Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02566166 2006-10-30
=
1
IMPROVED FLAVONOLS
FIELD OF THE INVENTION
The present invention relates generally to flavonol compounds with improved
properties
that may be useful in the treatment of conditions that require an anti-oxidant
effect such as
cardiovascular disease, compositions containing the compounds, methods of
treatment of
such conditions using the compounds, and the use of these compounds in the
preparation
of medicaments for the treatment of conditions of this type. In another
embodiment the
I.() invention relates to a method of reducing the vasodilatory effect of a
flavonol compound
whilst substantially maintaining or enhancing its anti-oxidant activity.
BACKGROUND OF THE INVENTION
There are a large number of conditions in which anti-oxidant activity has been
implicated
as being useful in the development of treatment regimes. These include acute
conditions
such as myocardial ischaemia, stroke, cardiac surgery (e.g. coronary bypass
surgery),
and chronic conditions such as diabetes, atherosclerosis and hypertension.
Notwithstanding the prevalence of disorders of this type there is still the
need to develop
new drugs that can be used in the treatment of these conditions.
For example one of the best known of the conditions of this type,
cardiovascular disease
(CVD), is currently the leading cause of mortality worldwide in adults aged 60
years and
above. While there are various types of CVD, the two most common causes of
fatality are
coronary heart disease and stroke. In 2002, the total number of deaths from
CVD globally
amounted to 16.7 million, of which approximately 7 million resulted from
coronary heart
disease and a further 6 million from stroke. In Australia, CVD is also the
leading cause of
death where 38% of all deaths in 2002 were a result of CVD. In addition, CVD
causes
long-term disability in 1.10 million Australians. Consequently, CVD represents
a heavy
economic burden with the direct costs of the disease estimated to be
approximately $5.4
billion in 2000-1 in Australia, and $286 billion in 1999 in the United States
of America, and
it is predicted that this figure will continue to rise due to the aging
population. Although
CVD has long been thought to be a disease predominantly occurring in developed
countries, it has become increasingly clear that it is also emerging in third-
world countries
and is already the leading cause of mortality in some regions of the
developing world. As
such, there is an urgent need to develop novel agents for the treatment or
prevention of
CVD.
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The underlying cause of CVD is atherosclerosis, which is the development of
fatty
deposits on normally-smooth blood vessels, which start to form in people from
a very
young age. There are a number of risk factors such as obesity, high blood
cholesterol
and high blood pressure that predispose individuals to the formation of these
fatty
deposits, which in turn places them in a high risk category for CVD. As the
fatty deposits
continue to develop, the vessel narrows and the wall thickens, hardens and
loses
elasticity. Blood flow through these vessels is disturbed resulting in
platelet activation,
causing the formation of a thrombus at the site of the lesion, which occludes
the vessel.
When this occurs in the heart or brain, ischaemic heart disease or stroke,
respectively,
result.
Oxygen supply may be restored after ischaemic injury, by dislodging or
dissolving the
thrombus. However, paradoxically, restoration of the oxygen supply can lead to
a
worsened secondary condition known as reperfusion injury. The reintroduction
of oxygen
causes the production of reactive oxygen species (ROS), which exacerbates and
accelerates the injury already produced by the ischaemia. ROS include free
radicals that
have an unpaired electron, such as 02-' and HO', as well as other reactive
species such
as H202. HO' is particularly reactive and reacts indiscriminately with
membrane lipids,
proteins and DNA, degrading them and causing cellular damage. There are
various
sources of ROS, including nitric oxide synthase, myeloperoxidase, superoxide
dismutase,
mitochondria; electron transport, metabolism of arachidonic acid by
cyclooxygenase and
xanthine oxidase. One of the pathways for production of ROS is shown below.
Fe" Fe"
oxidative enzymes superoxide _
=
02 ___________________ P, 02 -11" H2O2 -
HO + OH
(eg. myeloperoxidase) dismutase Fenton reaction
A diet high in cholesterol has long been known to be associated with CVD.
Epidemiological studies have indicated that the French population has a lower
than
predicted incidence of CVD given their comparatively high fat diet. This
anomaly is known
as "The French Paradox" (9-11). With other risk factors for CVD, such as
smoking and
obesity, comparable to other Western populations, it has been suggested that
the regular
consumption of red wine in the French diet holds the key to cardio-protection.
Later
studies have suggested that it is a non-alcoholic component of wine, the
flavonoids, that
contribute to protective effects in the cardiovascular system. In other
studies, flavonoids
have been found to possess many beneficial properties, such as anti-
inflammatory, anti-
allergic, anti-viral, anti-thrombotic and anti-carcinogenic effects.
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Flavonoids are polyphenolic compounds with many subclasses, and previous
studies
have shown a group of compounds called flavonols to be of particular interest
for
treatment of and protection against CVD. Flavonols possess 3 rings, with a
hydroxyl
group in the 3-position of the C ring. They are found in a large variety of
plant materials,
such as fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, tea
and red wine,
and have been consumed by humans since prehistoric times, suggesting that they
are
unlikely to possess significant adverse effects. Some structure-activity
relationship
studies have been performed to identify substituents on the flavonol ring
system that are
important for vasorelaxant and antioxidant activity. It has been found that
the 3-0H group
of the C ring was essential for endothelium-dependent vasorelaxant activity
and additional
hydroxyl groups at the 3' and 4' positions of the B ring further improves
biological activity.
For antioxidant activity, the 3-0H of the C ring, attached to the C2-C3 double
bond, which
is in conjugation with the 4-oxo group of the C ring, together with either a
4'-hydroxy or a
3',4'-catechol moiety on the B ring were shown to be important. Thus, the most
potent
flavonol for antioxidant and vasorelaxant activities described to date is
3',4'-
dihydroxyflavonol (diOHF).
5'
4'
8 B
3'
7
I A CI3 2'
6
OH
General structure of flavonols.
There is significant potential for flavonols or flavonol analogues to be
useful in the
treatment of conditions which can be treated by anti-oxidants due to the
strong anti-
oxidant activity demonstrated by compounds of this general structural type.
Unfortunately,
however, there are a number of problems encountered for compounds of this
general
structural type that lead to a reduction in their ability to be used in this
way. For example
one undesirable property of compounds of this type is that they are generally
insoluble in
water making their use as drugs impractical. In addition many of these
compounds
display multiple biological activities, which in many instances is undesirable
and limits
their broad spectrum use. For example many of the flavonols display both anti-
oxidant
and vasodilatory activity. This is generally undesirable as it is preferable
to be able to
administer a drug with a single activity in order to limit the possible
adverse side effects.
In relation to flavonols which have both anti-oxidant and vasodilatory
activity there are a
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number of instances in which such dual activity is undesirable. If the anti-
oxidant activity
is the desired end result then vasodilatory activity may lead to adverse side
effects such
as hypotension (excessively low blood pressure), postural hypotension
(dizziness and
possible collapse when moving from lying to standing), tachycardia (an
excessively high
heart rate to try to compensate for the low blood pressure) and arrhythmias.
As such the
fact that flavonols have both properties is undesirable. It would therefore be
desirable to
develop flavonols with improved specificity as current flavonols do not
possess any
selectivity of note.
It would therefore be desirable to overcome or ameliorate one or more of the
observed
problems with the flavonol compounds as discussed above.
The present invention is based on the finding by the present applicants that
modification
of flavonols or flavonol compounds in certain pre-defined ways leads to
improvements in
the functional performance of the compounds and addresses one or more of the
deficiencies identified above.
SUMMARY OF THE INVENTION
In a first aspect the present invention provides a compound of formula (I):
R1 (X)m
p 8 \ 2
(Y)p.-1
OR
0
Formula (I)
wherein
R is selected from the group consisting of H, alkyl, alkenyl, alkynyl,
heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl and acyl, each of which may be
optionally
substituted;
R1 is an organic moiety that is capable of being converted into a charged
group;
CA 02566166 2006-10-30
each X and Y is independently selected from the group consisting of H,
halogen, -
CN, -NO2, -CF3, -0CF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl,
heteroalkyl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,
heteroaryl,
cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl,
heteroarylalkyl, arylalkenyl,
5 cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl,
heteroarylheteroalkyl,
hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy,
alkynyloxy,
cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy,
phenoxy,
benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino,
sulfinylamino, -COOH, -COR2, -COOR2, -CONHR2, -NHCOR2, -NHCOOR2, -NHCONHR2,
C(=NOH)R2, alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl,
alkylsulfinyl,
arylsulfonyl, arylsulfinyl, aminosulfonyl, SR2 and acyl, each of which may be
optionally
substituted;
each R2 is independently selected from the group consisting of H, alkyl,
alkenyl,
alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
heteroaryl, cycloalkylalkyl,
heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, and acyl, each of which may
be optionally
substituted;
m is an integer selected from the group consisting of 0, 1, 2, 3, 4 and 5;
p is an integer selected from the group consisting of 0, 1, 2 and 3;
or a pharmaceutically acceptable salt or prodrug thereof.
In one embodiment of the compounds of the invention R is H.
In one embodiment of the compounds of the invention m is selected from the
group
consisting of 0, 1 and 2.
In one embodiment of the compounds of the invention X is OH.
In one embodiment of the invention R1 is an ionisable group. In one form of
this
embodiment R1 is selected from the group consisting of:
-L-CO2H, -L-S03H, -L-S02H, -L-P03H and -LCONH2OH;
wherein L is a linking moiety containing from 1 to 20 atoms in the normal
chain, more
preferably from 1 to 10 atoms in the normal chain, most preferably from 1 to 4
atoms in
the normal chain.
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In one embodiment R1 is a group of formula -L-CO2H. In one specific embodiment
R1 is a
group of formula ¨NHCO(CH2)2CO2H. In another specific embodiment R1 is a group
of
the formula ¨OCH2CO2H.
