A synthetic 7,8-dihydroxyflavone derivative promotes neurogenesis and exhibits potent antidepressant effect

Article abstract of DOI:10.1021/jm101206p

7,8-Dihydroxyflavone is a recently identified small molecular tropomyosin-receptor-kinase B (TrkB) agonist. Our preliminary structural-activity relationship (SAR) study showed that the 7,8-dihydroxy groups are essential for the agonistic effect. To improv

Full text of DOI:10.1021/jm101206p

8274 J. Med. Chem. 2010, 53, 8274–8286
DOI: 10.1021/jm101206p
A Synthetic 7,8-Dihydroxyflavone Derivative Promotes Neurogenesis and Exhibits Potent
Antidepressant Effect
Xia Liu,† Chi-Bun Chan,† Sung-Wuk Jang,† Sompol Pradoldej,† Junjian Huang,† Kunyan He,† Lien H. Phun,‡
Stefan France,‡ Ge Xiao,§ Yonghui Jia, Hongbo R. Luo, and Keqiang Ye*,†
†Department of Pathology and Laboratory Medicine Emory University School of Medicine, Room 141 Whitehead Building, 615 Michael Street,
Atlanta, Georgia 30322, United States , ‡School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, NW,
Atlanta, Georgia 30332-0400, United States, §Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, Georgia 30341,
United States, and Department of Pathology and Lab Medicine, Harvard Medical School and Children’s Hospital Boston,
10214 Karp Research Building, 1 Blackfan Circle, Boston, Massachusetts 02115, United States
Received April 27, 2010
7,8-Dihydroxyflavone is a recently identified small molecular tropomyosin-receptor-kinase B (TrkB)
agonist. Our preliminary structural-activity relationship (SAR) study showed that the 7,8-dihydroxy
groups are essential for the agonistic effect. To improve the lead compound’s agonistic activity, we have
conducted an extensive SAR study and synthesized numerous derivatives. We have successfully
identified 40-dimethylamino-7,8-dihydroxyflavone that displays higher TrkB agonistic activity than
that of the lead. This novel compound also exhibits a more robust and longer TrkB activation effect in
animals. Consequently, this new compound reveals more potent antiapoptotic activity. Interestingly, chronic
oral administration of 40-dimethylamino-7,8-dihydroxyflavone and its lead strongly promotes neurogenesis
in dentate gyrus and demonstrates marked antidepressant effects. Hence, our data support that the
synthetic 40-dimethylamino-7,8-dihydroxyflavone and its lead both are orally bioavailable TrkB
agonists and possess potent antidepressant effects.
Introduction
compound displays potent neurotrophic effect in stroke and
Parkinson disease animal models in a TrkB-dependent manner.
Thus, 7,8-dihydroxyflavone is a novel TrkB agonist4
Neurotrophins are growth factors that regulate the develop-
ment and maintenance of the peripheral and the central nervous
system.1 Brain-derived neurotrophic factor (BDNFa) is a
member of the neurotrophin family, which includes nerve
growth factors (NGF), NT-3, and NT-4/5.2 BDNF, like the
other neurotrophins, exerts its biological functions on neurons
through TrkB.3 BDNF binding to TrkB triggers its dimerization
through conformational changes and autophosphorylation of
tyrosine residues in its intracellular domain, resulting in activa-
tion of the three major signaling pathways involving mitogen-
activated protein kinase (MAPK), phosphatidylinositol 3-kinase
(PI3K), and phospholipase C-γ1. To pharmacologically mimic
BDNF, we have developed a cell-based assay and identified a
small molecule 7,8-dihydroxyflavone that binds the extracellular
domain of TrkB and triggers its dimerization and activation.
