Chemical modification of the multitarget neuroprotective compound fisetin

Article abstract of DOI:10.1021/jm2012563

Many factors are implicated in age-related central nervous system (CNS) disorders, making it unlikely that modulating only a single factor will provide effective treatment. Perhaps a better approach is to identify small molecules that have multiple biolog

Full text of DOI:10.1021/jm2012563

Article
pubs.acs.org/jmc
Chemical Modification of the Multitarget Neuroprotective
Compound Fisetin
Chandramouli Chiruta, David Schubert, Richard Dargusch, and Pamela Maher*
The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: Many factors are implicated in age-related
central nervous system (CNS) disorders, making it unlikely
that modulating only a single factor will provide effective
treatment. Perhaps a better approach is to identify small
molecules that have multiple biological activities relevant to
the maintenance of brain function. Recently, we identified an
orally active, neuroprotective, and cognition-enhancing mole-
cule, the flavonoid fisetin, that is effective in several animal
models of CNS disorders. Fisetin has direct antioxidant activity
and can also increase the intracellular levels of glutathione
(GSH), the major endogenous antioxidant. In addition, fisetin
has both neurotrophic and anti-inflammatory activity.
However, its relatively high EC50 in cell based assays, low
lipophilicity, high topological polar surface area (tPSA), and poor bioavailability suggest that there is room for medicinal chemical
improvement. Here we describe a multitiered approach to screening that has allowed us to identify fisetin derivatives with
significantly enhanced activity in an in vitro neuroprotection model while at the same time maintaining other key activities.
(GSH), the major intracellular antioxidant, via the activation of
transcription factors such as Nrf2.5 Fisetin can also maintain
mitochondrial function in the presence of oxidative stress. In
addition, it has anti-inflammatory activity against immune cells
and inhibits the activity of 5-lipoxygenase, thereby reducing the
production of lipid peroxides and their proinflammatory
byproducts.5 This wide range of actions suggests that fisetin
has the ability to reduce the loss of neurological function
associated with multiple disorders, including stroke.
Although fisetin has been shown to be effective in the rabbit
small clot embolism model of stroke,6 its relatively high EC50 in
cell based assays (2−5 μM) and its low lipophilicity (CLogP =
1.24), high tPSA (107 Å), high number of hydrogen bond
donors (HBD = 5), and poor bioavailability28 suggest that there
is room for medicinal chemical improvement if fisetin is to be
used therapeutically for treating neurological disorders such as
stroke. However, given its ability to activate multiple target
pathways related to neuroprotection, screening for improve-
ments is significantly more complicated than with the current
classical approach to the development of a single target drug. In
this paper, we describe a multitiered approach to screening that
has allowed us to identify fisetin derivatives with significantly
enhanced neuroprotective activity in an in vitro ischemia model
while at the same time maintaining other key actions including
anti-inflammatory and neurotrophic activity as well as the
ability to maintain GSH under conditions of oxidative stress.
INTRODUCTION
■
There are currently no drugs available that prevent the nerve
cell death associated with the majority of age-related disorders
of the CNS. A prime example of this problem is ischemic
stroke, which is the leading cause of adult disability and the
third leading cause of death in the U.S.1 Worldwide, approx-
imately 5 million people die each year of stroke, and the
mortality rates are estimated to double by the year 2020.2 The
nerve cell death associated with cerebral ischemia is due to
multiple factors resulting from the lack of oxygen to support
respiration and ATP synthesis, acidosis due to the buildup of
the glycolytic product lactic acid, the loss of neurotrophic
support, multiple metabolic stresses, and inflammation.3a,b
While the focus of current drug discovery paradigms is
primarily on the development of high affinity, single target
ligands, a drug directed against a single molecular target may
not be effective in treating the nerve cell death associated with
conditions such as stroke because of the multitude of insults that
contribute to the cell’s demise. This conclusion is supported by
the lack of drugs for the treatment of stroke. Indeed, the only
FDA-approved treatment to date is recombinant tissue-type
plasminogen activator (rt-PA),4 which is a vascular agent. An
alternative approach is to identify small molecules that have
multiple biological activities relevant to the maintenance of
neurological function.
Over the past few years, we have identified an orally active,
neuroprotective, and cognition-enhancing molecule, the
flavonol fisetin.5 Fisetin not only has direct antioxidant activity
but can also increase the intracellular levels of glutathione
Received: September 20, 2011
Published: December 20, 2011
© 2011 American Chemical Society
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CHEMISTRY
Scheme 2. Synthesis of Flavone Derivatives
■
The synthesis of substituted chalcones 013, 032, 033, 057, 063,
085, 086, 086A, 105−108, and 137 was carried out by con-
densation of 2′-hydroxyacetophenones with appropriately
substituted aldehydes using Ba(OH)2 in methanol7 (Scheme 1).
a
Scheme 1. Synthesis of Chalcone Derivatives
a
Reagents and conditions: (a) I2, DMSO, 130 °C, 6 h, 50−95%; (b)
BBr3, CH2Cl2, 0 °C to room temp, overnight, 30−70%; (c) H2, Pd/C,
1:1, EtOAc/MeOH, overnight, 60%.
a
Scheme 3. Synthesis of Flavonol Derivatives
a
Reagents and conditions: (a) Ba(OH)2, MeOH, 40 °C, overnight,
30−90%; (b) BBr3, CH2Cl2, 0 °C to room temp, overnight, 30−70%;
(c) pTSA, MeOH, room temp, 94%.
The trihydroxy chalcones 011, 034, and 087 were prepared
from the corresponding chalcones by treatment with BBr3 in
dicloromethane8 and the dihydroxychalcone 088 was synthe-
sized by THP deprotection using pTSA in methanol7 from the
corresponding chalcone. The suitably substituted flavones 018,
038, 058, 068, 089, 115, 116, 119, and 120 were synthesized
from the corresponding chalcones using I2 in DMSO9 (Scheme 2).
