A colormetric assay for catechol-O-methyltransferase

Article abstract of DOI:10.1016/j.bmc.2003.10.038

A series of catechol diazo dyes were synthesized and tested as substrates for the enzyme catechol-O-methyltransferase (COMT) with the aim of developing a sensitive HPLC assay method using visible wavelength light detection. A method was developed which al

Full text of DOI:10.1016/j.bmc.2003.10.038

Bioorganic & Medicinal Chemistry 12 (2004) 595–601
A colormetric assay for catechol-O-methyltransferase
Karl Bailey, Rebecca Cowling, Eng Wui Tan* and Daniel Webb
Department of Chemistry, University of Otago, PO Box 56, Dunedin, New Zealand
Received 7 August 2003; revised 14 October 2003; accepted 22 October 2003
Abstract—A series of catechol diazo dyes were synthesized and tested as substrates for the enzyme catechol-O-methyltransferase
(COMT) with the aim of developing a sensitive HPLC assay method using visible wavelength light detection. A method was
developed which allowed for the determination of the two regioisomeric methylated products of the COMT catalyzed reaction of
4-[(3,4-dihydroxyphenyl)azo]benzenesulfonate with S-adenosylmethionine (AdoMet). Separation of the assay components was
achieved by reverse phase chromatography using an isocratic mobile phase. No pre-preparation of the assay samples was required.
# 2003Elsevier Ltd. All rights reserved.
1. Introduction
problem by using 3,4-dihydroxybenzylamine as
a
The enzyme catechol-O-methyltransferase (COMT) has
been implicated in the ‘wearing off’ effect seen in
Parkinson’s disease patients who have been treated with
l-dopa based medications for an extended period of
time. The enzyme inactivates catechol based neuro-
transmitters by methylating either of the two aromatic
hydroxy groups of the catechol substructure.1 Extensive
research has been focused on developing inhibitors of
the enzyme,2ꢀ11 which in turn has led to the develop-
ment of numerous enzyme assays to determine the
activity of COMT. The most popular type of assays use
radiolabeled AdoMet substrates such as S-adenosyl-l-
[3H methyl] methionine or S-adenosyl-l-[14C methyl]
methionine.7,12ꢀ17 During the course of the enzyme
reaction the radiolabel is incorporated into the methyl-
ated products. Products and reactants are then
separated using solvent extraction methods and the
radiolabeled products in the organic phase are quanti-
fied using a photo-scintillation device. From the level of
products formed the enzyme activities can be
determined.
substrate.20 After the reaction with COMT the assay
sample is directly injected onto the HPLC machine for
analysis. UV detection can also be used in place of
electrochemical detection. One such method determines
the amount of product formed by determining the
amount of SAH produced during the course of the
assay.21 Direct measurement of the methylated product
has also been achieved by using methyl 3,4,5-trihydrox-
ybenzoate as the substrate with detection of the product
methyl 3,5-dihydroxy-4-methoxybenzoate.2 A short-
coming with the last method is that problems arise if
there are other components in the assay that also have
large UV absorbancies (at ꢁ280 nm) that have similar
retention times. Products that absorb in an entirely dif-
ferent region of the UV–vis spectrum would be an
advantage as interference effects would be diminished.
Diazo catechol dyes, which absorb in the visible range of
the spectrum, would be good substrates in this regard. A
paper by Haghbeen et al. describes the synthesis of a set
of diazo catechol dyes and their suitability as substrates
for the enzyme monooxygenase.22 In this paper, we
describe the synthesis of a series of diazo catechol dyes
which were tested as substrates for the enzyme COMT.
In addition these dyes were analyzed to see if they could
be incorporated into a new COMT assay.
Assays can also be performed without radiolabeled
substrates. One example uses HPLC with electro-
chemical detection.18,19 Most methods using this setup
require sample cleanup procedures before the
O-methylated products from the COMT reactions can
be analyzed. A method by Nissinen et. al. avoids this
2. Results
Three sets of diazo dyes were synthesized (Fig. 1). One
set (1) are potential COMT substrates while the other
two sets (2 and 3) are the expected methylated products
* Corresponding author. Tel.: +64-34-79-7926; fax: +64-34-70-7906;
e-mail: ewtan@alkali.otago.ac.nz
0968-0896/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.038

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K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
Figure 1. Diazo dye COMT substrates and expected products.
Scheme 1. Synthesis of guaiacol diazo dyes.
after the reaction of 1 with COMT. Compounds 2 and 3
were used as authentic sample standards to determine
product retention times for HPLC analysis.