In one embodiment of the compounds of the invention Y = H and p = 3.
Specific examples of compounds of the invention are selected from the group
consisting
of:
= 11101
9
0
I
OH
0 0
,OH
0
HO 0
I
OH
0 0
0 OH
* OH
0
OH
0 0
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7
OH
0
H0)() OH
OH
OH 0
In a further aspect the invention provides pharmaceutical compositions
including a
compound of the invention as described above and a pharmaceutically acceptable
carrier,
diluent or excipient.
In an even further aspect the invention provides a method of treatment of a
condition that
ro may be treated by administration of an anti-oxidant, the method
including administration of
a therapeutically effective amount of a compound of the invention.
In an even further aspect the invention provides the use of a compound of the
invention in
the preparation of a medicament for the treatment or prophylaxis of a
condition that can
be treated by administration of an anti-oxidant.
In an even further aspect the invention provides a method of reducing the
vasodilatory
activity of a compound of the formula (II) whilst substantially conserving or
enhancing the
antioxidant activity of the compound, the method including the step of
converting the
compound of formula (II)
---i--(X)m
0
410
OR
0
Formula (II)
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wherein
R is selected from the group consisting of H, alkyl, alkenyl, alkynyl,
heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl and acyl, each of which may be
optionally
substituted;
each X and Y is independently selected from the group consisting of H,
halogen, -
CN, -NO2, -CF3, -0CF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl,
heteroalkyl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,
heteroaryl,
cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl,
arylalkenyl,
cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl,
heteroarylheteroalkyl,
hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy,
alkynyloxy,
cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy,
phenoxy,
benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino,
sulfinylamino, -COOH, -COR2, -COOR2, -CONHR2, -NHCOR2, -NHCOOR2, -NHCONHR2,
C(=NOH)R2, alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl,
alkylsulfinyl,
arylsulfonyl, arylsulfinyl, aminosulfonyl, SR2 and acyl, each of which may be
optionally
substituted;
each R2 is independently selected from the group consisting of H, alkyl,
alkenyl,
alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
heteroaryl, cycloalkylalkyl,
heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, and acyl, each of which may
be optionally
substituted;
m is an integer selected from the group consisting of 0, 1, 2, 3, 4 and 5;
or a pharmaceutically acceptable salt or prodrug thereof
into a compound of formula (HI)
¨T--(X)m
1
(RN
OR
Y 0
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wherein R3 is selected from the group consisting of halogen, -CN, -NO2, -CF3, -
OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl,
cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl,
heterocycloalkylalkyl,
arylalkyl, heteroarylalkyl, arylalkenyl, cycloalkylheteroalkyl,
arylheteroalkyl,
heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl,
alkoxy,
alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy,
heterocycloalkyloxy,
aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino,
aminoalkyl,
acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR2, -COOR2, -
CONHR2,
-NHCOR2, -NHCOOR2, -NHCONHR2, C(=NOH)R2, alkoxycarbonyl, alkylaminocarbonyl,
sulfonyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsuffinyl,
aminosulfonyl, SR2 and acyl,
each of which may be optionally substituted,
or R3 is an organic moiety capable of being converted into a charged group;
and q is 1 or 2.
In one embodiment of the method the compound of formula (II) is converted into
a
compound of formula (111a)
--,--(X)m
R3 10 0
OR
0
Formula (111a).
In another embodiment of the method the compound of formula (II) is converted
into a
compound of formula (111b)
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-7¨(X)11,
1
R3 0
OR
0
Formula (111b).
In another embodiment of the method the compound is converted into a compound
of
5 formula (111c)
1
--T--(X)m
R3 At 0
R3
OR
0
Formula (111c).
In one embodiment of the method of the invention R is H.
In one embodiment of the methods of the invention m is selected from the group
consisting of 0, 1 and 2.
In one embodiment of the methods of the invention X is OH.
In one embodiment of the methods of the invention Y is H or OH.
In one embodiment of the method R3 is an ionisable group. In a further
embodiment R3 is
selected from the group consisting of:
-L-CO2H, -L-SO3H, -L-S02H, -L-P03H and -LCONH2OH;
wherein L is a linking moiety containing from 1 to 20 atoms in the normal
chain, more
preferably from 1 to 10 atoms in the normal chain, most preferably from 1 to 4
atoms in
the normal chain. In one embodiment of the method R3 is a group of formula -L-
CO2H. In
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= 11
another specific embodiment R3 is a group of formula ¨NHCO(CH2)2CO2H. In
another specific
embodiment R1 is a group of the formula ¨OCH2CO2H.
In yet an even further aspect the invention includes a method of achieving an
anti-oxidant effect
in a subject without eliciting a vasodilatory effect in the subject, the
method including
administering an effective amount of a compound of the invention to the
subject:
In accordance with an aspect of the present invention there is provided a
compound of the
formula (I):
17 i\23
HO 6 4
OR
z H
0
wherein, R is H; X is OH; m is an integer selected from the group consisting
of 0, 1, 2, 3, 4 and
5; z is an integer selected from 0, 1, 2 and 3; or a pharmaceutically
acceptable salt thereof
In a further aspect of the present invention there is provided a compound
selected from the group
consisting of:
00
HOL
OH
0 0
CA 02566166 2013-05-31
1 1 a
OH
0
0
OH
0 0
OH
o 0
HO
OH
0 0
In another aspect of the invention there is provided the use of an effective
amount of a compound
of formula (I) for achieving an anti-oxidant effect in a subject without
eliciting a vasodilatory
effect in the subject,
_______________________________________________________ oo,õ
0
17 21
HO 6 4 3
OR
0
wherein, R is H; X is OH; m is an integer selected from the group consisting
of 0, 1, 2, 3, 4 and
5; z is an integer selected from 0, 1, 2 and 3; or a pharmaceutically
acceptable salt thereof.
CA 02566166 2012-02-23
lib
DETAILED DESCRIPTION OF THE INVENTION
In this specification a number of terms are used which are well known to a
skilled
addressee. Nevertheless for the purposes of clarity a number of terms will be
defined.
As used herein, the term unsubstituted means that there is no substituent or
that the only
substituents are hydrogen.
The term "optionally substituted" as used throughout the specification denotes
that the
group may or may not be further substituted or fused (so as to form a
condensed
polycyclic system), with one or more substituent groups. Preferably the
substituent
groups are one or more groups independently selected from the group consisting
of
halogen, =0, =S, -CN, -NO2, -CF3, -0CF3, alkyl, alkenyl, alkynyl, haloalkyl,
haloalkenyl,
haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl,
aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl,
arylalkyl,
cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl,
heteroarylalkenyl,
cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl,
heteroarylheteroalkyl,
hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl,
alkoxyheterocycloalkyl,
alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy,
alkynyloxy,
cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy,
aryloxy,
phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl,
cycloalkylalkyl,
heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl,
arylamino,
sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl,
aminosulfonyl, sulfinyl,
alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -COOH, -COR2, -C(0)0R2, -
CONHR2, -
NHCOR2, -NHCOOR2, NHCONHR2, C(=NOH)R2, -SH, -SR2, -0R2, acyl, a group of
formula
¨N(R2)2 or ¨CON(R2)2 or a group of formula ¨NHCON(R2)2
"Alkyl" as a group or part of a group refers to a straight or branched
aliphatic hydrocarbon
group, preferably a C1¨C14 alkyl, more preferably C1-C10 alkyl, most
preferably C1-C6
unless otherwise noted. Examples of suitable straight and branched C1-C6 alkyl
substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-
butyl, hexyl, and
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the like. When alkyl is used as a bridging group it is typically (but not
exclusively) referred
to as alkylene. A similar convention applies to other bridging groups.
"Acyl" means an alkyl-CO- group in which the alkyl group is as described
herein.
Examples of acyl include acetyl and benzoyl. The alkyl group is preferably a
C1-C6 alkyl
group.
"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group
containing
at least one carbon-carbon double bond and which may be straight or branched
preferably
having 2-14 carbon atoms, more preferably 2-12 carbon atoms, most preferably 2-
6
carbon atoms, in the normal chain. The group may contain a plurality of double
bonds in
the normal chain and the orientation about each is independently E or Z.
Exemplary
alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl,
pentenyl,
hexenyl, heptenyl, octenyl and nonenyl.
"Alkoxy" refers to an ¨0-alkyl group in which alkyl is defined herein.
Preferably the alkoxy
is a C1-C6alkoxy. Examples include, but are not limited to, methoxy and
ethoxy.
"Alkynyr as a group or part of a group means an aliphatic hydrocarbon group
containing a
carbon-carbon triple bond and which may be straight or branched preferably
having from
2-14 carbon atoms, more preferably 2-12 carbon atoms, more preferably 2-6
carbon
atoms in the normal chain. Exemplary structures include, but are not limited
to, ethynyl
and propynyl.
"Cycloalkyl" refers to a saturated or partially saturated, monocyclic or fused
or Spiro
polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring,
such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless
otherwise specified. It
includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic
systems such
as decalin, and polycyclic systems such as adamantane.
"Heterocycloalkyl" refers to a saturated or partially saturated monocyclic,
bicyclic, or
polycyclic ring containing at least one heteroatom selected from nitrogen,
sulfur, oxygen,
preferably from 1 to 3 heteroatoms in at least one ring. Each ring is
preferably from 3 to 10
membered, more preferably 4 to 7 membered. Examples of suitable
heterocycloalkyl
substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl,
piperidyl, piperazyl,
tetrahydropyranyl, morpholino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane,
and
1,4-oxathiapane.