The hydroxyflavones 002, 028, 064, 072, and 094 were
obtained from the corresponding chalcones by demethylation/
deethylation or debenzylation using BBr3 in dicloromethane8 or
H2, Pd/C in EtOAc/methanol,10 respectively.
Substituted flavonols 025, 036, 037, 059, 065, 090, 091, 114,
117, 118, 122, and 139 were prepared (Scheme 3) using 5.4%
NaOH, 30% H2O2 in methanol11 from the corresponding
chalcones. The known compounds fisetin, 002, and 04P were
purchased from Indofine Chemicals, and the other hydroxy-
flavonols 027, 040, 041, 069, 070, 092, 093, and 140 were
obtained from their corresponding flavonols (Scheme 3) by
demethylation/deethylation (BBr3 in dicloromethane)8 or
debenzylation (H2, Pd/C in EtOAc/methanol)10 methods.
Finally the substituted quinolines 001, 004, 007, 017, 021−
024, 083, 084, 109−113, and 121 were synthesized (Scheme 4)
by condensation of 2′-aminoacetophenones with appropriately
substituted aldehydes using H2SO4 in methanol.12 Experimental
procedures and data for all of the compounds are reported below
or in the Supporting Information.
a
Reagents and conditions: (a) 5.4% NaOH, 30% H2O2, MeOH, 0 °C
to room temp, overnight, 40−90%; (b) BBr3, CH2Cl2, 0 °C to room
temp, overnight, 30−70%; (c) H2, Pd/C, 1:1, EtOAc/MeOH,
overnight, 60%.
RESULTS
■
The goals of this study were to improve the potency of fisetin
based upon the activation of multiple neuroprotective pathways
while at the same time altering its physicochemical properties
to be more consistent with those of successful CNS drugs
(molecular weight ≤ 400, CLogP ≤ 5, tPSA ≤ 90, HBD ≤ 3,
HBA ≤ 7)26,33 in order to increase the possibility of efficient
brain penetration and to better understand its SAR. We took
two different approaches to the improvement of fisetin. In the
first, we removed/modified/replaced the different hydroxyl
groups in a systematic manner. In the second approach, we
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fisetin. We found that removal of the 7 hydroxyl (04P) not only
improved the neuroprotective activity ∼6-fold over fisetin in
our primary screen of in vitro ischemia without loss of either
the GSH maintaining activity or PC12 cell differentiation but
also enhanced lipophilicity increasing the CLogP from 1.24 to
1.82 (Table 1). Furthermore, this modification did not alter the
anti-inflammatory activity of fisetin (Table 1). This finding
allowed us to replace the 7 hydroxyl with hydrophobic groups
in order to further improve the lipophilicity and tPSA to values
more consistent with typical CNS drugs.26,33 The addition of a
benzene ring (040) to the A ring further enhanced neuro-
protective activity ∼5.5-fold with a much more pronounced
effect seen with the α-naphtha derivative (040) as opposed to
the β-naphtha (041) derivative (Table 1). However, this
modification eliminated the ability of the derivative to maintain
GSH under conditions of oxidative stress. For this derivative,
the 3-hydroxyl was not important for neuroprotective activity
(040 vs 002) but did enhance anti-inflammatory activity.
We also examined the role of the B ring hydroxyls in
neuroprotection as well as the other key activities of the α-naphtha
derivative. Changing both hydroxyls to ethoxy groups (036,
038) not only greatly reduced neuroprotective activity but also
eliminated both the anti-inflammatory activity and the ability to
induce PC12 cell differentiation. Changing only one of the
hydroxyls to a methoxy group enhanced neuroprotective acti-
vity ∼2-fold over 040 in the absence of the 3-hydroxyl group
(072) but greatly reduced neuroprotective activity relative to
040 in the presence of the 3-hydroxyl (070). Furthermore, this
modification did not restore the ability to maintain GSH under
conditions of oxidative stress and the derivative without the 3
hydroxyl (072) also lacked anti-inflammatory activity and the
ability to induce PC12 cell differentiation. Surprisingly, chang-
ing the 4′-hydroxyl to a benzyloxy group (065) restored neuro-
protective activity in the presence of the 3-hydroxyl. Com-
pounds possessing tertiary nitrogen, a feature of many CNS
drugs, show a higher degree of brain permeation.26,33,34 With
this observation in mind, both hydroxyls on the B ring were
replaced with a single dimethylamino group at the 4′ position,
resulting in a highly neuroprotective compound in the presence
of the 3-hydroxyl group (118) and a somewhat less effective
compound in its absence (120). This modification also elimi-
nated two hydrogen bond donors. Although 118 regained the
ability to maintain GSH levels, it lacked both anti-inflammatory
and neurotrophic activity. Modification of the dimethylamine to
a pyrrolidine group at the 4′ position gave a compound that had
excellent neuroprotective activity in the presence of the 3-
hydroxyl (114) and could also induce PC12 cell differentiation
but had poor anti-inflammatory activity and did not maintain
GSH levels. However, addition of a 3′-hydroxyl to this deriva-
tive resulted in a compound with outstanding neuroprotective
activity (EC50 = 5 nM) (140) that could also maintain GSH
under conditions of oxidative stress, induce PC12 differ-
entiation and had reasonably good anti-inflammatory activity.
As a second approach, we replaced the benzene ring with two
methyl groups (027) in order to generate a derivative with a
similar CLogP and tPSA as 040 but with a less bulky addition
to the A ring (Table 1). Surprisingly, this derivative not only
showed significantly decreased neuroprotective activity com-
pared with 040 but also lost the ability to induce PC12 cell
differentiation along with the continued failure to maintain
GSH levels. Removal of the 3-hydroxyl enhanced neuro-
protective activity 2-fold (028) but did not restore the
induction of PC12 cell differentiation or the maintenance of
Scheme 4. Synthesis of Quinoline Derivatives
a
Reagents and conditions: (a) ROH, H2SO4, reflux, overnight, 15−
40%.
modified the flavone scaffold by changing it to a quinoline
while at the same time maintaining key structural elements.