The synthesis of dyes 1b and 1d have been previously
described.22 Using the same methodology, diazo dyes
1a, 1c and 1e were synthesized. Fortuitously, the inter-
mediates in the synthesis are the 3-methylated catechol
diazo dyes (2) that were required as product standards.
Thus, compounds 2a, 2c and 2e were made by the direct
coupling of diazotised aniline derivatives to guaiacol
(Scheme 1).
Compounds 1a, 1c and 1e were synthesized by the
removal of the 3-O-methyl group from 2a, 2c and 2e using
pyridine and AlCl3 dissolved in chloroform. In the case of
2e it was found that some of the 40-O-methyl group
was also removed resulting in lowered yields of 1e. By
replacing chloroform with toluene the amount of 1e
produced was increased.
The synthesis of the dyes 3(a, b, d, e) is as shown in
Scheme 2. The tosyl ester of catechol 4 was prepared by
the reaction of tosyl chloride with catechol in pyridine.
The coupling of 4 with the appropriate phenyldiazo-
nium derivatives gave the 3-O-tosyl catechol diazo dye
precursors. The 4-O-hydroxy groups were then methy-
lated to give compounds 6(a, b, d, e). Detosylation of
these compounds gave the 4-O-methyl dyes 3(a, b, d, e).
Scheme 2. Synthesis of 4-O-methyl 3,4-dihydroxyazobenzene
derivatives.
6c. The tosyl and phenyl groups of compound 6c were
removed to give the final product 3c.
The synthesis of the 4-O-methylsulfonic acid dye 3c is as
shown in Scheme 3. The synthesis of the starting material
N-acetyl 4-aminobenzenesulfonyl chloride has been
previously described in a book article by Blatt et al.23 N-
Acetyl 4-aminobenzenesulfonyl chloride was dissolved
into pyridine and reacted with phenol to form the
phenyl ester 7. The acetate group of 7 was hydrolyzed
by heating 7 in a dilute solution of HCl under reflux to
give 8. Compound 8 was then transformed into the
diazonium salt and reacted with 4 to form the diazo dye
which was subsequently methylated to give compound
3. Discussion
The buffers used in the mobile phase for the HPLC
analysis of the COMT assay reactions were chosen to
give good resolution between the substrate and
product(s) of the assay. For maximum sensitivity the
UV–vis detector was set to scan at the wavelength
corresponding to the absorption maxima of the sub-
strate and product dyes being analysed. The resulting
chromatograms of the assay mixtures are shown in

K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
597
Figure 2. In the case of substrate 1a, the expected
products were not detected. For the reaction of 1b with
COMT the resulting chromatogram of the assay shows
three peaks. Compound 1b was found to elute at 9.7
min and the enzyme products 2b and 3b were found to
elute at 16.4 min and 17.9 min, respectively. Some peak
tailing was observed which did not disappear with
higher concentrations of TFA in the mobile phase.
The assay containing 1c as the substrate was analyzed
by HPLC and the resulting chromatogram of the assay
showed three well-resolved and symmetrical peaks.
Compound 1c was found to elute at 5.4 min and the
enzyme products 2c and 3c were found to elute at 8.2
min and 8.9 min, respectively. The assay containing 1d
as the substrate was analyzed by HPLC and the result-
ing chromatogram showed two peaks. Compound 1d
was found to elute at 6.6 min and the product 3d was
found to elute at 12.9 min. The assay containing 1e as
the substrate was analyzed by HPLC and the resulting
chromatogram showed two peaks. Compound 1e was
found to elute at 9.3min and compound 3e was found
to elute at 9.3min. The absence of peaks corresponding
to 2d and 2e would indicate that COMT exclusively
methylates at the 4-O- position when substrates 1d and
1e were used.
Of the five dyes used in the COMT assays, 1c and the
corresponding products 2c and 3c had the shortest
elution times and also gave the most symmetrical peaks
with no tailing in HPLC analysis. In addition, this
Scheme 3. Synthesis of diazo COMT substrate 3c.
Figure 2. HPLC chromatograms of COMT reactions with 1b–1e.

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K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
compound was also the only water soluble dye which
meant an organic co-solvent was not required in the
enzyme assay. These results indicated that 1c was
the most suitable substrate for a COMT assay. The fact
that 1c produces both the 3- and 4-O-methylated
products was not considered to be overly detrimental as
the enzyme activity can be determined from the total
amount of both products in the assay.
inexpensive in comparison to the methods using
radioactive substrates while offering comparative
sensitivity.
