Synthesis and evaluation of bifunctional nitrocatechol inhibitors of pig liver catechol-O-methyltransferase

Article abstract of DOI:10.1016/j.bmc.2005.05.069

Bifunctional compounds were tested in vitro as potential inhibitors of pig liver catechol-O-methyltransferase (COMT) with respect to the catechol substrate 4-[(3,4-dihydroxyphenyl)azo]benzenesulfonate. The bifunctional compounds were a composite of either two nitrocatechols or one nitrocatechol and one phenol, linked by amide bonds to a spacer unit comprising two to five methylene groups. The unsymmetrical compounds N-[2-(4-hydroxybenzoylamine)ethyl]-3,4-dihydroxy-5- nitrobenzamide], N-[3-(4-hydroxybenzoyl-amine)propyl]-3,4-dihydroxy-5- nitrobenzamide] and N-[5-(4-hydroxybenzoylamine)pentyl]-3,4-dihydroxy-5- nitrobenzamide] demonstrated strong inhibitory action against COMT with K _i values in the 100 nM range. In comparison, the monofunctional nitrocatechol analogues of these compounds had K_i values that were significantly higher.

Full text of DOI:10.1016/j.bmc.2005.05.069

Bioorganic & Medicinal Chemistry 13 (2005) 5740–5749
Synthesis and evaluation of bifunctional nitrocatechol inhibitors
of pig liver catechol-O-methyltransferase
Karl Bailey and Eng Wui Tan*
Department of Chemistry, University of Otago, PO Box 56, Dunedin, New Zealand
Received 30 April 2005; accepted 31 May 2005
Available online 5 July 2005
Abstract—Bifunctional compounds were tested in vitro as potential inhibitors of pig liver catechol-O-methyltransferase (COMT)
with respect to the catechol substrate 4-[(3,4-dihydroxyphenyl)azo]benzenesulfonate. The bifunctional compounds were a composite
of either two nitrocatechols or one nitrocatechol and one phenol, linked by amide bonds to a spacer unit comprising two to five
methylene groups. The unsymmetrical compounds N-[2-(4-hydroxybenzoylamine)ethyl]-3,4-dihydroxy-5-nitrobenzamide], N-[3-(4-
hydroxybenzoyl-amine)propyl]-3,4-dihydroxy-5-nitrobenzamide]
and
N-[5-(4-hydroxybenzoylamine)pentyl]-3,4-dihydroxy-5-
nitrobenzamide] demonstrated strong inhibitory action against COMT with Ki values in the 100 nM range. In comparison, the
monofunctional nitrocatechol analogues of these compounds had Ki values that were significantly higher.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ure, in that they have duplicated substructures for enzyme
binding. Three such inhibitors are from the so-called first
generation inhibitors and include D-catechin, desmethyl-
papaverine and nordihydroxyguaiaretic acid (Fig. 1).10,11
Catechol-O-methyltransferase (COMT) catalyses the
transfer of a methyl group from S-adenosylmethionine
(AdoMet) to a catechol substrate. This ubiquitous
enzyme plays an important role in the extraneuronal
inactivation of catecholamine neurotransmitters.1
Effective inhibitors for this enzyme have been shown
to improve the effectiveness of current drug therapies
for Parkinsonꢀs disease.2
Recently, two new progressive series of bifunctional com-
pounds were synthesized and examined for their inhibi-
tion characteristics.12 The compounds were designed
with dual substituted catechols for enzyme binding sepa-
rated by linker sections of various lengths (Fig. 2). The
catechol derivatives were either 3,4-dihydroxybenzamide
or 3,4,5-trihydroxybenzamide groups, linked through the
The first series of inhibitors that were identified as having
limited clinical use were the so-called ꢁfirst generation
inhibitorsꢀ. Examples of these are pyrogallol,3 tropo-
lone,4 catechin,3 and gallic acid.5 These inhibitors had
Ki values in the 10ꢀ5 M range. The following so-called
ꢁsecond-generationꢀ inhibitors are characterized by hav-
ing electron withdrawing groups, such as nitro groups,
in the 3- and 5-position of the catechol ring.6 These fea-
tures have been incorporated into clinically useful inhib-
itors such as nitecapone,7 tolcapone8 and entacapone.9
Of the various COMT inhibitors that have been reported
in the literature, a small group are of a ꢁbifunctionalꢀ nat-
Keywords:
Bifunctional.
Catechol-O-methyltransferase;
Enzyme
inhibitors;
*
Corresponding author. Tel.: +6434797926; fax: +6434707906; e-mail:
ewtan@alkali.otago.ac.nz
Figure 1. Bifunctional ꢁfirst generationꢀ inhibitors.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.069

K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
5741
Vanillin was nitrated using a mixture of acetic acid
and nitric acid to give 5-nitrovanillin (9).6 Methylation
of the hydroxy group of 5-nitrovanillin using a two-
phase system containing the methylating reagent
dimethylsulfate gave 3,4-dimethoxy-5-nitrobenzalde-
hyde (10).14 The aldehyde was then oxidized to the
corresponding carboxylic acid (11) using acidified chro-
mium trioxide. Finally, the carboxylic acid was convert-
ed into the corresponding acid chloride (12) using
phosphorus pentachloride.
Figure 2. Previously evaluated polyhydroxybenzamide bifunctional
inhibitors.
nitrogen atoms to a spacer section consisting of a varying
number of methylene units. It was found that some of
these bifunctional inhibitors were significantly more
potent than their monofunctional counterparts.
To make the methylated precursors (13–16) of the dia-
mides 1–4, 3,4-dimethoxy-5-nitrobenzoyl chloride was
coupled to a set of variable length diamines, ranging
from 2 to 5 methylene units. The methyl groups were
then removed by treatment with boron tribromide to
give the bis-nitrocatechols (1–4).15 To make the diamides
5 and 6, a three step synthesis was used (Scheme 2). Ani-
soyl chloride was reacted with an excess of 1,2-diamino
ethane and 1,3-diaminopropane to form the monoacylat-
ed diamines 17 and 18, respectively.16 The monoacylated
diamines were then reacted with 3,4-dimethoxy-5-nitro-
benzoylchloride to give compounds 19 and 20. These
diamides were demethylated using boron tribromide to
give the first two compounds (5 and 6) that make up
the phenol–catechol conjugate inhibitor series.
In this paper, we report the preparation of a series of
inhibitors that incorporate the extra binding substruc-
ture of a bifunctional inhibitor, with aspects of the ꢁsec-
ond generationꢀ inhibitors in an attempt to create even
more potent inhibitors. In addition, we chose to investi-
gate the effect of these inhibitors with pig liver COMT,
as the structure and function of pig liver COMT has
not been as well established as rat and human COMT,13
both of which have very similar protein sequences.
In order to investigate the effect of having electron with-
drawing groups on the catechol rings, we synthesized a
series of bifunctional inhibitors of varying length that
incorporated nitro group substitutions at the 5 and 50
positions of the catechol rings (1–4) (Table 1). On the
basis of the results from using these bis-nitrocatechols
as COMT inhibitors, a second series of unsymmetrical
bifunctional compounds were also synthesized (5–8).
These inhibitors were composite of a nitrocatechol
and phenol linked by amide bonds to a variable length
spacer group.
The formation of the butyl and pentyl phenol–catechol
conjugate inhibitors (7 and 8) via the synthesis of
mono-acylated diamines was impractical due to the high
cost of the diamines and the low yields of the mono-
acylated diamines. Consequently, an alternative strategy
was used to synthesize compounds 7 and 8 (Scheme 3).