We chose to use for our primary screen an in vitro ischemia
model in combination with the HT22 hippocampal nerve cell
line.6 For this screen, we set a cutoff for the EC50 of 1 μM. To
induce ischemia in the HT22 cells, we used iodoacetic acid
(IAA), a well-known, irreversible inhibitor of the glycolytic
enzyme glyceraldehyde 3-phosphate dehydrogenase
(G3PDH).14 IAA has been used in a number of other studies
to induce ischemia in nerve cells,15a−e and we have used it in
several recent screens for neuroprotective molecules.16a,b The
changes following IAA treatment of neural cells are very similar
to those seen in animal models of ischemic stroke17 and include
alterations in membrane potential,18 breakdown of phospholip-
ids,19 loss of ATP,20a,b and an increase in reactive oxygen
species (ROS).21,19
We used three secondary screens that allowed us to assess
three key activities of fisetin that are highly relevant to stroke, as
well as other neurological disorders: maintenance of GSH, the
major endogenous cellular antioxidant, inhibition of bacterial
lipopolysaccharide (LPS) induced microglial activation, an
indicator of anti-inflammatory activity, and PC12 cell differ-
entiation, a measure of neurotrophic activity. All of these
activities are relevant to the nerve cell loss seen in stroke.22a−c
Previous and ongoing studies suggest that these activities of
fisetin are mediated via distinct pathways but that all three may
be important for the neuroprotective effects of fisetin in vivo.5
To assess GSH maintenance, we looked at total intracellular
GSH levels after a 24 h treatment with the compound both in
the absence and in the presence of glutamate, an inducer of
GSH loss and oxidative stress.23a,b Inhibition of LPS-induced
microglial activation was determined by treating N9 mouse
microglial cells with LPS alone and in the presence of the
compounds and assaying nitrite (spontaneously produced by
air oxidation of nitric oxide) levels in the medium 24 h later.24
PC12 cell differentiation was determined by treating PC12 cells
with the compounds and looking at neurite outgrowth after
24 h. In all cases, fisetin was used as a positive control.25
Structure−Activity Relationship. We began by assessing
the roles of the four different hydroxyl groups in the activity of
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Table 1
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Table 1. continued
a
Half maximal effective concentrations (EC50) for protection in the in vitro ischemia assay were determined by exposing HT22 cells to different
doses of each derivative in the presence of 20 μM IAA for 2 h (HT22/IAA). Cell viability was determined after 24 h by the MTT assay. The ability
to maintain GSH was determined by treating HT22 cells with different doses of each derivative (1−10 μM) in the presence of 5 mM glutamate.
After 24 h cell extracts were prepared and analyzed for total GSH. Fisetin (10 μM) was used as a positive control. The ability to induce PC12 cell
differentiation (PC12 diff’n) was determined by treating PC12 cells in N2 medium with different doses of each derivative (1−10 μM) for 24 h.
Differentiation was assessed by visual inspection with fisetin (10 μM) as a positive control. Anti-inflammatory activity (microglia) was assessed in N9
microglial cells treated with bacterial lipopolysaccharide alone or in the presence different doses of each derivative (1−10 μM) for 24 h. Fisetin was
used as a positive control. TEAC values, a measure of direct antioxidant activity, were determined using the ABTS+ decolorization assay.
GSH. Modification of the B ring hydroxyls produced mixed
092) improved neuroprotective activity approximately 10- to
results. Modification of one hydroxyl to a methoxy (064, 069,
20-fold but slightly reduced anti-inflammatory activity. Similar
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a
Table 2
a
Half maximal effective concentrations (EC50) for protection in the in vitro ischemia assay were determined by exposing HT22 cells to different
doses of each derivative in the presence of 20 μM IAA for 2 h (HT22/IAA). Cell viability was determined after 24 h by the MTT assay. The ability
to maintain GSH was determined by treating HT22 cells with different doses of each derivative (1−10 μM) in the presence of 5 mM glutamate.
After 24 h cell extracts were prepared and analyzed for total GSH. Fisetin (10 μM) was used as a positive control. The ability to induce PC12 cell
differentiation (PC12 diff’n) was determined by treating PC12 cells in N2 medium with different doses of each derivative (1−10 μM) for 24 h.
Differentiation was assessed by visual inspection with fisetin (10 μM) as a positive control. Anti-inflammatory activity (microglia) was assessed in N9
microglial cells treated with bacterial lipopolysaccharide alone or in the presence different doses of each derivative (1−10 μM) for 24 h. Fisetin was
used as a positive control. TEAC values, a measure of direct antioxidant activity, were determined using the ABTS+ decolorization assay.
to the results with the derivatives of 040, modification of both
the B ring hydroxyl groups to ethoxy groups (018, 025) did not
improve neuroprotective activity. Furthermore, none of these
derivatives regained the ability to maintain GSH or induce
PC12 cell differentiation and they also showed reduced anti-
inflammatory activity. While the methoxy, benzyloxydimethyl
derivative did have somewhat enhanced neuroprotective activ-
ity relative to 027 in the presence of the 3-hydroxyl (059), it
was deficient in anti-inflammatory activity. Furthermore,
separation of the B ring hydroxyls (093, 094) eliminated not
only neuroprotective activity but the other key activities as well.
However, similar to the results with the derivatives of 040,
replacement of the hydroxyls with a single dimethylamino
group at the 4′ position produced a compound with excellent
neuroprotective activity but only in the presence of the
3-hydroxyl (117 vs 119). This compound also regained the
ability to maintain GSH but lacked both neurotrophic and anti-
inflammatory activity. Addition of a single pyrrolidine group to
the 4′ position instead gave a compound that had excellent
neuroprotective activity only in the presence of the 3-hydroxyl
(122 vs 116) and could also maintain GSH levels and induce
PC12 cell differentiation but still had poor anti-inflammatory
activity. Addition of a 3′-hydroxyl to this derivative resulted in a
compound with outstanding neuroprotective activity (142) that
could also maintain GSH under conditions of oxidative stress
and induce PC12 differentiation but had lower anti-
inflammatory activity than 140.