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13
"Heteroalkyl" refers to a straight- or branched-chain alkyl group preferably
having from 2
to 14 carbons, more preferably 2 to 10 atoms in the chain, one or more of
which is a
heteroatom selected from S, 0, and N. Exemplary heteroalkyls include alkyl
ethers,
secondary and tertiary alkyl amines, alkyl sulfides, and the like.
"Aryl" as a group or part of a group denotes (i) an optionally substituted
monocyclic, or
fused polycyclic, aromatic carbocycle (ring structure having ring atoms that
are all carbon)
preferably having from 5 to 12 atoms per ring. Examples of aryl groups include
phenyl,
"Heteroaryl" either alone or part of a group refers to groups containing an
aromatic ring
25 thienyl.
The term "therapeutically effective amount" or "effective amount" is an amount
sufficient to
effect beneficial or desired clinical results. An effective amount can be
administered in
one or more administrations. An effective amount is typically sufficient to
palliate,
THE COMPOUNDS OF THE INVENTION
The compounds of the invention were developed with a view to probing ways of
increasing the water solubility (and hence bioavailability) of the flavonols
or flavonol
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14
Studies that have been carried out on structure activity relationships of
flavonols and
flavonol analogues suggest that the important portions of the molecule for
anti-oxidant
activity were the substitution pattern on the B and C rings. As such initial
studies into the
area were focussed on elaboration of the A ring or the A ring substituents.
Whilst there
are a vast number of chemical modifications that could be made there was some
concern
that if the elaboration was too extreme it would lead to a loss of activity.
As such in
making the desired modifications an attempt was made to make the modifications
as
simple as possible. As a model system for the flavonols and analogs thereof a
simple
member of this series was chosen along with its analogs namely compound A.
0
10 I OH
0
Compound A.
It was also appreciated that the presence of reactive oxygen species (ROS)
generated in
mitochondria are believed to play a significant role in a number of common
pathological
states including diabetes and ischaemia-reperfusion injury.
The present inventors have found that the incorporation of an organic moiety
containing a
group capable of being converted into a charged group significantly increases
the water
solubility of the molecules of this type. There are a number of organic
moieties that meet
this criteria as would be well known in the art. There are a number of
possible species
that can be incorporated that are able to be converted into a charged species.
A preferred
example of such a species are basic nitrogen containing moieties.
In an alternative embodiment the group R1 may be an ionisable group such that
the group,
under basic conditions, can be converted (ionised) into a negatively charged
species.
Alternatively, the group may be converted under acidic conditions into a
positively charged
species. Once again there are a number of possible moieties that would fit
either of these
[
CA 02566166 2006-10-30
descriptions with organic acids and the like being preferred examples of the
first type and
amine compounds being representative examples of the second.
A number of well known ionisable groups are well known in the art but it is
preferred that
-L-CO2H, -L-S03H, -L-S02H, -L-P03H and -LCONH2OH;
wherein L is a linking moiety containing from 1 to 20 atoms in the normal
chain. The
length of the linking moiety may be varied but it preferably has from 1 to 10
atoms in the
normal chain, more preferably from 1 to 4 atoms in the normal chain. The atoms
in the
10 chain may be only carbon atoms or the normal chain may also contain one or
more
heteroatoms.
It has been found that it is preferred that R1 is a group of formula -L-0O2H1
most
preferably a group of formula ¨NHCO(CH2)2CO2H
Following this general approach the following compounds were identified.
0 0
I
OH
0 0
Compound B
OH
0
9
HO
1
OH
0 0
Compound C
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16
OH
0 OH
HO 0
140 I
OH
0 0
Compound D.
It was found that compounds B, C and D had improved solubility in comparison
with the
unmodified moiety. Whilst flavonols are water insoluble, it was found that the
succinamic
acid flavonols readily dissolved in 0.1 M Na2CO3 solution. The maximum
concentrations
attained were 10'1 M for compounds B and C and 10.2 M for compound D. The
results
demonstrate that the incorporation of a carboxylic acid moiety of this type
can significantly
improve the drug delivery properties of the flavonol type compounds.
MODULATION OF VASODILATORY ACTIVITY
The compounds synthesized were tested in order to determine their anti-oxidant
and
vasodilatory activity. These studies demonstrated that the modification
proposed above
whilst not having any significant effect on anti-oxidant activity lead to a
reduction in
vasodilatory activity.
VASODILATORY ACTIVITY
Vascular activity can be tested in standard organ bath assays using rat
isolated thoracic
aorta. The synthesized flavonols were first assayed for their ability to
inhibit contractions
induced by phenylephrine (PE), since it has been previously shown that
flavonols can act
as functional antagonists of PE. Next, the efficacy of the various flavonols
would be
determined in a direct relaxation assay. The vascular activities of the
synthesized
compounds were compared to diOHF since this is the most potent flavonol
described to
date. It was expected that the flavonols should reduce the magnitude of PE-
induced
contractions in endothelium-intact aortic rings, thus reducing the maximum
response
(Rm.) for PE-induced contractions. Figure 1 shows the results from these two
assays.
As can be seen in Figure 1a, the succinamic acid-substituted flavonols B, C
and D were
less effective than diOHF at inhibiting PE-induced contractions. In the case
of D, which
bears a catechol moiety like diOHF, vascular activity was completely
abolished. This
CA 02566166 2006-10-30
17
correlates with the results from the relaxation assay (Figure 1 b), where two
of the
synthesized compounds B and C showed weak vasorelaxation activity, and slight
contractions were observed at high concentrations of D. The pEC50 value for
diOHF was
found to be 5.33 0.07 (n = 4) and pEC50 values for the various flavonols
were not
determined as the data did not fit a sigmoidal curve. The maximum relaxation
of the
various flavonols is shown in Table 1, and are significantly different from
each other (p <
0.001).
Interestingly, compounds B and C, which possessed fewer hydroxyl groups on the
B ring,
exhibited some vascular activity, albeit less than diOHF, indicating that the
introduction of
the succinamic acid side chain does not always completely ablate vascular
activity. While
the mechanism by which flavonols exert their vascular activity remains
controversial, there
have been several suggestions as to its mode of activity, such as interference
with second
messenger proteins, protein kinase C and cAMP-phosphodiesterase, as well as
inhibiting
influx of extracellular calcium.
Table 1: Maximum response by the flavonols in the relaxation assay.
Compound n Rmax (%)
6 47 2a'b
4 80 5"
4 -25 3a
diOHF 4 105 2
asignificant difference to diOHF;
bsignificant difference to D (p <0.001, Newman-Keuls test).
Since the vascular activity of the synthesized flavonols was attenuated, it
was of great
interest to test these compounds for antioxidant activity. Antioxidant
activity can be
assessed in a tissue-based lucigenin-enhanced chemiluminescence assay.
Isolated rat
aortic rings were incubated with NADPH and the various flavonols. NADPH, a
substrate
for NADPH oxidase in the vasculature, increases superoxide production.
Superoxide that
is produced reacts with lucigenin, leading to the emission of photons, which
can be
quantified to give a measure of superoxide levels. If the flavonols possess
antioxidant
activity, superoxide levels will be reduced, resulting in a decrease in photon
emission.
Again, in this assay, the level of antioxidant activity of the various
flavonols was compared
with diOHF, one of the most potent flavonol antioxidants identified to date,
and results are
shown in Figure 2.
CA 02566166 2006-10-30
18
As can be seen, compounds B and D possess moderate to good antioxidant
activity
whereas compound C had little effect. The activity of D is most promising,
where
superoxide levels were substantially reduced, approaching the activity of
diOHF. This
correlates with previous studies that have demonstrated that a catechol moiety
on the B
ring improves antioxidant activity. Thus, while modifications at the 6-
position of the A ring
showed vascular activity to be attenuated, antioxidant activity was shown to
be largely
retained especially in circumstances where there was a catechol moiety on the
B ring.
As the results seem to indicate that there was the ability to retain the anti-
oxidant activity
of the flavonol whilst at the same time mediating the vasodilatory effect it
has thus been
to
shown that there is a mechanism by which a flavonol may be modified to both
improve its
solubility and to attenuate the vasodilatory activity without having a
negative impact on the
anti-oxidant activity.
THE THERAPEUTIC APPROACH
The compounds of the present invention can therefore be administered in any
circumstance where it is desired to provide an anti-oxidant effect without a
vasodilatory
effect. As a general principle it is almost always desirable to administer a
compound that
has a single activity as it minimises the possibility of adverse side effects
being
encountered and at the same time allows the clinician to administer the
desired dosage of
the drug focussing solely on the desired outcome without having to be
concerned by the
possible negative implications of the treatment regimen. The present invention
therefore
provides the ability to achieve an anti-oxidant effect in a subject without
eliciting a
vasodilatory effect in the subject.
ADMINISTRATION OF COMPOUNDS
Administration of compounds within Formula I to humans can be by any of the
accepted
modes of administration well known in the art. For example they may be
administered by
enteral administration such as oral or rectal, or by parenteral administration
such as
subcutaneous, intramuscular, intravenous and intradermal routes. Injection can
be bolus
or via constant or intermittent infusion. The active compound is typically
included in a
pharmaceutically acceptable carrier or diluent and in an amount sufficient to
deliver to the
subject a therapeutically effective dose.
It is anticipated that the compounds of the invention will be useful in
treating a wide variety
of disorders that are amenable to treatment with anti-oxidants. These include
acute
conditions such as myocardial ischaemia, stroke, cardiac surgery (e.g.
coronary bypass
surgery), and chronic conditions such as diabetes, atherosclerosis and
hypertension.
õ
CA 02566166 2006-10-30
19
In using the compounds of the invention they can be administered in any form
or mode
which makes the complex bio-available. One skilled in the art of preparing
formulations
can readily select the proper form and mode of administration depending upon
the
particular characteristics of the compound selected, the condition to be
treated, the stage
of the condition to be treated and other relevant circumstances. We refer the
reader to
Remingtons Pharmaceutical Sciences, 19th edition, Mach Publishing Co. (1995)
for further
information.
