted with homologous arylalkyl
Grignard reagents (Ar(CH2)nMgX, n ) 0-3) to provide

8072 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Learmonth et al.
Table 1. Physical Constants, Percent Inhibition of COMT Activity in SK-N-SH Cells by Compounds 1-3 and 7a-d (100 nM),a and
IC50 Values for Inhibition of Rat Brain and Liver COMTb
% Inhibition
(SK-N-SH)
No.
n
mp (°C)
Formula
Anal.
IC50 Liver (µM)
IC50 Brain (nM)
1
97 ( 1
0.93
(0.55, 1.56)
2.32
2.2
(0.8, 6.4)
12.8
(4.0, 41.3)
3.7
2
77 ( 1
90 ( 1
98 ( 0
76 ( 1
95 ( 2
87 ( 4
(0.74, 7.26)
0.69
3
(0.36, 1.36)
0.13
(1.7, 8.1)
3
7a
7b
7c
7d
1
2
3
4
155-157
153-154
108-109
142-143
C
13H9NO5
C,H,N
C,H,N
C,H,N
C,H,N
(0.08, 0.2)
2.0
(2,4)
4
C14H11NO5
(1.4, 2.8)
0.13
(3,5)
4
C
C
15H13NO5
16H15NO5
(0.08, 0.2)
0.11
(3,5)
4
(0.08, 0.17)
(3,5)
a Results are mean SEMs of four experiments per group. b 95% confidence intervals in brackets.
Scheme 1. Synthesis of Homologous Ortho-Nitrated BIA 3-202 Analoguesa
a Reagents: (a) i. Ar(CH2)nMgX, ii. H3O+; (b) NaOtBu, cyclohexanone, PhCH3, ∆; (c) 30% HBr-AcOH, CH2Cl2; (d) Ac2O, pyridine,
CH2Cl2; (e) Cu(NO3)2, Ac2O; (f) 3 N NaOH, MeOH; (g) AlCl3, pyridine, Cl(CH2)2Cl, ∆.
the alcohols 9a-d (n ) 0-3) in good yields. Oppenauer
oxidation furnished the ketones 10a-d (R ) Bn) in
moderate to excellent yields. Deprotection of the O-
benzyl protecting groups proceeded smoothly on contact
with excess hydrogen bromide in acetic acid to provide
the phenols 10a-d (R ) H) which were then converted
to the O-acetates 10a-d (R ) COCH3) in quantitative
yields. Regioselective introduction of the nitro group at
C-2 proved elusive and could not be satisfactorily
achieved in acceptable yields using nitric acid under a
variety of conditions, principally due to predominant
formation of the undesired 6-nitro isomers 12a-d (R )
COCH3) or nitration occurring on the nonfunctionalized
aromatic ring. Fortunately, however, reaction of the
acetates 10a-d (R ) COCH3) with copper (II) nitrate
trihydrate in acetic anhydride gave better results, and
the 2-nitro compounds 11a-d (R ) COCH3) could be
obtained in moderate yields after separation from the
6-nitro regioisomers by chromatography or crystalliza-
tion. The acetates were then hydrolyzed to the corre-
sponding phenols 11a-d (R ) H) in aqueous methanolic
sodium hydroxide. Finally, scission of the methyl groups
was achieved using aluminum chloride and pyridine in
warm 1,2-dichloroethane,42 which furnished the target
compounds 7a-d (Table 1) in excellent yields.
Selected ortho-nitrated analogues (14a-l) of the
previously reported35 piperidines and piperazines from
the BIA 3-335 series were prepared by the Mannich
reaction of the nitrocatecholic acetophenone building
block 13 with appropriate cyclic secondary amines
(Scheme 2). Acetophenone (13) was prepared from
acetovanillone by acetylation, nitration, hydrolysis, and
methyl ether cleavage as in steps d-g of Scheme 1.

Inhibitors of Catechol-O-methyltransferase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8073
Table 2. Physical Constants and Percent Inhibition of COMT
Activity in SK-N-SH Cells by Compounds 4 and 14a-l (100
nM)a
Scheme 2. Synthesis of Ortho-Nitrated BIA 3-335
Analoguesa
a
i
Reagents: (a) HNR1R2, H2CO, c‚HCl, PrOH, 80 °C.
