Discovery of a long-acting, peripherally selective inhibitor of catechol- O -methyltransferase

Article abstract of DOI:10.1021/jm1001524

Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a 1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4, 6-dimethylpyridine 1-oxide (BIA 9-1067)) was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation as an adjunct to l-Dopa therapy of Parkinson's disease.

Full text of DOI:10.1021/jm1001524

3396 J. Med. Chem. 2010, 53, 3396–3411
DOI: 10.1021/jm1001524
Discovery of a Long-Acting, Peripherally Selective Inhibitor of Catechol-O-methyltransferase
†
Laszlo E. Kiss, Humberto S. Ferreira, Leonel Torrao, Maria Joao Bonifacio, P. Nuno Palma,
†
‡
‡
‡
ꢀ
Patrı
ꢀ
~
~
ꢀ
´
cio Soares-da-Silva,*,‡ and David A. Learmonth†
†
‡
Laboratory of Chemistry, Laboratory of Pharmacology, and Department of Research and Development, BIAL, A Avenida da Siderurgia
ꢁ
Nacional, 4745-457 S. Mamede do Coronado, Portugal
Received February 4, 2010
Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit
catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a
1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT
inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to
analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in
comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity
and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-
3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide (BIA 9-1067))
was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation
as an adjunct to L-Dopa therapy of Parkinson’s disease.
Introduction
Catechol-O-methyltransferase (COMTa)1 catalyzes the
transfer of a methyl group from S-adenosyl-L-methionine to
catecholicsubstratessuchasendogenouscatecholneurotrans-
mitters2 and xenobiotics3,4 including (S)-2-amino-3-(3,4-di-
hydroxyphenyl)propanoic acid (L-Dopa), the gold standard5
drug for treatment of Parkinson’s disease (PD).6,7 Coadmi-
Figure 1. Chemical structures of tolcapone 1, entacapone 2, and
nebicapone 3.
nistration of a peripheral amino acid decarboxylase (AADC)
inhibitor prevents breakdown of L-Dopa in the periphery by
blocking enzymatic decarboxylation,8 and inhibition of
COMT further improves its bioavailability by reducing the
formation of 3-O-methyl-L-Dopa (3-OMD).
“First-generation” COMT inhibitors3,9 such as pyrogallol,
tropolone, and gallic acid are poorly selective and have poor
efficacy in vivo. “Second-generation” inhibitors10,11 are exem-
plifiedby tolcapone 1,12 entacapone 2,13 and nebicapone(BIA
3-202) 314 (Figure 1). Structure-activity (SAR) studies ex-
Figure 2. Chemical structure of screening hit 4.
ploring the position of the nitro group15 and various side-
uncoupling of oxidative phosphorylation.20,21 Nebicapone 3
possesses a longer duration of peripheral COMT inhibition
than 2 and more limited access to the brain than 1, but due to
limited clinical experience, firm conclusions concerning safety
have not yet been established. Undoubtedly therefore, a
requirement exists for improved COMT inhibitors to address
the unmet medical needs of many PD patients.
Our group has recently focused on the design of longer
acting COMT inhibitors, and a cell viability assay was devel-
oped in order to assess the safety of the compounds. Pyrazole
4 (Figure 2) was found to possess activity in a preliminary in
vitro screen. Although 4 has an atypical structure compared to
classical COMT inhibitors, it offered scope for an optimiza-
tion program, since several fragments of the molecule were
amenable to structural modification. Introducing electron-
withdrawing groups onto the catechol ring would be expected
to increase COMT inhibition. The pyrazole could be replaced
chain substituents16 have been reported. Subtle differences in
the mode of COMT inhibition by 1-3 are thought to be
relevant in terms of efficacy. Entacapone 2 is a short-acting,17
peripherally selective inhibitor which is taken concomitantly
with every dose of L-Dopa. Albeit the most widely marketed
COMT inhibitor, the clinical efficacy of 2 has been ques-
tioned.18 Tolcapone 1 is a more potent, longer acting but
nonselective inhibitor of both cerebral and peripheral COMT.
Unlike 2, clinical use of 1 is severely restricted due to its
elevated hepatotoxicity risk,19 postulated to occur through
*To whom correspondence should be addressed. E-mail: Psoares.
silva@bial.com. Phone: þ351-22-9866100. Fax: þ351-22-9866192.
a Abbreviations: COMT, catechol-O-methyltransferase; PD, Parkinson’s
disease; AADC, amino acid decarboxylase; SAR, structure-activity rela-
tionship; ADMET, absorption, distribution, metabolism, excretion, toxicity;
L-Dopa, (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid; 3-OMD, 3-
O-methyl-L-Dopa.
r
pubs.acs.org/jmc
Published on Web 03/24/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3397
Scheme 1a
a Reagents and conditions: (a) 60% HNO3, AcOH, room temperature; (b) 48% HBr, 120-140 °C; (c) AlCl3, pyridine, DCE, reflux; (d) MeOH,
HCl(g); (e) Me2SO4 or BnBr, acetone, reflux; (f) NaOH, MeOH; (g) PhN(Me)3þBr3-, THF, 0 °C; (h) CuCN, NMP, 170 °C; (i) CH3COCl, AlCl3, DCE,
room temperature; (j) NH2OH HCl, NaOAc, water, 60 °C; (k) Ac2O, 140 °C; (l) NH2OH HCl, KOH, ethanol, 70 °C.
3
3
with other heterocycles of different size or containing other
heteroatoms in alternative spatial arrangements within the
ring. Finally, variation of the substituents on the central
heterocyclic ring could allow manipulation of the physico-
chemical properties of the compounds, important for influen-
cing ADMET properties.
benzoic acid (7d) was converted to the corresponding methyl
ester and then etherified by reaction with either Me2SO4 or
benzyl bromide (BnBr). Hydrolysis of the esters to carboxylic
acid derivatives 8d and 8e was accomplished using aqueous
sodium hydroxide in methanol at 60 °C. 5-Acetyl-2,3-di-
methoxybenzonitrile (10) was prepared via the 5-bromo inter-
mediate 9. Regioselective bromination of acetovanillone 5a
was followed by O-methylation of the phenolic group
(acetone, Me2SO4, K2CO3). The resulting intermediate 9 was
heated with copper(I) cyanide in N-methyl-2-pyrrolidinone
(NMP) to yield 10 in 40% yield. 1-Fluoro-2,3-dimethoxyben-
zene, which was prepared by exhaustive methylation of 3-
fluorocatechol 11, underwent Friedel-Crafts acylation with
acetyl chloride to furnish acetophenone 12 in 58% yield.
Synthesis of the amidoxime 15 began with 3,4-dimethyl-5-
nitrobenzaldehyde (8c), which reacted with hydroxylamine
Chemistry
The catechol intermediates 8a-e, 10, 12, and 15 containing
various electron-withdrawing groups (NO2, CN, F) were
prepared as summarized in Scheme 1. Vanillin derivatives
5a-d underwent regioselective nitration to the corresponding
5-nitro derivatives 6a-d in moderate to good yields
(55-95%). Attempts to directly protect the phenolic hydroxyl
groupthroughalkylation of6a-c toobtain 8a-c failed. Thus,
the monohydroxyl compounds 6b-d were demethylated in
boiling 48% aqueous hydrobromic acid (HBr). A milder
procedure was applied for 6a, wherein cleavage was effected
with aluminum chloride (AlCl3) and pyridine in refluxing 1,2-
dichloroethane (1,2-DCE).22 The nitrocatechols 7a-c thus
obtained were treated with dimethyl sulfate (Me2SO4) and
potassium carbonate (K2CO3) in warm acetone to provide the
fully protected intermediates 8a-c. 3,4-Dihydroxy-5-nitro-
hydrochloride (NH2OH HCl) to give the oxime 13 in 93%
3
yield. Dehydration of 13 occurred in hot acetic anhydride, and
the resulting nitrile 14 was converted to 15 on reaction with
NH2OH HCl.
3
Pyrazoles were prepared by the general method depicted in
Scheme 2. Condensation of appropriately substituted aceto-
phenones (5e-g, 8a,b, 10, 12) with dimethylformamide

3398 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
Scheme 2a
a Reagents and conditions: (a) DMF-dimethylacetal, neat, 140 °C; (b) R1-NH-NH2, EtOH, reflux; (c) 48% HBr, 140 °C; (d) PhCO2H, CDI, DMF,
room temperature, then 120 °C; (e) 48% aqueous HBr, 30% HBr in AcOH, 120 °C; (f) AlCl3, pyridine, DCE, reflux; (g) Ph-(CO)-NH-NH2, CDI, THF,
reflux; (h) POCl3, neat, 120 °C; (i) BBr3, DCM, -78 °C, then room temperature; (j) CDI, DMF, room temperature, then 100-157 °C; (k) CDI, DMF,
room temperature, then TBAF, THF, room temperature.
dimethylacetal (DMF-DMA) provided enaminones 16a-g in
good to excellent yield (67-96%), which were subsequently
reacted with selected hydrazines to provide the dimethoxy-
pyrazole derivatives.23 Removal of both O-methyl protecting
groups was achieved with aqueous 48% aqueous HBr to
furnish the final catechol products 17a-k and 17m. In the
case of the cyano-substituted pyrazole derived from the
acetophenone 10, deprotection of the methyl protecting
groups under the strongly acidic conditions caused concomi-
tant hydrolysis of the nitrile group, thereby forming the 5-
carboxyl-substituted catechol derivative 17l.
The synthetic routes employed to obtain the three possible
oxadiazole regioisomers are briefly outlined in Scheme 2.
Benzoic acid was condensed with amidoxime 15 using N,N0-
carbonyldiimidazole (CDI) as the coupling agent, followed by
thermal cyclization of the O-acylated intermediate to form the
oxadiazole ring. Intermediate 18 was monodemethylated in a
mixture of a 30% solution of HBr in AcOH and aqueous 48%
HBr. Compound 19 was then obtained by heating the above
mono-O-methyl intermediate (2-methoxy-6-nitro-4-(5-phen-
yl-1,2,4-oxadiazol-3-yl)phenol) with AlCl3 and pyridine. In
a similar fashion, O-protected 5-nitrovanillic acids 8d,e
were activated by CDI and then allowed to react with the
corresponding amidoximes. The O-acylated amidoxime inter-
mediates underwent thermal cyclization to provide oxadia-
zoles 22a-u.24 Alternatively, cyclization of certain isolated
O-acylated amidoximes was promoted by tetrabutylammo-
nium fluoride (TBAF) in THF.25 Cleavage of the protecting
groups of 22a-u involved a two-step procedure. First, 22a-u
were treated with boron tribromide (BBr3) in dichloro-
methane at -78 °C to furnish the intermediate monomethoxy
intermediates, which were then fully demethylated on treat-
ment with AlCl3 and pyridine to access the desired 1,2,4-
oxadiazolylnitrocatechols 23a-u. The regioisomeric 1,3,4-
oxadiazoles were prepared according to the following proce-
dure. Benzohydrazide was acylated with 3,4-dimethoxy-
5-nitrobenzoicacid(8d) using CDI inrefluxing THF. Without
purification, the intermediate thus obtained was dehydrated
with phosphorus oxychloride to give the 1,3,4-oxadiazole
20.26 Heating 20 in aqueous 48% HBr led to destruction of
the heterocyclic ring, while reaction of 20 with BBr3 stalled
after removal of only one methyl group. The second methyl
group could however be removed on further treatment of this
intermediate with AlCl3 and pyridine.
Representative syntheses of various heterocyclic analogues
are depicted in Scheme 3. Imidazole derivatives were prepared

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3399
Scheme 3a
pyridine N-oxide residues are outlined in Scheme 4 and are
similar to the methods employed for preparation of 1,2,4-
oxadiazoles (23a-u). Due to the difficulties encountered in
the demethylation of intermediates 22a-u, the more labile
O-benzyl group was chosen for protection of the catechol.