The compounds of the present invention can be administered alone or in the
form of a
pharmaceutical composition in combination with a pharmaceutically acceptable
carrier,
diluent or excipient.
The compounds are, however, typically used in the form of pharmaceutical
compositions
which are formulated depending on the desired mode of administration. As such,
in a
further embodiment the present invention provides a pharmaceutical composition
including a compound of Formula (I) and a pharmaceutically acceptable carrier,
diluent or
excipient. The compositions are prepared in manners well known in the art.
The invention in other embodiments provides a pharmaceutical pack or kit
comprising one
or more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. In such a pack or kit can be found a container
having a unit
dosage of the agent (s). The kits can include a composition comprising an
effective agent
either as concentrates (including lyophilized compositions), which can be
diluted further
prior to use or they can be provided at the concentration of use, where the
vials may
include one or more dosages. Conveniently, in the kits, single dosages can be
provided
in sterile vials so that the physician can employ the vials directly, where
the vials will have
the desired amount and concentration of agent(s). Associated with such
container(s) can
be various written materials such as instructions for use, or a notice in the
form prescribed
by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or
sale for human administration.
The compounds of the invention may be used or administered in combination with
one or
more additional drug (s) that are useful for the treatment of the
disorder/diseases
mentioned. The components can be administered in the same formulation or in
separate
CA 02566166 2006-10-30
formulations. If administered in separate formulations the compounds of the
invention may
be administered sequentially or simultaneously with the other drug(s).
Pharmaceutical compositions of this invention for parenteral injection
comprise
5 pharmaceutically acceptable sterile aqueous or nonaqueous solutions,
dispersions,
suspensions or emulsions as well as sterile powders for reconstitution into
sterile
injectable solutions or dispersions just prior to use. Examples of suitable
aqueous and
nonaqueous carriers, diluents, solvents or vehicles include water, ethanol,
polyols (such
as glycerol, propylene glycol, polyethylene glycol, and the like), and
suitable mixtures
10 thereof, vegetable oils (such as olive oil), and injectable organic
esters such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use of coating
materials
such as lecithin, by the maintenance of the required particle size in the case
of
dispersions, and by the use of surfactants.
15 These compositions may also contain adjuvants such as preservative, wetting
agents,
emulsifying agents, and dispersing agents. Prevention of the action of micro-
organisms
may be ensured by the inclusion of various antibacterial and antifungal
agents, for
example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also
be
desirable to include isotonic agents such as sugars, sodium chloride, and the
like.
20 Prolonged absorption of the injectable pharmaceutical form may be
brought about by the
inclusion of agents that delay absorption such as aluminium monostearate and
gelatin.
If desired, and for more effective distribution, the compounds can be
incorporated into
slow release or targeted delivery systems such as polymer matrices, liposomes,
and
microspheres.
The injectable formulations can be sterilized, for example, by filtration
through a bacterial-
retaining filter, or by incorporating sterilizing agents in the form of
sterile solid
compositions that can be dissolved or dispersed in sterile water or other
sterile injectable
medium just prior to use.
Solid dosage forms for oral administration include capsules, tablets, pills,
powders, and
granules. In such solid dosage forms, the active complex is mixed with at
least one inert,
pharmaceutically acceptable excipient or carrier such as sodium citrate or
dicalcium
phosphate and/or a) fillers or extenders such as starches, lactose, sucrose,
glucose,
mannitol, and silicic acid, b) binders such as, for example,
carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants
such as
CA 02566166 2006-10-30
21
glycerol, d) disintegrating agents such as agar-agar, calcium carbonate,
potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate, e) solution
retarding agents
such as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol monostearate,
h)
absorbents such as kaolin and bentonite clay, and i) lubricants such as talc,
calcium
stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and
mixtures thereof. In the case of capsules, tablets and pills, the dosage form
may also
comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft
and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as well as
high molecular
weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can
be prepared
with coatings and shells such as enteric coatings and other coatings well
known in the
pharmaceutical formulating art. They may optionally contain opacifying agents
and can
also be of a composition that they release the active ingredient(s) only, or
preferentially, in
a certain part of the intestinal tract, optionally, in a delayed manner.
Examples of
embedding compositions which can be used include polymeric substances and
waxes.
If desired, and for more effective distribution, the compounds can be
incorporated into
slow release or targeted delivery systems such as polymer matrices, liposomes,
and
microspheres.
The active compounds can also be in microencapsulated form, if appropriate,
with one or
more of the above-mentioned excipients.
Liquid dosage forms for oral administration include, pharmaceutically
acceptable
emulsions, solutions, suspensions, syrups and elixirs.
In addition to the active
compounds, the liquid dosage forms may contain inert diluents commonly used in
the art
such as, for example, water or other solvents, solubilizing agents and
emulsifiers such as
ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl
benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in
particular,
cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of
sorbitan, and
mixtures thereof.
CA 02566166 2006-10-30
22
Besides inert diluents, the oral compositions can also include adjuvants such
as wetting
agents, emulsifying and suspending agents, sweetening, flavouring, and
perfuming
agents.
Suspensions, in addition to the active compounds, may contain suspending
agents as, for
example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters,
microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and
tragacanth, and mixtures thereof.
io Compositions for rectal or vaginal administration are preferably
suppositories which can
be prepared by mixing the complexes of this invention with suitable non-
irritating
excipients or carriers such as cocoa butter, polyethylene glycol or a
suppository wax
which are solid at room temperature but liquid at body temperature and
therefore melt in
the rectum or vaginal cavity and release the active compound.
Dosage forms for topical administration of a compound of this invention
include powders,
patches, sprays, ointments and inhalants. The active compound is mixed under
sterile
conditions with a pharmaceutically acceptable carrier and any needed
preservatives,
buffers, or propellants which may be required.
The amount of compound administered will preferably treat and reduce or
alleviate the
condition. A therapeutically effective amount can be readily determined by an
attending
diagnostician by the use of conventional techniques and by observing results
obtained
under analogous circumstances. In determining the therapeutically effective
amount a
number of factors are to be considered including but not limited to, the
species of animal,
its size, age and general health, the specific condition involved, the
severity of the
condition, the response of the subject to treatment, the particular compound
administered,
the mode of administration, the bioavailability of the preparation
administered, the dose
regime selected, the use of other medications and other relevant
circumstances.
A preferred dosage will be a range from about 0.01 to 300 mg per kilogram of
body weight
per day. A more preferred dosage will be in the range from 0.1 to 100 mg per
kilogram of
body weight per day, more preferably from 0.2 to 80 mg per kilogram of body
weight per
day, even more preferably 0.2 to 50 mg per kilogram of body weight per day. A
suitable
dose can be administered in multiple sub-doses per day.
,
CA 02566166 2006-10-30
23
SYNTHESIS
The compounds of the various embodiments may be prepared using the reaction
routes
and synthesis schemes as described below, employing the techniques available
in the art
using starting materials that are readily available. The preparation of
particular
compounds of the embodiments is described in detail in the following examples,
but the
artisan will recognize that the chemical reactions described may be readily
adapted to
prepare a number of other agents of the various embodiments. For example, the
synthesis of non-exemplified compounds may be successfully performed by
modifications
apparent to those skilled in the art, e.g. by appropriately protecting
susceptible groups, by
changing to other suitable reagents known in the art, or by making routine
modifications of
reaction conditions. A list of suitable protecting groups in organic synthesis
can be found
in Protective Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd
Edition,
John Wiley & Sons, 1999. Alternatively, other reactions disclosed herein or
known in the
art will be recognized as having applicability for preparing other compounds
of the various
embodiments.
There are several different approaches to the synthesis of flavonols and in
principle any of
the known methodologies may be used to produce the compounds of the invention.
We
utilized a two-step reaction process namely, the Claisen-Schmidt condensation
reaction,
followed by the Algar-Flynn-Oyamada reaction (Scheme 1).
OH io OH R
R Claisen-Schmidt
AcHN OHC R condensation AcHN
0 0
Algar-Flynn-Oyamada
o
reaction
AcHN OH
Scheme 1: Two-step reaction process for synthesis of flavonols.
The first target flavonol 1 was a model compound for the development of the
chemistry for
the synthesis of more complex B-ring hydroxylated flavonols. While
benzaldehyde was
commercially available, 5-acetamido-2-hydroxyacetophenone was not and
therefore had
to be synthesized utilising the procedure outlined in Scheme 2. Thus, p-
anisidine was
acetylated by treatment with acetic anhydride in dichloromethane. Addition of
petroleum
spirits allowed isolation of 4-acetamidoanisole. Next, treatment of 4-
acetamidoanisole with
,
CA 02566166 2006-10-30
24
acetyl chloride and aluminium chloride effected Friedel-Crafts acylation.
Subsequently,
heating the reaction mixture at reflux resulted in demethylation, affording 5-
acetamido-2-
hydroxyacetophenone 4 in large amounts (20-30 g).
OMe OH
OMe 0 0
a
'AN AN
H2N
4
Scheme 2: Reagents/conditions: (a) Ac20, CH2Cl2, 78%; (b) AlC13, AcCI, CH2Cl2,
96%.
With substantial amounts of 4 in hand, the next steps were to form the 6-
acetamidoflavonol. Thus, the acetophenone 4 was treated with benzaldehyde and
base
ethanol at reflux, followed by neutralization with aqueous ammonia to give 6-
aminoflavonol.
With 6-aminoflavonol in hand the condensation with succinic anhydride was
investigated.