Chalcones are intermediates in the synthesis of flavonoids
and were used to determine the effect of opening up the C-ring
on activity (Table 2). Surprisingly, the chalcones of both the
naphtha (034) and dimethyl derivatives (011) had similar
(034) or enhanced (011) neuroprotective activity compared to
their flavone counterparts and also regained all of the key
activities including the ability to maintain GSH under con-
ditions of oxidative stress. In contrast, the chalcones where both
the B ring hydroxyls were modified had either no (032, 013,
086) or greatly reduced (063) neuroprotective activity.
Furthermore, splitting the B ring hydroxyls of 011 (087)
eliminated the ability to maintain GSH under conditions of
oxidative stress. The conversion of a hydroxyl to a methoxy
(088) also eliminated the ability to promote PC12 cell
differentiation. Thus, of the chalcones tested 011 and 034 are
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superior to fisetin by both the selection criteria and medicinal
chemistry properties.
key activities of the fisetin derivatives show any correlation with
antioxidant activity as defined by the TEAC value (Table 1).
Each of the key activities of the fisetin derivatives shows
distinct structural requirements. For example, within the
flavone structure (Table 1), the maintenance of GSH poses
the strictest structural requirements. It is highly sensitive to
modification of the A ring (040, 027). However, substitution of
the B ring hydroxyls with a single tertiary amino group is
compatible with the maintenance of GSH even in the presence
of A ring modifications (117, 118) as long as a 3-hydroxyl
group is present. In contrast, the anti-inflammatory activity of
the flavone-based derivatives is not particularly sensitive to
modification of the A ring, especially in the presence of a
3-hydroxyl group (e.g., 040 vs 04P). The anti-inflammatory
activity of the flavone-based derivatives, however, is not very
tolerant of modification of the B ring hydroxyls (e.g., 036, 072)
and is also not tolerant of the substitution of the tertiary amino
groups regardless of the presence of a 3-hydroxyl group (e.g.,
117, 119). However, the anti-inflammatory dampening effect of
the tertiary amino groups can be reduced by the readdition of a
hydroxyl group to the 3′ position (140). The PC12 differentia-
tion promoting activity of the flavone-based derivatives shows a
similar but less demanding set of structural requirements as the
GSH maintaining activity for it is somewhat more tolerant of
modifications to the A ring (e.g., 040 but not 027). In addition,
while this activity is sensitive to modifications of the B ring
hydroxyls, it tolerates limited modifications that eliminate the
GSH maintaining activity (e.g., 065).
Once the flavone structure is opened up to give the chalcone
(Table 2), only modification of the B ring hydroxyls affects the
GSH maintaining activity of the fisetin derivatives. The one
exception is 086 which has a methoxy and a benzyloxy group
on the B ring. The PC12 differentiation promoting activity of
the chalcone-based derivatives shows similar structural require-
ments as the GSH maintaining activity. Interestingly, while the
anti-inflammatory activity of the α-naphtha chalcone based
derivatives is eliminated by modification of the B ring hydro-
xyls, the anti-inflammatory activity of the dimethyl chalcone
based derivatives is much more tolerant of this type of
modification.
We have also identified a new quinoline scaffold that reserves
the key structural elements of the flavone but results in
enhanced neuroprotective activity (to >400×) while maintain-
ing the other key activities. Although these derivatives have
greatly reduced free radical scavenging activity relative to fisetin
based on TEAC values (Table 3), several are highly neuro-
protective in our in vitro assay. In addition, while the most
neuroprotective compounds with this scaffold do have hydroxyl
groups, they are not polyphenols. Interestingly, within the
context of this scaffold, the structural requirements for each key
activity are somewhat sharper. For the maintenance of GSH, a
catechol group on the B ring is essential. PC12 differentiation
promoting activity requires both a catechol group on the B ring
and a hydrophobic group on the 4-position of the C ring. The
requirements for anti-inflammatory activity are somewhat less
stringent but are sensitive to modifications of the B ring
hydroxyls in a manner similar to that of the flavone-based
derivatives.
As an alternative approach to improving fisetin, we modified
the flavone scaffold, changing it to a quinoline scaffold
(Table 3) in an attempt to further improve potency and
physiochemical properties while retaining the key structural
elements of the flavone in the quinoline scaffold. The simplest
version, 007, showed a ∼75-fold increase in neuroprotective
activity relative to fisetin, maintained GSH under conditions of
oxidative stress, and had strong anti-inflammatory activity.
However, it did not induce PC12 cell differentiation. We
explored a number of modifications to see if we could enhance
neuroprotective activity and/or restore the PC12 cell differ-
entiating activity. Interestingly, the substitution of an ethoxy
(023) or an isopropoxy (024) for the methoxy group on the
C ring did restore the differentiating activity while also slightly
improving (∼2-fold) the neuroprotective activity relative to
007. Importantly, replacement of the O-methyl group with an
O-cyclopentyl ring resulted in a compound with a >400-fold
decrease in EC50 relative to fisetin for neuroprotective activity
(121) and maintenance of all of the key activities. For all forms
of the quinoline-based derivative, removal of one (022) or both
(021) of the B ring hydroxyls or conversion of one or both of
these hydroxyls to methoxy (001, 017), ethoxy (004), nitro
(111), or chlorine or fluorine (not shown) greatly reduced or
eliminated neuroprotective activity. All of these changes also
reduced or eliminated all of the other key activities. Splitting
the two ring hydroxyls (083, 084) also reduced neuroprotective
activity and eliminated the ability to maintain GSH and induce
PC12 cell differentiation but did not impact anti-inflammatory
activity. In contrast to the derivatives based on the flavone
scaffold, the addition of a single dimethylamino (109, 112) or
pyrrolidine group (110, 113) to the 4′ position of the B ring did
not enhance neuroprotective activity relative to the 3′,4′-
dihydroxy derivative and generally resulted in a reduction or
elimination of the other key activities. Thus, in the presence of
the quinoline scaffold the catechol group on the B ring is
essential for activity.