5. Experimental
Melting points were recorded on a Gallenkamp capil-
lary melting point apparatus and are uncorrected.
Elemental analyses were performed at the Campbell
Microanalytical laboratory, Otago University. 1H
NMR and 13C NMR spectra were measured on a
Varian Unity Inova 300 MHz spectrometer. The d
values are reported in ppm downfield from TMS.
Column chromatography was performed with silica gel
(Sorbsil, partical size 32–63 mM). AdoMet as the p-
toluenesulfonate salt and COMT were purchased from
SigmaTM. Guaiacol, aniline, 4-methoxyaniline, 4-sulfa-
nilamide, 4-aminobenzenesulfonic acid and 4-nitroana-
line were purchased from Aldrich. Solvent and
inorganic bases were generally sourced.
The assay using 1c as the substrate, was then optimized
by varying the incubation time and AdoMet concen-
tration. Time dependence experiments were performed
to determine the optimal incubation time for the COMT
assay and to ensure a constant rate with time. A series
of reaction assay solution containing the substrate 1c
were set up and during the 2 h of the experiment an
assay solution was quenched with 4 M perchloric acid
every 15 min and analyzed by HPLC. A plot of
velocity (calculated as the sum of nmol of 2b and 2c
produced per mg of protein per min) versus time
showed that a linear relationship existed for reaction
times up to one h.
5.1. 4-[(4-Nitrophenyl)azo]-catechol (1a)
The kinetics of the enzyme reaction were studied by
running assays using various concentrations of 1c.
AdoMet was used at a concentration of 2 mM (saturation
kinetics) in the assay solution. The resulting two product
peaks (2b and 2c) were converted from area into velo-
city values. Maximum product formation was approa-
ched when the concentration of 1c was approximately
150 mM. This concentration can be taken as the satur-
ating concentration under these assay conditions. Vmax
and Km values were found to be 2.85 (nmol product/
min/mg protein)ꢀ1 and 41 mM, respectively, by Line-
weaver–Burke double reciprocal plots. The Km value for
1c is within the range of Km values that have been
determined for a range of COMT substrates.3,7,17,24 The
limits of detection for this assay were 150 and 255 pmol
in a 30 mL injection (signal to noise ratio of 10) for the
enzyme products 2b and 2c, respectively.
Into a two neck flask was placed 2a (1 g, 3.7 mmol),
CHCl3 (100 mL) and anhydrous AlCl3 (4.34 g, 0.033
mol). The mixture was stirred under nitrogen for 30 min
whilst adding distilled pyridine (10 mL, 0.12 mol). The
reaction vessel was then refluxed in an oil bath for 24 h.
Distilled water (100 mL) and concd HCl (10 mL, 0.11
mol) was added to hydrolyse the product. The crude
product was then isolated by vacuum filtration and
recrystallised from ethanol (60 mL) and HCl (10mL of 1
mol Lꢀ1). (0.2 g, 20%): mp 230 ꢂC; H NMR (DMSO-
1
d6) 8.48 (2H, d, J=9.0 Hz), 8.06 (2H, d, J=9.0 Hz),
7.56 (1H, dd, J=2.4, 8.4 Hz), 7.48 (1H, d, J=2.4 Hz),
7.06 (1H, dd, J=2.4, 8.4 Hz). Anal. calcd for
.
C12H9N3O4 1/2H2O: C, 53.57; H, 3.76; N, 15.67. Found
C, 53.52; H, 3.65; N, 15.33. HRMS m/z MH+,
260.0674. calcd for C12H10N3O4 260.0671.
5.2. 4-[(3,4-Dihydroxyphenyl)azo]benzenesulfonic acid (1c)
4. Conclusion
A mixture of 2c (1.15 g, 3.25 mmol), AlCl3 (4.34g, 32.5
mmol) and CHCl3 (100 mL) was stirred for 30 min
under a nitrogen atmosphere. Following the addition of
pyridine (10.28 g, 0.13mol) the mixture was stirred for a
further hour then heated under reflux for 23h. The
addition of 30 mL of water resulted in the formation of
a solid red precipitate. Concentrated HCl was added
until the solid redissolved. The chloroform layer was
removed and then concentrated HCl was added to the
aqueous layer until an orange precipitate formed. The
precipitate was filtered off and redissolved in a minimum
amount of water. Sodium bicarbonate was slowly added
to the solution until the sodium salt precipitated. The salt
was filtered off and recrystallized from water to give the
final product as flaky yellow crystals. (0.24 g, 23%): mp
The diazo dye 4-[(3,4-dihydroxyphenyl)azo]benzene
sulfonate (1c) was shown to be a good substrate for
COMT. As 1c is readily water soluble, assay conditions
could dispense with the use of an organic co-solvent. It
was the preferred substrate of the range of dyes tested.