Intraperitoneal administration of this compound elicits robust
TrkB activation in mouse brain. Moreover, like BDNF, this
Although BDNF/TrkB signaling is traditionally thought of
in terms of synaptic plasticity and neuroprotection,5,6 recent
evidence indicates that these molecules are also critical for neu-
rogenesis and antidepressant drug efficacy. Antidepressant
treatment induces BDNF mRNA expression as well as au-
tophosphorylation and activation of TrkB in the brain.7,8 The
behavioral effects of antidepressants in the forced swim test are
attenuated in mice with only one active BDNF allele (BDNF(
mice), completely lacking BDNF specifically in forebrain or
expressing a dominant negative form of TrkB (trkB.T1).8,9
However, in this work, TrkB activation and the behavioral
effects of these drugs are observed with acute antidepressant
treatment, leaving the questions concerning chronic antidepres-
sant efficacy unanswered. A breakthrough occurred recently
linking antidepressants, TrkB, and neurogenesis with the
publication of a landmark paper by Parada’s group.10 They
showed that ablation of TrkB specifically in hippocampal
neural progenitor cells prevents chronic antidepressant-induced
neurogenesis and renders the mice behaviorally nonresponsive
to chronic antidepressant treatment. Combined, these results
suggest a model whereby chronic antidepressant treatment
induces BDNF expression and long-term activation of TrkB,
leading to increased neurogenesis and an antidepressant
effect.11-13 As the result of a rash of recent data, a “neuro-
trophic” hypothesis of antidepressant activity has been sug-
gested. Administration of chronic, but not acute, monoamine
antidepressant drugs enhances adult neurogenesis in the sub-
granular zone of the dentate gyrus of rodents and nonhuman
*To whom correspondence should be addressed. Phone: 404-712-2814.
E-mail: kye@emory.edu.
a Abbreviations: 1NMPP1, 1-(1,1-dimethylethyl)-3-(1-naphthalenyl-
methyl)-1H-pyrazolo[3,4-d ]pyrimidin-4-amine; 40-DMA-7,8-DHF, 40-
dimethylamino-7,8-dihydroxyflavone; 7,8-DHF, 7,8-dihydroxyflavone;
BDNF, brain-derived neurotrophic factor; CBC, complete blood count;
DIV, day in vitro; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked
immunosorbent assay; FITC, Fluorescein isothiocyanate; ip, intraperito-
neal; KA, kainic acid; LDH, lactate dehydrogenase; MAPK, mitogen-
activated protein kinase; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; NGF, nerve growth factor; PBS, phosphate
buffered saline; PBST, phosphate buffered saline Tween-20; ppm, parts
per million; TMB, 3,30,5,50-tetramethylbenzidine; TrkB, tropomyosin-
receptor-kinase B.
r
pubs.acs.org/jmc
Published on Web 11/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8275
primates, while blocking neurogenesis by irradiation attenu-
ates the behavioral antidepressant-like effects of these drugs
in some rodent strains.14-17 These results suggest that
enhanced adult hippocampal neurogenesis is required for
some aspects of antidepressant drug efficacy and may help
to explain why chronic treatment is required for therapeutic
benefit.
In this study, we report that a hydrogen bond acceptor on
the 40-position of the flavone B ring is critical for 7,8-dihy-
droxyflavone’s TrkB agonistic effect. The synthetic 40-dimethy-
lamino-7,8-dihydroxyflavone possesses a more potent agonistic
effect on TrkB than the lead. Moreover, these two compounds
are orally bioavailable. They both strongly provoke neuro-
genesis and display robust antidepressant effects in a TrkB-
dependent manner.
To further explore the structure-activity relationships, we
examined the role of different hydroxy groups in each ring
(Supporting Information, Figure 1A,B). Akt ELISA anal-
ysis showed that an 8-hydroxy group on the A ring was
essential for the stimulatory effect, as 5,7-DHF, 5,6-DHF,
and 5,6,7-THF each lost the effect as compared to 7,8-DHF.
These compounds even decreased Akt activity as compared
to the DMSO control. The dihydroxy groups on the B ring
displayed relatively higher activity when compared to com-
pounds with dihydroxy groups on the A ring. However, they
were not significantly higher than the DMSO control. None
of the single hydroxy flavone derivatives exhibited a notable
stimulatory effect. Moreover, none of the tested trihydroxy
flavones or dimethoxy flavones demonstrated any substan-
tial effect on Akt activation. Similarly, neither coumarin deriva-
tives nor 4-Cl-7-hydroxy-8-methoxy-isoflavone had any effect
as compared to the DMSO control. It is noteworthy that 6,7-
DHF and 6,7,30-THF exhibited significant activity, suggest-
ing that 6,7-dihydroxy groups retain partial activity (Supporting
InformationFigure1C). Together,theSARstudy suggests that
the catechol group (7,8-dihydroxy on the A ring) might be
indispensable for the agonistic activity. A 40-hydroxy group
on the B ring reduces the activity, whereas a 30-hydroxy group
increases the activity.