The transcription factor Nrf2 plays a key role in regulating
GSH metabolism in many different cell types.27 We have shown
that fisetin can induce Nrf2 and that this correlates with its
ability to enhance GSH levels.5 To determine if the derivatives
that can maintain GSH levels do so by increasing Nrf2, we
looked at Nrf2 levels in the nuclei of derivative-treated cells
using fisetin as a positive control (Table 4). Surprisingly, not all
of the derivatives that maintain GSH levels induce Nrf2. This
was particularly true for the derivatives based on the quinoline
scaffold where none of them increased Nrf2 despite being very
effective at maintaining GSH levels.
DISCUSSION AND CONCLUSIONS
■
Several important findings emerge from this study. First, within
the flavone scaffold we were able to demonstrate SARs with
respect to four distinct biological activities and to improve
neuroprotective activity up to 600-fold (140). We also show
that while it is possible to maintain all of the biological activities
that are likely to be important for in vivo efficacy, each of these
activities has specific and unique structural requirements. Thus,
it is possible to balance enhanced neuroprotective activity with
the other key activities as well as the physical characteristics of
the compounds in order to arrive at compounds that have the
best chance for efficacy in vivo. An additional key finding is that
neither the neuroprotective activity nor any of the other three
We have also found that it is possible to separate
neuroprotective activity from the three other key activities of
fisetin. This result suggests that none of the three key activities
play a role in neuroprotection in our in vitro ischemia assay.
Both the differentiation-promoting and anti-inflammatory
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a
Table 3
a
Half maximal effective concentrations (EC50) for protection in the in vitro ischemia assay were determined by exposing HT22 cells to different
doses of each derivative in the presence of 20 μM IAA for 2 h (HT22/IAA). Cell viability was determined after 24 h by the MTT assay. The ability
to maintain GSH was determined by treating HT22 cells with different doses of each derivative (1−10 μM) in the presence of 5 mM glutamate.
After 24 h cell extracts were prepared and analyzed for total GSH. Fisetin (10 μM) was used as a positive control. The ability to induce PC12 cell
differentiation (PC12 diff’n) was determined by treating PC12 cells in N2 medium with different doses of each derivative (1−10 μM) for 24 h.
Differentiation was assessed by visual inspection with fisetin (10 μM) as a positive control. Anti-inflammatory activity (microglia) was assessed in N9
microglial cells treated with bacterial lipopolysaccharide alone or in the presence different doses of each derivative (1−10 μM) for 24 h. Fisetin was
used as a positive control. TEAC values, a measure of direct antioxidant activity, were determined using the ABTS+ decolorization assay.
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In summary, starting with the multitarget polyphenol fisetin,
we have generated a number of derivatives with greatly
enhanced neuroprotective activity (e.g., 011, 50 nM; 121,
7 nM; 140, 5 nM) in a cell culture-based model of ischemia.
Many of the more potent fisetin derivatives also have good
CNS medicinal chemical properties. In addition, some of these
derivatives maintain the other three key activities of fisetin
including anti-inflammatory, neurotrophic, and GSH-maintain-
ing activities, making them good candidates for further testing
in animal models of stroke as well as other neurological
diseases. In creating these derivatives, we have shown that it is
possible to enhance a primary activity of a polyphenol such as
fisetin while at the same time maintaining other key activities
that are not necessarily directly related to this primary activity.
Thus, we are able to maintain the multitarget qualities while
improving both the physiochemical and pharmacological
properties of the compound.
Table 4
compd
Nrf2
fisetin
04P
007
011
034
086
117
118
121
122
140
yes
yes
no
yes
yes
yes
no
no
no
no
no
no
142
a
The ability of the derivatives that maintain GSH levels to induce the
transcription factor Nrf2 was assayed by SDS−PAGE and Western
blotting of nuclear extracts of untreated and derivative-treated cells.
Fisetin treatment was used as a positive control.
EXPERIMENTAL SECTION
■
activities could have critical roles in maintaining CNS function
in vivo but are less likely to be relevant in an in vitro assay with
a single cell type. What is more surprising is that the ability to
maintain GSH is not essential for neuroprotection in the in
vitro ischemia assay, as GSH loss is a component of this cell
death paradigm.6 However, the compounds with the lowest
EC50 values for neuroprotection are all effective at maintaining
GSH levels. Furthermore, many of the effective neuroprotective
compounds that do not maintain GSH are also not good
antioxidants as defined by the TEAC assay, an in vitro assay for
antioxidant activity. Together, these results suggest that the
neuroprotection by these compounds is mediated by some
other, as yet undefined, action that is independent of GSH.
This is currently under investigation.
In addition, we also found that the ability to maintain GSH
levels did not correlate with the induction of Nrf2 by the
compounds. There are a number of other mechanisms for
maintaining GSH levels that could be modulated by these
compounds including reduction of GSH utilization or
inhibition of GSH export.27 These possibilities will be explored
in future studies.
Many of our most effective fisetin derivatives also have
improved medicinal chemical properties in terms of HBD,
CLogP, and tPSA, falling within the criteria for CNS drugs.26,33
Hydrogen bonding properties of drugs can significantly
influence their CNS uptake profiles. Polar molecules are
generally poor CNS agents, and low lipophilicity (CLogP) and
high hydrogen bonding decrease BBB penetration.33 Another
important aspect of our fisetin derivatives is that they all lack A
ring hydroxyl groups which are known to be subject to
modification following oral administration.28 Thus, they are less
likely to be metabolized in this way, leading to enhanced
bioavailability and brain penetration.