Pyridines containing electron-withdrawing groups (halogen,
trifluoromethyl) did not readily undergo N-oxidation with
either peroxyacetic acid or m-chloroperoxybenzoic acid
(MCPBA), and only trace amounts of the desired N-oxides
were detected. Therefore, a considerably stronger oxidant was
required to obtain compounds 36a-d and 32e-m. The pyr-
idine intermediates were oxidized using a mixture of urea-
hydrogen peroxide addition complex (UHP) and trifluoroa-
cetic anhydride (TFAA) in dichloromethane.31 In most cases,
conversions around 90% were achieved, even for those pyr-
idines containing electron-withdrawing groups. Either the
pyridyl N-oxide group could be introduced on a downstream
building block (32n-u) or, alternatively, the oxidation step
could be performed on the O-benzyl protected oxadiazole
derivatives 35a-d. The 2-trifluoromethylnicotinonitrile 32t
was prepared via a novel route32 from a readily available
fluorinated precursor. Accordingly, substituted nicotinoni-
triles 32a-m were converted to the corresponding amidox-
imes 33a-m on reaction with aqueous H2NOH, and these
were allowed to react with di-O-benzylnitrovanillic acid (8e)
activated by CDI. After isolation of the O-acylated amidox-
ime intermediates 34a-m, cyclization was performed either
thermally in DMF or by treatment with TBAF in THF.
Conversion of the protected pyridine N-oxides 36a-m to
the desired nitrocatechols 37a-m was readily achieved on
exposure to BBr3.
Results and Discussion
In vitro COMT inhibition by new compounds was evalu-
ated in rat liver homogenates by measuring the formation of
metanephrine (O-methylated adrenaline) as previously de-
scribed.33 All nonnitrated catechols were evaluated under
competitive conditions at a concentration of 30 μM, whereas
nitrocatechols were tested under tight-binding conditions at a
10-fold lower concentration of 3 μM. The results from a
representative selection of compounds (17a-m) are shown
in Table 1. The 3,4,5-trihydroxypyrogallol (17i) was more
active than the 2,3,4-trihydroxy isomers (4, 17j) and the
catechol 17m, as well as compounds incorporating weakly
electron-withdrawing groups (17k, fluorine, and 17l, carbo-
xyl). As expected, pyrazoles 17a-g containing a nitro group
exhibited the strongest inhibition.
Focus then shifted to the central pyrazole ring, and we
looked first at the effect of substitution of the nitrogen atom,
while maintaining a hydrogen atom at positionR2. The parent
compound 17g abolished COMT activity, as did compounds
containing various substituents on the phenyl group (17a-c
and 17e). Residual COMT activity was observed when the
phenyl group was replaced by smaller alkyl groups, such as
methyl (17d). The introduction of a second aryl group at
position R3 however led to a drastic reduction in COMT
inhibition (17f), which could be partially restored by insertion
of a carbonyl group between the nitrogen of the pyrazole and
the aryl ring (17h).
a Reagents and conditions: (a) Ph-NH2, ethanol reflux; (b) TOSMIC,
DME-MeOH, K2CO3, reflux; (c) 48% HBr, 140 °C; (d) R-C(dNH)-
NH2 HCl, KOtBu, ethanol, reflux; (e) Pb(OAc)4, AcOH, 110 °C; (f)
3
NH4OAc, AcOH, 120 °C; (g) PhN(Me)3þBr3-, THF, 0 °C; (h)
NH2-(CdS)NH2, ethanol, reflux.
by a known method.27 Thus aldehyde 8c was condensed with
aniline, and the resulting Schiff base was cyclocondensed with
tosylmethyl isocyanide (TOSMIC) under basic conditions to
providethe protected imidazole 24, which underwent cleavage
on treatment with 48% aqueous HBr to afford hydrobromide
salt 25. The pyrimidine 26 was prepared by cyclization of
benzamidine hydrochloride with enaminone 16a and potas-
sium tert-butoxide in ethanol.28 Demethylation of 26 in
aqueous 48% HBr furnished catechol 27 in 67% yield. The
oxazole analogue 29 was synthesized starting from ketone 8b.
Reaction of 8b with lead(IV) tetraacetate in acetic acid and
treatment of the intermediate with ammonium acetate in
AcOH29 gave oxazole 28. Finally, cleavage of both methyl
protecting groups occurred smoothly on reaction with 48%
aqueous HBr. Aminothiazole 31 was also prepared by a
literature method.30 Bromination of ketone 8b and treatment
of phenacyl bromide 30 with thiourea in ethanol led to the
cyclic thiazole intermediate, which underwent methyl ether
cleavage on reaction with 48% aqueous HBr.
Thereafter, the pyrazole ring itself was modified. Several
five- and six-membered heterocyclic analogues were prepared
in which the number and arrangement of the heteroatoms
varied. The majority of these compounds, as well as some of
The general synthetic routes to support SAR exploration
within the series of 1,2,4-oxadiazoles substituted with various

3400 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
Scheme 4a
a Reagents and conditions: (a) NH2OH, MeOH-H2O, 80 °C; (b) CDI, DMF, room temperature; (c) DMF, 110-155 °C; (d) TBAF, THF, room
temperature; (e) UHP, TFAA, DCM, room temperature; (f) BBr3, DCM, -78 °C; (g) BBr3, DCM, -78 °C, then AlCl3, pyridine, DCE, reflux.
the most active pyrazole derivatives from the initial in vitro
screen (17c, 17e, and 17h) were administrated orally (30 mg/
kg) to mice. Thereafter, at 6 h postadministration, the animals
were killed by decapitation, and the livers were removed and
used to determine COMT activity. Results are shown in
Table 2, which includes comparative data for reference com-
pounds 1-3. N-Phenyl-substituted pyrazole compounds (17c,
17e) showed slightly improved in vivo inhibition over both 2
and 3, but they were significantly less potent than 1, which
shows sustained COMT inhibition. Compound 17h, which
had performed reasonably well in vitro, did not display
promising COMT inhibition in vivo. Surprisingly, it was
discovered that replacement of pyrazole (17c) with an imida-
zole ring (25) led to an abrupt diminishment of COMT
inhibition. On the contrary, pyrimidine 27 performed better
than the pyrazoles and the reference standards 2 and 3.
Replacement of a nitrogen atom either with oxygen, as in
oxazole 29, or with sulfur, as in thiazole 31, provided com-
pounds with poor in vivo efficacy. Heterocyclic rings contain-
ing three heteroatoms were considered at this point, and
oxadiazoles represented an attractive option, as one could
envisage the synthesis of two 3,5-disubstituted 1,2,4-oxadia-
zolyl isomers as well as a third 1,3,4-oxadiazole isomer. While
all three prototype oxadiazoles 19, 21, and 23a showed greater
COMT inhibition than pyrazoles 17c and 17e, the 1,3,4-isomer
21 was clearly the least active of this family. The 1,2,4-isomer
19, which incorporates the nitrocatechol fragment at position
3 of the heterocyclic ring, was significantly more active and
reduced COMTactivity byapproximately75%. However, the
“reverse” 1,2,4-oxadiazole 23a, in which the nitrocatechol
group is located at position 5 of the heterocyclic ring, was
found to the most active of the trio and essentially equipotent
to 1. This hierarchy of activity, 5-(3-nitrocatechol-5-yl)-1,2,4-
oxadiazole > 3-(3-nitrocatechol-5-yl)-1,2,4-oxadiazole >
2-(3-nitrocatechol-5-yl)-1,3,4-oxadiazole, was observed consis-
tently in each family of three regioisomeric oxadiazoles.
The selectivity of COMT inhibition by 19, 21, and 23a was
determined in rats at a lower dose of 3 mg/kg and at 3 h pos-
tadministration (Table 3). As expected, 21 was poorly active and
uninteresting in terms of selectivity. Isomer 19 was found to be
essentially equipotent in both the liver and the brain, causing an
approximately 50% reduction of COMT activity. However, 23a
displayed a clear, 3-fold preference for peripheral COMT inhibi-
tion and was thus selected as a lead for further optimization.
The next round of SAR concentrated on substitution
at position 3 of the oxadiazole ring. Table 4 details results
(mice, 3 mg/kg, 3 h postdose) for new compounds and
includes comparative data for 1 and 2. First, with regard to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3401
Table 1. In Vitro COMT Inhibition by 4 and Pyrazoles 17a-m in Rat
Liver Homogenates
Table 2. In Vivo COMT Inhibition by Selected Heterocyclic Analogues
in Mouse Liver Homogenates
no.
R1
R2 R3
R4
R5 % of controlc
4
17a p-tolyl
15.6 ( 0.2a
H H
H H
H H
H H
H H
H
NO2
0 ( 0a
0 ( 0a
17b 3-chlorophenyl
17c phenyl
17d methyl
H
H
H
H
NO2
NO2
NO2
NO2
0 ( 0a
1.1 ( 0.1a
0.3 ( 0.1a
68.6 ( 2.3a
0 ( 0a
17e 4-cyanophenyl
17f 3-chlorophenyl
H phenyl H NO2
H H NO2
H phenyl H NO2
17g
H
H
17h 4-methylbenzoyl
17i phenyl
17j phenyl
2.8 ( 0.4a
0.8 ( 0.2b
26.8 ( 0.6b
32.3 ( 1.8b
H H
H H
H
OH H
OH
17k 4-(trifluoromethyl)phenyl H H
17l 3-chlorophenyl
17m p-tolyl
F
H
H H
H H
H
H
COOH 24.1 ( 0.3b
H
41.2 ( 1.2b
a Tight binding conditions, Cinhib = 3 μM. b Competitive conditions,
Cinhib = 30 μM. c Results are given as percentage (%) of metanephrine
formed relative to the control measured in the absence of inhibitor and
are expressed as the mean ( SEM of four experiments per group.
phenyl rings, lipophilic substituents, in particular halogens,
were beneficial in terms of potency (23a-d) whereas more
polar piperazine or carboxamide residues were clearly detri-
mental (23e,f). Calculated log P values (see Experimental
Section for details) for the most active oxadiazole compounds
(23a-d) (log P 4.03-5.11) are considerably higher than for 1
(log P 3.30) and 2 (log P 2.00). Thus, despite their promising
activity, 23a-d might raise toxicity concerns. To this end,
compounds 23a-f were subjected to an in vitro toxicity test in
a mouse neuroblastoma cell line as previously described.34
This assay was validated by the finding that over 80% of cells
remained viable after exposure to 2, whereas 1 markedly
reduced the viable cell count to less than 30%. Unfortunately,
the viable cell count was reduced to between 12% and 40% on
exposure to compounds 23a-d. Paradoxically, cell viability
was not significantly reduced on exposure to less lipophilic
analogues 23e,f, the least effective COMT inhibitors.
a All at 30 mg/kg, po, mice; COMT activity was determined 6 h after
administration. b Results are given as percentage (%) of metanephrine
formed relative to the control measured in the absence of inhibitor and
are expressed as the mean ( SEM of four experiments per group. c An
asterisk represents point of attachment.
Table 3. In Vivo COMT Inhibition of Oxadiazoles 19, 21, and 23a in
Homogenates of Rat Liver and Braina
Incorporating lipophilicity index as a filter in the design of
further compounds, several more hydrophilic compounds
containing heterocyclic or heteroaromatic rings at position 3
of the oxadiazole ring were prepared. The morpholine 23g and
piperazine analogues 23i,j, each having log P below 2, dis-
played higher inhibition than 2 and 23e,f and showed border-
line (23g) or low toxicity risk (23i,j). The more lipophilic
2-pyridyl-substituted piperazine 23h showed interesting inhi-
bition (32% of control) but proved to be as toxic as 1. The
phenyl ring was then replaced with various heterocyclic
residues giving compounds covering a range of log P values.
Bioisosteres of 23a such as thiophene 23l and isoxazole 23m
showed comparable activity and toxicity. Thiazole 23k and
pyrimidine 23q, which contain weakly basic nitrogen atoms,
both showed respectable COMT inhibition but borderline
toxicity. The three pyridyl-substituted regioisomers 23n-p
were similar in terms of inhibition (approximately 30% of
control), but only the 2-pyridyl analogue was relatively
nontoxic. The more lipophilic pyrimidyl analogue23q showed
no improvement. Substitution of the pyridyl ring was then
no.
braina,b
livera,b
19
21
55.6 ( 2.4
87.9 ( 21.2
71.7 ( 11.6
51.9 ( 4.8
67.8 ( 10.2
20.6 ( 4.4
23a
a Results are given as percentage (%) of metanephrine formed relative
to the control measured in the absence of inhibitor and are expressed as
the mean ( SEM of four experiments per group. b 3 mg/kg, po; COMT
activity was determined 3 h after administration.
examined. Compounds (23r-u) presented similar inhibitory
activity to that of 23a, yet despite having lower log P also
showed a relatively high toxicity risk.