After reaction, a sample of the assay mixture could be
injected directly onto an HPLC column for the analysis
and quantification of the dye products, 2c and 3c.
Interference effects from the assay solvents, buffer,
enzyme and the co-factor AdoMet are markedly
reduced as none of these components absorb light in the
visible region. No pre-purification of the assay sample is
necessary prior to HPLC analysis. This factor and the
short retention times of the dyes and good resolution by
HPLC would facilitate the rapid analysis of numerous
samples, which is especially advantageous for kinetic
studies of COMT. The preferred substrate 1c and
product dye standards were easily prepared in high
yields. The assay method using the diazo dye is
ꢂ
1
260–265 C (dec); H NMR (D2O) 7.65 (2H, d, J=8.8
Hz), 7.45 (2H, d, J=8.8 Hz), 6.99 (1H, dd, J=2.8, 8.5
Hz), 6.77 (1H, d, J=2.8 Hz), 6.37 (1H, d, J=8.5 Hz).
Anal. calcd for C12H9N2O5SNa: C, 45.57; H, 2.87; N,
8.86; S, 10.14. Found C, 45.39; H, 2.84; N, 8.87; S, 9.92.

K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
599
5.3. 4-[(4-Methoxyphenyl)azo]-catechol (1e)
65.10; H, 5.46; N, 10.85. Found: C, 65.37; H, 5.65; N,
11.06.
AlCl3 (4.34g, 32.5 mmol), 2e (0.84 g, 3.25 mmol) and
toluene (100 mL) were mixed together and stirred for 30
min under a nitrogen atmosphere. Dried pyridine (10.28
g, 130 mmol) was added and the mixture was heated
under reflux for 2 h, then stirred for a further 12 h under
a nitrogen atmosphere. During the course of the reac-
tion a yellow precipitate formed which turned red upon
the addition of 2 M HCl (40 mL). The solution con-
taining the red precipitate was dissolved in ethyl acetate
and methanol (1:1 50 mL). The organic layer was sepa-
rated and rotary evaporation of the solvent gave the
crude product as a purple tar. Recrystallization from
ethyl acetate and toluene gave the final product as
5.5. General procedure for the preparation of 4-o-methy-
lated catechol diazo dyes (3)
The appropriate diazo dye derivative was dissolved into
5% KOH w/w methanol solution and heated under
reflux for 30 min. A 5% aqueous HCl solution was
added and the solvent is removed by rotary evaporation
to give the crude product.
5.5.1. 4-Nitro [(4-methoxy, 3-hydroxyphenyl)azo]benzene
(3a). The dye 3a was prepared from 6a using the proce-
dure described above. Recrystallization from chloro-
form and hexane gave the product as red needle-like
crystals. (98 mg, 37%): mp 167–169 ꢂC; 1H NMR
(CDCl3) 8.37 (2H, d, J=9.0 Hz), 7.99 (2H, d, J=9.0
Hz), 7.65 (1H, dd, J=2.4, 8.7 Hz), 7.57 (1H, d, J=2.4
Hz), 7.03(1H, d, J=8.7 Hz), 5.75 (1H, s), 4.03(3H, s).
Anal. calcd for C13H11N3O4: C, 57.14; H, 4.06; N,
15.38. Found: C, 57.15; H, 3.95; N, 15.26.
ꢂ
1
purple crystals. (0.29 g, 31%): mp 45–46 C; H NMR
(CDCl3) 7.87 (2H, d, J=9.0 Hz), 7.50 (1H, dd, J=2.2,
8.3Hz), 7.46 (1H, d, J=2.2 Hz), 7.02 (1H, d, J=8.3
Hz), 7.00 (2H, d, J=9.0 Hz), 5.68 (1H, br s), 5.52 (1H,
.
br s), 3.90 (3H, s). Anal. calcd for C13H12N2O3 11/
4H2O: C, 58.52; H, 5.48; N, 10.50. Found: C, 58.81; H,
5.18; N, 10.58.