Scheme 3. Synthesis of Ortho-Nitrated
Ring-Constrained BIA 3-335 Analoguesa
a Reagents: (a) 48% HBr (aq), reflux; (b) BBr3, CH2Cl2, -78 °C
to room temperature; (c) BnBr, K2CO3, DMF, 80 °C; (d) 65% HNO3,
AcOH; (e) 30% HBr-AcOH, 48% HBr, 110 °C; (f) H2NOH‚HCl,
i
pyridine, EtOH, reflux; (g) HNR1R2, H2CO, c‚HCl, PrOH, 80 °C.
Constrained carbocyclic analogues 15 and 16 were
synthesized from 6,7-dimethoxy-1-tetralone (17) and
5,6-dimethoxy-1-indanone (18), respectively (Scheme 3).
Exhaustive demethylation of 17 was readily achieved
in boiling 48% aqueous hydrobromic acid, whereas
treatment of 18 with boron tribromide gave superior
yields of the corresponding catechol 20. The para
hydroxyl group of catechols 19 and 20 were regioselec-
tively benzylated via the Williamson reaction to provide
the para-O-benzyl ethers 21 and 22 in moderate yields,
each of which permitted a regioselective introduction
of the nitro substituent ortho to the carbonyl group upon
reaction with dilute nitric acid in acetic acid to give
exclusively 23 and 24. The benzyl groups of each were
then cleaved on brief contact with warm hydrobromic
acid to furnish the nitrocatechols 15 and 16. These were
subsequently further elaborated by the Mannich reac-
tion with selected secondary amines giving access to
27a-d, and the carbonyl group of the parent ketones
15 and 16 were modified by reaction with hydroxy-
lamine to give the oximes 25 and 26.
a Results are mean SEMs of four experiments per group.
synthesized ortho-nitrated catechols to inhibit COMT.
Homologous alkylaryl compounds 7a-d were further
tested for their ability to decrease O-methylation of
adrenaline to metanephrine in rat liver and whole brain
homogenates, as previously described,44 and were com-
pared with the standards 1-3 (Table 1). Compounds 7a,
c, and d exerted significant inhibition of COMT in SK-
N-SH cells (87-98%) and were found to be unexpectedly
potent inhibitors of both peripheral and cerebral COMT,
having noticeably lower IC50’s for inhibition of periph-
eral COMT (0.11-0.13 M) than 1 and 3 (0.93 and 0.69
M, respectively). Rather surprisingly however, the etha-
none 7b, which structurally represents the ortho-
nitrated analogue of BIA 3-202 3, was found to be a
significantly less potent inhibitor in the SK-N-SH assay
(76%). The lower potency of 7b, compared to those of
7a, c, and d, was confirmed by its IC50 value for liver
COMT (2 µM), which was closest to that of the weakest
inhibitor 2 tested.
Results and Discussion
In vitro screening in human neuroblastoma SK-N-SH
cells, as previously described,43 was employed to make
a preliminary assessment of the ability of the newly

8074 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Learmonth et al.
Table 3. Physical Constants and Percent Inhibition of COMT Activity in SK-N-SH Cells by Compounds 15, 16, 25, 26 and 27a-d
(100 nM)a
a Results are mean SEMs of four experiments per group.
Table 4. Percent Inhibition of COMT Activity by Compounds
1-3 and 7a-d in Homogenates of Rat Brain and Liver after
Administration by Gastric Tube (all at 30 mg/kg)a
On the other hand, all of the open-chain amino
analogues 14a-l maintained good inhibition of COMT
in SK-N-SH cells (86-94%) irrespective of the side-chain
substituent, and indeed, 14k was essentially equipotent
to its nitro-regioisomer 4 (Table 2). We then turned our
attention to five- and six-membered ring-constrained
carbocyclic analogues, and it was interesting to observe
that the unsubstituted tetralone 15 was significantly
less active than the lower indanoyl homologue 16 (Table
3). Modification of the carbonyl group, as in the corre-
sponding oximes 25 and 26, led to a further significant
decrease in inhibition, confirming previous findings34 in
another series. Substitution to the carbonyl group of
indanone 16 led to generally slight improvements in
activity, with restoration of respectable inhibition ob-
served only for the 2-(chlorophenyl)piperazine 27d from
this series. The 3-(trifluoromethylphenyl)piperazinyl
indanone 27c was found to be approximately 10% less
active than both the open-chain analogue 14k and the
nitro regioisomer 4. Of the three series tested, the cyclic
derivatives listed in Table 3 appeared least interesting
and were thus not studied further.