The pyridines were further functionalized through oxida-
tion of the nitrogen atom. The resulting N-oxides could be
expected to have significantly lower lipophilicity. Although
the very hydrophilic (log P 1.05) nicotinic N-oxide derivative
37f (Table 5) displayed slightly lower activity than 2 (3 mg/kg
po, rat, 3 h), encouragingly it was devoid of toxicity. It was
hypothesized that the low activity of 37f could be due to poor

3402 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
Table 4. In Vivo COMT Inhibition in Mice Liver Homogenates and Cell Viability Count for Selected Oxadiazoles

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3403
Table 4. Continued
a A, CDI/DMF, room temperature, then Δ; B, TBAF, THF, room temperature. b C, BBr3, DCM, -78 °C; D, AlCl3, pyridine, 1,2-DCE or NMP, Δ.
c All at 3 mg/kg, po; COMT activity was determined 3 h after administration; results are given as percentage (%) of metanephrine formed relative to the
control measured in the absence of inhibitor. d Results are mean ( SEMs of four experiments. e % of remaining viable cells. f An asterisk represents point
of attachment.
Table 5. In Vivo COMT Inhibition in Rat Liver Homogenates and Cell Viability Count for Selected Oxadiazolylpyridine N-Oxides
no.
R1
R2
R3
R4
cyclization method
% of controla,b
log P
cell viabilityb,c
1
23.8 ( 14.2
66.6 ( 25.6
13.9 ( 10.9
33.7 ( 7.7
7.9 ( 3.8
3.30
2.00
2.45
2.08
2.91
2.95
2.03
1.05
1.99
1.53
2.48
2.95
4.53
2.03
2.02
27.2 ( 5.6
81.4 ( 2.0
92.3 ( 2.8
76.3 ( 3.1
95.1 ( 6.2
86.6 ( 2.3
93.4 ( 3.0
93.8 ( 2.7
100.2 ( 3.2
93.7 ( 2.0
62.2 ( 3.9
37.2 ( 4.0
26.6 ( 2.8
96.4 ( 3.5
87.0 ( 3.1
2
37a
37b
37c
37d
37e
37f
37g
37h
37i
37j
37k
37l
37m
Me
Me
Me
Me
H
H
H
Me
Cl
H
H
H
H
H
H
H
H
H
Me
H
Cl
TBAF/THF
CDI/DMF
TBAF/THF
CDI/DMF
TBAF/THF
CDI/DMF
TBAF/THF
TBAF/THF
TBAF/THF
CDI/DMF
CDI/DMF
TBAF/THF
TBAF/THF
Br
Cl
Me
Me
CF3
H
Cl
H
0.7 ( 1.1
23.6 ( 13.3
77.0 ( 5.7
31.6 ( 10.9
71.6 ( 8.2
13.9 ( 7.2
1.3 ( 0.3
24.3 ( 30.9
5.5 ( 1.8
34.4 ( 13.2
H
H
Cl
Me
H
H
H
Cl
CF3
Me
Ph
H
H
Me
Me
Me
CF3
H
CF3
CF3
H
CF3
H
a All at 3 mg/kg, po; COMT activity was determined at 3 h after administration; results are given as percentage (%) of metanephrine formed relative to
the control measured in the absence of inhibitor. b Results are mean ( SEMs of four experiments. c % of remaining viable cells.
absorption caused by the very polar nature of the N-oxide
group and that oral bioavailability might be improved by
substituents on the pyridine N-oxide residue. Introduction of
a trifluoromethyl group (37e and 37 m) revealed that interest-
ing levels inhibition could be achieved, without toxicity risk.
Shifting the trifluoromethyl group to the 6-position gave 37l,
which was exceptionally active and devoid of toxicity. Further
substitution (37i, methyl; 37j, dimethyl; 37k, phenyl) was
investigated, and although 37j was found to be even more
potent than 37l, these more lipophilic compounds signifi-
cantly reduced the cell viability count.
Replacing the trifluoromethyl group chlorine as in 37h
failed to provide improvement over the parent 37f. However,
the introduction of one, two, and three methyl groups to 37h

3404 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
Figure 3. COMT activity in rat liver (closed symbols) and brain
(open symbols) homogenates, determined at 1, 3, 6, 9, and 24 h after
oral administration of 3 mg/kg 1 (squares) or 37d (circles). Each
point represents mean values ( SEM of n = 4-8.
Figure 5. Plasma levels of L-Dopa (A) and 3-OMD (B) determined
1 h after oral administration of L-Dopa/benserazide (12/3 mg/kg)
and 2, 7, or 24 h after oral administration of 3 mg/kg 1 (open circles)
or 37d (closed circles). Each point represents mean values ( SEM of
n = 4-16.
later (i.e., 2, 7, and 24 h after administration of 1 and 37d),
L-Dopa and 3-OMD levels were assayed by HPLC. As shown
in Figure 5A, 1 caused an abrupt, 3-fold increase in L-Dopa
levels after 2 h, which fell by one-third at 7 h and was followed
by a gradual return to baseline at 24 h. This rapid fluctua-
tion in L-Dopa levels could potentially cause undesired CNS-
related side effects. Conversely, 37d conferred a more
conservative 2-fold increase in L-Dopa levels at 2 h, but
importantly this increase was sustained virtually over the
entire 24 h period. The ability of 37d to deliver a consistent
increase in L-Dopa levels over time as a result of prolonged
peripheral COMT inhibition could potentially make it suita-
ble for once daily administration. A complementary trend
could be observed for 3-OMD levels in plasma (Figure 5B).
Two hours after administration of 1, significantly reduced 3-
OMD levels were observed, which then rose sharply up to 7 h
followed by a gradual return to baseline over 24 h. With 37d,
however, 3-OMD levels were initially reduced to just below 50%
of control and sustained essentially over the entire 24 h period.
Figure 4. Dose-dependent inhibition of rat liver COMT deter-
mined 3 h after oral administration of 0.03, 0.1, 0.3, 1, 3, and
10 mg/kg 1 (open circles) or 37d (closed circles). Each point
represents mean values ( SEM of n = 4-10.
resulted in consecutive increases in COMT inhibition (37g,
methyl; 37a, dimethyl; 37c, trimethyl) with no reduction in cell
viability. Replacing the 3-methyl substituent of compound
37c with a second chlorine atom gave 37d, which abolished
COMT activity while cell viability was not compromised. On
the basis of these findings, 37d was selected for further pharm-
acological studies.
Figure 3 compares the COMT inhibition profiles of 37d and
1 in rat liver and brain homogenates. Compounds 1 and 37d
achieve maximal inhibitory effect at 1 and 3 h, respectively,
and 1 is essentially inactive against peripheral COMT at 9 h
while central inhibition remains around 50%. Oxadiazole 37d
has no measurable effect on central COMT, but significant
peripheral inhibition (50%) is maintained up to 24 h. The
concentration-dependent peripheral inhibitory potency of 1
and 37d was then evaluated in rats given increasing doses of
each compound (0.03-3 mg/kg). Animals were killed 3 h after
administration, andCOMT activity was then determined. The
results are shown in Figure 4. Compound 37d was found to
be more potent than 1, with ED50’s of 1.05 ( 0.04 and 1.77 (
0.10 mg/kg, respectively.
Conclusion
Novel heterocyclic COMT inhibitors were derived from the
initial in vitro screening hit 4. 1,2,4-Oxadiazoles substituted
with a pyridine N-oxide motif (37a-m) were found to have
reduced toxicity risk and were endowed with longer duration
of inhibition than reference compounds 1 and 2. Oxadiazole
37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-
oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide, BIA 9-1067)
was selected for further pharmacological studies and was
found to be a purely peripheral inhibitor of COMT with a
unprecedented duration of action. In addition, 37d presents
Finally, the pharmacodynamic interaction of 37d with L-
Dopa was assessed35 and compared to 1. Both compounds
were administered to overnight fasted rats (3 mg/kg each);
thereafter, at defined intervals (1, 6, and 23 h) L-Dopa (12 mg/
kg) was coadministered with benserazide (3 mg/kg). One hour

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3405
favorable pharmacodynamics with L-Dopa, which results in
stable and sustained plasma L-Dopa levels over prolonged
periods, such that a single daily dose of 37d may be effective in
the clinical setting of PD.
dimethyl sulfate (55.21 g, 437.76 mmol) in acetone (350 mL) was
stirred at vigorous reflux for 1.5 h. The reaction mixture was
then cooled to room temperature and filtered through Celite,
and the filter cake was washed with acetone. The combined
filtrate was evaporated to leave an oil that crystallized from 2-
propanol to leave a pale yellow solid (12.29 g, 62%). 1H NMR
(CDCl3): δ 7.90 (1H, d, J = 2 Hz), 7.72 (1H, d, J = 2 Hz), 4.05
(3H, s), 3.98 (3H, s), 2.62 (3H, s). 13C NMR (CDCl3): δ 195.1,
154.2, 146.8, 144.2, 132.1, 117.1, 114.2, 62.1, 56.6, 26.3.
Experimental Section
Chemistry. 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: chemical shift
(ppm), number of protons, multiplicity (s, singlet; d, doublet;
dd, doublet of doublet; t, triplet; q, quartet; m, multiplet; br,
broad), approximate coupling constant (J) in hertz, and assign-
ment of a signal. Elemental analyses were performed on a CE
Instruments, EA 1110 CHNS analyzer. The purity of the
compounds in all cases was higher than 95%. Analytical TLC
was performed on precoated silica gel plates (Merck 60 Kieselgel
F 254) and visualized with UV light. Solvents and reagents were
purchased from Aldrich, Merck, and Fluka and were used
without further purification unless otherwise noted. Com-
pounds 32b,36 32c,37 32r,38 and 32s39 were synthesized by known
methods.
1-(4-Hydroxy-3-methoxy-5-nitrophenyl)ethanone (6a). To a
solution of acetovanillone 5a (20 g, 120.35 mmol) in acetic acid
(200 mL) was added 60% nitric acid (9.7 mL, 126.4 mmol)
dropwise. The reaction was allowed to stir at room temperature
for 0.5 h and then poured into ice-water (1 L). The resulting
precipitate was filtered off, washed with water, and dried under
vacuum to give the product as a yellow powder (20.37 g, 80%).
1H NMR (CDCl3): δ 11.14 (1H, s), 8.31 (1H, d, J = 2 Hz), 7.76
(1H, d, J = 2 Hz), 4.00 (3H, s), 2.63 (3H, s). 13C NMR (CDCl3):
δ 194.9, 150.3, 150, 132.9, 128.3, 117.7, 115.1, 56.8, 26.
1-(3,4-Dihydroxy-5-nitrophenyl)ethanone (7a). Compound 6a
(20.3 g, 96.13 mmol) was suspended in 1,2-dichloroethane (200
mL) at 0 °C and treated with AlCl3 (14.10 g, 105.74 mmol)
followed by addition of pyridine (30.41 g, 384.52 mmol) drop-
wise under argon. The red reaction mixture was heated at reflux
for 2 h, then was cooled to room temperature, and poured onto a
mixture of ice and 2 N HCl. The resulting yellow precipitate was
filtered off, washed with water, and dried under vacuum to give
a yellow solid (17.3 g, 91%). 1H NMR (DMSO-d6): δ 10.86 (2H,
br), 7.94 (1H, d, J = 2 Hz), 7.56 (1H, d, J = 2 Hz), 2.52 (3H, s).
13C NMR (DMSO-d6): δ 195.4, 147.6, 145.8, 137.1, 127.3, 117.1,
116.5, 26.3.
3,4-Dihydroxy-5-nitrobenzaldehyde (7c). A suspension of 6c
(19 g, 96.4 mmol) in 48% aqueous HBr (165 mL) was heated at
140 °C for 4 h; then the reaction was cooled to room temperature
and poured onto ice-water. The resulting dark yellow precipi-
tate was filtered off, washed with water, then dissolved in ethyl
acetate, and dried over MgSO4. The solution was filtered and
evaporated under vacuum. The residue was stirred with diethyl
ether, filtered, and dried in air to leave a yellow powder (10.65 g,
60%). 1H NMR (DMSO-d6): δ 11.0 (2H, br), 9.80 (1H, s), 7.98
(1H, d, J = 2 Hz), 7.46 (1H, d, J = 2 Hz). 13C NMR (DMSO-
d6): δ 190.2, 148, 147, 137, 126.7, 119.6, 115.5.