5.5.2. 4-[(3-Hydroxy, 4-methoxyphenyl)azo]benzene
sulfonamide (3b). The dye 3b was prepared from 6b
using the procedure described above. Recrystallization
from ethanol and water gave the product as small
orange crystals. (80 mg, 30%): mp 229–231 ꢂC; 1H
NMR (DMSO-d6) 9.66 (1H, s), 8.03(2H, d, J=9.0 Hz),
7.99 (2H, d, J=9.0 Hz), 7.58 (1H, dd, J=2.7, 8.7 Hz),
7.54 (2H, s), 7.40 (1H, d, J=2.7 Hz), 7.20 (1H, d, J=8.7
Hz), 3.93 (3H, s). Anal. calcd for C13H13N3O4S: C,
50.80; H, 4.26; N, 13.68; S, 10.43. Found: C, 50.83; H,
4.15; N, 13.62; S, 10.45.
5.4. General procedure for the preparation of diazo deri-
vatives of guaiacol (2)
A cooled (5 ꢂC) solution containing NaNO2 (2.1 g, 0.03
mol) and water (50 mL) was slowly added to a cooled
solution of the appropriate aniline derivatives (0.03
mol), 6M HCl (10 mL) and water (50 mL) to form the
corresponding diazonium salt.
The diazonium salt solution was then added drop-wise
to a cooled (5 ꢂC) solution of 2-methoxyphenol (3.7 g,
0.03mol) dissolved in dilute sodium hydroxide (1.2 g in
50 mL) to give the diazo dye. The resultant mixture was
stirred for a further 2 h.
5.5.3. 4-[(4-Methoxy, 3-hydroxyphenyl)azo]benzenesulfo-
nate sodium salt (3c). The dye 3c was prepared from 6c
using the procedure described above. Recrystallization
from water gave the product as orange flaky crystals.
5.4.1. 4-[(4-Hydroxy, 3-methoxyphenyl)azo]benzenesulfo-
nate disodium salt (2c). The dye 2c was prepared from
4-aminobenzenesulfonate using the procedure above.
To precipitate the dye from the reaction mixture the
solution was acidified with concentrated HCl. The
precipitate was filtered off and recrystallised from 1M
NaOH solution to give compound 2c as dark red
chunky crystals. (5.21 g, 49%): mp 258–262 ꢂC (dec); 1H
NMR(D2O) 7.84 (2H, d, J=9.0 Hz), 7.63(2H, d, J=9.0
Hz), 7.33 (1H, dd, J=2.1, 8.4 Hz), 7.22 (1H, d, J=2.1
Hz), 6.82 (1H, d, J=8.4 Hz), 3.76 (3H, s). Anal. calcd
for C13H10N2O5SNa2: C, 44.32; H, 2.86; N, 7.95; S,
9.10. Found: C, 44.66; H, 3.12; N, 7.86; S, 9.09.
(88 mg, 29%): mp 276–279 ꢂC (dec); H NMR (D2O)
1
7.87 (2H, d, J=9.0 Hz), 7.74 (2H, d, J=9.0 Hz), 7.14
(1H, dd, J=2.5, 8.3Hz), 6.98 (1H, d, J=2.5 Hz), 6.93
(1H, d, J=8.3 Hz), 3.78 (3H, s). Anal. calcd for
C13H11N2SO5Na: C, 47.27; H, 3.36; N, 8.48; S, 9.71.
Found: C, 46.97; H, 3.51; N, 8.44; S, 9.66.
5.5.4. 3-Hydroxy, 4-methoxyazobenezene (3d). The dye
3d was prepared from 6d using the procedure described
above. Recrystallization from ethanol and water gave
the product as small orange crystals. (0.27 g, 90%): mp
84–86 ꢂC; H NMR (CDCl3) 7.88 (2H, d, J=9.0 Hz),
1
7.57 (1H, dd, J=2.5, 8.5 Hz), 7.55 (1H, d, J=2.5 Hz),
7.50 (2H, m), 7.44 (1H, m), 6.98 (1H, d, J=8.5 Hz), 5.72
(1H, s), 3.98 (3H, s). Anal. calcd for C13H12N2O2: C,
68.40; H, 5.30; N, 12.28. Found: C, 68.49; H, 5.52; N,
12.30.
5.4.2. 4-[(4-Methoxyphenyl)azo]-2-methoxyphenol (2e).