Attention was then turned to evaluating the effects
of incorporation of the nitro group at the ortho position
on in vivo parameters such as the duration of COMT
inhibition and its ability to access the brain. Test
compounds were administered by gastric tube to over-
night-fasted rats (1-3 and 7a-d) or mice (4, 14a-g,
14k). Thereafter, at defined intervals, the animals were
sacrificed and the liver and brains removed and used
to assess COMT activity. Compounds 7a-d were all
found to have a rapid onset of action, with a maximum
inhibitory effect observed at 30 min after administra-
tion, and general trends could be observed from the time
course profiles (Table 4). In the brain, compounds 7b
and particularly 7c presented an inhibition pattern over
time similar to that of 3, which is itself placed between
1 and 2, and both retained still significant inhibition at
6 h post dose. Benzophenone 7a, on the other hand, was
essentially inactive at this latter time point. In this
Time Course % Inhibition
No.
0.5 h
1 h
3 h
6 h
9 h
Brain
1
99 ( 1
72 ( 7
84 ( 1
88 ( 0.3
81 ( 2
87 ( 1
87 ( 1
99 ( 1
45 ( 7
81 ( 3
75 ( 2
81 ( 1
82 ( 1
65 ( 2
97 ( 1
30 ( 6
65 ( 4
48 ( 2
63 ( 6
63 ( 1
48 ( 2
86 ( 8
20 ( 7
32 ( 3
7 ( 8
35 ( 4
39 ( 10
33 ( 3
78 ( 2
23 ( 3
2
3
22 ( 3
7a
7b
7c
7d
-2.3 ( 1
1.6 ( 3
13 ( 9
5 ( 10
Liver
1
100 ( 1
98 ( 1
99 ( 1
96 ( 1
97 ( 2
73 ( 1
92 ( 2
95 ( 1
70 ( 8
98 ( 1
86 ( 2
96 ( 1
48 ( 3
38 ( 11
34 ( 12
45 ( 4
94 ( 1
74 ( 5
76 ( 4
21 ( 15
8 ( 11
5 ( 14
8 ( 1
67 ( 4
25 ( 8
70 ( 4
-9 ( 7
-26 ( 9
4 ( 7
2
3
99 ( 1
7a
7b
7c
7d
98 ( 0.5
94 ( 1
99 ( 0.5
99 ( 0.1
18 ( 4
a Results are mean SEMs of four experiments per group.
sense, 7a has a much shorter duration of inhibition in
the brain than its previously reported meta-nitrated
analogue (97% at 3 h and 86% at 6 h), whereas 7b, as
mentioned above, is very similar in performance to its
regioisomer 3. However, more significant differences
were observed in the liver. All compounds 7a-d were
relatively short-acting (surprisingly even more so than
2), and 7b-d in particular were practically inactive
against liver COMT at 6 h post dose. The differences in
inhibition over time between the regioisomers 3 and 7b
are particularly striking. In practical terms, it can be
seen that 7a and, to a slightly lesser extent, d have very
similar inhibition patterns in both the brain and liver.
Although 7b and c are both highly active against both
liver and brain COMT at shorter time points (0.5 and 1
h), the rapid reduction in peripheral inhibition at 3-6
h post dose means that, in effect, they are more active
against brain COMT at these intermediate time points.
Accordingly, these compounds represent the first COMT

Inhibitors of Catechol-O-methyltransferase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8075
previous molecular modeling studies48 on the interaction
of 3 with COMT, molecular modeling efforts are cur-
rently being focused on analyzing the effects of the
binding conformations of ortho-nitrated catechols, such
as 7a, on their metabolism. The results of crystal-
lographic and molecular modeling studies will be re-
ported in due course.
Table 5. Percent Inhibition of COMT Activity by 4 and
Compounds 14a-k in Homogenates of Mouse Brain and Liver
after Administration by Gastric Tube (All at 30 mg/kg)a
% Inhibition
COMT Activity at 1 h
COMT Activity at 6 h
No.