Recent studies in our laboratory have shown that fisetin is
effective in multiple animal models of neurological disorders
including stroke6 and Huntington’s disease.29 Furthermore,
fisetin can reduce both the kidney and CNS complications of
diabetes in the Akita model of type 1 diabetes.30 Thus, it shows
promise for the treatment of multiple diseases for which there
are currently no good treatments. The identification and
characterization of more efficacious derivatives are the first
steps in moving this lead compound toward the clinic.
Biology. Cell Culture. Fetal calf serum (FCS) and dialyzed FCS
(DFCS) were from Hyclone (Logan, UT). Dulbecco’s modified
Eagle’s medium (DMEM) was purchased from Invitrogen (Carlsbad,
CA). HT22 cells6 were grown in DMEM supplemented with 10% FCS
and antibiotics. PC12 cells were grown in DMEM supplemented with
10% FCS, 5% horse serum, and antibiotics. N9 microglial cells were
grown in DMEM supplemented with 10% FCS, 1× nonessential
amino acids, 1× essential amino acids, and antibiotics.
Cytotoxicity Assay. Cell viability was determined by a modified
version of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay based on the standard procedure.6 Cells were
seeded onto 96-well microtiter plates at a density of 5 × 103 cells per
well. For the in vitro ischemia assay, the next day, the medium was
replaced with DMEM supplemented with 7.5% DFCS and the cells
were treated with 20 μM iodoacetic acid (IAA) alone or in the
presence of the different derivatives. After 2 h the medium in each well
was aspirated and replaced with fresh medium without IAA but
containing the derivatives. At 20 h later, the medium in each well was
aspirated and replaced with fresh medium containing 2.5 μg/mL
MTT. After 4 h of incubation at 37 °C, the cells were solubilized with
100 μL of a solution containing 50% dimethylformamide and 20%
SDS (pH 4.7). The absorbance at 570 nm was measured on the
following day with a microplate reader (Molecular Devices). Results
were confirmed by visual inspection of the wells. Controls included
compound alone to test for toxicity and compound with no cells to
test for interference with the assay chemistry. All of the derivatives
were tested twice in this assay, and those that showed a strongly
positive response (EC50 < 1 μM) were tested a third time for
confirmation.
Differentiation Assay. PC12 cells in N2 medium were treated
with the derivatives (1−10 μM) or fisetin (10 μM) as a positive
control for 24 h, at which time the cells were scored for the presence
of neurites. PC12 cells produce neurites much more rapidly when
treated in N2 medium than when treated in regular growth medium.
For each treatment, 100 cells in each of three separate fields were
counted. Cells were scored positive if one or more neurites with >1
cell body diameter in length were observed. All of the derivatives were
tested twice in this assay, and those that showed a positive response
were tested a third time for confirmation.
Anti-Inflammatory Assay. Mouse N9 microglial cells plated in
DME with 7.5% DFCS were treated with 10 μg/mL bacterial
lipopolysaccharide (Sigma) alone or in the presence of the fisetin
derivatives (1−10 μM) or fisetin (10 μM) as a positive control. After
24 h the medium was removed, spun briefly to remove floating cells
and 100 μL assayed for nitrite using 100 μL of the Griess reagent
(Sigma) in a 96-well plate. After incubation for 10 min at room
temperature the absorbance at 550 nm was read on a microplate
reader. All of the derivatives were tested twice in this assay, and those
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Journal of Medicinal Chemistry
Article
that showed a positive response were tested a third time for
confirmation.
Total Glutathione. Total intracellular glutathione was determined
by a chemical assay as described.31 All of the derivatives were tested
twice in this assay, and those that showed a positive response were
tested a third time for confirmation.
for 12 h at 40 °C. Methanol was evaporated, and the residue was diluted
with water, neutralized with 1 N HCl, and extracted with ethyl acetate.
The organic layer was washed with brine solution, dried (Na2SO4), and
evaporated. Solid residues were recrystallized from CH2Cl2/hexane and
liquid residues were purified by flash chromatography using silica gel
(230−400 mesh) with 10−30% EtOAc/hexane, giving chalcones with
30−90% yield.
General Procedure B (Methyl/Ethyl Deprotection) for the
Synthesis of Compounds 002, 011, 027, 028, 034, 041, 087,
093, 094, 140, and 142. To a stirred and cooled 0 °C solution of
suitably protected starting material (1 equiv) in CH2Cl2 (5 mL/mmol)
was added BBr3 (2 equiv/alkoxy group), and the mixture was stirred
overnight at room temperature under nitrogen atmosphere. The
reaction mixture was quenched by adding 5% Na2HPO4 solution and
extracted with CH2Cl2. The combined organic extracts were washed
with saturated NaHCO3, brine, dried (Na2SO4), and evaporated. The
resulting solids were recrystallized from methanol.
Method C for the Synthesis of Chalcone 088. To a stirred
solution of chalcone 086A (74.7 mg, 0.203 mmol) in MeOH (2 mL)
was added p-toluenesulfonic acid (77.3 mg, 0.407 mmol). The reaction
mixture was stirred for 3 h at room temperature. After completion of
the reaction, the solvent was evaporated and the residue was diluted
with water (20 mL), then neutralized with saturated NaHCO3, and
extracted with EtOAc. Combined extracts were washed with brine,
dried (Na2SO4), and evaporated. The residue was purified by flash
chromatography using silica gel (230−400 mesh) with 20% EtOAc/
hexane, giving 088 on 94% yield as a yellow solid.
General Procedure D (Debenzylation) for the Synthesis of
Compounds 64, 069, 070, 072, and 092. The benzyl protected
flavones and flavonols were dissolved in 1:1 EtOAc/methanol (10 mL/
mmol) and then treated with 5% palladium on charcoal (5% w/w). The
mixture was stirred under hydrogen atmosphere (balloon pressure)
overnight. The reaction mixture was filtered and the solvent was
evaporated. The resulting solids were recrystallized from dichloro-
methane/methanol.