It was found that rather than form the amino flavonol, the crude HCI salt 7 of
6-
aminoflavonol, after hydrolysis of the acetamide, could be readily isolated
simply by
diluting the reaction mixture with water. The crude salt 7 was treated with
succinic
anhydride in pyridine to afford the succinamic acid I in an excellent yield
(42% from 5)
(Scheme 3).
OH OH
=10
40 0 40
H a I
AcHN AcHN AcHN OH
0 0 0 0
4 5 6
0 el
0
0 ao
O
H3N OH H
Col 0 0 0
7 1
Scheme 3: Reagents/conditions: (a) NaOH, Et0H, 71%; (b) NaOH, H202, Et0H, 50%;
(c)
5 M HCI, Et0H; (d) succinic anhydride, pyridine, 42% over 2 steps.
CA 02566166 2006-10-30
Unfortunately the general synthetic scheme used in the formation of 1 was not
to
amenable to the formation of the compounds functionalised on the B ring.
Installation of a
p-methoxybenzyl protecting group was performed by refluxing a solution of 5-
acetamido-
2-hydroxyacetophenone 4 and p-methoxybenzyl chloride in the presence of K2CO3.
Use
5 of a common solvent, acetone, in the reaction resulted in low yields and
long reaction
times. When the solvent was changed to butanone, which boils 23 C higher than
acetone, the PMB ether 8 was obtained in an excellent yield (82%). With this
protected
acetophenone in hand, the Claisen-Schmidt condensation was investigated.
Gratifyingly,
treatment of the PMB ether 8 and 4-benzyloxybenzaldehyde with NaOH, under the
10 conditions used previously for the synthesis of the model compound afforded
the
protected chalcone 9 in good yield (86%) (Scheme 4).
OMe
a AcH
=
is OH =
40 OBn
AcH 40
OH
0 0
4 8
is OPMB so OBn
AcH
0
15 Scheme 4: Reagents/conditions: (a) PMBCI, K2CO3, butanone, reflux, 82%;
(b) NaOH,
Et0H, 86%.
The next task was to selectively reveal the 2-hydroxyl of the PMB-chalcone 9,
while
keeping the benzyl and acetamide groups of the molecule intact. This was
achieved by
20 refluxing the protected chalcone 9 in aqueous HCI. This reaction had to
be performed
with care, as it was already known from the synthesis of the model compound 1
that
aqueous HCI can also hydrolyze the acetamido group. Here, a lower
concentration of HCI
was used (2 M, rather than 5 M), and the reaction was continuously monitored
by t.l.c. for
completion. The crude deprotected product 10 was carried forward in the next
step, the
25 Algar-Flynn-Oyamada reaction, in a procedure similar to that used for
the synthesis of 1,
but with the inclusion of 1,4-dioxane to improve the solubility of starting
materials. This
gave the desired flavonol 11 in good yield (57% from 9) (Scheme 5).
CA 02566166 2006-10-30
26
OBn
OPMB OBn OH OBn =
40 Si
AcHN tir AcHN AcH OH
0 0 0
9 10 11
Scheme 5: Reagents/conditions: (a) 2 M HCI, Et0H, 70 C, 1 h; (b) NaOH, H202,
Et0H,
1,4-dioxane, 57% over 2 steps.
5
Refluxing the fully protected flavonol 11 in concentrated HCI and acetic acid
resulted in
clean conversion to the deprotected flavonol, isolated as the HCI salt 12.
Finally, reaction
of the crude salt 12 with succinic anhydride in pyridine, under the conditions
that were
used for the model compound 1, afforded the succinamic acid derivative of 4'-
10 hydroxyllavonol 2 (Scheme 6), which was purified by recrystallization
from DMF/water.
OBn OH
10 9 I a 0 =OH
0 10 0 io 0
AcH OH H3N OH HoyAN OH
0 cie 0 0
,11 12 2
Scheme 6: Reagents/conditions: (a) 36% HCI, AcOH, reflux, 2 h; (b) succinic
anhydride,
15 pyridine, 38% over 2 steps.
Using the procedure established for the synthesis of 2, the synthesis of the
succinamic
acid substituted 3',4'-dihydroxyflavonol 3 proved mostly uneventful. Thus,
while the
condensation of the protected acetophenone 8 with 3,4-dibenzyloxybenzaldehyde
did not
20 proceed at room temperature, upon heating the mixture to 40 C, an
excellent yield of the
protected chalcone 13 was obtained (71%), emphasizing the advantage of
protecting the
2-hydroxyl group of the acetophenone. The PMB group of the chalcone 13 was
selectively removed by careful treatment with refluxing 2 M HCI in ethanol and
the crude
2'-hydroxychalcone 14 treated immediately with alkaline hydrogen peroxide to
effect an
25 Algar-Flynn-Oyamada reaction, affording the flavonol 15 in 50%
yield. The benzyl ethers
and acetamido group of the flavonol 15 were hydrolyzed with concentrated
aqueous HCI
in acetic acid and the crude HCI salt 16 isolated by centrifugation. Finally,
the succinamic
acid moiety was introduced by treating the HCI salt 16 with succinic anhydride
in pyridine
to afford the target compound 3 in 19% yield (Scheme 7), purified by
recrystallization from
30 DMF/water.
CA 02566166 2006-10-30
,
27
0 OMe
AcH + OH IW righ, OBn a
___...
AcH OPMB 0 OBn
OBn
OBn
0 0
8 13
an OBn
b is OH a OBn c
oI 1.) OBn
AcH \IF OBn AcH OH
0 0
14 15
al OHAi OH
d
= r,
OH e 0 0 itr 4W) OH
H3N OH HO1HL
_____....
N OH
e 0 0 H
CI 0
16 3
Scheme 7: Reagents/conditions: (a) NaOH, Et0H, 40 C, 71%; (b) 2 M HCI, Et0H,
70 C,
1 h; (c) H202, NaOH, Et0H, 1,4-dioxane, 50% over 2 steps; (d) 36% HCI, AcOH,
reflux, 3
5 h; (e) succinic anhydride, pyridine, 19% over 2 steps.
In order to probe the generality of the modifications it was decided to make
an analog of
quercetin, another active flavonol. This was synthesised as shown in Scheme 8.
lei OAc
0 OAc
0
Ac0 40 0
I OAc 0)L() 0 0
I OAc
OAc
OAc
OAc 0
BrCH2CO2Et OAc 0
C 0
K23
21
1
i) NaOH
ii) HCI
0 OH
0
0 0 0 1
HO) OH
OH
OHO
22
,
CA 02566166 2006-10-30
28
Scheme 8
Thus quercetin pentaacetate (20) was subjected to alkylation conditions to
selectively
alkylate the hydroxyl moiety at the 7 position. Saponification of the acetate
groups and
the methyl ester lead to the formation of quercetin 7-0-acetic acid.
EXAMPLES
Reagents useful for synthesizing compounds may be obtained or prepared
according to
techniques known in the art.
In the examples described below, unless otherwise indicated, all temperatures
in the
following description are in degrees Celsius and all parts and percentages are
by weight,
unless indicated otherwise.
Various starting materials and other reagents were purchased from commercial
suppliers,
such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and used without
further
purification, unless otherwise indicated. Tetrahydrofuran (THF) and N,N-
dimethylformamide (DMF) were purchased from Aldrich in SureSeal bottles and
used as
received. All solvents were purified by using standard methods in the art,
unless
otherwise indicated.
Chemistry
General methods
Thin layer chromatography (t.l.c) was performed on aluminium sheets pre-coated
with
Merck Silica Gel 60, using mixtures of ethyl acetate and petroleum spirits, or
mixtures of
diethyl ether and dichloromethane. Detection was achieved by irradiation with
UV light.
NMR data was obtained on Varian Unity Plus 400 or 500 instruments in solutions
of d6-
DMS0 using residual solvent as internal standard (62.50 ppm for 1H, 639.51 ppm
for 13C)
or in CDCI3 using TMS as an internal standard (8 0.00 ppm). Evaporation of
solvents was
performed under reduced pressure using a rotary evaporator. Elemental analyses
were
performed by CMAS (Belmont, Victoria). Melting points were obtained using an
Electrothermal melting point apparatus or Riechert-Jung Hot-stage melting
point
apparatus and in the latter case are corrected. Low resolution mass spectra
were
obtained by electrospray ionization using a triple-quad Quattro 11 instrument
(The
University of Melbourne).
CA 02566166 2006-10-30
29
Example 1
4-Acetamidoanisole
Acetic anhydride (16.0 mL, 169 mmol) was added dropwise over 1 h to a mixture
of p-
anisidine (20.0 g, 162 mmol) and dichloromethane (60 mL), with moderation by
cooling in
a water bath. The mixture was stirred at room temperature for 1 h, during
which time a
solid formed. Petroleum spirit (190 mL) was added, and the mixture was stirred
for a
further 1 h. The mixture was filtered and washed with petroleum spirit to
afford 4-
acetamidoanisole as a pale grey solid (25.8 g, 96 %), m.p. 127-128 C. 1H NMR
(399.7
MHz, CDCI3) 8 2.13, 3.78 (2s, 2 X 3H, 2 X CH3); 6.83 (app. d, 2H, J 8.8 Hz,
BB'); 7.38
to (app. d, 2H, J 8.8 Hz, AA'); 7.59 (br s, 1H, NH).