3,4-Dimethoxy-5-nitrobenzoic Acid (8d). Acid 7d (16.4 g,
82.36 mmol) was dissolved in MeOH (300 mL), and dry HCl
gas was bubbled through the mixture. After 30 min, the HCl
stream was shut off, and the mixture was stirred at room
temperature for 1 h. The methanol was removed by evaporation,
and the residue was dissolved in a mixture of 2-propanol/
dichloromethane (1:9). After being washed with water, the
organic phase was dried over MgSO4 and filtered. Evaporation
of the solvent afforded 16.68 g of a yellow solid. The thus
obtained methyl 3,4-dihydroxy-5-nitrobenzoate (16.65 g, 78.1
mmol) was taken up in acetonitrile (250 mL), and freshly
powdered K2CO3 (54.0 g, 391 mmol) was added, followed by
dimethyl sulfate (49.31 g, 391 mmol). The resulting mixture was
heated at 100 °C for 1.5 h and then allowed to cool to room
temperature. The insoluble material was filtered off, and the
filter cake was washed with acetonitrile. The combined filtrate
was evaporated to leave an orange oil that solidified on standing
overnight. Recrystallization from 2-propanol gave a white solid
(9.5 g, 50%). The thus obtained methyl 3,4-dimethoxy-5-nitro-
benzoate (9.5 g, 39.39 mmol) was suspended in a mixture of
methanol (150 mL) and water (30 mL). Aqueous sodium
hydroxide solution (3 N) (39 mL, 117 mmol) was added in one
portion, and the mixture was stirred at 60 °C for 1 h. The
resulting deep red solution was cooled to room temperature. The
methanol was removed by evaporation, and water (200 mL) was
added to the residue. The mixture was cooled in ice-water, and
37% aqueous HCl was added until pH 2 was reached. The
copious white precipitate was filtered off, washed with water,
and dried under vacuum (8.2 g, 92%). 1H NMR (CDCl3): δ 8.11
(1H, s), 7.81 (1H, s), 4.08 (3H, s), 4.02 (3H, s). 13C NMR
(CDCl3): δ 169.0, 153.7, 146.9, 144.3, 124.2, 118.4, 116.4, 62.3,
56.7.
1-(3-Bromo-4-hydroxy-5-methoxyphenyl)ethanone (9). To an
ice-cooled solution of acetovanillone 5a (5.0 g, 30.12 mmol) in
THF (50 mL) was added phenyltrimethylammonium tribro-
mide (12.49 g, 33.23 mmol), allowing each portion to dissolve
before adding the next. After being stirred for 15 min in the cold,
the reaction was poured onto cold water (400 mL). The resulting
precipitate was filtered off, washed with water, and dried in air.
Recrystallization from 2-propanol afforded beige crystals
(3.98 g, 54%). 1H NMR (CDCl3): δ 7.75 (1H, d, J = 1.8 Hz), 7.48
(1H, d, J = 1.8 Hz), 6.45 (1H, s, exch), 3.98 (3H, s), 2.57 (3H, s).
5-Acetyl-2,3-dimethoxybenzonitrile (10). Compound 9 (3.92
g, 16 mmol) was converted to 1-(3-bromo-4,5-dimethoxyphe-
nyl)ethanone by a similar procedure described for 8a. Recrys-
tallization from dichloromethane-petroleum ether afforded a
white solid (3.15 g, 76%). A mixture of the thus obtained 1-(3-
bromo-4,5-dimethoxyphenyl)ethanone (1.69 g, 6.52 mmol) and
Cu(I)CN (642 mg, 7.18 mmol) was heated in NMP (10 mL) at
170 °C for 5 h. The reaction mixture was cooled to room
temperature and then poured onto water. The brown precipitate
was filtered off and washed with water. The solid was stirred in a
dichloromethane-2-propanol mixture (9:1) and left in an ultra-
sound bath at room temperature for 10 min. After the suspended
material was allowed to sediment, the mixture was then filtered
through Celite. The filtrate was washed with water and brine
and then dried over MgSO4. Evaporation of the solvent left a
brown solid, which was recrystallized from dichloromethane-
2-propanol to leave a pale brown solid (512 mg, 40%). 1H NMR
(CDCl3): δ 7.74 (1H, d, J = 1.8 Hz), 7.73 (1H, d, J = 1.8 Hz),
4.16 (3H, s), 3.96 (3H, s), 2.60 (3H, s). 13C NMR (CDCl3): δ
3,4-Dihydroxy-5-nitrobenzoic Acid (7d). 6d (18.5 g, 86.8
mmol) was placed in a mixture of aqueous 48% HBr (120 mL)
and 30% HBr in acetic acid (120 mL). The reaction mixture was
heated at 120 °C for 3.5 h, then cooled to room temperature, and
poured onto ice-water. The precipitated solid was filtered off
and washed with water. The filtrate was extracted by ethyl
acetate, and the extracts were washed with brine, then dried
over MgSO4, filtered, and evaporated leaving a yellow solid.
Both solids were combined and dried by azeotropic distillation
1
with toluene twice to leave a yellow solid (16.42 g, 95%). H
NMR (DMSO-d6): δ 10.7 (1H, br), 7.85 (1H, s br), 7.57 (1H, s
br). 13C NMR (DMSO-d6): δ 166.1, 147.9, 145.8, 137.3, 121.2,
119.3, 117.1.
1-(3,4-Dimethoxy-5-nitrophenyl)ethanone (8a). A mixture of
6a (17.26 g, 87.55 mmol), K2CO3 (42.3 g, 306.52 mmol), and

3406 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
194.8, 155.1, 152.1, 132.6, 125.8, 115.6, 115.2, 105.6, 61.8, 56.3,
26.4.
cooled to room temperature, then poured onto water (100 mL),
and extracted with dichloromethane. The organic phase was
washed with water and brine, then dried over MgSO4, filtered,
and evaporated. Chromatography (petroleum ether-ethyl acet-
ate, 4:1) gave 5-(3,4-dimethoxy-5-nitrophenyl)-1-p-tolyl-1H-
pyrazole as a thick yellow oil (510 mg, 68%). A solution of this
intermediate (490 mg, 1.44 mmol) in 48% aqueous HBr (16 mL)
was heated at 130 °C for 3.5 h. The reaction mixture was then
allowed to cool to room temperature and poured onto ice--
water (150 mL). The resulting orange precipitate was filtered off,
washed with water, and dried under vacuum to give the product
(312 mg, 69%). 1H NMR (DMSO-d6): δ 10.45 (2H, s, br), 7.71
(1H, s), 7.24 (2H, d, J = 8 Hz), 7.23 (1H, s), 7.17 (2H, d, J =
8 Hz), 6.82 (1H, s), 6.64 (1H, s), 2.33 (3H, s). 13C NMR (DMSO-
d6): δ 147.4, 141.5, 140.4, 139.8, 137.0, 136.9, 136.9, 129.4, 124.8,
120.4, 119.0, 114.4, 107.6, 20.8.
1-(2-Fluoro-3,4-dimethoxyphenyl)ethanone (12). 3-Fluoroca-
techol 11 (5.0 g, 39.06 mmol) was converted to 1-fluoro-2,3-
dimethoxybenzene by a similar procedure as described for 8a,
and the product was then taken up in 1,2-dichloroethane
(50 mL), whereupon acetyl chloride (5.685 g, 72.42 mmol) was
added. After the mixture was cooled in an ice-water bath, AlCl3
(12.37 g, 92.77 mmol) was added in portions. The mixture was
stirred in the cold for 1 h and then at room temperature for 0.5 h.
The reaction was then cooled again and carefully quenched with
a mixture of ice and 2 N HCl. The phases were separated, and the
organic phase was washed with water and brine, then dried over
Na2SO4, filtered, and evaporated. The residue was chromato-
graphed (petroleum ether-ethyl acetate, 4:1) to give 4.45 g of a
mobile oil that solidified on standing (58%). 1H NMR (CDCl3):
δ 7.66 (1H, dd, J = 8.2 and 9 Hz), 6.77 (1H, dd, J = 1.6 and 9
Hz), 3.95 (3H, s), 3.94 (3H, d, J = 1 Hz), 2.62 (3H, d, J = 5.5
Hz). 13C NMR (CDCl3): δ 194.3 (d, J = 3.5 Hz), 157.6 (d, J =
5 Hz), 156.4 (d, J = 254 Hz), 136.8 (d, J = 15 Hz), 125.1 (d, J =
4.5 Hz), 119.6 (d, J = 12.5 Hz), 107.1 (d, J = 3 Hz), 61.7 (d, J =
3.3 Hz), 56.4, 31.7 (d, J = 7 Hz).
3,4-Dimethoxy-5-nitrobenzaldehyde Oxime (13). A mixture of
aldehyde 8c (1.5 g, 7.11 mmol), hydroxylamine hydrochloride
(2.07 g, 29.79 mmol), and sodium acetate (5.21 g, 63.53 mmol)
was heated in water (55 mL) at 60 °C for 1 h and then cooled to
room temperature. The resulting precipitate was filtered off,
washed with water, and dried under vacuum to give the product
as off-white crystals (1.5 g, 93%). 1H NMR (DMSO-d6): δ 11.53
(1H, s), 8.17 (1H, s), 7.61 (1H, s, br), 7.57 (1H, s, br), 3.92 (3H, s),
3.88 (3H, s). 13C NMR (DMSO-d6): δ 153.3, 146, 144.3, 141.7,
129.2, 113.3, 113.3, 61.7, 56.5.
3,4-Dimethoxy-5-nitrobenzonitrile (14). Oxime 13 (1.45 g, 6.41
mmol) was suspended in acetic anhydride (10 mL) and the
mixture then heated at 140 °C for 24 h. After being cooled to
room temperature, the mixture was poured onto ice-water. The
resulting precipitate was filtered off, washed with water, and
dried under vacuum. Trituration in a mixture of petroleum ether
and diethyl ether followed by filtration and drying gave the title
compound as an off-white powder (1.15 g, 86%). 1H NMR
(CDCl3): δ 7.66 (1H, d, J = 2 Hz), 7.31 (1H, d, J = 2 Hz), 4.06
(3H, s), 3.98 (3H, s). 13C NMR (CDCl3): δ 154.4, 146.5, 144.6,
120.3, 118.1, 116.6, 107.2, 62.5, 56.9.
3,4-Dimethoxy-5-nitrobenzamidoxime (15). To a stirred mix-
ture of nitrile 14 (1.10 g, 5.29 mmol) and hydroxylamine hydro-
chloride (1.481 g, 21.30 mmol) in ethanol (35 mL) was added 12
mL of 10% aqueous KOH solution. The reaction was heated 70
°C for 1 h and then allowed to cool to room temperature. The
ethanol was removed by vacuum, the residue was dissolved in
ethyl acetate, dried over MgSO4, and filtered, and the solvent
was evaporated. Trituration in a mixture of petroleum ether and
diethyl ether followed by filtration and drying gave the title
compound as an off-white powder (1,15 g, 90%). 1H NMR
(DMSO-d6): δ 9.88 (1H, s), 7.69 (1H, s), 7.62 (1H, s), 6.05 (2H, s),
3.93 (3H, s), 3.38 (3H, s). 13C NMR (DMSO-d6): δ 152.8, 148.6,
143.9, 141.4, 129.2, 113.3, 112, 61.6, 56.6.
4-(5-(3,4-Dihydroxy-5-nitrophenyl)-1H-pyrazol-1-yl)benzo-
nitrile (17e). Compound 16a (400 mg, 1.43 mmol) was cyclized
with4-cyanophenylhydrazine hydrochloride (267 mg, 1.57 mmol)
in ethanol (5 mL) as described for 17a. A solution of the
obtained pyrazole derivative (340 mg, 0.97 mmol) in dichlor-
omethane (5 mL) was cooled to -78 °C and treated with BBr3
(2.43 g, 9.70 mmol). The resulting deep purple mixture was
allowed to stir at room temperature for 5 h, then cooled to
-20 °C, and carefully quenched with ice-water. The mixture
was then extracted with a dichloromethane-2-propanol mix-
ture (7:3), and the extracts were washed with water, then dried
over MgSO4, filtered, and evaporated to leave an orange solid.
