Synthesis and in vitro evaluation of two progressive series of bifunctional polyhydroxybenzamide catechol-O-methyltransferase inhibitors

Article abstract of DOI:10.1021/jm9605187

Two progressive series of molecules with two polyhydroxybenzamide substructures were synthesized and tested as potential inhibitors of catechol-O-methyltransferase (COMT). These compounds were designed for the purpose of enhanced enzyme binding with dupli

Full text of DOI:10.1021/jm9605187

J . Med. Chem. 1997, 40, 2035-2039
2035
Syn th esis a n d in Vitr o Eva lu a tion of Tw o P r ogr essive Ser ies of Bifu n ction a l
P olyh yd r oxyben za m id e Ca tech ol-O-m eth yltr a n sfer a se In h ibitor s
Sharon E. Brevitt and Eng Wui Tan*
Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand
Received J uly 18, 1996X
Two progressive series of molecules with two polyhydroxybenzamide substructures were
synthesized and tested as potential inhibitors of catechol-O-methyltransferase (COMT). These
compounds were designed for the purpose of enhanced enzyme binding with duplicated
substructures separated by a linker section of various lengths. Our results show that potency
and mode of inhibition observed with the “bifunctional” compounds were a reflection of their
bifunctional nature. Furthermore, potency and mode of inhibition were dependent on the length
of the linker section. Of the assayed compounds, the optimum linker was found to be
diaminopropane. For example, N,N′-1,3-propanediylbis(3,4-dihydroxybenzamide) and N,N′-
1,3-propanediylbis(3,4,5-trihydroxybenzamide) demonstrated strong inhibitory action against
COMT, with apparent Ki values of 0.3 and 6.0 µM, respectively.
In tr od u ction
“bifunctional” nature, in that they have duplicated
substructures for enzyme binding.
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is
a magnesium dependent enzyme which catalyzes the
transfer of a methyl group from S-adenosylmethionine
(AdoMet) to endogenous and exogenous catechols,1,2
forming O-methylated products and S-adenosylhomocys-
teine (SAH).3 COMT is responsible for the extraneu-
ronal inactivation of catecholamines and the detoxifi-
cation of many xenobiotic catechols.4-7 Inhibitors of
COMT are of interest for preventing the inactivation of
exogenously administered catechols and, therefore, have
potential as drugs to combat disorders of catecholamine
metabolism such as Parkinson’s disease (PD). Orally
administered L-dopa (3-(3,4-dihydroxyphenyl)-L-ala-
nine), taken in conjunction with dopa decarboxylase
inhibitors8,9 (DCI’s) such as carbidopa and benserazide,
is currently used in the treatment of PD. The methy-
lation of L-dopa by COMT results in the formation of
the O-methylated metabolite 3-O-methyldopa (3-OMD).
When a DCI is used in conjunction with L-dopa, the level
of 3-OMD increases and it becomes the main circulating
metabolite in Parkinsonian patients.10,11 3-OMD ac-
cumulates in the tissues and plasma and competes with
L-dopa and other large neutral amino acids for the large
neutral amino acid transport system8,10,13,14 at the
blood-brain barrier. Inhibition of COMT would provide
a new therapeutic approach for increasing the bioavail-
ability of L-dopa especially when used in conjunction
with a DCI.15
D-Catechin (1), desmethylpapaverine (2) and nordi-
hydroguaiaretic acid (3) are first generation inhibi-
tors7,30 while 2,5-bis(3,4-dihydroxy-5-nitrobenzylidene)cy-
clopentanone (4) and OR-441 (5) are new generation
inhibitors of high potency.9,21,31 All five molecules have
two substituted catechols for potential enzyme binding,
but no correlation has been drawn between their
potency and bifunctional nature.
In order to investigate the contribution from a second
binding substructure, we synthesized two progressive
series of bifunctional compounds and examined their
inhibition characteristics. These compounds were
designed with dual substituted catechols for enzyme
binding separated by a linker section of various lengths.