Example 2
5-Acetamido-2-hydroxyacetophenone (4)
Aluminium chloride (56.0 g, 420 mmol) was added in four portions over 45 min
to a
mixture of 4-acetamidoanisole (20.0 g, 121 mmol) and acetyl chloride (25.8 mL,
363
mmol) in dichloromethane (190 mL). After addition of the first portion, the
mixture became
clear, and after addition of all four portions, a suspension formed again. The
mixture was
then heated at reflux for 4.5 h, after which it was cooled and poured into
ice/water and
vigorously stirred for 30 min. The resultant slurry was filtered and washed
with water and
the solid was dried to afford the acetophenone (4) as a light green powder
(18.2 g, 78%),
m.p 163-167 C. 1H NMR (399.7 MHz, CDCI3) 8 2.18 (s, 3H, CH3CON); 2.62 (s, 3H,
CH3COAr); 6.93 (d, 1H, J3.4 9.0 Hz, H3); 7.34 (dd, 1H, J3,4 9.0, Jo 2.6 Hz,
H4); 8.17 (d,
1H, J4,6 2.6 Hz, H6); 12.10 (s, 1H, NH).
Example 3
5'-Acetamido-2'-hydroxychakone (5)
Aqueous NaOH (12 mL of 25.2 g/100 mL) was added to a mixture of 5-acetamido-2-
hydroxyacetophenone (4) (1.00 g, 5.18 mmol) and benzaldehyde (0.79 mL, 7.77
mmol) in
ethanol (12 mL), and the mixture was stirred at room temperature for 6 h. The
mixture was
acidified with 30% aqueous acetic acid, with cooling on ice. The mixture was
stirred for 1 h
and filtered to afford the chalcone (5) as a brown solid (1.20 g, 82%), m.p.
162-165 C. 1H
NMR (399.7 MHz, CDCI3) 8 2.21 (s, 3H, CH3); 6.98 (d, 1H, J3.,4. 8.8 Hz, H3');
7.34 (dd, 1H,
8.8, 46 2.4 Hz, H4'); 7.41-7.44 (m, 3H, H3,4,5); 7.61-7.69 (m, 3H, H2,6,C=CH);
7.92
(d, 1H, Arms 15.6 Hz, CH=C); 8.41 (d, 1H, 4.6. 2.4 Hz, H3').
CA 02566166 2006-10-30
Example 4
6-Acetamidoflavonol (6)
Aqueous hydrogen peroxide (30% w/v, 4 mL) was added to an ice-cold suspension
of 5'-
acetamido-2'-hydroxychalcone (5) (2.00 g, 7.11 mmol) and 1 M NaOH (20 mL) in
ethanol
5 (60 mL). The mixture was allowed to warm to room temperature and was
vigorously
stirred overnight. The mixture was acidified with 1 M HCI and the precipitate
formed was
collected by filtration to afford the flavonol (6) as a bright yellow powder
(1.04 g, 50%),
m.p. 241-242 C. 1H NMR (399.7 MHz, d6-DMS0) 8 2.10 (s, 3H, CH3); 7.48-7.59
(m, 3H,
H3',4',5'); 7.73 (d, 1H, J7,5 9.2 Hz, H8); 7.89 (dd, 1H, J5,7 2.8, J7,5 9.2
Hz, H7); 8.21 (app. d,
10 2H, J 7.6 Hz, H2',6'); 8.44 (d, 1H, J5,7 2.8 Hz, H5), 9.60 (br s, 1H,
OH); 10.28 (s, 1H, NH).
Example 5
6-Aminoflavonol
Aqueous HCI (5 M, 20 mL) was added to a suspension of 6-acetamidoflavonol (6)
(1.009,
15 3.39 mmol) in ethanol (30 mL) and the mixture was heated under reflux
for 1.5 h. The
mixture was cooled and made basic with aqueous NH3 (litmus) and the
precipitate formed
was collected by filtration to afford 6-aminoflavonol as a bright yellow
powder (0.437 g,
51%), m.p. 211-212 C. 1H NMR (399.7 MHz, d6-DMS0) 8 5.48(s, 1H, NH); 7.08
(dd, 1H,
J5,7 2.8, J7,5 9.2 Hz, H7); 7.14 (d, 1H, J5,7 2.8 Hz, H5); 7.47-7.57 (m, 4H,
H3',4',5',8); 8.17
20 (app. d, 2H, J7.6 Hz, H2',6'); 8.62 (br s, 1H, OH).
Example 6
6-(Hydroxycarbonylethylcarbonylamino)flavonol (1)
Method A: A mixture of 6-aminoflavonol (100 mg, 0.395 mmol) and succinic
anhydride (47
25 mg, 0.47 mmol) in pyridine (2 mt.) was stirred at room temperature for 4
h. Water (1 mL)
was added and the mixture was acidified with 2 M HCI. The resulting suspension
was
filtered to afford the succinamic acid (1) as a yellow solid, which was
recrystallised from
THF/petroleum spirits (72.0 mg, 52%).
Method B: A mixture of aqueous HCI (5 M, 1 mL) and 6-acetamidoflavonol (0.50
mg,
30 0.169 mmol) in ethanol (1.5 mL) was heated under reflux for 1.5 h. The
mixture was
cooled and diluted with water and the precipitate that formed was collected by
filtration to
afford the hydrochloride salt as a bright yellow powder. The crude product and
succinic
anhydride (14.1 mg, 0.141 mmol) was dissolved in pyridine (2 mL), and the
mixture was
stirred at room temperature for 4 h. Water (1 mL) was added and the mixture
was acidified
with 2 M HCI. The mixture was left to stand at room temperature for 30 min,
and the
resulting suspension was filtered and recrystallised from THF/petroleum
spirits to afford
the succinamic acid (1) as a yellow powder (24.5 mg, 42%), m.p. 220-223 C.
Anal. Calc.
CA 02566166 2006-10-30
=
31
for Ci9H15N06: C, 64.59; H, 4.28; N, 3.96. Found: C, 64.51; H, 4.19; N, 4.08.
1H NMR
(399.7 MHz, d6-DMS0) 52.55-2.61 (m, 4H, CH2CH2); 7.49-7.59 (m, 3H, H3',4',5');
7.73 (d,
1H, J7,8 9.2 Hz, H8); 7.87 (dd, 1H, J5,7 2.8, J7,8 9.2 Hz, H7); 8.20 (app. d,
2H, J 8.4 Hz,
H2',6'); 8.47 (d, 1H, J5,7 2.8 Hz, H5); 9.61 (br s, 1H, OH); 10.31 (s, 1H,
NH); 12.18 (br s,
1H, CO2H).
Example 7
5-Acetamido-2-(4-methoxybenzyloxy)acetophenone (8)
A mixture of 5-acetamido-2-hydroxyacetophenone (4) (3.00 g, 15.5 mmol), 4-
methoxybenzyl chloride (3.20 mL, 31.1 mmol) and K2CO3 (3.21 g, 23.3 mmol) in
butanone
(45 mL) was heated under reflux overnight. The mixture was filtered and the
filtrate was
concentrated in vacuo, giving a yellow residue. The residue was triturated
with petroleum
spirit and immediately recrystallised from THF/petroleum spirit to afford the
protected
acetophenone (8) as a white powder (4.02 g, 82%), m.p. 169-171 C. Anal.
Calcd. for
C18F119N04: C, 68.99; H, 6.11; N, 4.47. Found: C, 68.86; H, 6.19; N, 4.53%. 1H
NMR
(399.7 MHz, CDCI3) 8 2.16 (s, 3H, CH3CON); 2.57 (s, 3H, CH3COAr); 3.83 (s, 3H,
CH30);
5.08 (s, 2H, CH2); 6.92 (d, 2H, J 8.4 Hz, H2',6'); 7.02 (d, 1H, J3,4 8.8 Hz,
H3); 7.35 (app. d,
2H, J 8.4 Hz, H3',5'); 7.52 (d, 1H, J4,6 3.2 Hz, H6); 7.97 (dd, 1H, 44 8.8,
4,6 3.2 Hz, H4).
13C NMR (100.5 MHz, CDCI3) 8 24.58, 32.45, 55.52, 71.03 (4C, CH2,0CH3,2 x
CH3);
113.86, 114.28, 122.09, 126.52, 128.30, 128.43, 129.59, 131.47, 155.23, 159.82
(10C,
Ar); 168.63, 199.52 (2C, 2 x C=0).
Example 8
5'Acetamido-4-benzyloxy-2'-(4-methoxybenzyloxy)chalcone (9)
A mixture of aqueous NaOH (25.5 mL of 25.2 g/100 mL), the protected
acetophenone (8)
(3.00 g, 9.57 mmol) and 4-benzyloxybenzaldehyde (2.03 g, 9.57 mmol) in ethanol
(25.5
mL) was stirred at room temperature overnight. The mixture was filtered to
afford the
protected chalcone (9) as a light yellow solid (3.54 g, 73%), m.p. 201-202 C.
Anal. Calcd.
for C32H29N05: C, 75.72; H, 5.76; N, 2.76. Found: C, 75.60; H, 5.74; N, 2.73%.
1H NMR
(399.7 MHz, CDCI3) 8 2.17 (s, 3H, CH3CON); 3.77 (s, 3H, CH30); 5.06, 5.09 (2s,
2 x 2H, 2
x CH2Ar); 6.82 (app. d, 2H, J 8.8 Hz, BB'); 6.88 (app. d, 2H, J 8.8 Hz, BB');
7.04 (d, 1H,
9.0 Hz, H3'); 7.27 (app. d, 2H, J 8.8 Hz, AA'); 7.31-7.45 (m, 8H,
AA',C=CH,Ph); 7.51
(d, 1H, 4,6, 2.8 Hz, H6'); 7.58 (d, 1H, Arans 15.6 Hz, C=CH); 7.98 (dd, 1H,
J3.. 9.0, J4,62.8
Hz, H4'). '3C NMR (100.5 MHz, d6-DMS0) 8 23.85, 55.05 (2C, 2 X CH3); 69.38,
70.13 (2C,
2 x CH2); 79.20, 113.82, 113.91, 115.14, 120.54, 123.94, 124.80, 127.39,
127.79, 128.00,
CA 02566166 2006-10-30
32
128.45, 128.53, 129.94, 130.30, 132.73, 136.70, 142.17, 153.06, 159.10, 160.24
(20C, Ar,
CH=CH); 168.06, 190.69 (2C, 2 x C=0).