The thus obtained 4-(5-(4-hydroxy-3-methoxy-5-nitrophenyl)-
1H-pyrazol-1-yl)benzonitrile (330 mg, 0.98 mmol) was sus-
pended in 1,2-dichloroethane (4 mL) and treated with AlCl3
(142 mg, 1.07 mmol), followed by addition of pyridine (307 mg,
3.88 mmol). The reaction was stirred at reflux for 1.5 h and then
allowed to cool to room temperature. After being poured onto a
mixture of 2 N HCl and ice, the resulting precipitate was filtered
off, washed with water, and dried under vacuum. The product
was obtained as a yellow solid (284 mg, 91%). 1H NMR
(DMSO-d6): δ 10.42 (2H, s), 7.93 (2H, d, J = 8.0 Hz), 7.84
(1H, s), 7.51 (2H, d, J = 8.0 Hz), 7.29 (1H, s), 6.82 (1H, s), 6.72
(1H, s). 13C NMR (DMSO-d6): δ 147.5, 142.5, 141.7, 141.1,
140.9, 137.1, 133.2, 125.0, 119.7, 118.9, 118.1, 114.6, 109.7, 109.1.
3-(3,4-Dimethoxy-5-nitrophenyl)-5-phenyl-1,2,4-oxadiazole (18).
CDI (294 mg, 1.81 mmol) was added to a stirred solution of
benzoic acid (201 mg, 1.65 mmol) in DMF (8 mL). The colorless
reaction mixture was stirred at room temperature for 1 h under
argon, whereupon the amidoxime 15 (361 mg, 1.5 mmol) was
added, and the reaction was stirred for further 2 h at room
temperature. After addition of a second aliquot of CDI (294 mg,
1.81 mmol), the reaction was then heated at 120 °C for 2 h. After
being cooled to room temperature, the mixture was slowly
poured onto water, and the resulting precipitate was filtered
off, washed with water, and dried under vacuum. Chromatog-
raphy using neat dichloromethane gave the title compound as a
white powder (244 mg, 50%). 1H NMR (CDCl3): δ 8.23 (2H, d,
J = 8.3 Hz), 8.19 (1H, d, J = 1.8 Hz), 7.89 (1H, d, J = 1.8 Hz),
7.65 (1H, t, J = 7.2 Hz), 7.58 (2H, t, J = 7.5 Hz), 4.07 (3H, s),
4.05 (3H, s). 13C NMR (CDCl3): δ 175.9, 166.9, 154.2, 144.9,
144.7, 132.9, 129, 128.1, 123.7, 122.6, 115.4, 113.9, 62.2, 56.8.
3-Nitro-5-(5-phenyl-1,2,4-oxadiazol-3-yl)benzene-1,2-diol (19).
Compound 18 (197 mg, 0.60 mmol) was heated at 120 °C in a
mixture of aqueous 48% HBr (3 mL) and 30% HBr in acetic acid
(2 mL) for 30 min. The reaction was then allowed to cool to
room temperature and poured onto ice-water. The resulting
yellow precipitate was filtered off, washed with water, and dried
under vacuum. The thus obtained 2-methoxy-6-nitro-4-(5-
phenyl-1,2,4-oxadiazol-3-yl)phenol (156 mg, 0.50 mmol) was
suspended in 1,2-dichloroethane (8 mL) and treated with AlCl3
(73 mg, 0.55 mmol), followed by addition of pyridine (291 mg,
3.68 mmol). The red reaction mixture was heated at reflux for 2 h
under argon, then cooled to room temperature, and poured onto
1-(3,4-Dimethoxy-5-nitrophenyl)-3-(dimethylamino)prop-2-en-
1-one (16a). A suspension of acetophenone 8a (12.22 g, 54.26 mmol)
in dimethylformamide dimethylacetal (80 mL) was stirred at 140 °C
for 5 h and then allowed to cool to room temperature. The resulting
orange precipitate was filtered off, washed with diethyl ether, and
dried in air to give the product (13.12 g, 86%). 1H NMR (CDCl3):δ
7.84 (1H, d, J=12.3 Hz), 7.81 (1H, d, J=2Hz), 7.72(1H, d, J=2
Hz), 5.64 (1H, d, J = 12.3 Hz), 4.00 (3H, s), 3.98 (3H, s), 3.19 (3H,
s), 2.96 (3H, s). 13C NMR (CDCl3): δ 184.7, 155, 153.9, 144.8, 144,
135.9, 115.1, 114.9, 90.7, 62, 56.5, 45.3, 37.5.
3-Nitro-5-(1- p-tolyl-1H-pyrazol-5-yl)benzene-1,2-diol (17a).
A mixture of enaminone 16a (522 mg, 1.86 mmol) and p-
tolylhydrazine hydrochloride (310 mg, 1.95 mmol) in ethanol
(10 mL) was heated at reflux for 1.5 h. The reaction mixture was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3407
a mixture of ice and 1 N HCl solution. The resulting yellow
precipitate was filtered off, washed with water, and dried under
vacuum (72 mg, 40%). 1H NMR (DMSO-d6): δ 10.94 (2H, br),
8.17 (2H, d, J = 7.5 Hz), 8.0 (1H, d, J = 2 Hz), 7.74 (2H, m), 7.65
(2H, t, J = 7.8 Hz). 13C NMR (DMSO-d6): δ 175.1, 166.6, 148.2,
144.4, 137.2, 133.3, 129.4, 127.8, 123.0, 116.3, 116.1, 114.0.
2-(3,4-Dimethoxy-5-nitrophenyl)-5-phenyl-1,3,4-oxadiazole (20).
A mixture of acid 15 (532 mg, 2.34 mmol) and CDI (418 mg,
2.57 mmol) was heated in THF (10 mL) at reflux for 3 h under
argon and then cooled to room temperature. Benzohydrazide
(351 mg, 2.57 mmol) was added in one portion, and the yellow-
ish mixture was stirred at reflux overnight. After being cooled
to room temperature, the mixture was poured onto water
(100 mL), and the copious precipitate was filtered off, washed
water, and dried under vacuum (630 mg, 78%). The thus
was dried over MgSO4, filtered, and evaporated to leave a yellow
solid (259 mg, 95%). The thus obtained 2-methoxy-6-nitro-4-(3-
phenyl-1,2,4-oxadiazol-5-yl)phenol intermediate was taken up
in 1,2-dichloroethane (15 mL) and treated with AlCl3 (117 mg,
0.88 mmol) followed by pyridine (300 mg, 3.80 mmol). The red
reaction mixture was heated at reflux for 2 h under argon, then
cooled to room temperature, poured onto a mixture of ice and
37% aqueous HCl, and extracted with ethyl acetate. The organic
phase was dried over MgSO4, filtered, and evaporated to leave a
colorless oil, which crystallized as a white powder on trituration
with diethyl ether (157 mg, 60%). 1H NMR (DMSO-d6): δ 11.10
(2H, br), 8.09 (1H, s), 8.07 (2H, d, J = 7 Hz), 7.76 (1H, s), 7.60
(3H, m). 13C NMR (DMSO-d6): δ 173.7, 167.8, 148.2, 145.5,
137.4, 131.4, 129.0, 126.8, 125.8, 116.5, 115.0, 113.1.
5-(3-(4-Bromophenyl)-1,2,4-oxadiazol-5-yl)-3-nitrobenzene-
1,2-diol (23d). The monomethyl ether 22d (500 mg, 1.27 mmol)
was placed in NMP (2 mL) and treated with AlCl3 (211 mg,
1.58 mmol), followed by the addition of pyridine (403 mg,
5.10 mmol). The red reaction mixture was heated at reflux for
0.5 h under argon, then cooled to room temperature, and poured
onto a mixture of ice and 37% aqueous HCl. The resulting
yellow precipitate was filtered off, washed with water, and dried
obtained
N0-benzoyl-3,4-dimethoxy-5-nitrobenzohydrazide
(359 mg, 1.04 mmol) was heated in phosphorus oxychloride
(10 mL) at 120 °C for 3 h. The pale yellow solution was then
allowed to cool to room temperature and carefully poured onto
ice-water (200 mL). After being stirred for 1 h at room tempera-
ture, the resulting precipitate was filtered off and washed with
plenty of water. Drying under vacuum afforded the title com-
pound as a white solid (309 mg, 91%). 1H NMR (CDCl3): δ 8.16
(2H, d, J = 7.6 Hz), 8.04 (1H, d, J = 2 Hz), 7.93 (1H, d, J =
2 Hz), 7.58 (3H, m), 4.09 (3H, s), 4.07 (3H, s).
3-Nitro-5-(5-phenyl-1,3,4-oxadiazol-2-yl)benzene-1,2-diol (21).
A solution of compound 20 (200 mg, 0.61 mmol) in dichlor-
omethane (8 mL) was cooled to -78 °C and treated with BBr3
(766 mg, 3.06 mmol) under argon. The resulting deep purple
mixture was allowed to reach room temperature for 1 h, then
cooled again to -78 °C, and carefully quenched by the addition
of methanol. After removal of the solvents by evaporation, the
residual yellow solid was triturated with diethyl ether, then
filtered, and dried under vacuum. The thus obtained mono-
methyl ether (200 mg, 0.69 mmol) was suspended in 1,2-dichlor-
oethane (5 mL) and treated with AlCl3 (180 mg, 1.355 mmol)
followed by pyridine (385 mg, 4.88 mmol). The reaction was
stirred at reflux for 4 h under argon. The reddish suspension was
then cooled to room temperature and poured onto a mixture of
2 N HCl and ice. After being stirred for 30 min at room tempera-
ture, the resulting precipitate was filtered off, washed with
water, and dried under vacuum to give the product as a yellow
powder (112 mg, 59%). 1H NMR (DMSO-d6): δ 11.0 (2H, s, br),
8.11 (2H, d, J = 7.3 Hz), 8.05 (1H, s, br), 7.76 (1H, s, br), 7.63
(3H, m). 13C NMR (DMSO-d6): δ 163.6, 162.5, 148.2, 144.4,
137.5, 131.8, 129.2, 126.4, 123.0, 115.8, 113.5, 113.4.
4-(5-(3,4-Bis(benzyloxy)-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-
morpholine (22g). A stirred solution of acid 8e (727 mg, 1.92 mmol)
in DMF (5 mL) was stirred with CDI (358 mg, 2.21 mmol) for 1.5 h
under argon. Thereupon, 4-morpholinoamidoxime (320 mg,
2.21 mmol) was added, and the mixture was stirred for additional
3 h and then poured onto water. The resulting precipitate was
filtered off, washed with water, and dried under vacuum. The thus
obtained O-acylated amidoxime intermediate (858 mg, 1.69 mmol)
was dissolved in THF (10 mL) and treated with a 1 N solution of
TBAF in THF (1.7 mL). The reaction was allowed to stir at room
temperature for 3 h under argon and then poured onto water. The
resulting precipitate was filtered off, washed with water, and dried
under vacuum. Recrystallization from ethanol gave 22g as a white
powder (527 mg, 56%). 1H NMR (CDCl3): δ 8.09 (1H, d, J = 2
Hz), 7.86 (1H, d, J = 2 Hz), 7.52-7.29 (10H, m), 5.27 (4H, s), 3.84
(4H, m), 3.54 (4H, m). 13C NMR (CDCl3): δ 172.0, 170.4, 153.4,
145.4, 144.7, 135.4, 134.8, 128.7 (2 signals), 128.6, 128.5, 128.3,
127.7, 120.0, 116.3, 115.6, 76.4, 71.9, 66.2, 46.3.
1
under vacuum (460 mg, 96%). H NMR (DMSO-d6): δ 11.17
(2H, br), 8.09 (1H, d, J = 2 Hz), 7.99 (2H, d, J = 8.5 Hz), 7.79
(2H, d, J = 8.5 Hz), 7.73 (1H, d, J =2 Hz). 13C NMR (DMSO-
d6): δ 174.3, 167.5, 148.6, 146.0, 137.7, 132.4, 129.1, 125.3, 116.7,
115.4, 113.1.