Liver
Brain
Liver
Brain
4
82 ( 7
58 ( 6
85 ( 3
87 ( 2
69 ( 5
76 ( 6
88 ( 3
91 ( 3
91 ( 3
14 ( 14
25 ( 3
74 ( 4
7 ( 17
14 ( 11
8 ( 4
14a
14b
14c
14d
14e
14f
14g
14k
Conclusions
8 ( 14
35 ( 7
-24 ( 12
-10 ( 8
-72 ( 8
-45 ( 18
-12 ( 9
-19 ( 10
17 ( 16
18 ( 4
21 ( 14
10 ( 15
-10 ( 10
80 ( 2
87 ( 3
36 ( 4
Several novel regioisomeric ortho-nitrated catechols
structurally related to BIA 3-202 (3) and BIA 3-335 (4)
have been synthesized and evaluated for their ability
to inhibit COMT. Altering the position of the nitro group
from the meta- to the ortho-position relative to the side-
chain substituent of the nitrocatechol pharmacophore
was found to have a profound effect on the in vitro
potency and in vivo selectivity/duration of COMT inhi-
bition. Compounds 7a, c, and d of the alkylaryl series
were found to be extremely potent inhibitors in vitro
with significantly lower IC50’s than their meta-nitrated
regioisomers and had a rapid onset but shorter duration
of action in vivo than their previously reported regioi-
somers. This may reflect subtle differences in the
metabolic profiles of ortho- and meta-nitrated catechols,
and this aspect is currently under further investigation.
On the other hand, ethanone 7b displayed reversed
selectivity over 3 at 3 and 6 h post dose, thus exhibiting
a degree of preferential inhibition of central COMT. In
the amino-substituted series, regioisomer 14k was found
to be much less peripherally selective than 4 and
endowed with a shorter duration of action, whereas the
decahydroquinoline 14g displayed an unprecedented
combination of almost totally selective peripheral inhi-
bition and excellent duration of action. Ethanone 7b
could be a useful tool to probe the pharmacological
utility of short-acting but centrally selective COMT
inhibitors in the treatment of depression in Parkinso-
nian patients. 14g is also presented as a promising
candidate for clinical evaluation as an adjunct to L-Dopa
therapy for the treatment of the symptoms of Parkin-
son’s disease.
-27 ( 14
-11 ( 15
26 ( 18
7 ( 8
78 ( 5
a Results are mean SEMs of four experiments per group.
inhibitors presenting some degree of selectivity for
central rather than peripheral COMT.
The in vivo time course results for 4, 14a-g, and 14k
in the mouse were equally revealing (Table 5). Whereas
4 has been pharmacologically characterized as a potent,
long-acting, and peripherally selective inhibitor of COMT,
the ortho-nitrated regioisomer 14k was endowed with
a much shorter duration of action, exhibiting only 50%
of the peripheral activity of 4 at the 6 h time point. In
contrast to 4, 14k was also a potent inhibitor of cerebral
COMT at 1 h post dose. In analogy with 7b, 14k seems
to be able to penetrate the BBB more effectively than
its meta-nitro regioisomer 4 but is less able to sustain
inhibition of COMT. Morpholine 14a was poorly selec-
tive at the shorter time point and short acting, whereas
piperidine 14b and pyrrolidine 14c were peripherally
selective but still appreciably short acting compared to
4. Adding substituents to the piperidine ring, such as
3-diethylamide 14d and 3-methyl 14e, abolished central
activity at the cost of a further reduction in the duration
of action. However, shifting the methyl group from C-3
to C-4 of the piperidine ring, as in 14f, effectively
restored both peripheral selectivity and duration of
action, such that 80% inhibition was maintained at 6 h
after administration. Annelation of a lipophilic ring to
the piperidine substituent, as in the decahydroquinoline
14g, resulted in a COMT inhibitor possessing thus far
unprecedented properties, in that 14g is virtually devoid
of central action even shortly after administration (1 h),
yet maintains almost 90% inhibition of peripheral
COMT at 6 h post dose.