3,4-Dimethoxy-5-nitrobenzoylchloride was reacted with
4-aminobutanol and 5-aminopentanol to give com-
pounds 21 and 22, respectively. The hydroxy groups of
these amide compounds were converted into amino
groups via a three-step process. Compounds 21 and 22
were transformed into tosylate esters (23 and 24) which
were then converted into the corresponding phthala-
mides 25 and 26. Removal of the phthaloyl groups with
hydrazine hydrate gave the monoacylated diamines,
2. Chemistry
3,4-Dimethoxybenzoyl chloride was prepared from van-
illin in a straightforward four-step process (Scheme 1).
Table 1. Structure, COMT inhibitory pattern, and kinetic inhibition constants of the compounds synthesized
Compound
R1
R2
n
Inhibition pattern
Kinetic inhibition constant, Ki (lM)
1
2
3
4
5
6
7
8
NO2
NO2
NO2
NO2
H
OH
OH
OH
OH
H
2
3
4
5
2
3
4
5
Ca
C
NCb
NC
C
3.48 0.30
5.47 1.08
3.94 0.60
4.33 0.76
0.64 0.15
0.77 0.03
11.8 0.60
0.97 0.06
H
H
C
H
H
C
H
H
C
a Competitive mode of inhibition.
b Noncompetitive mode of inhibition.

5742
K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
Scheme 1. Reagents: (a) HNO3, AcOH; (b) Me2SO4, Bu4NBr, NaOH, H2O/CH2Cl2; (c) aqueous H2SO4, CrO3; (d) PCl5.
Scheme 2. Reagents: (a) CH2Cl2; (b) 12, K2CO3, H2O/EtOAc; (c) BBr3.
which were in turn reacted with anisoyl chloride to give
the diamide products 27 and 28. Removal of the methyl
groups by treatment with boron tribromide gave the
desired compounds 7 and 8.
The Ki values for the bis-nitrocatechol inhibitors (1–4)
range from 3.48 to 5.47 lM. The length of the variable
spacer group does not contribute to any trend in the
potency of inhibition. In comparison, the monofunction-
al analogues 31 and 32 gave Ki values of 2.56 and
3.41 lM, respectively (see Table 2), which are within
the same order of magnitude as the Ki values obtained
with the bis-nitrocatechol inhibitors. This would suggest
that the additional functionality of the bis-nitrocatechol
inhibitors 1–4 played no significant part in enhancing the
potency of inhibition. However, compounds 3 and 4
showed noncompetitive modes of inhibition with respect
to the catechol substrate, whereas the monofunctional
analogues 31 and 32 and the bifunctional compounds 1
and 2 were competitive inhibitors. Presumably, the long-
er tethers in compounds 3 and 4 alter the interaction of
the inhibitor with the enzyme. It was hoped that the
bis-nitrocatechol inhibitors would show an increase in
potency of at least one order of magnitude greater that
the monofunctional inhibitors, because this increase in
binding was observed for the bis-catechol compound
33 when compared to its monofunctional analogue 34.
The monofunctional inhibitors (31 and 32) (Fig. 4) that
were used for comparison were made by reacting 3,4-di-
methoxy-5-nitrobenzoylchloride with methylamine and
ethylamine to give the corresponding amides (29 and
30), which were then treated with boron tribromide to
give the final products.
3. Biological results
Initial observations showed that the inhibitors tested in
this study with pig liver COMT do not exhibit the tight
binding behaviour usually associated with nitrocatechol
compounds in human and rat COMT. A lower affinity
of pig COMT relative to the human and rat enzyme of
ꢁ10- to 100-fold has been reported for nitrocatechol
derivatives.17 Accordingly, Ki values were derived using
double reciprocal plots of 1/V versus 1/[S] at different
inhibitor concentrations. Figure 3 shows a double reci-
procal plot that is representative of the plots we obtained.
It was postulated that for inhibitor 33, one end of the
bifunctional inhibitor binds to the active site of COMT,

K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
5743
Scheme 3. Reagents: (a) triethylamine, MeCN; (b) tosyl chloride, pyridine; (c) potassium phthalamide, DMSO; (d) hydrazine hydrate, EtOH;
(e) anisoyl chloride, K2CO3, H2O/EtOAc; (f) BBr3.
Figure 3. Burke–Lineweaver plot for inhibitor 2.
Table 2. Structure, inhibition pattern, and kinetic inhibition constants
of the monofunctional derivatives
the bis-nitrocatechol inhibitors (1–4), there was no
correlation between the spacer length and the degree
of inhibition. This could suggest that while one end of
Compound
R1
Inhibition
pattern
Kinetic inhibition
constant, Ki (lM)
the bifunctional inhibitor binds to the active site
of COMT, the additional nitrocatechol functionality
does not interact at all with the proposed secondary
binding site. The large reported increase in binding
observed for bifunctional inhibitor 33 when compared
to the monofunctional inhibitor 3412 would suggest that
a secondary binding is taking place, but this interaction
is more favorable between the secondary binding
site and an unsubstituted catechol. As this interaction
is not evident with a nitrocatechol, the pKa (and hence
31
32
Me
Et
Ca
C
2.56 0.67
3.41 0.68
whereas the other end interacts with some other part of
the enzyme. Binding at the active site and the magnitude
of the secondary interaction was optimal when the spac-
er group was three methylene units long. In the case of

5744
K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
Figure 4.
ionization state) of the hydroxy groups may be the
important factor that determines the magnitude of this
secondary interaction. It is likely that an appropriate
pKa of the 4-hydroxy group of catechol is required for
binding at the secondary site, as this hydroxy group
would experience the largest change in pKa with the
incorporation of a nitro group at the 5-position. Hence,
it was postulated that a nitrocatechol group at the end
of a bis-nitrocatechol inhibitor could be replaced with
a phenol to produce a better binding molecule. To test
the validity of this theory, a new group of unsymmetri-
cal inhibitors (5–8), as discussed earlier, were made and
evaluated for inhibitory strength. Three of these phenol–
catechol conjugate inhibitors (5, 6, and 8) had Ki values
in the 100 nM range. As these three compounds gave
values that were almost one order of magnitude lower
than the bis-nitrocatechol series 1–4, the proposed sec-
ondary binding interaction may be responsible for the
increase in potency. Interestingly, the butyl linked inhib-
itor 7 has the highest Ki of all the inhibitors tested. This
is perhaps due to unfavorable interactions of the butyl
linker with the enzyme, which outweighs or disallows
the secondary binding interaction.
acid per hour at 37 ꢁC using AdoMet as the methyl do-
nor (SigmaTM)] in buffer solution; 3 lL of a stock solu-
tion of catechol dye (0.5–150 lM) and 3 lL of inhibitor
(0–21 lM) in buffer solution. The mixture was incubated
for 1 h at 37 ꢁC, stopped by the addition of 20 lL of 4 M
perchloric acid, and then diluted with 200 lL of buffer.
An aliquot of 30 lL of the assay mixture was injected
into a Microsorb-MV column (C18, 5 mm particle size,
4.6 · 250 mm2), and the components of the mixture were
eluted with a mobile phase which consisted of water/
MeOH/trifluoroacetic acid (30:70:0.2, v/v/v), at a flow
rate of 0.45 mL/min. Detection was at 370 nm, and the
products, 4-[(4-hydroxy-3-methoxyphenyl)azo]benzene-
sulfonate and 4-[(3-hydroxy-4-methoxyphenyl)azo]ben-
zenesulfonate, had retention times of 8.2 and 8.9 min,
respectively. The enzyme activity was calculated as nano-
mole of product per minute per milligram of protein. As-
says were performed in duplicates, and each assay had
two 30 lL aliquots analysed. Conversion of substrate
to product was less than 5%. The concentrations of Ado-
Met and Mg2+ used were saturating, and the product for-
mation was linear with respect to protein concentration
and reaction time. Bifunctional inhibitors were tested
at concentrations up to 21 lM. Compounds which
exhibited inhibitory activity less than the standard devi-
ation, at this concentration, were classified as having no
inhibitory action.