Chemical Synthesis of 7,8-Dihydroxyflavone Derivatives.
The above SAR suggests that the 7,8-dihydroxy groups on
the A ring should be kept intact. Hence we chose to leave the
A ring intact and focused on modifying the B and C rings. To
optimize the lead compound and conduct a more thorough
SAR study, we synthesized a few more derivatives of 7,8-DHF.
The synthetic pathways are outlined in Figure 2. In Figure 2A,
2-hydroxy-3,4-dimethoxyacetophenone (1) was first treated
with pyridine and various benzoyl chlorides under refluxing
conditions, followed by an acid-induced dehydrative cyclization
to generate the desired unprotected flavones. To synthesize the
NH-replaced flavones in the middle C ring, a mixture of ethyl
3-(4-fluorophenyl)-3-oxopropanoate and 2,3-dimethoxybenze-
namine were refluxed in the presence of AcOH (cat.) and
CaSO4 in EtOH (100 mL) at 75 °C under N2, followed by
cyclization and deprotection to generate the desired products
in good yields (Figure 2B). The yield for each step in the
synthesis was indicated.
40-Dimethylamino-7,8-dihydroxyflavone Displays More Potent
Stimulatory Effect on TrkB Receptor than the Lead. To compare
the TrkB activation by these synthetic compounds, we prepared
primary cortical cultures and treated them with 500 nM of
various compounds for 15 min and collected the cell lysates.
Phospho-Akt ELISA analysis revealed that 40-dimethyl-
amino-7,8-dihydroxyflavone (compound 6, 40-DMA-7,8-DHF)
and 7,8-dihydroxy-2(pyrimidin-5-yl)-4H-chromen-4-one (com-
pound 8) strongly activated Akt, followed by 40-fluoro-7,8-DHF
(compound 7). However, 2-(4-fluoro-phenyl)-7,8-dihydroxy-
quinolin-4(1H)-one (compound 13) and 7,8-dihydroxy-2-phe-
nylquinolin-4(1H)-one (compound 14) failed to provoke Akt
activation, indicating that the oxygen in the middle C ring is
essential for 7,8-DHF’s agonistic effect to TrkB (Figure 3A,B).
These data suggest that replacing the H-bond accepting oxygen
with H-bond donating NH abolishes its stimulatory effect. To
quantitatively compare the activity between 40-DMA-7,8-DHF
and 7,8-DHF, we conducted a titration assay. Both 7,8-DHF
and 40-DMA-7,8-DHF triggered Akt activation at concentra-
tions as low as 10 nM, and Akt activity gradually increased
as drug concentration escalated. However, 40-DMA-7,8-DHF
displayed more robust activity than 7,8-DHF at 10 and 50 nM.