Previously, we reported details45,46 on the structure
of the ternary complex between recombinant rat liver
S-COMT, the cosubstrate SAM, and compound 4, as
determined by X-ray crystallography. More recently, we
disclosed preliminary details on the crystallization and
crystallographic data of the ternary complex between
S-COMT, SAM, and 7a.47 The crystals of this complex
are not isomorphous to those of 4, which may reflect
different crystal contacts due to the nature of the side-
chain substituent or position of the nitro group. Further
studies are currently in progress to further characterize
this and other S-COMT-SAM-ortho-nitrated inhibitor
complexes, which are expected to reveal the effect on
binding conformations of the inhibitors described herein
caused by altering the position of the nitro group from
the meta- to the ortho-position. Furthermore, following
Experimental Section
Chemistry. Melting points were measured in open capillary
tubes on an Electrothermal model 9100 hot stage apparatus
and are uncorrected. NMR spectra were recorded on a Bruker
Avance DPX (400 MHz) spectrometer with solvent used as
internal standard, and data are reported in the order: chemi-
cal shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad), number of protons, ap-
proximate coupling constant (J) in hertz, and assignment of a
signal. IR spectra were measured with a Bomem Hartmann
& Braun MB Series FTIR spectrometer using KBr disks.
Analytical TLC was performed on precoated silica gel plates
(Merck 60 Kieselgel F254) and visualized with UV light.
Elemental analyses were performed on a Fisons EA 1110
CHNS instrument, and all analyses are consistent with
theoretical values to within (0.4%, unless otherwise indicated.
Solvents and reagents were purchased from Aldrich, Merck,
and Fluka and used as received unless otherwise noted.
The following details are representative procedures for the
synthesis of alkylaryl compounds 7a-d. Compounds 10a-d
(R ) H) were prepared as previously described.34
1-(4-Acetyloxy-3-methoxyphenyl)-2-phenyl-methanone
(10a, n ) 0, R ) Ac). To a stirred solution of the phenol 10a
(n ) 0, R ) H) (14.9 g, 65 mmol) in dichloromethane (150 mL)
at room temperature was added pyridine (9.17 g, 117 mmol)
and DMAP (spatula tip) followed by acetic anhydride (10.2 g,

8076 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Learmonth et al.
117 mmol) dropwise. The resulting solution was allowed to stir
for 15 min and then washed by cold 2 N hydrochloric acid,
saturated aqueous sodium bicarbonate solution, water, and
brine and then dried over anhydrous sodium sulfate, filtered,
and evaporated to leave a pale yellow solid. Recrystallization
from dichloromethane-diethyl ether-petroleum ether gave
) 7.5, 1.4 Hz, Ar-H), 7.60 (tt, 1H, J ) 7.5, 1.4 Hz, Ar-H), 7.52
(td, 2H, J ) 7.5, 1.4 Hz, Ar-H), 7.32 (d, 1H, J ) 8.2 Hz, H-6),
6.92 (d, 1H, J ) 8.2 Hz, H-5) and 6.41 (br, 1H, OH). 13C NMR
(CDCl3) δ 193.2, 148.1, 143.2, 134.1, 132.8, 129.1, 129.6, 129.2,
120.8, and 120.7.
1-(3,4-Dihydroxy-2-nitrophenyl)-3-[4-[3-(trifluoromethyl)-
phenyl]-1-piperazinyl]-1-propanone Hydrochloride (14k).
A mixture of 3,4-dihydroxy-2-nitroacetophenone (13) (0.099 g,
0.5 mmol), 35% aqueous formaldehyde solution (0.2 mL, 2.5
mmol), and concentrated hydrochloric acid (0.25 mL, 3 mmol)
in 2-propanol (2.5 mL) was heated at reflux for 24 h. The
reaction mixture was allowed to cool to room temperature, and
the resulting precipitate was filtered off and dried in air.
Recrystallization from acetic acid gave yellow crystals (0.19
g, 80%). νmax (KBr disk)/cm-1 3471 (OH), 1687 (CO), 1543
(NO2). 1H NMR (DMSO-d6) δ 9.49 (2H, br, OH), 7.61 (1H, d, J
) 8.5 Hz, H-6), 7.51-7.23 (m, 4H, Ar-H), 7.11 (1H, d, J ) 8.5
Hz, H-5), 3.65 (t, 2H, COCH2), 3.42 (t, 2H, CH2), and 3.39-
3.2 (m, 8H, 4 × CH2). 13C NMR (DMSO-d6) δ 194.1, 153.7,
150.9, 140.0, 139.7, 131.2, 130.9, 125.4 (J ) 272.3 Hz), 123.6,
120.3, 119.8, 116.8, 115.9, 112.8, 51.8, 51.5, 45.9, and 34.0.