Although this study has provided new insight to the
enhanced binding observed with these bifunctional
inhibitors, further work would be required to fully
elucidate the nature of the secondary interaction.
4.2. Analysis of kinetic data
4. Experimental
All kinetic data were analyzed graphically using double
reciprocal plots, where the reciprocal velocities versus
the reciprocal substrate concentrations gave a linear
relationship by which Km and Vmax could be calculated.
Inhibition constants for competitive and noncompetitive
inhibition were calculated from data fitting to equations
4.1. COMT assay
The activity of pig liver COMT was measured using a
method described in a previous paper.18 Accordingly,
the activity of COMT was measured by determining
the summed amount of the dyes 4-[(4-hydroxy-3-meth-
oxyphenyl)azo]benzenesulfonate and 4-[(3-hydroxy-4-
methoxyphenyl)azo]benzenesulfonate formed over time
using HPLC with visible light detection. The buffer used
in the COMT assays consisted of 0.2 M NaH2PO4 and
5 mM MgCl2Æ6H2O with the pH adjusted to 7.45. All
incubation mixtures were made up to a total volume of
60 lL, each consisting of 6 lL of 20 mM AdoMet in buff-
er solution; 48 lL of COMT [125 U/mL, where one unit
is defined as the methylation of 1 nmol of protocatechuic
v = Vmax[S]/(Km(1 + [I]/Ki) + [S])
and
v = Vmax[S]/
(([S] + Km)(1 + [I]/Ki)), respectively. Standard devia-
tions were obtained from fitting all available data for a
given compound. All calculations were performed with
Microsoft Excel (Redmond, WA, USA).
4.3. Chemical methods
Elemental analyses were performed at the Campbell
Microanalytical laboratory, University of Otago. The
analytical results obtained were within 0.4% of the

K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
5745
theoretical values. 1H NMR spectra were measured on a
Varian INOVA-300 MHz spectrometer. The signals are
given in ppm relative to tetramethylsilane (TMS),
HOD, CHD2SOCD3 or CHD2COCD3 as internal stan-
dards in CDCl3, D2O, DMSO-d6 and acetone-d6, respec-
tively. The peak patterns are shown with the following
abbreviations: br, broad; d, doublet; m, multiplet; t, trip-
let; q, quartet. Column chromatography was performed
with silica gel (Sorbsil, particle size 32–63 mM). AdoMet
as the p-toluenesulfonate salt, and pig liver COMT were
purchased from Sigma. Boron tribromide, 1,2-diamino-
ethane, 1,3-diaminopropane, 1,4-diaminobutane and
1,5-diaminopentane, 4-aminobutanol and 5-aminopro-
panol were purchased from Aldrich. Solvent and
inorganic bases were generally sourced.
4.4.4. N,N0-1,5-Pentanediylbis(3,4-dihydroxy-5-nitrobenz-
amide) (4). The pentyl derivative was prepared from
N,N0-1,5-pentanediylbis-(3,4-dimethoxy-5-nitro-benzam-
ide) (16) (1 g, 1.9 mmol) and BBr3 (20 mL) using the pro-
cedure described above. Recrystallization from water and
ethanol gave the product as flaky orange crystals (0.24 g,
26%): mp 226–228 ꢁC; 1H NMR (acetone-d6) d 8.17 (2H,
d, J = 2.1 Hz), 7.96 (2H, br t), 1.51 (2H, m), 7.78 (2H, d,
J = 2.1 Hz), 3.45 (4H, q, J = 6.9 Hz), 1.71 (4H, quint,
J = 6.9 Hz). Anal. Calcd for C19H20N4O10: C, 49.14; H,
4.34; N, 12.07. Found: C, 49.44; H, 4.58; N, 11.76.
4.4.5.
N-[2-(4-Hydroxybenzoylamino)ethyl]-3,4-dihy-
droxy-5-nitrobenzamide (5). An excess of BBr3 (1.7 mL,
15 mol equiv) was added to a solution of N-[2-(4-meth-
oxybenzoylamino)ethyl]-3,4-dimethoxy-5-nitrobenzamide
(19) (0.5 g, 1.2 mmol) in dried CH2Cl2 (50 mL) under
N2. The mixture was left stirring for 24 h at room tem-
perature. Water was carefully added to eliminate any
unreacted BBr3 and then removed along with the
CH2Cl2 by rotary evaporation under reduced pressure
to yield the crude product. The product was purified
by recrystallization from acetone and water solution to
give the final product as small yellow crystals (0.21 g,
48%): mp 268–270 ꢁC (dec); 1H NMR (acetone-d6) d
8.30 (1H, br s), 8.20 (1H, d, J = 2.1 Hz), 7.96 (1H, br
s), 7.84 (2H, d, J = 8.7 Hz), 7.77 (1H, d, J = 2.1 Hz),
6.91 (2H, d, J = 8.7 Hz), 3.63 (4H, m). Anal. Calcd for
C16H15N3O7: C, 53.18; H, 4.19; N, 11.63. Found: C,
53.23; H, 4.09; N, 11.55.
4.4. General procedure for the preparation of N,N0-
bis(3,4-dihydroxy-5- nitrobenzamide) derivatives
BBr3 (15 mol equiv) was added to a cooled (0–5 ꢁC)
solution of the appropriate N,N0-bis(3,4-dimethoxy-5-
nitrobenzamide) derivative suspended in CH2Cl2. The
reaction was left to stir overnight and then water was
carefully added to eliminate any unreacted BBr3. The
resulting yellow precipitate was removed by filtration
and recrystallized from the appropriate solvent.
4.4.1. N,N0-1,2-Ethanediylbis(3,4-dihydroxy-5-nitrobenz-
amide) (1). The ethyl derivative was prepared from N,N0-
1,2-ethanediylbis-(3,4-dimethoxy-5-nitro-benzamide) (13)
(1 g, 2.1 mmol) and BBr3 (2 mL) using the procedure
described earlier. Recrystallization from methanol and
water gave the product as small yellow crystals (0.84 g,
81%): mp 248–251 ꢁC; 1H NMR (acetone-d6) d 9.82
(4H, br s) 8.27 (2H, br t), 8.19 (2H, d, J = 2.1 Hz),
7.79 (2H, d, J = 2.1 Hz), 3.67 (4H, br s). Anal. Calcd
for C16H14N4O10Æ4H2O: C, 38.87; H, 4.48; N, 11.36.
Found: C, 38.92; H, 4.50; N, 11.06.
4.4.6.
N-[3-(4-Hydroxybenzoylamino)propyl]-3,4-dihy-
droxy-5-nitrobenzamide (6). N-[3-(4-Methoxybenzo-
ylamino)propyl]-3,4-dimethoxy-5-nitrobenzamide (20)
(0.5 g, 1.2 mmol) was used in the BBr3 procedure de-
scribed above. The resulting crude material was recrys-
tallized from water to give the final product as bright
1
yellow crystals (0.27 g, 60%): mp 236–239 ꢁC (dec); H
NMR (acetone-d6) d 8.31 (1H, br s), 8.25 (1H, d,
J = 2.1 Hz), 7.92 (1H, br s), 7.87 (2H, d, J = 8.7 Hz),
7.83 (1H, d, J = 2.1 Hz), 6.92 (2H, d, J = 8.7 Hz), 3.52
(4H, m), 1.86 (2H, m). Anal. Calcd for C17H17N3O7:
C, 54.25; H, 4.82; N, 11.17. Found: C, 54.52; H, 4.58;
N, 11.14.