Results
Structure-Activity Relationship Study. 7,8-Dihydroxyfla-
vone (7,8-DHF) is a small molecular TrkB agonist. Our pre-
liminary SAR supports that the 7,8-catechol moiety is essential
for the agonistic effect by 7,8-DHF.4 To explore the SAR in
depth, we examined the TrkB stimulatory activity by numer-
ous flavonoid derivatives. The numeric positions and each
ring’s nomenclature were designed (Figure 1A). The com-
pounds were dissolved in dimethyl sulfoxide (DMSO), then
diluted into 500 μM with 1ꢀ PBS (final vehicle contains 10%
DMSO/phosphate buffered saline (PBS)) (Figure 1B). The
primary rat cortical neurons (13 day in vitro (DIV)) were
treated with 500 nM compounds for 20 min. The cell lysates
were analyzed by immunoblotting. The positive control
BDNF (100 ng/mL) strongly activated TrkB, as TrkB
was robustly phosphorylated. As expected, 7,8,20-trihydroxy-
flavone, 5,7,8-trihydroxyflavone, 3,7,8,20-tetrahydroxyfla-
vone, 7,8-DHF, and 3,7-dihydroxyflavone all stimulated TrkB
activation as compared to control (Figure 1C, top panel lane 3
and 7-10). Interestingly, 7,30-dihydroxyflavone and 7,8,30-
trihydroxyflavone displayed an even more robust stimula-
tory effect on TrkB phosphorylation than the lead 7,8-DHF
(Figure 1C, top panel lane 4-5), indicating that the 30-
hydroxy group in B ring can effectively escalate 7,8-DHF’s
agonistic activity. By contrast, 7,8,40-trihydroxyflavone,
7-hydroxy-40methoxyflavone, and 8-hydroxy-7-methoxyfla-
vone barely activated TrkB as compared to vehicle control
(Figure 1C, top panel, lanes 6, 11, and 12). Strikingly, the
highly hydroxylated flavone derivative, 3,5,7,8,30,40-hexahy-
droxylatedflavone, completely blocked TrkB phosphoryla-
tion (Figure 1C, top panel, last lane), suggesting the hyper-
hydroxylated compound even antagonizes the TrkB activity.
Immunoblotting analysis demonstrated Akt phosphoryla-
tion tightly coupled to TrkB activation (Figure 1C, bottom
panel). Because no quantitative approach for mouse or rat
TrkB phosphorylation is currently available, we monitored
Akt activity by enzyme-linked immunosorbent assay (ELISA) in
the primary neurons instead. In alignment with the immuno-
blotting results, Akt phosphorylation by these compounds
exhibited the similar activation patterns. 7,8,30-Trihydroxy-
flavone displayed a more than 2-fold higher activity than
vehicle control, and the 40-hydroxylated derivatives failed to
activate Akt (Figure 1D). Therefore, the hydroxy groups on
the B ring can regulate 7,8-DHF’s stimulatory activity on
TrkB receptor. The 20-hydroxy and, especially, the 30-hydroxy
groups elevate the agonistic effect, whereas the 40-hydroxy
group diminishes its stimulatory effect.

8276 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Liu et al.
Figure 1. Structure-activity relationship study. (A) 7,8-Dihydroxyflavone chemical structure with every position in each ring labeled.
(B) Chemical structures of flavonoids from Indofine, Inc. (C) Immunoblotting analysis with neuronal lysates. Primary rat cortical neurons
from E17 embryos (13 DIV) were treated with 500 nM various chemicals for 15 min. The neuronal cell lysates were collected and resolved on
10% SDS-PAGE. Immunoblotting was conducted with various antibodies. p-TrkB Y817 antibody was employed at 1:20000-40000 dilution.
(D). Phospho-Akt 473 ELISA. The cell lysates (20 μg/sample) from the neurons treated with various indicated drugs were analyzed by p-Akt
ELISA. The quantitative p-Akt activity in ELISA correlated with TrkB activity. Results were expressed as mean ( SEM (*P < 0.05, compared
with the vehicle control group, Student t test, n=3).
At concentration of 100 nM or higher, p-Akt ELISA by
40-DMA-7,8-DHF was slightly decreased and displayed com-
parable activity as that of 7,8-DHF. This effect might be caused
by other unknown pathways activated by 40-DMA-7.8-DHF
that subsides Akt activation at higher concentration (Figure 3B,
right panels). The immunoblotting of TrkB phosphorylation
correlated with the observed Akt activation pattern (Supporting
Information Figure 2). To further study 40-DMA-7,8-DHF’s
kinetics on TrkB activation, we treated the primary neurons for
various time points. 40-DMA-7,8-DHF swiftly activated TrkB
as soon as 5 min, and the signal slightly reduced at 10 min and
decayed back to baseline at 30-180 min (Figure 3C, top left
panel). Once again, Akt activation pattern appears temporally
coupled to TrkB activation (Figure 3C, third left panel). How-
ever, MAPK phosphorylation peaked at 10 min (Figure 3C,
fourth left panel). To compare the stimulatory effect on TrkB
receptor in mouse brain, we orally administrated 1 mg/kg of
these compounds into C57BL/6J mice and monitored TrkB

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8277
Figure 2. Organic synthesis of various 7,8-dihydroxyflavone derivatives. (A) and (B) Schematic diagram of synthetic pathways for various
7,8-DHF derivatives.