Example 9
6-Acetamido-4'-benzyloxyflavonol (11)
A solution of 4-methoxybenzyloxychalcone (9) (1.20 g, 1.70 mmol) in aqueous
HCI (2 M,
66 mL) and ethanol (290 mL) was heated at reflux for 1 h. The mixture was
cooled to
room temperature, and evaporated in vacuo to approximately half the volume.
The
resultant suspension was filtered to afford the crude deprotected chalcone
(10) as a
yellow solid. The crude deprotected chalcone was dissolved in 1,4-dioxane
(19.2 mL),
ethanol (24 mL) and NaOH (5.4% w/v, 7.8 mL). The resultant solution was cooled
in an
ice bath and H202 (30%, 1.2 mL) was added. The solution was stirred at 0 C
for 2 h, and
subsequently at room temperature overnight. The solution was then acidified
with 2 M HCI
and the precipitate that formed was filtered, then recrystallised from
THF/petroleum spirit
to afford the flavonol (11) as a bright yellow solid (341 mg, 36%), m.p. 255-
258 C. Anal.
Calcd. for C241-118N06: C, 71.81; H, 4.77; N, 3.49. Found: C, 71.90; H, 4.80;
N, 3.51%. 1H
NMR (499.7 MHz, d6-DMS0) 8 2.09 (s, 3H, CH3); 5.19 (s, 2H, CH2); 7.19 (app. d,
2H, J
9.0 Hz, H2',6'); 7.32-7.50 (m, 5H, Ph); 7.69 (d, 1H, J7,8 9.0 Hz, H8); 7.86
(dd, 1H, J5,7 2.5,
J7,5 9.0 Hz, H7); 8.18 (app. d, 2H, J9.0 Hz, H3',5'); 8.41 (d, 1H, J5,72.5 Hz,
H5); 10.28 (br
s, 1H, NH). 13C NMR (100.5 MHz, d6-DMS0) 8 24.00 (1C, CH3); 69.37 (1C, CH2);
112.63,
114.88, 118.83, 121.41, 123.84, 125.27, 127.84, 127.98, 128.49, 129.35,
135.89, 136.68,
137.93, 145.41, 150.45, 159.48 (16C, Ar); 168.54, 172.46 (2C, 2 X C=0).
Example 10
4cHydroxy-6-(hydroxycarbonylethylcarbonylamino)flavonol (2)
A mixture of the protected flavonol (11) (600 mg, 1.49 mmol) in aqueous HCI
(36%, 38
mL) and acetic acid (38 mL) was heated under reflux for 2 h. The mixture was
then cooled
on ice and diluted with water. The resulting suspension was centrifuged and
the collected
solid washed with water, then freeze-dried to afford the salt as a crude
yellow solid (457
mg). A mixture of the crude yellow product and succinic anhydride (179 mg,
1.79 mmol) in
pyridine was stirred at room temperature for 4 h. Water (1 mL) was added and
the mixture
was acidified with 2 M HCI. The mixture was left to stand at room temperature
for 30 min,
and the resulting suspension was centrifuged and the collected solid was
washed with
water, freeze-dried and recrystallised from DMF/water to afford the succinamic
acid (2) as
a brown powder (211 mg, 38.2%), m.p. 256-257 C. Anal. Calcd. for
C19H16N07.1/2H20:
C, 60.32; H, 4.26; N, 3.70. Found: C, 59.99; H, 4.53; N, 3.90. 1H NMR (399.7
MHz, d6-
DMS0) 6 2.55-2.60 (m, 4H, CH2CH2); 6.93 (app. d, 2H, J 9.2 Hz, H3',5'); 7.69
(d, 1H, J7,8
I I
CA 02566166 2006-10-30
33
8.8 Hz, H8); 7.85 (dd, 1H, J5,7 2.8, J7,5 8.8 Hz, H7); 8.09 (app. d, 2H, J 9.2
Hz, H2',6'); 8.43
(d, 1H, J5,7 2.8 Hz, H5); 9.33 (s, 1H, NH); 10.11, 10.31 (2 br s, 2 x 1H, 2 x
OH); 12.21 (br
s, 1H, CO2H). 13C NMR (100.5 MHz, d6-DMS0) 8 25.16, 28.78 (2C, CH2CH2); 31.06,
67.05, 112.65, 115.46, 118.80, 121.45, 122.06, 125.11, 129.55, 135.80, 137.57,
146.01,
Example 11
5'-Acetamido-3,4-dibenzyloxy-2'-(4-methoxybenzyloxy)-chalcone (13)
A mixture of aqueous NaOH (3.8 mL of 25.2 g/100 mL), the protected
acetophenone (8)
129.06, 129.51, 132.78, 136.93, 137.07, 142.74, 148.31, 150.46, 152.73, 158.96
(25C,
Ar,CH=CH); 168.15, 191.48 (2C, 2 x C=0).
Example 12
A solution of 4'-methoxybenzyloxychalcone (13) (300 mg, 0.489 mmol) in aqueous
HCI (2
M, 16 mL) and ethanol (66 mL) was heated at reflux for 1 h. The mixture was
cooled to
room temperature, and evaporated in vacuo to approximately half the volume.
The
resultant suspension was filtered to afford the crude deprotected chalcone
(14) as a dark
30 yellow solid. The deprotected chalcone was dissolved in 1,4-dioxane (4.8
mL), ethanol (6
mL) and NaOH (5.4% w/v, 1.9 mL) and the resultant solution was cooled in an
ice bath
and H202 (30%, 0.3 mL) was added. The solution was stirred at 0 C for 2 h,
and
subsequently at room temperature overnight. The solution was then acidified
with 2 M HCI
and the precipitate that formed was filtered, and then recrystallised from
THF/petroleum
35 spirits to afford the flavonol (15) as a yellow solid (136 mg, 55%),
m.p. 229-230 C. Anal.
Calcd. for C311-126N06: C, 73.36; H, 4.96; N, 2.76. Found: C, 73.38; H, 4.98;
N, 2.68%. 1H
CA 02566166 2006-10-30
34
NMR (399.7 MHz, d6-DMS0) 8 2.09 (s, 3H, CH3); 5.21, 5.24 (2s, 2 x 2H, 2 x
CH2); 7.26 (d,
1H, 46, 8.8 Hz, H5'); 7.33-7.52 (m, 11H, 2 X Ph); 7.71 (d, 1H, th,8 9.2 Hz,
H8); 7.84-7.92
(m, 3H, H2',6',7); 8.40 (d, 1H, i5,7 2.4 Hz, H5); 9.48 (br s, 1H, OH); 10.27
(s, 1H, NH). 13C
NMR (100.5 MHz, d6-DMS0) 8 24.03 (1C, CH3); 69.91, 70.43, 112.65, 112.69,
113.79,
118.89, 121.38, 121.91, 124.06, 125.32, 135.91, 136.88, 137.08, 138.14,
145.14, 147.77,
149.77, 150.42 (18C, Ar); 168.59, 172.47 (2C, 2 x C=0).
Example 13
3',4'-Dihydroxy-6-(hydroxycarbonylethylcarbonylamino)flavonol (3)
A mixture of the protected flavonol (15) (500 mg, 0.985 mmol) in aqueous HCI
(36%, 37
mL) and acetic acid (37 mL) was heated under reflux for 3 h. The mixture was
then cooled
on ice and diluted with water. The resulting suspension was centrifuged and
the collected
solid washed with water, then freeze-dried to afford the salt as a crude
yellow solid (322
mg). A mixture of the crude yellow product and succinic anhydride (118 mg,
1.18 mmol) in
pyridine was stirred at room temperature for 4 h. Water (1 mL) was added and
the mixture
was acidified with 6 M HCI. The mixture was left to stand at room temperature
for 30 min,
and the resulting suspension was centrifuged, and the collected solid was
washed with
water, freeze-dried and recrystallised from DMF/water to afford the succinamic
acid (3) as
a yellow powder (70.0 mg, 19%), m.p 257-258 C. 1H NMR (399.7 MHz, d6-DMS0) 8
2.54-2.60 (m, 4H, CH2CH2); 6.89 (d, 1H, J5.0 8.5 Hz, H5'); 7.57 (dd, 1H,
J2.,6. 2.5, J5.,6, 8.5
Hz, H6'); 7.66 (d, 1H, J7,5 9 Hz, H8); 7.73 (d, 1H, J2,6. 2.5 Hz, H2'); 7.85
(dd, 1H, J5,7 2.5,
J7,5 9.0 Hz, H7); 8.43 (d, 1H, J5,7 2.5 Hz, H5); 9.29, 9.32, 9.59 (3 br s, 3 x
1H, 3 x OH);
10.29 (s, 1H, NH); 12.20 (br s, 1H, CO2H). 13C NMR (100.5 MHz, de-DMS0) 8
28.83,
31.11 (2C, CH2CH2); 112.7, 115.25, 115.65, 118.76, 120.00, 121.43, 122.39,
125.17,
135.80, 137.67, 145.12, 146.04, 147.63, 150.38 (14C, Ar); 170.46, 172.32,
173.93 (3C, 3
x C=0). Low resolution mass spectrum (ESI) miz 384.4 [C19F116N08 (M-H)4
requires
384.081
Example 14
Penta-0-acetylquercetin (20)
A suspension of quercetin (10.0 g, 33.2 mmol) in acetic anhydride (50 mL, 530
mmol)
and pyridine (25 mL) was stirred at room temperature for 15 min. The mixture
was
poured into ice- water (500 mL) and stirred for 15 min, and the solid that
formed was
collected by vacuum filtration and washed with ice-cold ethanol (20 mL). The
crude
material was recrystallised from Et0Ac/petroleum spirits to afford the
pentaacetate as
pale beige needles (12.3 g, 72%), m.p. 195-196 C. 1H NMR (399.7 MHz, CDCI3) 6
CA 02566166 2006-10-30
2.32, 2.33, 2.34, 2.43 (4s,15H, 5 x Me); 6.88, 7.33 (2d, J6,8 2.4 Hz, H6,8);
7.35 (d, J5',6'
8.4 Hz, H5'); 7.7 (d, J2',6' 2.0 Hz, H2'); 7.63 (dd, H6'). 13C NMR (100.5 MHz,
CDCI3) 6
20.43, 20.56, 20.94, 21.06 (5C, Me);108.94, 113.82, 114.63, 123.75, 123.86,
126.33,
127.62, 133.93, 142.09, 144.29, 150.26, 153.65, 164.17, 156.73 (14C, Ar);
167.71,
5 167.78, 169.19, 169.95 (6C, C=0).