4.4.2. N,N0-1,3-Propanediylbis(3,4-dihydroxy-5-nitrobenz-
amide) (2). The propyl derivative was prepared from
N,N0-1,3-propanediylbis-(3,4-dimethoxy-5-nitro-benzam-
ide) (14) (1 g, 2 mmol) and BBr3 (2 mL) using the proce-
dure described above. Recrystallization from ethanol and
water gave the product as a bright yellow powder (0.48 g,
54%): mp 185–180 ꢁC (dec); 1H NMR (acetone-d6) d 8.23
(2H, d, J = 1.8 Hz), 8.19 (2H, br t), 7.85 (2H, d,
J = 1.8 Hz), 3.59 (4H, dt, J = 3.6, 6.0 Hz), 1.92 (2H,
quint, J = 6.0 Hz). Anal. Calcd for C17H16N4O10: C,
46.79; H, 3.70; N, 12.84. Found: C, 46.79; H, 3.78; N,
12.62.
4.4.7.
droxy-5-nitrobenzamide (7). N-[4-(4-Methoxybenzo-
ylamino)butyl]-3,4-dimethoxy-5-nitrobenzamide (21)
N-[4-(4-Hydroxybenzoylamino)butyl]-3,4-dihy-
(0.5 g, 1.2 mmol) was used in the BBr3 procedure de-
scribed above. The resulting crude material was recrys-
tallized from methanol and water to give the final
product as chunky orange/brown crystals (0.09 g,
20%): mp 251–255 ꢁC (dec); 1H NMR (acetone-d6) d
8.17 (1H, d, J = 2.1 Hz), 8.01 (1H, br s), 7.84 (2H, d,
J = 9.0 Hz), 7.71 (1H, d, J = 2.1 Hz), 7.65 (1H, br s),
6.89 (2H, d, J = 9.0 Hz), 3.47 (4H, m), 1.71 (4H, m).
Anal. Calcd for C18H19N3O7: C, 55.52; H, 4.92; N,
10.79. Found: C, 55.43; H, 4.77; N, 10.75.
4.4.3. N,N0-1,4-Butanediylbis(3,4-dihydroxy-5-nitroben-
zamide) (3). The butyl derivative was prepared from
N,N0-1,4-butanediylbis-(3,4-dimethoxy-5-nitro-benzam-
ide) (15) (1 g, 2 mmol) and BBr3 (2 mL) using the proce-
dure described above. Recrystallization from ethanol
and water gave the product as small orange crystals
(0.62 g, 69%): mp 260–285 ꢁC (dec); 1H NMR (ace-
tone-d6) d 8.18 (2H, d, J = 2.1 Hz), 8.06 (2H, br t),
7.81 (2H, d, J = 2.1 Hz), 3.67 (4H, m), 1.74 (4H, m).
Anal. Calcd for C18H18N4O10Æ0.25H2O: C, 47.52; H,
4.09; N, 12.32. Found C, 47.71; H, 3.95; N, 11.98.
4.4.8.
N-[5-(4-Hydroxybenzoylamino)pentyl]-3,4-dihy-
droxy-5-nitrobenzamide (8). N-[5-(4-Methoxybenzo-
ylamino)pentyl]-3,4-dimethoxy-5-nitrobenzamide (22)
(0.5 g, 1.1 mmol) was used in the BBr3 procedure

5746
K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
described above. The resulting crude material was
recrystallized from methanol and water to give the final
product as bunched orange crystals (0.15 g, 31%): mp
107–110 ꢁC; 1H NMR (acetone-d6) d 10.57 (1H, s),
9.16 (1H, s), 8.94 (1H, s), 8.18 (1H, d, J = 1.8 Hz),
8.02 (1H, t, J = 5.9 Hz), 7.82 (1H, d, J = 1.8 Hz), 7.80
(2H, d, J = 8.7 Hz), 7.63 (1H, t, J = 5.9 Hz), 6.88 (2H,
d, J = 8.7 Hz), 3.43 (4H, m), 1.68 (4H, quint,
J = 7.2 Hz), 1.51 (2H, quint, J = 7.2 Hz). Anal. Calcd
for C19H21N3O7Æ1Æ5H2O: C, 53.02; H, 5.62; N, 9.76.
Found: C, 53.36; H, 5.74; N, 9.63.
(1H, d, J = 2.0), 7.81 (1H, d, J = 2.0), 4.08 (3H, s),
4.01 (3H, s). Anal. Calcd for C9H9NO6: C, 47.58; H,
3.99; N, 6.17. Found: C, 47.58; H, 3.96; N, 6.20.
4.4.12. 3,4-Dimethoxy-5-nitrobenzoylchloride (12). 3,4-
Dimethoxy-5-nitrobenzoic acid (5.2 g, 23 mmol), PCl5
(9.5 g, 45 mmol) and CH2Cl2 (60 mL) were carefully
mixed together and stirred overnight. The CH2Cl2 was
quickly washed with cold water and then dried with
MgSO4. Rotary evaporation under reduced pressure of
the CH2Cl2 solution gave the product as a cream colored
1
solid (4.95 g, 88%): mp 68–70 ꢁC; H NMR (CDCl3) d
8.14 (1H, d, J = 2.2 Hz), 7.74 (1H, d, J = 2.2 Hz), 4.09
(3H, s), 4.01 (3H, s).
4.4.9. 4-Hydroxy-3-methoxy-5-nitrobenzaldehyde (9).
Fuming HNO3 (2 mL) was carefully added to a cooled
(5 ꢁC) solution of vanillin (5 g, 33 mmol) and acetic acid
(50 mL) over a period of 30 min. The gold colored pre-
cipitate that formed was filtered, washed with water, and
allowed to dry (5.21 g, 80%): mp 171 ꢁC; 1H NMR
(CDCl3) d 9.88 (1H, s), 8.17 (1H, d, J = 1.8 Hz), 7.61
(1H, d, J = 1.8 Hz), 4.01 (3H, s). Anal. Calcd for
C8H7NO5: C, 48.73; H, 3.58; N, 7.11. Found: C,
48.64; H, 3.31; N, 7.32.
4.5. General procedure for the synthesis of N,N0-bis(3,4-
dimethoxy-5-nitrobenzamide) derivatives
Freshly prepared 3,4-dimethoxy-5-nitrobenzoyl chloride
(10 g scale) and potassium carbonate (1.2 mol equiv)
was dissolved in H2O and ethyl acetate (1:1, 350 mL
total). The appropriate amount of diamine (0.45 mol
equiv) was introduced into the acid chloride mixture
and stirred overnight. The product precipitated out dur-
ing the course of the reaction.
4.4.10. 3,4-Dimethoxy-5-nitrobenzaldehyde dihydrate
(10). A mixture of CH2Cl2 (67.5 mL), water (67.5 mL),
4-hydroxy-3-methoxy-5-nitrobenzaldehyde (5.0 g, 25
mmol), sodium hydroxide (2 g, 50 mmol), tetrabutylam-
monium bromide (0.81 g, 2.5 mmol), and dimethylsul-
fate (12.5 mL, 132 mmol) were mixed together and
stirred vigorously for 24 h under a nitrogen atmosphere.
The aqueous layer was separated and washed with
CH2Cl2. Rotary evaporation under reduced pressure
of the pooled CH2Cl2 fractions gave a yellow oil which
was redissolved in diethyl ether. The ether was washed
with water, 2 M ammonia solution, and 2 M NaOH
solution to remove unreacted phenol and dimethylsul-
fate. Rotary evaporation under reduced pressure of the
dried (Na2SO4) ether solution gave a brown oil that
solidified on cooling. Purification by recrystallization
from acetone and water gave the product as long nee-
dle-like cream colored crystals (5.21 g, 84%): mp 62–
4.5.1. N,N0-1,2-Ethanediylbis(3,4-dimethoxy-5-nitrobenz-
amide) (13). The ethyl derivative was prepared from 3,4-
dimethoxy-5-nitrobenzoyl chloride (10 g, 40 mmol),
K2CO3 (6.8 g, 1.2 mol equiv) and 1,2-diaminoethane
(1.2 g, 20 mmol) using the procedure described above.