1
colorless crystals (16.57 g, 94%) of mp 103-104 °C. H NMR
(CDCl3) δ 7.62 (d, 1H, J ) 2.0 Hz, H-2), 7.60 (dd, 1H, J ) 8.0,
2.0 Hz, H-6), 7.40-7.21 (5H, m, Ar-H), 7.10 (d, 1H, J ) 8.0
Hz, H-5), 3.9 (s, 3H, OCH3), and 2.40 (s, 3H, COCH3). 13C NMR
(CDCl3) δ 198.4, 169.0, 151.8, 144.1, 141.6, 136.0, 128.9, 128.8,
126.6, 123.2, 121.8, 111.9, 56.5, and 21.1.
1-(4-Acetyloxy-3-methoxy-2-nitro-phenyl)-2-phenyl-
methanone (11a, n ) 0, R ) Ac) and 1-(4-Acetyloxy-3-
methoxy-6-nitro-phenyl)-2-phenyl-methanone (12a, n )
0, R ) Ac). To a stirred solution of the O-acetate obtained
above (16.4 g, 60.7 mmol) in acetic anhydride (170 mL) at room
temperature was added copper (II) nitrate trihydrate (18.3 g,
75.87 mmol) in one portion. After approximately 10 min, an
exothermic reaction began, which was controlled by occasion-
ally cooling the reaction mixture in an ice-water bath over
30 min. Once the exotherm had subsided, the reaction mixture
was poured onto water (1.2 L), and the resulting precipitate
was filtered off, washed by water, and dried in air. Recrystal-
lization from dichloromethane-petroleum ether gave 12a (n
) 0, R ) Ac) as a pale-yellow solid (3.75 g, 20%) of mp 210.2-
6-Benzyloxy-7-hydroxy-1-tetralone (21, n ) 2). To a
stirred solution of 6,7-dihydroxy-1-tetralone (19, n ) 2) (1.0
g, 5.62 mmol) in dimethylformamide (25 mL) at room temper-
ature was added anhydrous potassium carbonate (0.78 g, 5.62
mmol) followed by benzyl bromide (0.96 g, 5.62 mmol) drop-
wise. The resulting suspension was stirred at 90 °C for 3 h
and then allowed to cool to room temperature. Inorganic
material was filtered off, and the filter cake was washed with
dimethylformamide (5 mL). The solvent was then evaporated
off and the residue partitioned between 2 N aqueous sodium
hydroxide solution (25 mL) and diethyl ether (25 mL). The
aqueous phase was separated and carefully acidified to pH 2
by the addition of 2 N hydrochloric acid. After extraction with
ethyl acetate, the organic phase was washed with water and
brine and then dried over anhydrous sodium sulfate, filtered,
and evaporated to leave a brownish solid. Recrystallization
from 2-propanol gave long beige needles (0.86 g, 57%) of mp
1
211.1 °C. H NMR (CDCl3) δ 7.85-7.75 (m, 5H, Ar-H), 7.71
(s, 1H, H-5), 7.20 (s, 1H, H-2), 3.91 (s, 3H, OCH3), and 2.42 (s,
3H, COCH3). 13C NMR (CDCl3) δ 193.8, 169.0, 154.0, 148.3,
144.8, 134.7, 129.7, 129.6, 129.2, 111.8, 111.4, 57.5, and 21.1.
The mother liquors were evaporated, and the residue was
chromatographed over silica gel (dichloromethane-petroleum
ether, 1:1). Homogeneous fractions were pooled and evaporated
to leave an oil that solidified on standing. Recrystallization
from dichloromethane-diethyl ether-petroleum ether gave
11a (n ) 0, R ) Ac) as a very pale-yellow solid (9.89 g, 52%)
1
of mp 84.5-86 °C. H NMR (CDCl3) δ 7.80-7.54 (m, 5H, Ar-
H), 7.33 (d, 1H, J ) 8.5 Hz, H-6), 7.22 (d, 1H, J ) 8.5 Hz,
H-5), 4.02 (s, 3H, OCH3), and 2.41 (s, 3H, COCH3). 13C NMR
(CDCl3) δ 192.6, 169.0, 144.7, 144.1, 140.6, 134.0, 130.5, 129.1,
127.4, 125.8, 117.8, 56.5, and 21.1.