The dye 2e was prepared from p-anisidine using the
procedure above. To precipitate the dye from the reaction
mixture the solution was acidified with concentrated
HCl. The precipitate was filtered off and recrystallised
from water and ethanol to give compound 2e aꢂs dark
5.5.5. 3-Hydroxy-4,40-dimethoxyazobenzene (3e). The
dye 3e was prepared from 6e using the procedure
described above. Recrystallization from methanol and
water gave the product as flaky gold crystals. (80 mg,
1
blue chunky crystals. (6.51 g, 84%): mp 104–106 C; H
NMR (CDCl3) 7.89 (2H, d, J=8.7 Hz), 7.56 (1H, dd,
J=2.4, 8.4 Hz), 7.50 (1H, d, J=2.4 Hz), 7.05 (1H, d,
J=8.4 Hz). 7.01 (2H, d, J=8.7 Hz), 5.91 (1H, s), 4.00
(3H, s), 3.9 (3H, s). Anal. calcd for C14H14N2O3: C,
ꢂ
1
26%): mp 171–172 C; H NMR (CDCl3) 7.88 (2H, d,
J=9.0 Hz), 7.51 (2H, d, J=9.0 Hz), 7.01 (2H, d, J=9.0

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K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
Hz), 6.97 (1H, d, J=9.0 Hz), 5.68 (1H, s), 3.99 (3H, s),
3.90 (3H, s). Anal. calcd for C14H14N2O3: C, 65.10; H,
5.46; N, 10.85. Found: C, 65.29; H, 5.51; N, 11.10.
give the product 6b using the procedure described above.
Purification by column chromatography (chloroform)
followed by recrystallization from hexane and chloro-
form gave the product as small orange crystals. (1.02 g,
79%): mp 190–191 ꢂC; H NMR (acetone-d6) 8.09 (2H,
1
5.5.6. Catechol mono O-p-toluenesulfonate (4). Catechol
(4.44 g, 40 mmol), p-toluenesulfonyl chloride (9.53g, 50
mmol) and pyridine (9.5 mL) were mixed together in a
100 mL conical flask and heated on a water bath for 15
min with occasional stirring. Cold water (100 mL) was
added and the mixture stirred until a precipitate formed.
The precipitate was extracted with chloroform and
washed with 1 M HCl to remove any dissolved pyridine.
Rotary evaporation under reduced pressure of the dried
(Na2SO4) chloroform solution gave the crude product.
Purification by recrystallization from acetone and water
gave the final product as small white cubic crystals.
(4.84 g, 46%): mp 90 ꢂC; 1H NMR (CDCl3) 7.76 (2H, d,
J=8.1 Hz), 7.34 (2H, d, J=8.1 Hz), 7.14 (1H, m), 7.01
(1H, d, J=7.8 Hz), 6.77 (2H, m), 6.00 (1H, s), 2.47 (3H,
s). Anal. calcd for C13H12NSO4: C, 59.07; H, 4.57; S,
12.13. Found: C, 58.98; H, 4.65; S, 12.11.
d, J=8.5 Hz), 8.02 (2H, d, J=8.5 Hz), 7.98 (1H, dd,
J=2.5, 9.0 Hz), 7.79 (1H, d, J=2.5 Hz), 7.78 (2H, d,
J=8.5 Hz), 7.48 (2H, d, J=8.5 Hz), 7.25 (1H, d, J=9.0
Hz), 6.76 (2H, s), 3.68 (3H, s), 2.48 (3H, s). Anal. calcd
for C20H19N3S2O6: C, 52.05; H, 4.15; N, 9.11; S, 13.89.
Found: C, 51.94; H, 4.24; N, 9.11; S, 13.75.
5.5.10. 3-Hydroxy, 4-methoxy, 40-phenoxysulfonylazo-
benzene 3-O-p-toluenesulfonate (6c). The dye 5c was
prepared from 4-phenoxysulfonylaniline (8) and then
methylated to give the product 6c using the procedure
described above. Purification by column chromato-
graphy (chloroform) followed by recrystallization from
ethanol and water gave the product as chunky bright
red crystals. (2.31 g, 42%): mp 177–178 ꢂC; H NMR
1
(CD2Cl2) 7.96 (2H, d, J=8.9 Hz), 7.93(2H, d, J=8.9
Hz), 7.93(1H, dd, J=2.5, 9.0 Hz), 7.80 (1H, d,
J=2.5 Hz), 7.73(2H, d, J=8.5 Hz), 7.33 (2H, d,
J=8.5 Hz), 7.28 (3H, m), 7.01 (2H, d, J=8.5 Hz),
7.00 (1H, d, J=9.0 Hz), 3.63 (3H, s), 2.44 (3H, s).
Anal. calcd for C26H22N2S2O7. 12H2O: C, 57.03; H, 4.23;
N, 5.11; S, 11.71. Found: C, 56.96; H, 3.86; N, 4.73; S,
11.78.