Recrystallization from methanol and DMSO gave the
product as small white crystals (5.41 g, 57%): mp
1
235 ꢁC; H NMR (CDCl3) d 8.33 (2H, br t), 7.61 (2H,
d, J = 1.9 Hz), 7.47 (2H, d, J = 1.9 Hz), 3.68 (6H, s),
3.66 (6H, s), 3.30 (4H, m). Anal. Calcd for
C20H22N4O10: C, 50.21; H, 4.64; N, 11.71. Found: C,
50.16; H, 4.52; N, 11.60.
4.5.2.
N,N0-1,3-Propanediylbis(3,4-dimethoxy-5-nitro-
benzamide) (14). The propyl derivative was prepared
from 3,4-dimethoxy-5-nitrobenzoyl chloride (10 g,
40 mmol), K2CO3 (6.8 g, 1.2 mol equiv) and 1,3-diami-
nopropane (1.48 g, 20 mmol) using the procedure de-
scribed above. Recrystallization from methanol gave
the product as long needle-like crystals (2.36 g, 24%):
mp 200–201 ꢁC; 1H NMR (CDCl3) d 7.77 (2H, d,
J = 2.0 Hz), 7.74 (2H, d, J = 2.0 Hz), 7.28 (2H, t,
J = 5.9 Hz), 4.01 (6H, s), 3.99 (6H, s), 3.53 (4H, quint,
J = 5.9 Hz), 1.87 (2H, quint, J = 5.9 Hz). Anal. Calcd
for C21H24N4O10: C, 51.22; H, 4.91; N. 11.38. Found:
C, 51.35; H, 4.89; N, 11.44.
1
65 ꢁC; H NMR (CDCl3) d 9.92 (1H, s), 7.84 (1H, d,
J = 1.9 Hz), 7.63 (1H, d, J = 1.9 Hz), 4.09 (3H, s), 4.01
(3H, s). Anal. Calcd for C9H9NO5Æ2H2O: C, 43.72; H,
5.30; N, 5.67. Found: C, 43.47; H, 4.97; N, 5.56.
4.4.11. 3,4-Dimethoxy-5-nitrobenzoic acid (11). A solu-
tion of CrO3 (6 g, 60 mmol), concentrated H2SO4
(5 mL), and water (100 mL) was added drop-wise to a
stirring solution of 3,4-dimethoxy-5-nitrobenzaldehyde
(10 g, 47 mmol), acetone (100 mL), and water
(100 mL). The solution was stirred for a further 24 h
and then isopropanol was added to eliminate any unre-
acted Cr(VI) species. Rotary evaporation, under re-
duced pressure, of the acetone and water mixture gave
the crude product as a green sludge. The crude product
was extracted into ethyl acetate and washed with 1 M
HCl to remove any remaining Cr(III) species. Rotary
evaporation under reduced pressure of the dried
(Na2SO4) ethyl acetate solution gave the crude product.
Purification by recrystallization from water and ethanol
gave the final product as long needle-like white crystals
4.5.3.
N,N0-1,4-Butanediylbis(3,4-dimethoxy-5-nitro-
benzamide) (15). The butyl derivative was prepared from
3,4-dimethoxy-5-nitrobenzoyl chloride (10 g, 40 mmol),
K2CO3 (6.8 g, 1.2 mol equiv) and 1,4-diaminobutane
(1.76 g, 20 mmol) using the procedure described above.
Recrystallization from methanol and DMSO gave the
product as a white crystalline powder (5.15 g, 51%):
1
mp 230–231 ꢁC; H NMR (DMSO-d6) d 8.14 (2H, t,
J = 5.6 Hz), 7.83 (2H, d, J = 2.0 Hz), 7.66 (2H, d,
J = 2.0 Hz), 3.86 (6H, s), 3.84 (6H, s), 3.33 (4H, quint,
1
(7.4 g, 69%): mp 194–195 ꢁC; H NMR (CDCl3) d 8.10

K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
5747
J = 5.6 Hz), 1.57 (4H, m). Anal. Calcd for C22H26N4O10:
C, 52.17; H, 5.17; N, 11.07. Found: C, 52.13; H, 5.26; N,
10.89.
removed by filtration and recrystallized from water
and ethanol to give the product as small white crystals
(0.67 g, 27%): mp 177 ꢁC; H NMR (DMSO-d6) d 8.87
1
(1H, s), 8.52 (1H, s), 7.94 (1H, d, J = 2.1 Hz), 7.87
(2H, d, J = 8.8 Hz), 7.84 (1H, d, J = 2.1 Hz), 7.51 (2H,
d, J = 8.8 Hz), 4.00 (3H, s), 3.95 (3H, s), 3.84 (3H, s),
3.48 (4H, br s). Anal. Calcd for C19H21N3O7: C, 56.57;
H, 5.25; N, 10.42. Found: C, 56.53; H, 5.13; N, 10.19.
4.5.4. N,N0-1,5-Pentanediylbis(3,4-dimethoxy-5-nitroben-
zamide) (16). The pentyl derivative was prepared from
3,4-dimethoxy-5-nitrobenzoyl chloride (10 g, 40 mmol),
K2CO3 (6.8 g, 1.2 mol equiv) and 1,5-diaminopentane
(2.04 g, 20 mmol) using the procedure described above.
Recrystallization from methanol gave the product as
long needle-like crystals (4.79 g, 46%): mp 198–199 ꢁC;
1H NMR (CDCl3) d 7.63 (2H, d, J = 2.0 Hz), 7.61
(2H, d, J = 2.0 Hz), 6.34 (2H, t, J = 6.4 Hz), 3.99 (6H,
s), 3.94 (6H, s), 3.45 (4H, quint, J = 6.4 Hz), 1.70 (4H,
quint, J = 6.4 Hz), 1.49 (2H, quint, J = 6.4 Hz). Anal.
Calcd for C23H28N4O10: C, 53.07; H, 5.42; N, 10.77.
Found: C, 52.82; H, 5.38; N, 10.64.
4.5.8. N-[3-(4-Methoxybenzoylamino)propyl]-3,4-dime-
thoxy-5-nitrobenzamide (20). Freshly prepared 3,4-dime-
thoxy-5-nitrobenzoyl chloride (1.42 g, 5.8 mmol) and
potassium carbonate (0.80 g, 5.8 mmol) were dissolved
into a two phase system consisting of H2O (30 mL)
and ethyl acetate (30 mL). 3-(4-Methoxybenzamide)pro-
pylamine (1 g, 4.8 mmol) was added to the solution
which was then stirred overnight. The crude product
was extracted with ethyl acetate and washed with 1 M
NaOH to remove any carboxylate byproduct. Rotary
evaporation under reduced pressure of the dried
(Na2SO4) ethyl acetate solution gave the crude product,
which was purified by recrystallization from water and
ethanol to give the final product as small white flaky
4.5.5. 2-(4-Methoxybenzamide)ethylamine (17). A solu-
tion of 4-methoxybenzoylchloride (1 g, 5.7 mmol) and
CH2Cl2 (100 mL) was added drop-wise into a vigorously
stirred and cooled (ꢀ78 ꢁC) solution of 1,2-diamino-
ethane (4 mL) and CH2Cl2 (200 mL). The CH2Cl2 mix-
ture was washed with 1 M HCl solution to extract the
monosubstituted amine and unreacted diamine. The
pH of the combined aqueous extracts was adjusted to
14 by the addition of sodium hydroxide pellets and the
amine product was extracted into ethyl acetate. Rotary
evaporation under reduced pressure of the dried
(Na2SO4) ethyl acetate solution gave the crude product
as an oil, which eventually solidified to a crystalline
white solid (0.42 g, 38%): mp 81–85 ꢁC; 1H NMR
(CDCl3) d 7.76 (2H, d, J = 8.8 Hz), 6.91 (2H, d,
J = 8.8 Hz), 6.72 (1H, t, J = 5.9 Hz), 3.84 (3H, s), 3.48
(2H, q, J = 5.9 Hz), 2.93 (2H, t, J = 5.9 Hz).