(4-(5-(3,4-Dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)phenyl)-
(4-methylpiperazin-1-yl)methanone Hydrobromide (23e). A solu-
tion of compound 22e (269 mg, 0.59 mmol) in dichloromethane
(10 mL) was cooled to -78 °C and treated with BBr3 (7.08 g,
28.25 mmol). The resulting purple mixture was allowed to reach
room temperature overnight under argon, then cooled again to
-78 °C, and carefully quenched with methanol. The mixture
was evaporated to dryness and azeotroped twice with etha-
nol-toluene to leave a yellowish orange solid (112 mg, 37%). 1H
NMR (DMSO-d6): δ 11.10 (2H, br), 9.94 (1H, br), 8.17 (2H, d,
J = 8.4 Hz), 8.12 (1H, d, J = 2 Hz), 7.78 (1H, d, J = 2 Hz), 7.68
(1H, d, J = 8.4 Hz), 3.47-3.16 (8H, br), 2.84 (3H, s). 13C NMR
(DMSO-d6): δ 174.0, 167.9, 167.3, 148.3, 145.7, 137.4, 127.9,
127.1, 116.5, 115.2, 113.0, 52.1, 44.1, 42.3.
5-(3,4-Dimethoxy-5-nitrophenyl)-1-phenyl-1H-imidazole (24).
A mixture of aniline (232 mg, 2.49 mmol), aldehyde 8c (527 mg,
2.50 mmol), and acetic acid (0.25 mL) was stirred in refluxing
ethanol (12.5 mL) for 2 h. The reaction was then cooled to room
temperature, and the ethanol was removed by vacuum dis-
tillation. The residue was dissolved in a mixture of methanol
(17.5 mL) and 1,2-dimethoxyethane (7.5 mL). This solution was
treated with K2CO3 (690 mg, 5 mmol) followed by TOSMIC
(731 mg, 3.74 mmol). The mixture was stirred at reflux for 3 h
under argon and then allowed to cool to room temperature, and
the solvents were evaporated off. The residue was dissolved in
dichloromethane, and the organic phase was washed with water,
then dried over MgSO4, filtered, and evaporated. The crude
product was purified by chromatography (dichloromethane-
methanol, 99:1). Trituration of the crude product with diethyl
ether and filtration provided 24 as a white powder (363 mg,
45%). 1H NMR (CDCl3): δ 7.74 (1H, s), 7.49 (3H, m), 7.34 (1H,
s), 7.24 (2H, m), 7.18 (1H, s), 6.70 (1H, s), 3.95 (3H, s), 3.60 (3H,
s). 13C NMR (CDCl3): δ 153.5, 144.6, 141.6, 139.3, 135.8, 130.6,
129.6, 129.3, 128.6, 125.7, 125.2, 114.9, 114.9, 62.1, 56.1.
3-Nitro-5-(1-phenyl-1H-imidazol-5-yl)benzene-1,2-diol Hydro-
bromide (25). Compound 24 (289 mg, 0.89 mmol) was heated
in 48% aqueous HBr (5 mL) at 140 °C for 2.5 h, whereupon the
reaction mixture was cooled to room temperature, and the
volatiles were removed by distillation under vacuum. The
resulting yellow crystalline mass was dried under high vacuum.
Trituration of the crude product with diethyl ether and filtration
provided 25 as a yellow powder (285 mg, 85%). 1H NMR (DMSO-
d6): δ 10.60 (2H, br), 9.54 (1H, s), 8.12 (1H, d, J = 1.5 Hz), 7.58
3-Nitro-5-(3-phenyl-1,2,4-oxadiazol-5-yl)benzene-1,2-diol (23a).
A solution of compound 22a (283 mg, 0.86 mmol) in DCM
(12 mL) was cooled to -78 °C and treated with BBr3 (1.65 g, 6.58
mmol). The resulting dark mixture was allowed to reach room
temperature over 0.5 h under argon, then carefully poured onto
ice-water, and extracted with ethyl acetate. The organic phase

3408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
1
(2H, m), 7.57 (1H, m), 7.51 (2H, m), 7.25 (1H, d, J = 2 Hz), 6.83
(1H, d, J = 2 Hz). 13C NMR (DMSO-d6): δ 147.6, 142.4, 136.9,
136.8, 133.4, 132.1, 130.2, 129.6, 126.2, 119.1, 118.3, 115.7, 115.5.
4-(3,4-Dimethoxy-5-nitrophenyl)-2-phenylpyrimidine (26). A
suspension of the enaminone 16a (300 mg, 1.07 mmol), benza-
midine hydrochloride (186 mg, 1.18 mmol), and potassium tert-
butoxide (264 mg, 1.47 mmol) in ethanol (6 mL) was heated at
100 °C in a sealed tube for 1 h. The reaction mixture was then
allowed to cool to room temperature, and water was added. The
resulting yellow precipitate was filtered off, washed with water,
and dried under vacuum (195 mg, 58%). 1H NMR (CDCl3): δ
8.89 (1H, d, J = 5.2 Hz), 8.55 (2H, m), 8.10 (2H, m), 7.58 (1H, d,
J = 5.2 Hz), 7.54 (3H, m), 4.09 (3H, s), 4.08 (3H, s). 13C NMR
(CDCl3): δ 164.4, 160.9, 158.1, 154.2, 144.8, 144.7, 137.1, 132.4,
130.9, 128.5, 128.1, 114.7, 114.1, 114.0, 62.2, 56.7.
2:1) afforded 30 (1.75 g, 69%) as a pale yellow oil. H NMR
(CDCl3): δ 7.95 (1H, d, J = 2 Hz), 7.75 (1H, d, J = 2 Hz), 7.53
(2H, d, J = 8.5 Hz), 7.44-7.34 (3H, m), 6.29 (1H, s), 4.05
(3H, s), 3.96 (3H, s). 13C NMR (CDCl3): δ 188.1, 154.0, 147.0,
143.9, 134.9, 129.3, 129.0, 128.9, 128.7, 117.3, 115.6, 62.3, 56.7,
50.2.
5-(2-Amino-5-phenylthiazol-4-yl)-3-nitrobenzene-1,2-diol Hydro-
bromide (31). A mixture of compound 30 (500 mg, 1.32 mmol) and
thiourea (110 mg, 1.44 mmol) was heated in ethanol (5 mL) at
reflux for 15 min. The reaction was allowed to cool to room
temperature, and the solvent was removed by distillation under
vacuum. The residue was added to 48% aqueous HBr (11 mL) and
heated at 140 °C for 2.5 h. After being cooled to room temperature,
the mixture was poured onto water, and the resulting yellow
precipitate was filtered off, washed with water, and dried under
vacuum (450 mg, 83%). 1H NMR (DMSO-d6): δ 10.50 (1H, br),
8.69 (1H, br), 7.43 (1H, d, J = 2.2 Hz), 7.41-7.32 (3H, m), 7.28
(2H, dd, J = 2.0 and 8.0 Hz), 7.02 (1H, d, J = 2.2 Hz). 13C NMR
(DMSO-d6): δ 166.9, 147.4, 142.1, 136.8, 134.7, 129.6, 129.0, 128.6,
128.4, 120.5, 119.3, 118.2, 115.2.
3-Cyano-4-(trifluoromethyl)pyridine 1-Oxide (32e). To a stir-
red mixture of 4-(trifluoromethyl)nicotinonitrile 32n (343 mg,
1.99 mmol) and urea-hydrogen peroxide addition complex
(UHP) (564 mg, 6.00 mmol) in dichloromethane (30 mL) was
added trifluoroacetic anhydride (TFAA) (1.26 g, 6.0 mmol) at
0 °C under argon. The reaction was allowed to stir at room
temperature for 0.5 h. Excess peroxide was destroyed by the
addition of 10% aqueous potassium iodide solution (30 mL). The
organic phase was washed with saturated 10% aqueous Na2S2O3
solution and water, then dried over MgSO4, and filtered. The
solvent was removed by evaporation, and the residue was tritu-
rated by diethyl ether to give 32e (156 mg, 42%) as a white solid.
1H NMR (CDCl3): δ 8.49 (1H, m), 8.39 (1H, m), 7.66 (1H, d,
J = 7 Hz). 13C NMR (CDCl3): δ 143.1, 142.5, 126.9 (q, J =
35.5 Hz), 124.0 (q, J = 4.5 Hz), 120.8 (q, J = 273 Hz), 110.7,
110.5 (br).
2-Chloro-4,6-dimethylnicotinamidoxime (33a). To a stirred
suspension of 2-chloro-4,6-dimethylnicotinonitrile 32a (623 mg,
3.74 mmol) in ethanol (15 mL) was added a 50% aqueous
solution of hydroxylamine (1.23 mL, 20.07 mmol). The reaction
was allowed to stir at reflux overnight and then cooled to room
temperature, whereupon the solvent was evaporated off. The
residue was triturated with diethyl ether. The resulting white
precipitate was filtered off, washed with diethyl ether, and dried
under vacuum (574 mg, 77%). 1H NMR (DMSO-d6): δ 9.39 (1H,
s), 7.17 (1H, s), 5.88 (2H, s), 2.41 (3H, s), 2.26 (3H, s). 13C NMR
(DMSO-d6): δ 157.7, 150.1, 148.7, 147.8, 126.8, 123.3, 23.3, 18.9.
Nicotinamidoxime 1-Oxide (33f). To a stirred solution of
3-cyanopyridine 1-oxide 32g (600 mg, 5.0 mmol) in a mixture
of ethanol (50 mL) and water (10 mL) was added hydroxylamine
hydrochloride (1.564 g, 22.5 mmol) followed by sodium acetate
(2.05 g, 25 mmol). The reaction was heated at 70 °C for 1 h and
then cooled to room temperature, and the solvent was evapo-
rated off. The resulting white precipitate was filtered off, washed
with water, and dried under vacuum (395 mg, 52%). 1H NMR
(DMSO-d6): δ 10.16 (1H, s), 8.47 (1H, s), 8.23 (1H, d, J = 6.3
Hz), 7.61 (1H, d, J = 8.0 Hz), 7.43 (1H, dd, J = 6.3 and 8.0 Hz),
6.09 (2H, s). 13C NMR (DMSO-d6): δ 146.9, 138.4, 135.3, 132.4,
126.0, 122.1.
3-Nitro-5-(2-phenylpyrimidin-4-yl)benzene-1,2-diol Hydrobro-
mide (27). Compound 26 was synthesized by a similar procedure
as described for 7c. Treatment of compound 26 (182 mg,
0.54 mmol) in a mixture of 48% aqueous HBr (8 mL) and
30% HBr in acetic acid (8 mL) at 150 °C for 6 h afforded 27
(142 mg, 67%) as a yellow solid. 1H NMR (DMSO-d6): δ 10.80
(2H, br), 8.92 (1H, d, J = 5.4 Hz), 8.49 (2H, m), 8.29 (1H, d, J =
2 Hz), 8.14 (1H, d, J = 2 Hz), 7.97 (1H, d, J = 5.4 Hz), 7.57 (3H,
m). 13C NMR (DMSO-d6): δ 162.8, 160.8, 158.3, 147.8, 143.8,
137.8, 136.9, 130.9, 128.6, 127.6, 126.2, 116.8, 114.3, 113.8.
4-(3,4-Dimethoxy-5-nitrophenyl)-2-methyl-5-phenyloxazole (28).
A suspension of 8b (1.00 g, 3.32 mmol) and Pb(OAc)4 (1.68 g,
3.80 mmol) in acetic acid (10 mL) was heated at 110 °C for 2 h.