The catechol derivatives were either 3,4-dihydroxy-
benzamide or 3,4,5-trihydroxybenzamide. These were
linked by a spacer section consisting of varying
numbers of methylene units. Although it is known that
electron-withdrawing substituents (e.g. NO2) potentiate
First generation inhibitors were either competitive
substrates for COMT16-18 or compounds that were
isosteric with the catechol ring.18,19 Some examples
were pyrogallol, tropolone, catechin, and gallic acid, all
of which had Ki’s in the 10-5 M range.2 They generally
lacked specificity of action, had low efficacy in vivo, and
were toxic.2,20,21 New generation inhibitors, especially
nitecapone,10,22-24 tolcapone,25 and entacapone,10,22,26,27
have been shown in clinical tests to be selective and
potent COMT inhibitors with IC50’s in the low nanomo-
lar range.1,28,29 Of the numerous COMT inhibitors that
have been reported in the literature, only five are of a
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00518-3 CCC: $14.00 © 1997 American Chemical Society

2036 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Brevitt and Tan
Sch em e 1
the methodology outlined above with n-propylamine in
place of diamine.
Biologica l Resu lts
The compounds synthesized were tested for in vitro
inhibitory activity with COMT from porcine liver (Sigma,
C4512) using methyl 3,4,5-trihydroxybenzoate (methyl
gallate) as the methyl group acceptor. The Km values
obtained for AdoMet and methyl gallate were 44.6 and
25.5 µM, respectively. These Km values are well within
the range of reported values for AdoMet and various
polyphenol substrates previously described in the lit-
erature.34 Table 1 shows the structure of the bifunc-
tional compounds tested and their inhibition constants
with respect to methyl gallate.
Within the 3,4-dihydroxybenzamide bifunctional se-
ries (compounds 6-9), the molecule with a spacer
section of three methylene units (7) was the only
inhibitor. It exhibited a competitive inhibition pattern
with a Ki of 0.3 µM, which is comparable to many
nitrocatechol inhibitors. This result shows that, in this
particular series, the length of linker between the
aromatic substructures is critical for interaction with
COMT. In comparison, compound 14, with only one 3,4-
dihydroxybenzamide moiety and a propyl substituent
on the amide nitrogen, was not an inhibitor of COMT
at concentrations up to 43.5 µM. The lack of activity
displayed by 14 toward COMT, with respect to methyl
3,4,5-trihydroxybenzoate, indicates that the observed
inhibition by compound 7 is due to its bifunctional
nature.
inhibition,9,21,31-34 it was decided that the effect of the
bifunctional nature of the inhibitors be determined
without the addition of enhancement substituents. The
results obtained show that maximum potency occurred
when the aromatic systems were separated by three
methylene spacer groups. Comparison with results
obtained with N-propyldi- and -trihydroxybenzamides
indicates that the bifunctional nature of these com-
pounds greatly affects their potency and mode of action.
Ch em istr y
All of the compounds tested in the 3,4,5-trihydroxy-
benzamide bifunctional series (compounds 10-13)
showed an uncompetitive inhibition pattern with respect
to methyl gallate. The most potent inhibitor in this
series was compound 11 with a spacer section of three
methylene units. The Ki measured was 6.6 µM. By
comparison, the N-propyl analogue 15 demonstrated a
purely competitive mode of inhibition with a Ki value
of 0.5 µM. Although 15 is a more potent inhibitor than
its bifunctional analogue 11 it has a distinctly different
mode of action.
Within the two families of bifunctional compounds
studied, the linker which confers the greatest inhibitory
activity is n-propyl. However, the two most potent
inhibitors from each group (compounds 7 and 11) exhibit
different modes of inhibition, presumably due to distinct
interactions from 3,4-dihydroxy- and 3,4,5-trihydroxy-
benzamides. Interestingly, the control molecules 14 and
15 interact differently with the active site of COMT than
their bifunctional analogues. Presumably, the second
tethered binding substructure alters the interaction of
the molecule with the enzyme. These results show that
duplication of a binding substructure within one mol-
ecule can have a profound effect on interaction with the
active site of COMT. Furthermore, the effect is greatly
affected by the nature of the linking structure.