General Procedure E for the Synthesis of Flavone
Derivatives 018, 038, 058, 068, 089, 115, 116, 119, and
120. A solution of chalcone (1 equiv) and iodine (0.01 equiv) in
DMSO (1 mL/mmol) was heated at 130 °C for 3−6 h. The reaction
mixture was cooled and diluted with water, extracted with CH2Cl2,
washed with aqueous saturated Na2S2O3, dried (Na2SO4), and
evaporated. Solid residues were recrystallized from CH2Cl2/hexane
and liquid residues were purified by flash chromatography using silica
gel (230−400 mesh) with 30−80% EtOAc/hexane, giving flavones
with 50−95% yield.
General Procedure F for the Synthesis of Flavonol
Derivatives 025, 036, 037, 059, 065, 090, 091, 114, 117, 118,
122, 139, and 141. To a stirred and cooled 0 °C solution of
chalcone in MeOH (5 mL/mmol) was added 5.4% NaOH (3.2
mL/mmol) followed by 30% H2O2 (0.37 mL/mmol) dropwise, and the
mixture was stirred for 3 h at 0 °C. Then the ice bath was left in place
but not recharged, and stirring was continued overnight. The reaction
mixture was neutralized with 2 M HCl, and the resulting precipitate was
collected by filtration and washed with water and recrystallized from
dichloromethane to give flavonols with 40−90% yield.
General Procedure G for the Synthesis of Quinoline
Derivatives 001, 004, 007, 017, 021−024, 083, 084, 109−113,
and 121. To a stirred solution of 2′-aminoacetophenone (1 equiv)
and aromatic aldehyde (1 to 3 equiv) in alcohol (3 mL/mmol) was
added H2SO4 (0.75 equiv), and the mixture was refluxed for 12−24 h.
The reaction mixture was cooled, and the solvent was evaporated. The
residue was diluted with water, neutralized with 5% NaHCO3 solution,
and extracted with ethyl acetate. The organic layer was washed with
brine, dried (Na2SO4), and evaporated. Flash chromatography of the
residue over silica gel using 10−50% EtOAc/hexane gave 4-alkoxy-2-
arylquinolines with 15−50% yield.
Analytical Data for Selective Compounds (011, 121, and
140). (E)-3-(3,4-Dihydroxyphenyl)-1-(2-hydroxy-4,5-
dimethylphenyl)prop-2-en-1-one (011). Following general proce-
dure B, 011 was obtained from chalcone 013 as an orange solid
(95% yield): mp 174−177 °C; LCMS purity 99%; 1H NMR (DMSO-d6,
SDS−PAGE and Immunoblotting. For immunoblotting of Nrf2,
nuclear extracts were prepared as described32 from untreated cells and
cells treated with the fisetin derivatives for 1, 2, and 4 h. Fisetin was
used as a positive control. For each derivative, the concentration that
was most effective at preventing cell death was used. Protein con-
centrations were determined using the BCA protein assay (Pierce).
Equal amounts of protein were solubilized in 2.5× SDS sample buffer,
separated on 10% SDS−polyacrylamide gels, and transferred to
nitrocellulose. Equal loading and transfer of the samples were
confirmed by staining the nitrocellulose with Ponceau-S. Transfers
were blocked for 1 h at room temperature with 5% nonfat milk in
TBS/0.1% Tween 20 and then incubated overnight at 4 °C in the
primary antibody diluted in 5% BSA in TBS/0.05% Tween 20. The
1
primary antibodies used were anti-Nrf2 (no. SC13032, /1000) from
Santa Cruz Biotechnology (Santa Cruz, CA) and anti-β-actin (no.
1
5125, /20000) from Cell Signaling (Beverly, MA). The transfers were
rinsed with TBS/0.05% Tween 20 and incubated for 1 h at room
temperature in horseradish peroxidase−goat anti-rabbit or goat anti-
1
mouse (Biorad, Hercules, CA) diluted /5000 in 5% nonfat milk in
TBS/0.1% Tween 20. The immunoblots were developed with the
Super Signal reagent (Pierce, Rockford, IL). All of the derivatives were
tested twice in this assay, and those that showed a positive response
were tested a third time for confirmation.
Determination of the Trolox Equivalent Activity Concen-
tration (TEAC). TEAC values for the flavonoids were determined as
described.31 Briefly, 250 μL of 2,2′-azinobis(3-ethylbenzothiazoline 6-
sulfonate) (ABTS) treated overnight with potassium persulfate and
diluted to an OD of ∼0.7 at 734 nm was added to 2.5 μL of a
derivative solution in ethanol. The change in absorbance due to the
reduction of the ABTS radical cation was measured at 734 nm for
4 min. To calculate the TEAC, the gradient of the plot of the
percentage inhibition of absorbance vs concentration for the derivative
in question was divided by the gradient of the plot for Trolox.
Chemistry: General Methods. All reagents and anhydrous
solvents were obtained from commercial sources and used as received.
1H NMR and 13C NMR were recorded at 500 and 125 MHz,
respectively, on a Varian VNMRS-500 spectrometer using the
indicated solvents. Chemical shift (δ) is given in parts per million
(ppm) relative to tetramethylsilane (TMS) as an internal standard.