The reaction was then allowed to cool to room temperature and
poured onto water. The mixture was extracted with ethyl
acetate, and the organic extracts were washed by water, satu-
rated aqueous NaHCO3 solution, and brine, then dried over
MgSO4, filtered, and evaporated to leave a pale yellow oil. Chro-
matography (petroleum ether-ethyl acetate, 4:1) gave 2-(3,4-
dimethoxy-5-nitrophenyl)-2-oxo-1-phenylethyl acetate (917 mg,
77%) as a pale yellow oil that solidified on standing. A mixture
of this intermediate (860 mg, 2.34 mmol) and ammonium
acetate (554 mg, 7.19 mmol) in acetic acid (10 mL) was heated
at 120 °C overnight. The reaction was allowed to cool to room
temperature and then poured onto water. The mixture was
extracted with ethyl acetate, and the organic extracts were
washed by water, saturated aqueous NaHCO3 solution, and
brine, then dried over MgSO4, filtered, and evaporated to leave
an orange oil. Chromatography (petroleum ether-ethyl acetate,
4:1) gave 28 (633 mg, 78%) as a pale yellow oil that slowly
1
solidified on standing. H NMR (CDCl3): δ 7.63 (1H, d, J =
2 Hz), 7.60 (2H, m), 7.45-7.35 (4H, m), 4.01 (3H, s), 3.84 (3H, s),
2.57 (3H, s). 13C NMR (CDCl3): δ 160.3, 153.7, 145.9, 144.8,
141.9, 132.6, 129.0, 128.7, 128.6, 127.9, 126.7, 114.7 (2 signals),
62.1, 55.4, 14.1.
5-(2-Methyl-5-phenyloxazol-4-yl)-3-nitrobenzene-1,2-diol Hydro-
bromide (29). Compound 29 was synthesized by a similar pro-
cedure as described for 7c. Treatment of compound 28 (551 mg,
1.62 mmol) with 48% aqueous HBr (13 mL) at 140 °C for 3 h
afforded 29 (220 mg, 34%) as an orange solid. 1H NMR
(DMSO-d6): δ 10.40 (2H, br), 7.55 (1H, d, J = 2 Hz), 7.55
(m, 2H), 7.47 (2H, m), 7.42 (1H, m), 2.49 (3H, s). 13C NMR
(DMSO-d6): δ 160.4, 147.8, 144.6, 141.8, 137.2, 132.6, 129.1,
129.1, 128.2, 126.7, 122.4, 117.9, 113.3, 13.6.
2-Bromo-1-(3,4-dimethoxy-5-nitrophenyl)-2-phenylethanone (30).
To an ice-cooled solution of 8b (2.0 g, 6.64 mmol) in THF
(68 mL) was added a solution of phenyltrimethylammonium
tribromide (2.87 g, 7.63 mmol) in THF (80 mL) dropwise. After
being stirred for 15 min in the cold, the reaction was allowed to
stir at room temperature for 1 h. The resulting white precipitate
was filtered off and washed with diethyl ether. The combined
filtrate was evaporated under vacuum, and the residue was
dissolved in dichloromethane. The organic layer was washed
with water and brine, then dried over MgSO4, filtered, and
evaporated. Chromatography (petroleum ether-ethyl acetate,
5-(3,4-Bis(benzyloxy)-5-nitrophenyl)-3-(2-chloro-4,6-dimethyl-
pyridin-3-yl)-1,2,4-oxadiazole (35a). A stirred solution of acid 8e
(758 mg, 2.0 mmol) in DMF (10 mL) was stirred with CDI
(340 mg, 2.1 mmol) for 1.5 h under argon. The amidoxime 33a
(399 mg, 2.0 mmol) was then added, and the mixture was stirred
for an additional 2 h, whereupon it was poured onto water. The
resulting precipitate was filtered off, washed with water, and
dried under vacuum. Recrystallization from dichloromethane-
2-propanol gave 34a (632 mg, 56%) as an off-white solid. This
intermediate 34a (614 mg, 1.09 mmol) was dissolved in THF
(40 mL) and treated with a 1 N solution of TBAF (2 mL) in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3409
THF. The reaction was allowed to stir at room temperature for
3 h under argon, then poured onto water, and extracted with
dichloromethane. The organic phase was dried over MgSO4 and
filtered, and the solvent was removed by evaporation. The
residue was chromatographed (dichloromethane-methanol,
99:1), and the crude product was recrystallized from 2-propa-
nol-diethyl ether to give 35a as a white powder (288 mg, 48%).
1H NMR (CDCl3): δ 8.22 (1H, m), 8.00 (1H, d, br, J = 1.8 Hz),
7.50 (2H, d, J = 7.8 Hz), 7.47-7.41 (3H, m), 7.39-7.30 (5H, m),
7.11 (1H, br), 5.30 (4H, s), 2.59 (3H, s), 2.28 (3H, s). 13C NMR
(CDCl3): δ 173.8, 166.1, 160.5, 153.7, 150.6, 149.8, 145.5, 145.2,
135.3, 134.6, 128.7, 128.7, 128.7, 128.6, 128.4, 127.7, 123.6,
119.9, 119.2, 116.5, 115.8, 76.4, 72, 24.3, 20.1.
5-(3,4-Bis(benzyloxy)-5-nitrophenyl)-3-(2-bromo-6-methylpyr-
idin-3-yl)-1,2,4-oxadiazole (35b). To a stirred solution of acid 8e
(500 mg, 1.32 mmol) in DMF (5 mL) was added CDI (246 mg,
1.52 mmol) in one portion. The resulting mixture was stirred at
room temperature for 1 h under argon; then the amidoxime 33b
(357 mg, 1.52 mmol) was added in one portion. The mixture was
allowed to stir at room temperature overnight and then poured
onto water (100 mL). Brine (10 mL) was added, and a white
precipitate formed that was filtered off and washed with water.
The solid was dissolved in dichloromethane and washed with
water. The organic phase was dried over MgSO4, filtered, and
evaporated. Recrystallization from dichloromethane-2-propa-
nol afforded 34b (601 mg, 76%) as a white powder. This
intermediate 34b (590 mg, 0.99 mmol) was treated with CDI
(178 mg, 1.097 mmol) in DMF (10 mL) at 120 °C for 3 h. The
reaction was cooled to room temperature and poured onto
water (100 mL). The resulting orange precipitate was filtered
off, washed with water, and dried in air. Recrystallization from
dichloromethane-ethanol mixture gave 35b (261 mg, 38%) as a
pale yellow solid. 1H NMR (CDCl3): δ 8.21 (1H, d, J = 2 Hz),
8.12 (1H, d, J = 7.8 Hz), 7.99 (1H, d, J = 2 Hz), 7.51 (2H, d, J =
7.9 Hz), 7.45 (3H, m), 7.40-7.29 (6H, m), 5.31 (2H, s), 5.30 (2H,
s), 2.66 (3H, s). 13C NMR (CDCl3): δ 173.2, 167.3, 161.8, 153.6,
145.5, 145.2, 140.0, 139.9, 135.3, 134.7, 128.8, 128.7 (2 signals),
128.5, 128.4, 127.7, 122.5, 122.1, 119.1, 116.5, 115.8, 76.4, 72.0,
24.5.
Animal Treatments. Animals were kept without bedding
(gridded cages) for 48 h before the experiments. Animals
fasted overnight were administered orally (rat 4 mL/kg; mice
8 mL/kg) with the test compounds (3 or 30 mg/kg) suspended
in carboxymethylcelulose (0.5% w/v). Then at the required
time the animals were anesthetized by ip administration of
sodium pentobarbital (60 mg/kg) and then were perfused
with NaCl (0.9%) by cardiac route. Liver and brain samples
were collected and stored at -80 °C in 5 mM sodium
phosphate buffer.
Homogenate Preparation. Tissues were thawed at room
temperature and homogenized using a Diax homogenizer
(Heidolph, 2 cycles of 20 s in position 5, on ice). Total protein
content in homogenates was measured with Bio-Rad stan-
dard protein assay using a standard curve of bovine serum
albumin (50-250 μg/mL). Samples were then diluted to
obtain a concentration of 4 mg/mL.
COMT Activity Determination. The reactionmixture (total
volume of 1 mL) contained homogenate (2 mg/mL), NCE
(3 μM for nitrocatechols or 30 μM for non-nitrocatechols),
100 μM MgCl2, 1 mM EGTA, 100 μM pargyline, and
S-adenosyl-L-methionine (500 μM) in phosphate buffer
(5 mM, pH 7.8). After a 20 min preincubation period at
37 °C, the reaction was initiated with adrenaline (1000 μM
when testing nitrocatechols or 25 μM when testing non-
nitrocatechols) and proceeded for 5 min at 37 °C. Reac-
1
tions were terminated with the addition of /10 volume of
2 N perchloric acid. Samples were centrifuged (16000g for
3 min at 4 °C) and filtered through 0.22 μm pore size
Spin-X 200 filters. Metanephrine was then quantified
by HPLC with electrochemical detection as previously
detailed.32 Evaluation of COMT activity in tissues of
animals administered with the NCEs was performed as
described above with changes in the S-adenosyl-L-meth-
ionine concentration (500 μM for rat liver, 100 μM for
rat brain, and 250 μM for mice tissues), adrenaline con-
centration (1000 μM for rat liver, 100 μM for rat brain,
and 50 μM for mice tissues), and incubation times (5 min
for rat liver, 15 min for rat brain, and 10 min for mice
tissues).
Toxicity Studies. Neuro-2A cells seeded in 96-well plastic
culture clusters (Costar) were grown in minimum essential
medium (Sigma-Aldrich, St. Louis, MO) supplemented with
1.5 g L-1 sodium bicarbonate, 1.0 mM sodium pyruvate,
10% fetal bovine serum, 100 units mL-1 penicillin G, 0.25 μg
mL-1 amphotericin B, 100 μg mL-1 streptomycin, and
25 mM N-(2-hydroxyethyl)piperazine-N0-2-ethanesulfonic
acid (HEPES) under a CO2/air (5%/95%) humidified atmo-
sphere at 37 °C. Five days after seeding, cell cultures were
incubated with NCEs (30 μM) for 24 h. Negative controls (no
NCE) and positive controls (cells incubated with ethanol)
were run in parallel. After treatment, cells were washed twice
with Hanks’ medium (medium composition, in mM: NaCl,
137; KCl, 5; MgSO4, 0.8; Na2HPO4, 0.33; KH2PO4, 0.44;
CaCl2, 0.25; MgCl2, 1.0; Tris-HCl, 0.15; sodium butyrate,
1.0; pH 7.4) and loaded with 2 μM calcein-AM in Hanks
medium at room temperature for 30 min. Fluorescence was
measured at 485 nm excitation and 530 nm emission wave-
lengths in a multiplate reader (Spectromax Gemini; Molecu-
2-Chloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-
3-yl)-4,6-dimethylpyridine 1-Oxide (37a). A solution of com-
pound 36a (175 mg, 0.31 mmol) in dichloromethane (8 mL) was
cooled to -78 °C and treated with BBr3 (314 mg, 1.25 mmol)
under argon. The resulting dark mixture was allowed to reach
room temperature for 0.5 h and then cooled again to -20 °C,
whereupon water was carefully added to quench the reaction
mixture. After being stirred for 1 h at room temperature, the
resulting yellow precipitate was filtered off, washed with water,
and dried under vacuum. Recrystallization from a dichloromet-
hane-2-propanol mixture gave compound 37a (57 mg, 48%) as
a yellow solid. 1H NMR (DMSO-d6): δ 11.15 (2H, br), 8.11 (1H,
s), 7.74 (1H, s), 7.61 (1H, s), 2.48 (3H, s), 2.19 (3H, s). 13C NMR
(DMSO-d6): δ 174.5, 164.2, 150.7, 148.3, 145.8, 139.8, 137.5,
135.2, 125.8, 123.0, 116.4, 115.3, 112.6, 18.7, 18.3.
Pharmacology
Animals. Adult male Wistar rats and NMRI mice, sup-
plied by Harlan (Barcelona, Spain), were kept five per cage
under controlled environmental conditions (12 h light/dark
cycle, room temperature 22 ( 1 °C and humidity 50 ( 5%,
food and tap water ad libitum). Rats weighed 250-300 g and
mice weighed 27-40 g. All animal procedures were con-
ducted in the strict adherence to the European Directive for
Protection of Vertebrate Animals Used for Experimental
and Other Scientific Purposes (86/609CEE), Portuguese
legislation, and the rules of the “Guide for the Care and
Use of Laboratory Animals”, 7th ed., 1996, Institute for
Laboratory Animal Research, Washington, DC.
lar Devices). Minimun staining for calcein-AM (calceinmin
)
was determined by treating six wells with ethanol 15 min
beforecalcein-AM addition. The viability (percent) was then
calculated as [(calceinsample - calceinmin)/(calceincontrol
-
calceinmin)] ꢀ 100.35

3410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Kiss et al.