activation at different time points. Clearly, both compounds
elicited TrkB activation (p-TrkB 706) at 1 h and the activity of
TrkB gradually escalated with time. 7,8-DHF-triggered TrkB
activation climaxed at about 1-2 h, whereas 40-DMA-7,8-DHF
peaked at 4 h and partially decayed at 8-16 h. The downstream
effectors of Akt and MAPK were potently activated by both
compounds. As expected, both p-Akt and p-MAPK activated
by 40-DMA-7,8-DHF were stronger than 7,8-DHF, and the
signals lasted longer for 40-DMA-7,8-DHF than 7,8-DHF
(Figure 3C, middle panels). Quantitative analysis with p-Akt
473 ELISA also supported that 40-DMA-7,8-DHF was more
potent than 7,8-DHF in triggering Akt activation in mouse brain
(Figure 3C, right panel). Hence, 40-DMA-7,8-DHF possesses a
higher agonistic effect on TrkB than the parental compound
7,8-DHF and its stimulatory effect sustained longer in animals
as well.
with a 7-methoxy group slightly reduced its agonistic effect.
Switching both hydroxy groups to dimethoxy groups sub-
stantially decreased the agonistic effect (Figure 3D). Thus,
the 7,8-dihydroxy groups are essential for the flavonoid’s
agonistic activity.
40-Dimethylamino-7,8-dihydroxyflavone Possesses More
Robust Antiapoptotic Activity than the Lead Compound. To
quantitatively compare the antiapoptotic activity of these
two TrkB agonists, we prepared cortical neurons and pre-
treated the cells with various indicated concentrations of 40-
DMA-7,8-DHF and 7,8-DHF for 30 min, followed by 50 μM
glutamate for 16 h. The cell lysates were quantitatively analyzed
with an active caspase-3 ELISA. Glutamate-provoked caspase-
3 activation was substantially blocked by both compounds
at 50 nM or higher concentrations. However, at 10 nM,
40-DMA-7,8-DHF displayed a more robust inhibitory effect
than 7,8-DHF (Figure 4A,B). These results fit with the TrkB
receptor activation status by 40-DMA-7,8-DHF and 7,8-DHF
(Figure 3B). To investigate whether these compounds exert
any neuroprotective effects in animals, we conducted a time
To further examine the role of each hydroxy group in
40-DMA-7,8-DHF’s agonistic activity, we prepared the
7-methoxy and 7,8-dimethoxy derivatives. Immunoblotting
analysis demonstrated that replacement of the 7-hydroxy

8278 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Liu et al.
Figure 3. 40-Dimethylamino-7,8-dihydroxyflavone displays more potent TrkB stimulatory effect than parental 7,8-dihydroxyflavone.
(A) Chemical structures of various 7,8-DHF derivatives. (B) Phospho-Akt ELISA assay by the synthetic compounds in cortical neurons.