The diamide compounds 6-13 were prepared by
standard procedures: by coupling 3,4-dimethoxybenzoic
acid or 3,4,5-trimethoxybenzoic acid with the appropri-
ate diamine followed by demethylation of the polyphe-
nols (Scheme 1). Carboxylic acid to amine coupling was
achieved via formation of the corresponding benzoyl
chloride. Thus, 3,4-dimethoxybenzoic acid and 3,4,5-
trimethoxybenzoic acid, both obtained commercially,
were reacted with phosphorous pentachloride in dichlo-
romethane to obtain the corresponding benzoyl chlo-
rides. Reaction with an appropriate amount of amine
or diamine gave the amides and diamides, respectively.
Initial attempts to synthesize the diamide compounds
using coupling reagents such as dicyclohexylcarbodiim-
ide (DCC),35-37 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihyd-
roquinoline (EEDQ),38 and ethyl chloroformate under
basic conditions,39 using both methoxy and acetyl pro-
tecting groups, were unsuccessful. Coupling reactions
with 1,4-diaminobutane using all of the methods de-
scribed above invariably gave only the monosubstituted
product. This result was inexplicable given that in all
the other cases diamide products were readily obtained
in high yields. Due to this complication, it was decided
to proceed without the butyl-linked bifunctional com-
pound in the series. Demethylation to give the hy-
droxylated derivatives 6-13 was achieved through the
use of the Lewis acid boron tribromide40,41 in dichlo-
romethane. An excess was used to allow for complex-
ation of boron tribromide to the ethers and amides.
Compounds 6-13 were collected from the reaction
mixture by filtration and purified by recrystallization
from methanol and water. N-Propyl-3,4-dihydroxyben-
zamide (14) and N-propyl-3,4,5-trihydroxybenzamide
(15) were synthesized for use as control molecules, using
Exp er im en ta l Section
COMT Assa y. The activity of COMT was measured by
determining the amount of methyl 3,5-dihydroxy-4-methoxy-
benzoate formed over time using HPLC with ultraviolet
detection. The standard incubation mixture was 60 µL
containing 2 mM AdoMet, 0.02-0.27 mM methyl 3,4,5-
trihydroxybenzoate, COMT (100 units, where one unit is
defined as the methylation of 1 nmol of protocatechuic acid

Polyhydroxybenzamide COMT Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2037
Ta ble 1. Structure, COMT Inhibitory Pattern, and Kinetic Inhibition Constants of the Compounds Synthesized
inhibition
kinetic inhibition
compd
R1
x
% yield
mp, °C
formula
C16H16N2O6
C17H18N2O6
anal.
pattern
constant, Ki/µM
6
7
8
H
H
H
H
OH
OH
OH
OH
2
3
5
6
2
3
5
6
27
45
57
34
16
26
58
28
236a
244
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
NAb
Cc
0.30 ( 0.013
210
C
19H22N2O6‚1/2H2O
NA
NA
UCd
UC
UC
UC
9
234a
230a
210a
210a
210a
C20H24N2O6
10
11
12
13
C16H16N2O8‚21/4H2O
C17H18N2O8‚4H2O
C19H22N2O8‚2H2O
C20H24N2O8‚21/2H2O
136 ( 10.5
6.6 ( 0.32
26.7 ( 1.6
35.0 ( 3.2
a
b
d
Decomposes. No inhibitory action. c Competitive mode of inhibition. Uncompetitive mode of inhibition.
Ta ble 2. Structure, Inhibition Pattern, and Kinetic Inhibition
Constants of the N-Propyl Derivatives
were recorded on a Biorad FTS-7 spectrometer using KBr
disks. 1H NMR and 13C NMR spectra were measured on a
Varian Gemini-200 MHz spectrometer. The δ values are
reported in ppm downfield from TMS. Column chromatograpy
was performed with silica gel (Sorbsil, particle size 32-63 µM)
and Dowex ion-exchange resin (counterion H, 100-200 mesh).