Coupling constants (J) are expressed in hertz (Hz), and conventional
abbreviations used for signal shape are as follows: s = singlet; d =
doublet; t = triplet; m = multiplet; dd, doublet of doublets; brs = broad
singlet. Liquid chromatography−mass spectrometry (LCMS) was
carried out using a Shimadzu LC-20AD spectrometer and electrospray
ionization (ESI) mass analysis with a Thermo Scientific LTQ
Orbitrap-XL spectrometer. Melting points were determined with a
Thomas-Hoover capillary melting point apparatus and are uncor-
rected. All final compounds were characterized by LCMS and 1H
NMR and gave satisfactory results in agreement with the proposed
structure. All of the tested compounds have a purity of at least 95%
which was determined by analysis on a C18 reverse phase HPLC
column [Phenomenex Luna (50 mm × 4.60 mm, 3 μm)] using 10−
90% CH3CN/H2O containing 0.02% AcOH with a flow rate of 1 mL/
min (5 min gradient) and monitoring by a UV detector operating at
254 nm. Mass spectra were acquired in the positive mode scanning
over the mass range of 50−1000. LCMS M + H signals were
consistent with the expected molecular weights for all of the reported
compounds. Thin layer chromatography (TLC) used EMD silica gel
F-254 plates (thickness of 0.25 mm). Flash chromatography used
EMD silica gel 60, 230−400 mesh.
General Procedure A for the Synthesis of Chalcone
Derivatives 013, 032, 033, 057, 063, 085, 086, 105−108, 137,
and 138. A mixture of 2′-hydroxyacetophenone (1 equiv), arylaldehyde
(1 equiv), and Ba(OH)2 (1 equiv) in MeOH (3 mL/mmol) was stirred
387
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Journal of Medicinal Chemistry
Article
500 MHz) δ ppm 2.23 (s, 3H), 2.24 (s, 3H), 6.78 (s, 1H) 6.82 (d, J = 8.0
Hz, 1H), 7.23 (dd, J = 8.5, 2.0 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.72 (q,
J = 15.5 Hz, 2H), 8.02 (s, 1H), 9.11 (br s, OH), 9.81 (br s, OH), 12.79 (s,
OH); 13C NMR (DMSO-d6, 125 MHz) δ ppm 18.72, 20.46, 116.18,
116.36, 117.94, 118.45, 118.69, 123.19, 126.65, 127.59, 130.91, 146.04,
146.12, 146.92, 149.59, 161.23, 193.36. LCMS: m/z 285 ([M + H]+). MS
(ESI): m/z calcd for C17H16NO4 ([M + H]+) 285.1082; found 285.1001
([M + H]+).
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NMR, nuclear magnetic resonance; Nrf2, NF-E2
related factor 2; p-TSA, p-toluenesulfonic acid; rtPA,
recombinant tissue-type plasminogen activator; ROS, reactive
oxygen species; SAR, structure−activity relationship; SDS,
sodium dodecyl sulfate; SDS−PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; TBS, Tris buffered saline;
TMS, tetramethylsilane; THP, tetrahydropyran; tPSA, topo-
logical polar surface area; TEAC, Trolox equivalent activity
concentration
4-(4-(Cyclopentyloxy)quinolin-2-yl)benzene-1,2-diol (121).
Following general procedure G, 121 was obtained as a dark yellow
1
solid (16% yield): mp 199−201 °C; LCMS purity 98%; H NMR
(DMSO-d6, 500 MHz) δ ppm 1.66 (m, 2H), 1.79 (m, 2H), 1.89 (m,
2H), 2.07 (m, 2H), 5.30 (m, 2H), 6.85 (d, J = 8.5 Hz, 1H), 7.31 (s,
1H), 7.44 (t, J = 8.0 Hz, 1H), 7.54 (dd, J = 8.5, 2.0 Hz, 1H), 7.67 (t,
J = 8.0 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H),
8.04 (d, J = 7.5 Hz, 1H), 9.21 (brs, OH); 13C NMR (DMSO-d6, 125
MHz) δ ppm 24.19, 32.71, 80.12, 99.28, 115.02, 115.99, 119.41,
120.60, 121.97, 125.21, 128.93, 130.30, 131.11, 145.88, 147.71, 149.11,
157.92, 160.73. LCMS: m/z 322 ([M + H]+). MS (ESI): m/z calcd for
C20H19NO3 ([M + H]+) 322.1437; found 322.1412 ([M + H]+).
3-Hydroxy-2-(3-hydroxy-4-(pyrrolidin-1-yl)phenyl)-4H-
benzo[h]chromen-4-one (140). Following general procedure B,
140 was obtained from compound 139 as an orange red solid (50%
yield): mp 223−225 °C; LCMS purity 98%; 1H NMR (DMSO-d6, 500
MHz) δ ppm 1.88 (s, 4H), 3.45 (s, 4H), 6.74 (d, J = 8.5 Hz, 1H), 7.85
(m, 5H), 8.04 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H), 8.68 (d,
J = 7.5 Hz, 1H), 9.37 (s, 1H); 13C NMR (DMSO-d6, 125 MHz) δ ppm
25.16, 50.19, 114.38, 114.69, 117.94, 120.61, 120.74, 120.99, 122.54,
124.10, 124.87, 128.06, 128.87, 129.77, 135.37, 139.19, 140.29, 146.34,
146.44, 151.65, 172.19. LCMS: m/z 374 ([M + H]+). MS (ESI):
m/z calcd for C23H19NO4 ([M + H]+) 374.1386; found 374.1402
([M + H]+).
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ASSOCIATED CONTENT
* Supporting Information
Detailed spectral analysis for all other compounds, structures,
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spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
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ABBREVIATIONS USED
■
ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid);
BCA, bicinchoninic acid assay; BSA, bovine serum albumin;
CNS, central nervous system; DFCS, dialyzed fetal calf serum;
DME, Dulbecco’s modified Eagle’s; DMEM, Dulbecco’s
modified Eagle’s medium; DMSO, dimethylsulfoxide; EC50,
half maximal effective concentration; EtOAc, ethyl acetate; ESI,
electrospray ionization; FCS, fetal calf serum; FDA, Food and
Drug Administration; G3PDH, glycolytic enzyme glyceralde-
hyde 3-phosphate dehydrogenase; GSH, glutathione; HBA,
hydrogen bond acceptor; HBD, hydrogen bond donor; IAA,
iodoacetic acid; LPS, lipopolysaccharide; LCMS, liquid
chromatography−mass spectrometry; MS, mass spectrometry;
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