Assay of L-Dopa and 3-OMD in Plasma. Plasma samples
were deproteinized by adding to 600 μL of plasma, 300 μL of
perchloric acid (1 N), and 100 μL of 0.1 N HCl. After an
incubation of 10 min at 4 °C, samples were centrifuged at
16000g for 5 min at 4 °C, and supernatants were filtered
through 0.22 μm filters (Costar Spin-X). L-Dopa and
3-OMD were quantified on the filtrate samples (50 μL) by
HPLC with electrochemical detection as previously de-
scribed.40
(16) Learmonth, D. A.; Palma, P. N.; Vieira-Coelho, M. A.; Soares-da-
Silva, P. Synthesis, biological evaluation, and molecular modeling
studies of a novel, peripherally selective inhibitor of catechol-O-
methyltransferase. J. Med. Chem. 2004, 47, 6207–6217.
(17) Keranen, T.; Gordin, A.; Harjola, V.-P.; Karlsson, M.; Korpela,
K.; Pentikainen, P. J.; Rita, H.; Seppala, L.; Wikberg, T. Inhibition
of soluble catechol-O-methyltransferase and single dose pharma-
cokinetics after oral and intraveneous administration of entaca-
pone. Eur. J. Clin. Pharmacol. 1994, 46, 151–157.
(18) Parashos, S. A.; Wielinski, C. L.; Kern, J. A. Frequency, reasons
and risk factors of entacapone discontinuation in Parkinson dis-
ease. Clin. Neuropharmacol. 2004, 27 (3), 119–123.
Log P Calculation. Octanol/water partition coefficients
were calculated using the log P fragmentation algorithm
implemented in ACD Laboratories v.10 software (Advan-
ced Chemistry Development, Inc.). The standard log P
model was corrected with ACD/PhysChem Accuracy Ex-
tender, by supplementing the internal database of fragment
contributions with experimentally determined log D (pH =
7.4) values of selected oxadiazolylnitrocatechol com-
pounds.
(19) Assal, F.; Spahr, L.; Hadengue, A.; Rubbici-Brandt, L.; Burkhard,
P. R. Tolcapone and fulminant hepaptitis. Lancet 1998, 352, 958.
(20) Nissinen, E.; Kaheinen, P.; Penttila, K. E.; Kaivola, J.; Linden, I. B.
Entacapone, a novel catechol-O-methyltransferase inhibitor for
Parkinson’s disease, does not impair mitochondrial energy produc-
tion. Eur. J. Pharmacol. 1997, 340, 287–294.
(21) Hassio, K.; Koponen, A.; Penttila, K. E.; Nissinen, E. Effects of
entacapone and tolcapone on mitochondrial membrane potential.
Eur. J. Pharmacol. 2002, 453, 21–26.
(22) Learmonth, D. A.; Alves, P. C. Improved method for demethyla-
tion of nitro-catechol methyl ethers. Synth. Commun. 2002, 32 (4),
641–649.
(23) Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.;
Collina, P. W.; Docter, S.; Graneto, M. J.; Lee, L. F.; Malecha, J.
W.; Miyashiro, J. M.; Rogers, R. S.; Rogier, D. J.; Yu, S. S.;
Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.;
Zhang, Y. Y.; Isakson, P. C. Synthesis and biological evaluation of
the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identi-
fication of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyra-
zole-1-yl]benzenesulfonamide (SC-58635, Celecoxib). J. Med.
Chem. 1997, 40, 1347–1365.
(24) Liang, G-B; Feng, D. D. An improved oxadiazole synthesis using
peptide coupling reagents. Tetrahedron Lett. 1996, 37 (37), 6627–
6630.
(25) Gangloff, A. R.; Litvak, J.; Shelton, E. J.; Sperandio, D.; Wang, V.
R.; Rice, K. D. Synthesis of 3,5-disubstituted-1,2,4-oxadiazoles
using tetrabutylammonium fluoride as a mild and efficient catalyst.
Tetrahedron Lett. 2001, 42 (8), 1441–1443.
Supporting Information Available: Further experimental de-
tails, NMR, and elemental analyses data. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Axelrod, J.; Senoh, S.; Witkop, B. O-Methylation of catechola-
mines in vivo. J. Biol. Chem. 1958, 233, 697–701.
(2) Guldberg, H.; Marsden, C. Catechol-O-methyltransferase: phar-
macological aspects and physiological role. Pharmacol. Rev. 1975,
27, 135–206.
(3) Zhu, B. T.; Ezell, E. L.; Liehr, J. G. Catechol-O-methyltransferase-
catalysed rapid O-methylation of mutagenic flavanoids. Metabolic
inactivation as a possible reason for their lack of carcinogenicity
in vitro. J. Biol. Chem. 1994, 269, 292–299.
(4) Burba, J. V.; Becking, G. C. Effect of the antioxidant norhydro-
guaiaretic acid on the in vitro activity of catechol-O-methyltrans-
ferase. Arch. Int. Pharmacodyn. Ther. 1969, 180, 323–329.
(5) Hauser, R. A.; Zesiewicz, T. A. Management of early Parkinson’s
disease. Med. Clin. N. Am. 1999, 83 (2), 393–414.
(26) Bentiss, F.; Lagrenee, M. A new synthesis of symmetrical 2,5-
disubstituted 1,3,4-oxadiazoles. J. Heterocycl. Chem. 1999, 36 (4),
1029–1032.
(27) Van Leusen, A. M.; Wildeman, J.; Oldenziel, O. H. Synthesis of
1,5-disubstituted and 1,4,5-trisubstituted imidazoles from aldi-
mines and imidoyl chlorides. J. Org. Chem. 1977, 42 (7), 1153–1159.
(28) Bennett, G. B.; Mason, R. B.; Alden, L. J.; James, B.; Roach, J. B.,
Jr. Synthesis and antiinflammatory activity of trisubstituted pyr-
imidines and triazines. J. Med. Chem. 1978, 21 (7), 623–628.
(29) Hashimoto, H.; Imamura, K.; Haruta, J.; Wakitani, K. 4-(4-
Cycloalkyl/aryl-oxazol-5-yl)benzenesulfonamides as selective cy-
clooxygenase-2 inhibitors: enhancement of the selectivity by intro-
duction of a fluorine atom and identification of a potent, highly
selective, and orally active COX-2 inhibitor JTE-522. J. Med.
Chem. 2002, 45 (7), 1511–1517.
(30) Taurins, A.; Blaga, A. Synthesis of pyridyl- and quinolyl-substi-
tuted 2-aminothiazoles. J. Heterocycl. Chem. 1970, 7, 1137–1141.
(31) Caron, S.; Do, N. M.; Sieser, J. E. A practical, efficient, and rapid
method for the oxidation of electron deficient pyridines using
trifluoroacetic anhydride and hydrogen peroxide-urea complex.
Tetrahedron Lett. 2000, 41 (14), 2299–2302.
(6) Mannisto, P. T.; Kaakkola, S. New selective COMT inhibitors:
useful adjuncts for Parkinson’s disease? Trends Pharmacol. Sci.
1989, 10, 54–56.
(7) Calne, D. B. Treatment of Parkinson’s disease. N. Engl. J. Med.
1993, 329, 1021–1027.
(8) Messiha, F.; Hsu, T.; Bianchine, J. Peripheral aromatic L-amino
acid decarboxylase inhibitor in parkinsonism. I. Effect on O-
methylated metabolites of L-Dopa-2-14C. J. Clin. Invest. 1972, 51,
452–455.
(9) Ericson, A. D. Potentiation of the L-dopa effect in man by the use of
catechol-O-methyltransferase inhibitors. J. Neurol. Sci. 1971, 24,
193–197.
(10) Borgulya, J.; Bruderer, H.; Bernauer, K.; Zurcher, G.; Da Prada,
M. Catechol-O-methyltransferase-inhibiting pyrocatechol deriva-
tives: synthesis and structure-activity studies. Helv. Chim. Acta
1989, 72, 952–968.
(11) Backstrom, R.; Honkanen, E.; Pippuri, A.; Kairisalo, P.; Pystynen,
(32) Kiss, L. E.; Ferreira, H. S.; Learmonth, D. A. Efficient synthesis of
2-(trifluoromethyl)nicotinic acid derivatives from simple fluori-
nated precursors. Org. Lett. 2008, 10 (9), 1835–1837.
ꢀ
J.; Heinola, K.; Nissinen, E.; Linden, I.-B.; Mannisto, P. T.;
Kaakkola, S.; Pohto, P. Synthesis of some novel potent and
selective catechol-O-methyltransferase inhibitors. J. Med. Chem.
1989, 32, 841–846.
ꢀ
(33) Bonifacio, M. J.; Vieira-Coelho, M. A.; Soares-da-Silva, P. Kinetic
inhibitory profile of BIA 3-202, a novel fast tight-binding, rever-
sible and competitive catechol-O-methyltransferase inhibitor. Eur.
J. Pharmacol. 2003, 460, 163–170.
(12) Borgulya, J.; Da Prada, M.; Dingemanse, J.; Scherschlicht, R.;
Schlappi, B.; Zurcher, G. Ro 40-7592. Catechol-O-methyltransfer-
ase (COMT) inhibitor. Drugs Future 1991, 16, 719–721.
(34) Pedrosa, R; Soares-da-Silva, P. Oxidative and non-oxidative me-
chanisms of neuronal cell death and apoptosis by L-3,4-dihydrox-
yphenylalanine (L-DOPA) and dopamine. Br. J. Pharmacol. 2002,
137, 1305–1313.
ꢀ
(13) Nissinen, E.; Linden, I.-B.; Schultz, E.; Pohto, P. Biochemical and
pharmacological properties of a peripherally acting catechol-O-
methyltransferase inhibitor entacapone. Naunyn-Schmiedeberg’s
Arch. Pharmacol. 1992, 346, 262–266.
~
(35) Soares-da-Silva, P.; Parada, A.; Serrao, P. The O-methylated
derivative of L-DOPA, 3-O-methyl-L-DOPA, fails to inhibit neu-
ronal and non-neuronal aromatic L-amino acid decarboxylase.
Brain Res. 2000, 863, 293–297.
(14) Learmonth, D. A.; Vieira-Coelho, M. A.; Benes, J.; Alves, P. C.;
Borges, N.; Freitas, A. P.; Soares-da-Silva, P. Synthesis of 1-(3,4-
dihydroxy-5-nitrophenyl)-2-phenyl-ethanone and derivatives as
potent and long-acting peripheral inhibitors of catechol-O-methyl-
transferase. J. Med. Chem. 2002, 45, 685–695.
(36) Kato, Y.; Okada, S.; Tomimoto, K.; Mase, T. A facile bromination
of hydroxyheteroarenes. Tetrahedron Lett. 2001, 42 (29), 4849–
4851.
ꢀ
(15) Learmonth, D. A.; Bonifacio, M. J.; Soares-da-Silva, P. Synthesis
(37) Narsaiah, B.; Sivaprasad, A.; Venkataratnam, R. V. An improved
synthetic route to trifluoromethyl-6-substituted-2(1H)-pyridones.
Org. Prep. Proced. Int. 1993, 25, 116–117.
and biological evaluation of a novel series of “ortho-nitrated”
inhibitors of catechol-O-methyltransferase. J. Med. Chem. 2005,
48, 8070–8078.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3411
(38) Yamaguchi, Y.; Katsuyama, I.; Funabiki, K.; Matsui, M.;
Shibata, K. A new expedient route to 2,6-diaryl-3-cyano-4-(tri-
fluoromethyl)pyridines. J. Heterocycl. Chem. 1998, 35, 805–
810.
synthesis of 4-trifluoromethylpyridines. Synthesis 1997, 11, 1321–
1324.
(40) Parada, A.; Soares-Da-Silva, P. BIA 3-202, A novel catechol-O-
methyltransferase inhibitor, reduces the peripheral O-methylation
of L-DOPA and enhances its availability to the brain. Pharmaco-
logy 2003, 68, 29–37.
(39) Katsuyama, I.; Ogawa, S.; Yamaguchi, Y.; Funabiki, K.; Matsui,
M.; Muramatsu, H.; Shibata, K. A convenient and regioselective