AdoMet as the p-toluenesulfonate salt, COMT, and DL-
dithiothreitol (DL-DTT) were purchased from Sigma. 3,4-
Dimethoxybenzoic acid, 3,4,5-trimethoxybenzoic acid, methyl
3,4,5-trihydroxybenzoate (methyl gallate), boron tribromide,
1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane,
1,5-diaminopentane, and 1,6-diaminohexane were purchased
from Aldrich. Solvent and inorganic bases were generally
sourced.
inhibition
pattern
kinetic inhibition
constant, Ki/µM
compd
R1
14
15
H
OH
NAa
Cb
0.5 ( 0.022
a
b
Gen er al P r ocedu r e for th e P r epar ation of N,N′-Bis(3,4-
d ih yd r oxyben za m id e) a n d N,N′-Bis(3,4,5-tr ih yd r oxyben -
za m id e) Der iva tives. 3,4-Dimethoxybenzoic acid or 3,4,5-
trimethoxybenzoic acid (recrystallized, 1.0-5.0 g scale) and
phosphorus pentachloride (1 molar equiv) were stirred in
dichloromethane (100 mL) overnight. The reaction mixture
was washed with a saturated solution of sodium hydrogen
carbonate, and the organic phase was dried and evaporated
under reduced pressure to afford the acid chloride. Freshly
prepared acid chloride (2.0-6.0 g scale) and potassium carbon-
ate (1.2 molar equiv) were dissolved in a mixture of water and
ethyl acetate (1:1, 70 mL total) and treated with the appropri-
ate diamine (0.5 molar equiv). The resulting biphasic solution
was stirred overnight. The solid N,N′-bis(3,4-dimethoxyben-
zamide) product was collected by filtration and dried. Boron
tribromide (10 molar equiv) was added to a cooled (0-5 °C)
and stirring solution of the appropriate N,N′-bis(3,4-dimethox-
ybenzamide) derivative (0.4-1.0 g scale) in CH2Cl2 under
anhydrous conditions. Stirring overnight at room temperature
resulted in the separation of a white solid. Filtration and
washing with copious quantities of CH2Cl2 afforded the deriva-
tive as a white solid.
N,N′-1,2-Eth a n ed iylbis(3,4-d ih yd r oxyben za m id e) (6).
The ethylene derivative 6 was prepared from 3,4-dimethoxy-
benzoic acid (2.0 g) and ethylenediamine using the procedure
described above. Recrystallization from MeOH/H2O gave the
desired product as fine white crystals (0.49 g, 27%): mp 235-
236 °C dec; IR 3500, 3438, 3376, 3145, 1573, 1559, 1521, 1391,
1327, 1194, 1124 cm-1; 1H NMR (CD3OD) δ 3.55 (4H, bs), 6.80
(2H, d, J ) 8.3 Hz), 7.23 (2H, dd, J ) 2.1, 8.3 Hz), 7.29 (2H,
d, J ) 2.1 Hz); 13C NMR (CD3OD) δ 41.00, 115.75, 115.88,
120.64, 126.92, 146.28, 150.18, 170.79.
No inhibitory action. Competitive mode of inhibition.
per hour at 37 °C using AdoMet as the methyl donor (Sigma)),
buffer23 (0.2 M NaH2PO4, 5 mM MgCl2‚6H2O, pH adjusted to
7.45 with NaOH). The mixture was incubated for 1 h at 37
°C, stopped by the addition of 20 µL of 4 M perchloric acid,
and then diluted with 200 µL of buffer. A 30 µL aliquot of the
assay mixture was injected onto a Microsorb-MV column (C18
,
5 µm particle size, 4.6 × 250 mm), and the components of the
mixture were eluted with a mobile phase which consisted of
buffer (50 mM NaH2PO4, 20 mM citric acid)-MeOH-THF (36:
20:1 v/v/v)23 at pH ) 2.0, at a flow rate of 0.8 mL/min.