1
crystals (0.83 g, 34%): mp 139 ꢁC; H NMR (CDCl3) d
7.89 (1H, d, J = 2.0 Hz), 7.83 (1H, t, J = 6.0 Hz), 7.78
(2H, d, J = 8.8 Hz), 7.77 (1H, d, J = 2.0 Hz), 6.94 (2H,
d, J = 8.8 Hz), 6.70 (1H, t, J = 6.0 Hz), 4.01 (3H, s),
4.00 (3H, s), 3.86 (3H, s), 3.58 (2H, q, J = 6.0 Hz),
3.53 (2H, q, J = 6.0 Hz), 1.84 (2H, quint, J = 6.0 Hz).
Anal. Calcd for C20H23N3O7: C, 57.55; H, 5.55; N,
10.07. Found: C, 57.32; H, 5.52; N, 10.03.
4.5.9. 4-(3,4-Dimethoxy-5-nitrobenzamide)butan-1-ol (21).
3,4-Methoxy-5-nitrobenzoyl chloride (3 g, 12.2 mmol),
triethylamine (3 mL), 4-aminobutanol (1.25 g, 14 mmol)
and acetonitrile (50 mL) were mixed together and heated
under reflux for 5 h. Removal of the solvent by rotary
evaporation under reduced pressure gave the crude prod-
uct, which was then extracted into ethyl acetate. The ethyl
acetate was washed with 10% aqueous HCl and 2 M
NaOH to remove triethylamine and carboxylate byprod-
ucts. Rotary evaporation under reduced pressure of the
dried (Na2SO4) ethyl acetate solution gave the crude
product, which was purified by column chromatography
(eluting with chloroform/hexane 1:1) and then recrystal-
lized from chloroform to give the final product as a white
crystalline powder (2.84 g, 78%): mp 91–93 ꢁC; 1H NMR
(CDCl3) d 7.73 (1H, d, J = 2.1 Hz), 7.68 (1H, d,
J = 2.1 Hz), 6.88 (1H, t, J = 5.9 Hz), 4.80 (1H, br s),
4.04 (3H, s), 4.00 (3H, s), 3.78 (2H, t, J = 5.9 Hz), 3.52
(2H, q, J = 5.9 Hz), 1.74 (4H, m). Anal. Calcd for
C13H18N2O6: C, 52.34; H, 6.08; N, 9.40. Found: C,
52.50; H, 5.89; N, 9.49.
4.5.6. 3-(4-Methoxybenzamide)propylamine (18). A solu-
tion of 4-methoxybenzoyl chloride (1 g, 5.7 mmol) and
CH2Cl2 (100 mL) was added drop-wise into a vigorously
stirred and cooled (ꢀ78 ꢁC) solution of 1,3-diaminopro-
pane (4 mL) and CH2Cl2 (200 mL). The CH2Cl2 mixture
was washed with 1 M HCl solution to extract the mon-
osubstituted amine and unreacted diamine. The pH of
the combined aqueous extract was adjusted to 14 by
the addition of sodium hydroxide pellets and the amine
product was extracted into ethyl acetate. Rotary evapo-
ration under reduced pressure of the dried (Na2SO4) eth-
yl acetate solution gave the crude product as an oil.
Further evaporation of the product under high vacuum
gave the product as a crystalline white solid (0.84 g,
1
71%): mp 89–90 ꢁC; H NMR (CDCl3) d 7.76 (2H, d,
J = 8.8 Hz), 7.56 (1H, t, J = 6.0 Hz), 6.91 (2H, d,
J = 8.84 Hz), 3.85 (3H, s), 3.57 (2H, q, J = 6.0 Hz),
2.91 (2H, t, J = 6.0 Hz), 1.74 (2H, quint, J = 6.0 Hz).
4.5.10. 5-(3,4-Dimethoxy-5-nitrobenzamide)pentan-1-ol
(22).
4.5.7.
N-[2-(4-Methoxybenzoylamino)ethyl]-3,4-dime-
3,4-Methoxy-5-nitrobenzoyl
chloride
(3 g,
thoxy-5-nitrobenzamide (19). Freshly prepared 3,4-dime-
thoxy-5-nitrobenzoyl chloride (1.5 g, 6.1 mmol) and
potassium carbonate (0.84 g, 6.1 mmol) were dissolved
in a two phase system consisting of H2O (20 mL) and
ethyl acetate (20 mL). 2-(4-Methoxybenzamide)ethyl-
amine (1 g, 5.1 mmol) was added to the solution which
was stirred overnight. The precipitate that formed was
12.2 mmol), triethylamine (3 mL), 5-aminopropanol
(1.44 g, 14 mmol) and acetonitrile (50 mL) were mixed
together and heated under reflux for 5 h. Removal of
the solvent by rotary evaporation under reduced pres-
sure gave the crude product, which was then extracted
into ethyl acetate. The ethyl acetate was washed with
10% aqueous HCl and 2 M NaOH to remove triethyl-

5748
K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
amine and carboxylate byproducts. Rotary evaporation
under reduced pressure of the dried (Na2SO4) ethyl ace-
tate solution gave the crude product, which was purified
by column chromatography (eluting with chloroform/
hexane 1:1) followed by recrystallization from water
and ethanol to give the final product as flaky white crys-
DMSO. Rotary evaporation under reduced pressure of
the dried (Na2SO4) chloroform solution gave the crude
product which was purified by column chromatography
(eluting with chloroform/hexane 3:1) to give the final
product as a cream colored powder (0.74 g, 53%): mp
1
144 ꢁC; H NMR (CDCl3) d 7.85 (2H, m), 7.73 (2H,
1
tals (3.09 g, 81%): mp 86–88 ꢁC; H NMR (CDCl3) d
m), 7.72 (H, d, J = 2.0 Hz), 7.71 (H, d, J = 2.0 Hz),
6.63 (H, t, J = 6.8 Hz), 4.02 (3H, s), 3.98 (3H, s), 3.76
(2H, t, J = 6.8 Hz), 3.54 (2H, q, J = 6.8 Hz), 1.76 (4H,
m). Anal. Calcd for C21H21N3O7: C, 59.01; H, 4.95; N,
9.83. Found: C, 58.87; H, 5.04; N, 9.71.
7.68 (1H, d, J = 1.8 Hz), 7.62 (1H, d, J = 1.8 Hz), 6.35
(1H, t, J = 6.6 Hz), 4.02 (3H, s), 3.98 (3H, s), 3.56
(2H, t, J = 6.6 Hz), 3.45 (2H, q, J = 6.6 Hz), 1.84 (2H,
quint, J = 6.6 Hz), 1.66 (2H, quint, J = 6.6 Hz), 1.55
(2H, quint, J = 6.6 Hz). Anal. Calcd for C14H20N2O6:
C, 53.84; H, 6.45; N, 8.97. Found: C, 53.90; H, 6.62;
N, 8.95.