1
135-137 °C. H NMR (CDCl3) δ 7.61 (s, 1H, H-8), 7.41-7.28
(m, 5H, Ar-H), 6.75 (s, 1H, H-5), 5.6 (br, 1H, OH), 5.25 (s, 2H,
CH2Ph), 2.92 (t, 2H, J ) 6.3 Hz, CH2), 2.55 (t, 2H, J ) 6.3 Hz,
COCH2), and 1.91 (m, 2H, CH2). 13C NMR (CDCl3) δ 202.2,
150.4, 145.0, 137.4, 135.9, 129.3, 129.1, 128.3, 125.9, 113.6,
111.1, 71.5, 39.3, 37.4, and 26.0.
1-(4-Hydroxy-3-methoxy-2-nitro-phenyl)-2-phenyl-meth-
anone (11a, n ) 0, R ) H). A stirred suspension of 11a (n )
0, R ) Ac) obtained above (11.3 g, 35.8 mmol) in methanol
(120 mL) at room temperature was treated with aqueous 3 N
sodium hydroxide solution (36 mL, 108 mmol) dropwise. The
resulting red solution was stirred for 15 min and then cooled
in an ice-water bath and acidified to pH 2 by the addition of
concentrated hydrochloric acid. The resulting yellow precipi-
tate was filtered off, washed with water, and dried to give a
yellow solid (9.12 g, 93%) of mp 168.9-170 °C. 1H NMR
(CDCl3) δ 7.80 (dt, 2H, J ) 7.5, 1.7 Hz, Ar-H), 7.71 (tt, 1H, J
) 7.5, 1.7 Hz, Ar-H), 7.55 (td, 2H, J ) 7.5, 1.7 Hz, Ar-H), 7.32
(d, 1H, J ) 8.5 Hz, H-6), 7.21 (d, 1H, J ) 8.5 Hz, H-5), 6.35
(br, 1H, OH), and 4.02 (s, 3H, OCH3). 13C NMR (CDCl3) δ 192.5,
153.1, 144.7, 140.5, 136.5, 134.0, 130.4, 129.0, 127.3, 125.9,
117.6, and 63.4.
6-Benzyloxy-7-hydroxy-8-nitro-1-tetralone (23, n ) 2).
To a stirred suspension of the O-benzyl-1-tetralone (21, n )
2) obtained above (0.81 g, 3.02 mmol) in glacial acetic acid (8
mL) in a water cooling bath was added 65% nitric acid
dropwise (0.32 mL, 4.54 mmol), during which time the initial
colorless mixture rapidly became deep-brown followed by the
formation of a copious reddish precipitate. After stirring at
room temperature for 40 min, the mixture was poured onto
ice-water (50 mL), and the dark orange-red precipitate was
filtered off, washed with water, and dried in air. Recrystalli-
zation from dichloromethane-ethyl acetate afforded yellow
1
crystals (0.60 g, 63%) of mp 232-233 °C. H NMR (CDCl3) δ
7.48-7.27 (m, 5H, Ar-H), 6.81 (s, 1H, H-6), 6.06 (br, 1H, OH),
5.22 (s, 2H, CH2Ph), 2.95-2.91 (t, 2H, J ) 6.2 Hz, CH2), 2.52-
2.28 (t, 2H, J ) 6.2 Hz, CH2), and 1.98-1.95 (m, 2H, CH2).
13C NMR (CDCl3) δ 199.3, 150.8, 138.2, 138.1, 135.0, 129.7,
129.6, 128.5, 117.6, 112.3, 72.5, 39.5, 36.8, and 26.4.