Detection was at 270 nm, and the product had a retention time
of 21 min. The enzyme activity was calculated as nmol of
methyl 3,5-dihydroxy-4-methoxybenzoate/min/µg of protein.
Assays were performed in duplicate, and each assay had two
30 µL aliquots analyzed. Conversion of methyl 3,4,5-trihy-
droxybenzoate to methyl 3,5-dihydroxy-4-methoxybenzoate
was less than 5%. The concentrations of AdoMet and Mg2+
used were saturating, and product formation was linear with
respect to protein concentration and reaction time. Bifunc-
tional inhibitors were tested at concentrations up to 33.9 µM.
Compounds which exhibited inhibitory activity less than the
standard deviation at this concentration were classified as
having no inhibitory action (Table 1). The highest concentra-
tions of compounds 14 and 15 tested were 43.5 and 47.3 µM,
respectively.
An a lysis of Kin etic Da ta . 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 uncompetitive inhibi-
tion were calculated from data fitting to equations v ) Vmax[S]/
(Km(1 + [I]/Ki) + [S]) and v ) Vmax[S]/(Km + [S](1 + [I]/Ki)),
respectively. Standard deviations were obtained from fitting
of all available data for a given compound.
N,N′-1,3-P r op a n ed iylbis(3,4-d ih yd r oxyben za m id e) (7).
The propane derivative 7 was prepared from 3,4-dimethoxy-
benzoic acid (2.0 g) and 1,3-diaminopropane using the proce-
dure described above. Recrystallization from MeOH/H2O gave
the desired product as a white solid (0.86 g, 45%): mp 243-
244 °C; IR 3373, 3198, 1623, 1590, 1546, 1519, 1435, 1311,
Ch em ica l Meth od s. Melting points were recorded on a
Gallenkamp capillary melting point apparatus and are uncor-
rected. Elemental analyses were performed at the Campbell
Microanalytical laboratory, Otago University. Infrared spectra
1
1119 cm-1; H NMR (CD3OD) δ 1.84 (2H, m), 3.43 (4H, bm),
6.80 (2H, d, J ) 8.2 Hz), 7.22 (2H, dd, J ) 2.2, 8.2 Hz), 7.31

2038 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Brevitt and Tan
(2H, d, J ) 2.2 Hz); 13C NMR (CD3OD) δ 30.56, 38.17, 115.73,
116.00, 120.68, 127.02, 146.25, 150.11 170.48.
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N,N′-1,5-P en ta n ed iylbis(3,4-d ih yd r oxyben za m id e) Hy-
d r a te (8). The pentane derivative 8 was prepared from 3,4-
dimethoxybenzoic acid (5.0 g) and 1,5-diaminopentane using
the procedure described above. Recrystallization from MeOH/
H2O gave the desired product as a white powder (2.8 g, 57%):
mp 210-211 °C; IR 3532, 3376, 3118, 1575, 1515, 1455, 1319,
1117 cm-1; 1H NMR (CD3OD) δ 1.44 (2H, bm), 1.64 (4H, bm),
3.35 (4H, m), 6.78 (2H, d, J ) 8.2 Hz), 7.19 (2H, dd, J ) 2.2,
8.2 Hz), 7.28 (2H, J ) 2.2 Hz); 13C NMR (CD3OD) δ 25.38,
30.26, 40.77, 115.50, 115.91, 120.47, 126.58, 126.77, 146.58,
146.68, 150.71, 170.40.
N,N′-1,6-Hexa n ed iylbis(3,4-d ih yd r oxyben za m id e) (9).