4.5.14. N-(5-Phthalimidopentyl)-3,4-dimethoxy-5-nitro-
benzamide (26). 5-(3,4-Dimethoxy-5-nitro-benzam-
ide)pentan-1-ol, p-toluenesulfonate ester (24) (1.8 g,
3.98 mmol), potassium phthalamide (0.88 g, 4.77 mmol)
and DMSO (30 mL) were mixed together and heated at
100 ꢁC with stirring for 12 h. Enough water was added
to the mixture to allow the formation of a precipitate
and then the mixture was reheated until the precipitate
dissolved. The pure product crystallized as the mixture
cooled (1.61 g, 96%): mp 118–119 ꢁC; 1H NMR (CDCl3)
d 7.83 (2H, m), 7.73 (6H, m), 6.43 (H, t, J = 6.6 Hz), 4.04
(3H, s), 3.99 (3H, s), 3.75 (2H, t, J = 6.6 Hz), 3.49 (2H,
q, J = 6.6 Hz), 1.75 (4H, m), 1.45 (2H, quint,
J = 6.6 Hz). Anal. Calcd for C22H23N3O7: C, 59.86; H,
5.25; N, 9.52. Found: C, 59.81; H, 5.30; N, 9.42.
4.5.11. 4-(3,4-Dimethoxy-5-nitrobenzamide)butan-1-ol, p-
toluenesulfonate ester (23). To a solution of 4-(3,4-di-
methoxy-5-nitrobenzamide)butan-1-ol
(21)
(2.5 g,
8.4 mmol) and dry pyridine (30 mL) cooled to below
5 ꢁC was added p-toluenesulfonyl chloride (1.93 g,
10.1 mmol). After stirring for 12 h, the mixture was
extracted into CH2Cl2 and washed with ice-cold 1 M
HCl solution to remove any dissolved pyridine. Rotary
evaporation under reduced pressure of the dried
(Na2SO4) CH2Cl2 solution gave the crude product
which was purified by column chromatography (eluting
with chloroform/hexane 1:2) to give the final product as
a white solid (2.04 g, 54%): mp 129 ꢁC; 1H NMR
(CDCl3) d 7.79 (2H, d, J = 8.1 Hz), 7.65 (2H, m), 7.36
(2H, d, J = 8.1 Hz), 6.37 (1H, t, J = 5.8 Hz), 4.10 (2H,
t, J = 5.8 Hz), 4.03 (3H, s), 3.99 (3H, s), 3.46 (2H, q,
J = 5.8 Hz), 2.45 (3H, s), 1.75 (4H, m). Anal. Calcd
4.5.15. N-[4-(4-Methoxybenzoylamino)butyl]-3,4-dimeth-
oxy-5-nitrobenzamide (27). N-(4-Phthalimidobutyl)-3,4-
dimethoxy-5-nitrobenzamide (25) (0.65 g, 1.5 mmol),
hydrazine monohydrate (0.22 mL, 4.5 mmol) and etha-
nol (30 mL) were mixed together and stirred for 12 h.
The precipitate that formed was removed by filtration
and the ethanol and hydrazine were removed by high
vacuum to yield the crude amine. The crude amine was
resuspended in a mixture of 4-methoxybenzoyl chloride
(0.31 g, 1.8 mmol), potassium carbonate (0.25 g,
1.8 mmol), H2O (25 mL) and ethyl acetate (25 mL) and
stirred overnight. The ethyl acetate mixture was separat-
ed and washed with 1 M NaOH to remove any carboxyl-
ate byproduct. Rotary evaporation under reduced
pressure of the dried (Na2SO4) ethyl acetate solution
gave the crude product, which was purified by column
chromatography (eluting with chloroform) to give the fi-
nal product as a white powder (0.23 g, 36%): mp 128 ꢁC;
1H NMR (CDCl3) d 7.93 (1H, d, J = 2.1 Hz), 7.81 (1H,
d, J = 2.1 Hz), 7.77 (2H, d, J = 9.0 Hz), 7.51 (1H, t,
J = 6.0 Hz), 6.95 (2H, d, J = 9.0 Hz), 6.40 (1H, t,
J = 6.0 Hz), 4.03 (3H, s), 4.02 (3H, s), 3.88 (3H, s), 3.59
(2H, q, J = 6.0 Hz), 3.54 (2H, q, J = 6.0 Hz), 1.75 (4H,
m). Anal. Calcd for C21H25N3O7: C, 58.46; H, 5.84; N,
9.74. Found: C, 58.08; H, 5.86; N, 9.90.
for C20H24N2SO8Æ0.5H O: C, 52.05; H, 5.46; N, 6.07;
2
S, 6.95. Found: C, 51.94; H, 5.30; N, 6.10; S, 6.57.
4.5.12. 5-(3,4-Dimethoxy-5-nitrobenzamide)pentan-1-ol,
p-toluenesulfonate ester (24). To a solution of 4-(3,4-
methoxy-5-nitrobenzamide)pentan-1-ol (22) (2.5 g,
8.0 mmol) and dry pyridine (30 mL) cooled to below
5 ꢁC was added p-toluenesulfonyl chloride (1.83 g,
9.6 mmol). After stirring for 12 h, the mixture was
extracted into CH2Cl2 and washed with ice-cold 1 M
HCl solution to remove any dissolved pyridine. Rotary
evaporation under reduced pressure of the dried
(Na2SO4) CH2Cl2 solution gave the crude product,
which was purified by column chromatography (eluting
with chloroform/hexane 1:2) to give the final product as
1
a clear oil (2.39 g, 64%): H NMR (CDCl3) d 7.79 (2H,
d, J = 8.4 Hz), 7.69 (1H, d, J = 2.1 Hz), 7.66 (1H, d,
J = 2.1 Hz), 7.36 (2H, d, J = 8.4 Hz), 6.35 (1H, t,
J = 6.6 Hz), 4.08 (2H, t, J = 6.6 Hz), 4.04 (3H, s), 4.00
(3H, s), 3.45 (2H, q, J = 6.6 Hz), 2.47 (3H, s), 1.75
(2H, quint, J = 6.6 Hz), 1.65 (2H, m), 1.50 (2H, m).
4.5.13.
benzamide
N-(4-Phthalimidobutyl)-3,4-dimethoxy-5-nitro-
(25). 4-(3,4-Dihydroxy-5-nitro-benzam-
4.5.16.
N-[5-(4-Methoxybenzoylamino)pentyl]-3,4-di-
methoxy-5-nitrobenzamide (28). N-(5-Phthalimidopen-
tyl)-3,4-dimethoxy-5-nitrobenzamide (26) (1 g, 2.3
mmol), hydrazine hydrate (0.35 mL, 6.9 mmol) and eth-
anol (30 mL) were mixed together and stirred for 12 h.
The precipitate that formed was removed by filtration
and the ethanol and hydrazine were removed by high
vacuum to yield the crude amine. The crude amine
was resuspended in a mixture of 4-methoxybenzoyl
ide)butan-1-ol, p-toluenesulfonate ester (23) (1.48 g,
3.27 mmol), potassium phthalamide (0.73 g, 3.93 mmol)
and DMSO (30 mL) were mixed together and heated at
100 ꢁC with stirring for 12 h. Water (150 mL) was added
and the mixture stirred until a precipitate formed. The
precipitate was extracted with chloroform and washed
with saturated salt solution to remove any residual

K. Bailey, E. W. Tan / Bioorg. Med. Chem. 13 (2005) 5740–5749
5749
chloride (0.48 g, 2.8 mmol), potassium carbonate
(0.39 g, 2.8 mmol), H2O (25 mL) and ethyl acetate
(25 mL) and stirred overnight. The ethyl acetate solution
was separated and washed with 1 M NaOH to remove
any carboxylate byproduct. Rotary evaporation under
reduced pressure of the dried (Na2SO4) ethyl acetate
solution gave the crude product, which was purified by
column chromatography (eluting with chloroform) to
the crude product. The product was purified by recrys-
tallization from an acetone and water solution to give
the final product as long yellow crystals (80 mg, 45%):
mp 217–219 ꢁC; H NMR (acetone-d6) d 10.40 (1H, br
s), 8.92 (1H, br s), 8.00 (1H, d, J = 2.1 Hz), 7.73 (1H,
d, J = 4.8 Hz), 7.60 (1H, d, J = 2.1 Hz), 2.74 (3H, d,
J = 4.8 Hz). Anal. Calcd for C8H8N2O5: C, 45.28; H,
3.80; N, 13.21. Found: C, 45.35; H, 3.82; N, 12.91.