Primary cortical cultures from E17 rat embryos were treated with 500 nM of various 7,8-DHF derivatives. The cell lysates were analyzed by the
ELISA (left panel) (*, P < 0.05; ***, P < 0.001 vs vehicle, Student’s t test). Different doses of 40-DMA-7,8-DHF and 7,8-DHF were incubated
with primary cortical neurons for 15 min. The cell lysates (20 μg) were analyzed with p-Akt ELISA (right panel) (*, P < 0.05; **, P < 0.01; ***,
P < 0.001 vs control; one-way ANOVA: b, P < 0.01; c, P < 0.001 vs 7,8-DHF at same concentration, Student’s t test). The data were from two
sets of replicated experiments (mean ( SEM). (C) Time course assay with 40-DMA-7,8-DHF. Rat primary neurons were treated with 500 nM
40-DMA-7,8-DHF from various time points. The neuronal lysates were analyzed with various antibodies. 40-DMA-7,8-DHF rapidly activated
TrkB and its downstream signaling cascades (left panels). 40-DMA-7,8-DHF revealed longer period of TrkB activation in mouse brain. One
mg/kg of 40-DMA-7,8-DHF and 7,8-DHF were orally administrated into C57 BL/6J mice and TrkB phosphorylation and its downstream
signaling cascades including Akt and MAPK in mouse brain were analyzed by immunoblotting at various time points. TrkB activation by
40-DMA-7,8-DHF peaked at 4 h, whereas the maximal TrkB activation by 7,8-DHF in mouse brain occurred at 1-2 h (middle panels). P-Akt
4734 ELISA in drug treated mouse brain was analyzed (right panel) (***,P < 0.001 vs control; one-way ANOVA: a, P < 0.05; c, P < 0.001 vs
7,8-DHF at same concentration, Student’s t test). The data were from two sets of replicated experiments (mean ( SEM). (D) 7,8-Dihydroxy
groups are essential for the flavone’s agonistic effect. Different methoxy replaced derivatives were tested on primary neurons by
immunoblotting assays.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8279
Figure 4. 40-Dimethylamino-7,8-dihydroxyflavone prevents neurons from apoptosis in a TrkB-dependent manner. (A,B) Active caspase-3
ELISA assay. Cortical neurons were prepared from E16 rat embryonic. The neurons were pretreated with different doses of compounds as
indicated for 30 min, followed by 50 μM glutamate for 16 h. The cell lysates were analyzed by active caspase-3 ELISA. (C) 40-DMA-7,8-DHF
and 7,8-DHF prevent KA-elicited neuronal cell death. C57BL/6J mice were orally administrated with 5 mg/kg of 40-DMA-7,8-DHF and 7,
8-DHF; at different time points, the mice were ip injected with 20 mg/kg KA for 2 h. The brain lysates were analyzed by immunoblotting with
anti-p-TrkB, antiactive caspase-3 antibodies, respectively. (D) TrkB activation is indispensable for the neuroprotective effect of 40-DMA-7,
8-DHF. 40-DMA-7,8-DHF and 7,8-DHF suppressed KA-induced caspase-3 activation in TrkB F616A mutant knockin mice, which can not be
blocked by 1NMPP1 (top panel). TrkB F616A was strongly activated by 40-DMA-7,8-DHF and 7,8-DHF, which was blocked by 1NMPP1
(middle panel).
course assay. A titration assay revealed that 7,8-DHF induced
TrkB activation in a dose-dependent manner upon oral admin-
istration (Supporting Information Figure 3). Our previous
study suggested that 7,8-DHF displayed a robust neuropro-
tective effect at a dose of 5 mg/kg.4 Hence, we administrated
both compounds (5 mg/kg) orally by gavage. At either 0, 2,
or 6 h, we intraperitoneally administrated kainic acid (KA)
(20 mg/kg). After 2 h, we sacrificed the mice and prepared the
hippocampal regions. Immunoblotting with mouse brain
lysates demonstrated that KA-induced caspase-3 activation
was slightly reduced with time lapse, which inversely corre-
lated with TrkB activation by 40-DMA-7,8-DHF (Figure 4C,
left panels). Nonetheless, KA-induced caspase-3 activation was
reduced by 7,8-DHF at 4 h, and active caspase-3 was slightly
increased at 8 h. This kinetic spectrum tightly coupled to the
TrkB activation status by 7,8-DHF (Figure 4C, right panel).
To explore whether the neuroprotective actions of these
small molecules is dependent on TrkB activation in vivo,
we employed TrkB F616A knock-in mice, where it has
been shown that TrkB F616A can be selectively blocked by
1-(1,1-dimethylethyl)-3-(1-naphthalenylmethyl)-1H-pyra-
zolo[3,4-d ]pyrimidin-4-amine (1NMPP1), a TrkB F616A
inhibitor, resulting in effective TrkB-null phenotypes.18
Because 1NMPP1 selectively inhibits TrkB F616A activa-
tion by 7,8-DHF, we reasoned that blockade of TrkB F616A
signaling by 1NMPP1 in mice would make the neurons
vulnerable to KA-provoked neuronal cell death. As expected,
KA caused significant caspase-3 activation, which was reduced
by 40-DMA-7,8-DHF and 7,8-DHF pretreatment. Interestingly,
1NMPP1 pretreatment abolished 40-DMA-7,8-DHF and
7,8-DHF’s protective effect in F616A mice (Figure 4D, top
panel). Accordingly, TrkB phosphorylation by 40-DMA-7,
8-DHF and 7,8-DHF was notably blocked by 1NMPP1
pretreatment (Figure 4D, second panel). Hence, these data
demonstrate that 40-DMA-7,8-DHF and 7,8-DHF selec-
tively activate the TrkB receptor and enhance neuronal
survival in mice.