The hexane derivative 9 was prepared from 3,4-dimethoxy-
benzoic acid (4.0 g) and 1,6-diaminohexane using the procedure
described above. Recrystallization from MeOH/H2O gave the
desired product as a white solid (1.4 g, 34%): mp 234-235 °C
dec; IR 3499, 3370, 3196, 1613, 1575, 1542, 1504, 1431, 1164,
1106 cm-1; 1H NMR (CD3OD) δ 1.43 (4H, bm), 1.62 (4H, bm),
3.35 (4H, bt), 6.79 (2H, d, J ) 8.2 Hz), 7.19 (2H, dd, J ) 2.1,
8.2 Hz), 7.27 (2H, d, J ) 2.1 Hz); 13C NMR (CD3OD) δ 27.72,
30.50, 40.81, 115.71, 115.82, 120.48, 127.29, 146.25, 149.98.
N,N′-1,2-Eth an ediylbis(3,4,5-tr ih ydr oxyben zam ide) Hy-
d r a te (10). The ethylene derivative 10 was prepared from
3,4,5-trimethoxybenzoic acid (2.0 g) and ethylenediamine using
the procedure described above. Recrystallization from MeOH/
H2O gave the desired product as a white solid (0.30 g, 16%):
mp 230-231 °C; IR 3509, 3322, 1581, 1531, 1452, 1365, 1032
cm-1; 1H NMR (CD3OD) δ 3.49 (4H, m), 6.98 (4H, s), 8.16 (2H,
bs); 13C NMR (CD3OD) δ 39.04, 105.48, 123.95, 135.13, 144.20,
166.58.
N,N′-1,3-P r op a n ed iylb is(3,4,5-t r ih yd r oxyb en za m id e)
Hyd r a te (11). The propane derivative 11 was prepared from
3,4,5-trimethoxybenzoic acid (2.0 g) and 1,3-diaminopropane
using the procedure described above. Recrystallization from
H2O (pH ) 1) gave the desired as an off-white solid (0.50 g,
26%): mp 210-211 °C dec; IR 1594, 1523, 1308, 1179, 1041
1
cm-1; H NMR (CD3OD) δ 1.76 (2H, bm), 3.39 (4H, m), 7.01
(4H, s), 8.13 (2H, bt); 13C NMR (CD3OD) δ 12.05, 34.60, 105.01,
123.75, 134.84, 144.04, 165.56.
N,N′-1,5-P en t a n ed iylb is(3,4,5-t r ih yd r oxyb en za m id e)
Hyd r a te (12). The pentane derivative 12 was prepared from
3,4,5-trimethoxybenzoic acid (2.0 g) and 1,5-diaminopentane
using the procedure described above. Recrystallization from
MeOH/H2O gave the desired product as a white solid (1.1 g,
58%): mp 210-211 °C dec; IR 1584, 1530, 1440, 1337, 1239,
1215 cm-1; 1H NMR (CD3OD) δ 1.40 (2H, bm), 1.61 (4H, bm),
3.29 (4H, m), 6.97 (4H, s), 7.66 (2H, bt); 13C NMR (CD3OD) δ
23.25, 28.08, 38.61, 105.74, 124.63, 135.04, 144.25, 166.52.
N,N′-1,6-Hexan ediylbis(3,4,5-tr ih ydr oxyben zam ide) Hy-
d r a te (13). The hexane derivative 13 was prepared from
3,4,5-trimethoxybenzoic acid (2.0 g) and 1,6-diaminohexane
using the procedure described above. Recrystallization from
MeOH/H2O gave the desired product as a white solid (0.57 g,
28%): mp 210-211 °C dec; IR 1582, 1538, 1445, 1348, 1041
1
cm-1; H NMR (CD3OD) δ 1.37 (4H, bm), 1.57 (4H, bm), 3.31
(4H, m), 6.98 (4H, s), 7.91 (2H, t, J ) 5.50 Hz); 13C NMR
(CD3OD) δ 24.85, 27.84, 37.81, 105.18, 124.15, 134.74, 144.01,
165.69.
Ack n ow led gm en t. This work was supported by a
grant from the University of Otago Science Division.
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