Example 15
3,3',4',5-Tetra-0-acetyl-7-0-(ethoxycarbonyOmethylquercetin (21)
A mixture of pentaacetate (20) (1.02 g, 1.98 mmol), ethyl bromoacetate (0.97
mL, 8.75
10 mmol), potassium iodide (0.1 g, 0.60 mmol), anhydrous potassium
carbonate (2.5 g)
and anhydrous acetone (25 mL) was heated under reflux for 19 h under an
atmosphere of nitrogen. The mixture was filtered to remove undissolved salts
and the
filtrate concentrated to yield an oil. The crude residue was purified by flash
chromatography (40% Et0Acitoluene) and recrystallised from Et0Aclpetroteum
15 spirits to afford the ethyl ester as a colourless crystalline powder
(0.322 g, 29%), m.p.
151-152 C. 1H NMR (399.7 MHz, CDCI3) 6 1.31 (t, 3H, J 7.2 Hz, CH2CH3); 2.17,
2.33, 2.43 (3s, 12H, 4 x Me); 4.29 (q, 2H, CH2CH3); 4.71 (s, 2H, CH2C0); 6.69,
6.81
(2d, J6,8 2.4 Hz, H6,8); 7.34 (d, J5',6' 8.4 Hz, H5'); 7.67 (d, J2',6' 2.0 Hz,
H2'); 7.70 (dd,
H6').
Example 16
7-0-(Hydroxycarbonyl)methylquercetin (22)
Aqueous sodium hydroxide (10%, 0.25 mL) was added to a suspension of the ethyl
ester (21) (252 mg, 0.453 mmol) in methanol (2.5 mL) and the mixture was
heated in a
water bath (60 C) for 5 min. Water (1 mL) was then added and heating
continued for 5
min. The mixture was acidified with concentrated hydrochloric acid (0.2 mL)
and heated
for a further 50 min. The crystalline solid that separated was collected and
recrystallised (Et0Acipetroleum spirits) to yield the acid as a yellow
crystalline powder
(54.0 mg, 33%), m.p. 241-243 C (dec.). 1H NMR (399.7 MHz, CD30D) 6 1.29 (s,
2H,
CH2); 6.28, 6.49 (2d, J6,8 2.4 Hz, H6,8); 6.86 (d, J5',6' 8.4 Hz, H5'); 7.62
(dd, J2',6' 2.4
Hz, H6'); 7.73 (d, H2'). 13C NMR (100.5 MHz, CD300) 6 52.42 (CH2); 93.16,
98.42,
105.47, 115.75, 115.87, 121.45, 123.57, 137.23, 145.85, 148.17, 148.57,
157.43, 161.93,
164.54 (Ar); 170.18, 176.98 (2 x C=0). High resolution mass spectrum (ESI)
nilz
397.0525 [C17H1 0K09 (M+K)- requires 396.9962].
CA 02566166 2006-10-30
36
Pharmacology
Drugs and chemicals used
Acetylcholine perchlorate was obtained from BDH Chemicals (Poole, Dorset,
England).
Phenylephrine and propranolol was purchased from Sigma-Aldrich Pty. Ltd.
(Castle Hill,
NSW, Australia). DiOHF was purchased from lndofine Chemical Co. Inc. (Belle
Mead, NJ,
U.S.A.). All other flavonols were synthesized in this study. U46619 was
purchased from
Cayman Chemical (Ann Arbor, MI, U.S.A.). All chemicals were dissolved in
distilled water,
except the following. DiOHF was dissolved in 10% dimethyl sulphoxide (DMSO),
90%
methanol, with subsequent dilutions in 50% methanol (10-3 n), and distilled
water (104-10-
7 M). Succinamic acid-substituted flavonols were dissolved in 0.1 M Na2CO3 as
a stock
solution (B and C at 101 and D at 104 M), and further diluted in distilled
water as required.
Preparation of rat aortic rings
Male Sprague-Dawley rats (200-400 g) were euthanised by exposure to 80% CO2,
20%
02, and their chests opened to isolate the thoracic aortae. After the removal
of superficial
connective tissues, the aorta was cut into ring segments, of approximately 2-3
mm in
length. The aortic rings were then mounted between two stainless steel wires,
one of
which was linked to an isometric force transducer connected to a chart
recorder, and the
other end anchored to a glass rod submerged in a standard 10 mL organ bath.
The organ
bath was filled with Krebs-bicarbonate solution [composition (mM): NaCI,
118.0; KCI, 4.7;
KH2PO4, 1.2; MgSO4.7H20, 1.2; glucose, 5.0; NaHCO3, 25.0; CaC12.2H20, 2.5].
The bath
medium was maintained at 37 C, pH 7.4 and continuously aerated with 95% 02,
5% CO2.
Equilibration of aortic rings & testing of endothelial integrity
Aortic rings were equilibrated for 1 h at a resting tension of 1 g, then were
precontracted
with an isotonic, high potassium physiological salt solution (KPSS) in which
all of the NaCI
of the normal Krebs solution was replaced with KCI (122.7 mM), so as to
achieve maximal
contraction. The solution was then washed out and replaced with normal Krebs
solution.
After re-equilibration, the rings were submaximally contracted with
phenylephrine (PE, 10"
8-104 M) and endothelial integrity was tested by a single dose of
acetylcholine (Ach, 10-5
M). Only rings that responded to Ach 90% relaxation) were judged endothelium
intact
and were used in the subsequent experiments. The aortic rings were then re-
equilibrated
(15 min) before the subsequent experiment.
Effect of flavonols on phenylephrine-induced vasoconstriction
Aortic rings were incubated with a p-adrenoceptor antagonist, propranolol (10-
5 M), and
either vehicle or flavonol (104-10-5 M) for 15 min before cumulative doses of
CA 02566166 2006-10-30
37
phenylephrine (10-9-1045 M) were added to generate a concentration-response
curve to
phenylephrine. Contraction responses were expressed as a percentage of KPSS-
induced
tension.
Vasorelaxallon
Aortic rings were precontracted submaximally with phenylephrine (10-8-1C M)
and the
thromboxane mimetic, 9,11-dideoxy-9a,11a-epoxymethano-prostaglandin F2a
(U46619,
g
lu
10-- M) to approximately 50% of KPSS-induced contraction. After stabilization
of the
contraction, cumulative doses of the flavonol or vehicle (10-10-10-4 M) were
added to
generate a concentration-response curve. Relaxant responses were expressed as
a
percentage of the precontraction tension.
Effect of flavonols on superoxide concentrations
Superoxide concentrations were measured in isolated aortic rings by lucigenin-
enhanced
chemiluminescence. Prior to assaying, the aortic rings were incubated at 37 C
for 1 h in
Krebs-HEPES buffer containing DETCA (3 x 10 M), which inactivates superoxide
dismutase, NADPH (10-4 M) and either vehicle, succinamic acid-substituted
flavonol (10'4
or 10-5 M) or diOHF (104 or 10-5 M). Assay solutions consisting of lucigenin
(5 X 10-6 M),
NADPH (104 M) and either vehicle (DMSO), succinamic acid-substituted flavonol
(10-4 or
10-5 M) or 3',4'-dihydroxyflavonol (diOHF, 10-4 or 10-5 M), as a positive
control, were
prepared in Krebs-HEPES buffer [composition (mM): NaCI, 99.0; KCI, 4.7;
KH2PO4, 1.0;
MgSO4.7H20, 1.2; glucose, 11.0; NaHCO3, 25.0; CaC12.2H20, 2.5; Na-HEPES,
20.0]. 300
1.1 aliquots of the assay solution were placed into separate wells on a 96-
well Optiplate,
which was loaded into a TopCount single photon counter (Packard Bioscience) to
determine the background emission (12 cycles). After background counting was
completed, one aortic ring was added per well, and photon emission was counted
(12
cycles). Superoxide levels were reported as a percentage of photon emission
for +NADPH
control. At the conclusion of the assay, the aortic rings were dried for 48 h
at 80 C for
normalization of superoxide production to dry tissue weight.
Data presentation and statistical analysis
The results are expressed as the mean s.e. mean and n indicates the number
of
experiments (rats). Relaxation concentration-response curves for diOHF were
computer
fitted to a sigmoidal curve using non-linear regression (Prism version 4) to
enable
calculation of pEC5,3 of the flavonols. However, as in most cases the data did
not fit a
sigmoidal curve, pEC50 was not calculated. Maximum relaxation responses were
compared using a one way analysis of variance (ANOVA) with post-hoc multiple
CA 02566166 2013-05-31
38
comparison using Newman-Keuls test. Superoxide levels were compared using a
one way
analysis of variance (ANOVA) with post hoc multiple comparison using Dunnett's
test.