1
1
give the final product as a clear oil (0.49 g, 47%): H
NMR (CDCl3) d 7.75 (1H, d, J = 2.1 Hz), 7.69 (1H, d,
J = 2.1 Hz), 7.65 (2H, d, J = 9.0 Hz), 6.88 (2H, d,
J = 9.0 Hz), 6.86 (1H, t, J = 6.9 Hz), 6.25 (1H, t,
J = 6.9 Hz), 4.01 (3H, s), 3.94 (3H, s), 3.84 (3H, s),
3.47 (4H, q, J = 6.9 Hz), 1.68 (4H, quint, J = 6.9 Hz),
1.45 (2H, quint, J = 6.9 Hz).
4.5.20. N-Ethyl-(3,4-dihydroxy-5-nitrobenzamide) (32).
The ethyl derivative was prepared using N-ethyl-(3,4-di-
methoxy-5-nitro-benzamide) (30) (0.2 g, 0.8 mmol) and
BBr3 (1.2 mL, 15 mol equiv) using the procedure de-
1
scribed above (96 mg, 54%): mp 176–177 ꢁC; H NMR
(acetone-d6)
d 10.60 (2H, br s), 8.00 (1H, d,
J = 2.1 Hz), 7.78 (1H, br s), 7.62 (1H, d, J = 2.1 Hz),
3.28 (2H, dq, J = 5.6, 7.2 Hz), 1.05 (3H, t, J = 7.2 Hz).
Anal. Calcd for C9H10N2O5: C, 47.79; H, 4.45; N,
12.38. Found: C, 48.02; H, 4.43; N, 12.09.
4.5.17. N-Methyl-(3,4-dimethoxy-5-nitrobenzamide) (29).
Methylamine in ethanol (33% w/w) (1.3 mL, 11 mmol)
was added to a solution of 3,4-dimethoxy-5-nitrobenzoyl-
chloride (2.5 g, 10.1 mmol), triethylamine (2 mL) and eth-
yl acetate (50 mL) and the mixture was stirred overnight.
The ethyl acetate was washed with 10% aqueous HCl
solution and 2 M NaOH solution. Rotary evaporation
under reduced pressure of the dried (MgSO4) ethyl acetate
solution gave the crude product, which was purified by
column chromatography (3:1 chloroform/hexane) to give
the final product as a white powder (2.1 g, 86%): mp 158–
162 ꢁC (dec); 1H NMR (CDCl3) d 7.66 (1H, d,
J = 2.1 Hz), 7.60 (1H, d, J = 2.1 Hz), 6.20 (1H, br s),
4.01 (3H, s), 3.96 (3H, s), 3.01 (3H, d, J = 7.1 Hz). Anal.
Calcd for C10H12N2O5: C, 50.00; H, 5.04; N, 11.66.
Found: C, 50.24; H, 4.97; N, 11.34.
References and notes
1. Axelrod, J.; Tomchick, R. J. Biol. Chem. 1958, 233, 702.
2. Woodard, W. R.; Nutt, J. G. Catechol-O-Methyltransfer-
ase Inhibitors in the Treatment of Parkinsonism. In
Therapy of Parkinsonꢀs Disease; Koller, W. C., Paulson,
G., Eds., 2nd ed.; Marcel Dekker: New York, 1995, pp
287–297.
3. Guldberg, H. C.; Marsden, C. H. Pharmacol. Rev. 1975,
27, 135.
4. Belleau, B.; Burba, J. Biochim. Biophys. Acta 1961, 54, 195.
5. Lehman, A. J.; Fitzhugh, O. G.; Nelson, A. A.; Woodard,
G. Adv. Food Res. 1951, 3, 197.
6. Borgulya, J.; Bruderer, H.; Bernauer, K.; Zurcher, G.;
Prada, M. D. Helv. Chim. Acta 1989, 72, 952.
7. Linden, I. B.; Nisssinen, E.; Etemadzableh, E.; Kaakkola,
S.; Mannisto, P.; Pohto, P. J. Pharmacol. Exp. Ther. 1988,
247, 289.
8. Roberts, J. W.; Cora-Locatelli, G.; Bravi, D.; Amantea,
M. A.; Mouradian, M. M.; Chase, T. N. Neurology 1993,
43, 2685.
9. Cedarbaum, J. M.; Leger, G.; Guttman, M. Clin. Neuro-
pharmacol. 1991, 14, 330.
10. Burba, J. V.; Becking, G. C. Arch. Int. Pharmacodyn.
Ther. 1969, 180, 323–329.
4.5.18. N-Ethyl-(3,4-dimethoxy-5-nitrobenzamide) (30).
Ethylamine in ethanol (33% w/w) (1.95 mL, 11 mmol)
was added to a solution of 3,4-dimethoxy-5-nitro-
benzoylchloride (2.5 g, 10.1 mmol), triethylamine
(2 mL) in ethyl acetate (50 mL) and the mixture was stir-
red overnight. The reaction mixture was washed with
10% aqueous HCl and 2 M NaOH solution. Rotary
evaporation under reduced pressure of the dried
(MgSO4) ethyl acetate solution gave the crude product,
which was recrystallized from water and ethanol to give
the product as white flaky crystals (1.4 g, 54%): mp
11. Burba, J. V.; Murnagham, M. F. Biochim. Pharmacol.
1965, 14, 823–829.
1
111 ꢁC; H NMR (CDCl3) d 7.68 (1H, d, J = 2.1 Hz),
7.61 (1H, d, J = 2.1 Hz), 6.20 (1H, br s), 4.02 (3H, s),
3.98 (3H, s), 3.51 (2H, dq, J = 5.6, 7.1 Hz), 1.27 (3H,
t, J = 7.1 Hz). Anal. Calcd for C11H14N2O5: C, 51.96;
H, 5.55; N, 11.02. Found: C, 52.07; H, 5.46; N, 10.79.
12. Brevitt, S. E.; Tan, E. W. J. Med. Chem. 1997, 40, 2035–
2039.
13. Mannisto, P. T.; Kaakkola, S. Pharmacol. Rev. 1999, 51,
¨
¨
593.
14. Andrew, R. G.; Raphael, R. A. Tetrahedron 1987, 43,
4803.
15. Press, J. B. Synth. Commun. 1979, 9, 407–410.
16. Jacobson, A. R.; Makris, A. N.; Sayre, L. M. J. Org.
Chem. 1987, 52, 2592.
4.5.19. N-Methyl-(3,4-dihydroxy-5-nitrobenzamide) (31).
An excess of BBr3 (1.2 mL, 15 mol equiv) was added to a
solution of N-methyl (3,4-dihydroxy-5-nitrobenzamide)
(29) (0.2 g, 0.8 mmol) in dried CH2Cl2 (20 mL) under
N2. The mixture was left stirring for 24 h at room tem-
perature. Water was carefully added to eliminate any
unreacted BBr3. The mixture was evaporated to dryness
by rotary evaporation under reduced pressure to yield
´
17. Bonifacio, M. J.; Archer, M.; Rodrigues, P. M.; Matias, P.
M.; Learmonth, D. A.; Carrondo, M. A.; Soares-da-silva,
P. Mol. Pharmacol. 2002, 62, 795.
18. Bailey, K. B.; Tan, E. W.; Cowling, R. M.; Webb, D. J.
Biochem. Med. Chem. 2004, 12, 595.