1-(3,4-Dihydroxy-2-nitro-phenyl)-2-phenyl-methanone
(7a, n ) 0). To a stirred solution of the methyl ether 11a (n )
0, R ) H) obtained above (0.27 g, 1 mmol) in 1,2-dichloroethane
(3 mL) at room temperature was added aluminum chloride
(0.15 g, 1.1 mmol) in one portion followed by pyridine (0.33 g,
4 mmol) dropwise. The resulting deep-red suspension was
stirred at 100 °C for 30 min and then allowed to cool to room
temperature. The mixture was poured onto ice-water (20 mL)
and acidified by the addition of 2 N hydrochloric acid (4 mL).
The phases were separated, and the aqueous phase was
extracted with 10% 2-propanol-dichloromethane. The com-
bined organic phases were washed with water and brine and
then dried over anhydrous sodium sulfate, filtered, and
evaporated to leave a yellow-orange solid. Recrystallization
from dichloromethane-petroleum ether gave orange crystals
(0.77 g, 81%). νmax (KBr disk)/cm-1 3246 (OH), 1656 (CO), 1550
6,7-Dihydroxy-8-nitro-1-tetralone (15, n ) 2). A suspen-
sion of the nitro-phenol (23, n ) 2) obtained from above (0.56
g, 1.79 mmol) in 30% solution of hydrogen bromide in acetic
acid (5 mL) and 48% hydrobromic acid (5 mL) was heated at
110 °C for 30 min. The dark solution was then allowed to cool
to room temperature and poured onto ice-water (100 mL). The
resulting precipitate was filtered off, washed with water, and
dried in air. Recrystallization from ethyl acetate gave yellow
crystals (0.23 g, 58%). νmax (KBr disk)/cm-1 3477 (OH), 1663
(CO), 1543 (NO2). 1H NMR (DMSO-d6) δ 10.61 (br, 2H 2 ×
OH), 6.81 (s, 1H, H-5), 2.85 (t, 2H, J ) 5.8 Hz, CH2), 2.53 (t,
2H, J ) 6.2 Hz, COCH2), and 2.02 (t, 2H, J ) 6.1 Hz, CH2).
1
(NO2). H NMR (CDCl3) δ 10.41 (1H, br, OH), 7.83 (dt, 2H, J

Inhibitors of Catechol-O-methyltransferase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8077
13C NMR (DMSO-d6) δ 194.6, 153.3, 139.7, 139.4, 138.0, 115.9,
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115.7, 39.3, 29.5, and 23.6.
6,7-Dihydroxy-8-nitro-1-hydroxyiminotetralone (25,
n ) 2). A suspension of the tetralone (15, n ) 2) obtained from
above (0.2 g, 0.89 mmol), hydroxylamine hydrochloride (0.2 g,
2.87 mmol), and pyridine (0.25 g, 3.14 mmol) in ethanol (10
mL) was stirred at 85 °C for 4 h and then allowed to cool to
room temperature. The solvent was evaporated and the residue
partitioned between ethyl acetate and water. The phases were
separated, and the aqueous layer was extracted with ethyl
acetate. The combined organic extracts were washed with 1
N hydrochloric acid, water, and brine and then dried over
anhydrous sodium sulfate, filtered, and evaporated to leave a
red-orange solid. Recrystallization from toluene-ethanol gave
orange crystals (0.16 g, 75%). νmax (KBr disk)/cm-1 3507(NOH),
1
3412 (OH), 1531 (NO2). H NMR (DMSO-d6) δ 10.92 (br, 1H,
NOH), 9.85 (br, 2H, 2 × OH), 6.51 (s, 1H, H-5), 2.35-2.20 (m,
4H, 2 × CH2), and 1.4 (q, 2H, J ) 5.8 Hz, CH2). 13C NMR
(DMSO-d6) δ 150.8, 148.0, 139.1, 138.0, 132.9, 116.4, 114.1,
30.1, 24.4, and 21.8.
Pharmacology. Complete experimental protocols for the
evaluation of in vitro and in vivo COMT inhibitory activity of
43-44
new compounds have been previously reported.34,
All
animal interventions were performed in accordance with
European Directive number 86/609, and the rules of the
National Institute of Health’s Guide for the Care and Use of
Laboratory Animals (http://oacu.od.nih.gov/regs/guide/gui-
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Supporting Information Available: Spectroscopic data
for compounds 7b-d, 14a-j, 14l, 15, 16, 25, 26, and 27a-d
and microanalytical data for all test compounds. This material
is available free of charge via the Internet at http://pubs.ac-
s.org.
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