40-Dimethylamino-7,8-dihydroxyflavone and 7,8-Dihydroxy-
flavone Promote Neurogenesis. Administration of chronic, but
not acute, monoamine antidepressant drugs enhances adult

8280 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Liu et al.
neurogenesis in dentate gyrus of rodents and nonhuman pri-
mates, while blocking neurogenesis by irradiation attenuates the
behavioral antidepressant-like effects of these drugs in some
rodent strains.14-17 This suggests that enhanced adult hippo-
campal neurogenesis is required for the efficacy of antidepres-
sant drugs. Ablation of TrkB specifically in hippocampal neural
progenitor cells prevents chronic antidepressant-induced neuro-
genesis and renders the mice behaviorally nonresponsive to
chronic antidepressant treatment.10 To test whether increasing
TrkB activation by these small agonists would elevate the neu-
rogenesis, we administrated either vehicle, 7,8-DHF and 40-
DMA-7,8-DHF (5 mg/kg), orally by gavage to adult male
C57BL/6J mice for 21 days. At the end of treatment (day 21),
the animals were injected with BrdU (50 mg/kg, intraperitoneal
(ip)) to label the dividing cells and were sacrificed 2 h later. BrdU
immunohistochemistry was then used to assess progenitor pro-
liferation (Figure 5A). We observed that long-term (21 days)
TrkB agonists treatment significantly increased neurogenesis as
compared to the vehicle control. Immunohistochemistry stain-
ing demonstrated that TrkB was markedly activated by these
two chemicals in dentate gyrus (Figure 5B) after 3 weeks of
treatment. Therefore, chronic treatment with TrkB agonists
promotes neurogenesis in the hippocampus of mice.
40-Dimethylamino-7,8-dihydroxyflavone and 7,8-Dihy-
droxyflavone Demonstrate Antidepressant Effect in a TrkB-
Dependent Manner. Accumulating evidence supports that
BDNF plays an essential role in mediating antidepressants’
therapeutic effects.8,9,19,20 Infusion of exogenous BDNF into
hippocampus or brain stem has antidepressant-like behav-
ioral effect.21,22 A forced swim test is broadly used for
screening of potential antidepressant drugs and is widely
used to measure antidepressant activity.23,24 To explore
whether 40-DMA-7,8-DHF and 7,8-DHF have any antide-
pressant effect like BDNF, we conducted a forced swim test after
chronic treatment of the mice for 21 days via oral admin-
istration. When mice were treated with 7,8-DHF (5 mg/kg), the
swimming immobility was significantly decreased. Interest-
ingly, 40-DMA-7,8-DHF (5 mg/kg) also evidently reduced
the immobility (Figure 6A), suggesting that 7,8-DHF and its
derivative imitate BDNF and exert potent antidepressant
effects. Immunoblotting analysis revealed that both com-
pounds evidently provoked TrkB but not TrkA activation in
mouse brain (Figure 6B).
heterocyclic ring (the C ring). Our previous structure-activity
relationship study and the current work both support that 7,
8-dihydroxy groups are critical for the lead compound’s
agonistic effect. Chrysin and 5,7-dihydroxyflavone have no
effect at all, supporting that the ortho-dihydroxy groups have
to be integral.4 Hence, when we synthesized new derivatives,
we kept the ortho-hydroxy groups on the A ring intact. In this
report, we show that the oxygen atom in the C ring is also
essential for the stimulatory activity. Replacement of this O
atom with NH group abolishes the agonistic activity
(Figure 3). However, the B ring might tolerate some modifica-
tion. We have demonstrated thatB-ring-modified compounds
bearing either a 40-dimethylamino, 20- or 30-hydroxy-group
(and, specifically, not 40-hydroxy) display robust TrkB stimu-
latory effects. Interestingly, 40-DMA-7,8-DHF exhibits a higher
agonistic activity and prolonged effect in mouse brain than the
lead. Fitting with its robust agonistic effect to TrkB receptor,
this derivative reveals potent neuroprotective activities. No-
tably, theseagonists exert a prominent neurogenesis effect and
significant antidepressant activity, in alignment with the
physiological actions of BDNF/TrkB signalings.