5.5.7. General preparation for the 4-O-Methyl, 3-O-tosyl
catechol diazo dyes (6). A cooled (5 ꢂC) solution made
up of the appropriate aniline derivative dissolved in
concd HCl (5 mL) and water (50 mL) was prepared. To
this is slowly added a cooled (5 ꢂC) solution of NaNO2
(1 molar equivalent) dissolved in water (15 mL). The
resulting mixture was stirred for an hour and then
added drop-wise to a stirred cooled (5 ꢂC) solution of
catechol mono p-toluenesulfonate (4) (1 molar equiv),
DMF (40 mL), NaOH (1 molar equiv) and water (5
mL) to bring about the formation of the dye. The mix-
ture was stirred for a further hour and then 5% aqueous
HCl was added to precipitate the dye, which was then
extracted with CH2Cl2 (60 mL). To this solution was
added NaOH (2 molar equiv), tetrabutylammonium
bromide (0.1 molar equiv), water (60 mL) and dime-
thylsulfate (5 molar equiv). The mixture was then stirred
vigorously under a nitrogen atmosphere for 24 h. The
CH2Cl2 layer was then separated and the solvent
removed by rotary evaporation under reduced pressure.
Residual DMF was removed by bulb to bulb distillation
to give the crude product.
5.5.11. 4-Methoxy, 3-hydroxyazobenzene 3-O-p-toluene-
sulfonate (6d). The dye 5d was prepared from aniline
and then methylated to give the product 6d using the
procedure described above. Purification by column
chromatography (chloroform) followed by recrystalli-
zation from hexane and chloroform gave the product as
ꢂ
1
small orange crystals. (0.64 g, 60%): mp 99–100 C; H
NMR (CDCl3) 7.86 (2H, d, J=7.5 Hz), 7.85 (1H, dd,
J=2.5, 8.5 Hz), 7.79 (2H, d, J=8.0 Hz), 7.77 (1H, d,
J=2.5 Hz), 7.5 (3H, m), 7.31 (2H, d, J=7.5 Hz), 6.96
(1H, d, J=8.5 Hz), 3.67 (3H, s), 2.45 (3H, s). Anal.
calcd for C20H18N2SO4: C, 62.81; H, 4.74; N, 7.33; S,
8.38. Found: C, 62.64; H, 4.66; N, 7.20; S, 8.33.
5.5.12. 4,40-Dimethoxy, 3-hydroxyazobenzene 3-O-p-to-
luenesulfonate (6e). The dye 5e was prepared from p-ani-
sidine and then methylated to give the product 6e using
the procedure described above. Purification by column
chromatography (chloroform) followed by recrystalliza-
tion from hexane and chloroform gave the product as
5.5.8. 4-Nitro[(4-methoxy, 3-hydroxyphenyl)azo]benzene
3-O-p-toluenesulfonate (6a). The dye 5a was prepared
from 4-nitroaniline and then methylated to give the
product 6a using the procedure described above.
Purification by column chromatography (chloroform)
followed by recrystallization from hexane and chloro-
form gave the final product as chunky dark red crystals.
yellow flaky crystals. (0.81 g, 70%): mp 127–129 ꢂC; H
1
NMR (CDCl3) 7.86 (2H, d, J=9.0 Hz), 7.81 (1H, dd,
J=2.1, 8.8 Hz), 7.80 (2H, d, J=8.1 Hz), 7.74 (1H, d,
J=2.1 Hz), 7.32 (2H, d, J=8.1 Hz), 7.04 (2H, d, J=9.0
Hz), 6.95 (1H, d, J=8.8 Hz), 3.90 (3H, s), 3.67 (3H, s),
2.47 (3H, s). Anal. calcd for C21H20N2SO5: C, 61.15; H,
4.89; N, 6.79; S, 7.77. Found: C, 60.93; H, 4.95; N, 6.83;
S, 7.68.
(0.67 g, 56%): mp 162 ꢂC; H NMR (CDCl3) 8.38 (2H,
1
d, J=9.3Hz), 7.98 (2H, d, J=9.3Hz), 7.94 (1H, dd,
J=2.5, 8.8 Hz), 7.86 (1H, d, J=2.5 Hz), 7.81 (2H, d,
J=8.6 Hz), 7.34 (2H, d, J=8.6 Hz), 7.01 (1H, d, J=8.8
Hz), 3.70 (3H, s), 2.48 (3H, s). Anal. calcd for
C20H17N3SO6: C, 56.20; H, 4.01; N, 9.83; S, 7.50.
Found: C, 56.11; H, 3.98; N, 9.81; S, 7.34.
5.5.13. 4-Phenoxysulfonylacetanilide (7). N-acetyl 4-
aminobenzenesulfonyl chloride (12.78 g, 55 mmol),
phenol (5.17 g, 56 mmol) and pyridine (12.5 mL) were
mixed together in a 100 mL conical flask and heated in a
water bath for 15 min with occasional stirring. Cold
5.5.9. 4-[(4-Methoxy, 3-hydroxyphenyl)azo]benzenesulfon-
amide 3-O-p-toluenesulfonate (6b). The dye 5b was
prepared from sulphanilamide and then methylated to

K. Bailey et al. / Bioorg. Med. Chem. 12 (2004) 595–601
601
water (100 mL) was added to the mixture and then
stirred until a precipitate formed. The precipitate was
extracted with chloroform and then washed with 1M
HCl to remove any dissolved pyridine. A second wash
with 2M NaOH solution removed any unreacted
phenol. Rotary evaporation of the dried (Na2SO4)
chloroform solution gave the crude product. Purifica-
tion by recrystallization from acetone and water gave
the finalꢂproduct as small brown crystals. (7.49 g, 47%):
size, 4.6ꢃ250 mm) in conjunction with a dual wave-
length UV–Vis detector. During the course of a
chromatography run, the detector was switched between
wavelengths depending on the elution time and lmax
values of the compounds being analyzed. For the assays
containing 1b and 1c as COMT substrates the detector
was set at 370nm for the duration of the HPLC run. For
the assay containing 1d as the COMT substrate the
detector was set to 450 nm for the first 10 min and then
set to 358 nm for the remainder of the run. For the
assay containing 1e as the COMT substrate the detector
was set to 450 nm for the first 8.5 min and then set to
363 nm for the remainder of the run.
1
mp 138 C; H NMR (CDCl3) 7.76 (2H, d, J=8.8 Hz),
7.67 (2H, d, J=8.8 Hz), 7.42 (1H, s), 7.28 (3H, m), 6.98
(2H, dd, J=1.7, 8.3Hz), 2.23(3H, s). Anal. calcd for
C14H13NSO4: C, 57.72; H, 4.50; N, 4.81; S, 11.00.
Found: C, 57.62; H, 4.67; N, 4.80; S, 10.88.
5.5.14. 4-Phenoxysulfonylaniline (8). A mixture of 4-
phenoxysulfonylacetanilide (5 g, 17 mmol) in 100 mL of
1M HCl was heated under reflux until the acetanilide
derivative dissolved. The product crystallized as the
solution cooled. (4.01 g, 94%): mp 134–135 ꢂC; 1H
NMR (CDCl3) 7.56 (2H, d, J=8.8 Hz), 7.27 (2H, m),
7.00 (3H, m), 6.64 (2H, d, J=8.8 Hz), 4.28 (2H, br s).
References and notes
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5.6. COMT assays
For all five catechol dyes the following incubation
mixture was used to determine the suitability of the dye
as a COMT substrate. The buffer used in the assay
consisted of 0.2 M NaH2PO4 and 5 mM MgCl2.6H2O
with the pH adjusted to 7.45. All of the incubation
mixtures were made up to a total volume of 60 mL and
consisted of 6 mL of 20 mM AdoMet in buffer solution,
48 mL of 125 Units/mL of COMT in buffer solution, 3
mL of a stock solution of catechol dye and 3mL of buffer
solution. For dyes 1a, 1b, 1d, and 1e the stock solution
solvent was made up of a 50/50 mixture of ethanol and
water to allow the dyes to completely dissolve. In the
case of 1c the dye was soluble in water. The final
concentrations in the assay mixture were AdoMet, 2
mM; COMT, 100 units; catechol dye, 0.150 mM The
mixtures were incubated for 1 h at 37ꢂC; the assay was
stopped by the addition of 40 mL of 4 M HClO4, then
diluted with 200 mL of buffer to give a final volume of
300 mL. A 30 mL aliquot of the assay mixture of the
assay was analyzed by HPLC.
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The assays containing 1a, 1d and 1e as the COMT
substrates were analyzed by HPLC using a mobile phase
of buffer (25 mM borate) and methanol (30:70 v/v)
adjusted to a pH of 8.5 with a flow rate of 0.75 mL/min.
The assays containing 1b and 1c as the substrates were
analyzed by HPLC using a mobile phase made up of
water, methanol and trifluoroacetic acid (TFA)
(30:70:0.2 v/v) with a flow rate of 0.75 mL/min.
All analytical HPLC chromatography was performed
using a Microsorb- MVTM Column (C18, 5 mm particle