Flavonoids represent oneof the largest and the mostdiverse
class of plant secondary metabolites. These compounds are
naturally present in vegetables, fruits, and beverages and thus
are important components of the daily diet. Flavonoids have
been known for a long time to exert diverse biological effects
(bioflavonoids) and in particular to act as antioxidants and
preventive agents against cancer.25 Accumulating evidence
suggests that flavonoids have the potential to improve human
memory and neurocognitive performance via their ability to
protect vulnerable neurons, enhance existing neuronal func-
tion, and stimulate neuronal regeneration.26 Flavonoids exert
effects on LTP, one of the major mechanisms underlying
learning and memory, and consequently memory and cogni-
tive performance through their interactions with the signaling
pathways including PI3K/Akt,27 MAPK,28,29 protein kinase
C,30 etc. The pleiotropic activity profile often results in a
complicated structure-activity relationship particularly in
cell-based and in vivo systems where metabolism adds further
complexity in data interpretation. Our data support that 40-
DMA-7,8-DHF possesses more robust neuroprotective ac-
tion than the lead compound. Oral administration of these
two compounds into mice elicits robust TrkB activation in
mouse brain, suggesting that these compounds are orally bio-
available. In addition, 40-DMA-7,8-DHF displays a longer
TrkB stimulatory activity in mouse brain (Figure 3). Presum-
ably, this modified derivative or its metabolites have a longer
half-life in animals. However, it has to be pointed out that we
did not determine drug levels either systemically or centrally
and it is unclear if the differential effects are simply a reflection
of differential in vivo exposures of the analogs. To explore
whether these compounds have any intolerable toxicity, we
have analyzed all of the major organs from the mice, which
were treated with both compounds at 5 mg/kg for 3 weeks. No
appreciable adverse pathological change was detectable in the
drug-treated mice (Supporting Information Figure 4). In
addition, a complete blood count (CBC) analysis shows that
there is no significant difference between drug-treated and the
control saline-treated mice (Supporting Information Table 1).
Hence, these data support that both compounds are not toxic
to the mice at5 mg/kg dose over the 3-weekchronic treatment.
The titration experiment also supports that neither compound
is toxic for HEK293 cell proliferation at a dose up to 50 μM.
Nonetheless, at 100 μM, 7,8-DHF displays demonstrable
To assess whether the behavior responses by 7,8-DHF and
its derivative are mediated by the TrkB receptor, we utilized
TrkB F616A knockin mice. The transgenic mice were sub-
jected to saline or 1NMPP1 pretreatment, respectively. No
significant difference was observed in the immobility time
between saline and 1NMPP1 treated control groups. In saline
group, both 7,8-DHF and 40-DMA-7,8-DHF substantially
reduced the immobility time; in contrast, neither 40-DMA-
7,8-DHF nor 7,8-DHF had any significant effect on the
immobility time after 1NMPP1 treatment (Figure 6C), sug-
gesting that inhibition of TrkB signaling cascade blocks the
antidepressant effect by the TrkB agonists. Thus, these data
demonstrate that 40-DMA-7,8-DHF and its parental lead
mimic BDNF and act as potent antidepressant drugs in mice
through activating TrkB receptor.
Discussion
Flavonoids are a large group of polyphenolic compounds
containing a basic flavan nucleus with two aromatic rings
(the A and the B rings) interconnected bya three-carbon-atom

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8281
Figure 5. 40-Dimethylamino-7,8-dihydroxyflavone and 7,8-dihydroxyflavone promote neurogenesis. (A) Neurogenesis assay. Male C57BL/6J mice
were orally administrated with 5 mg/kg 40-DMA-7,8-DHF and 7,8-DHF and vehicle solvent for 21 days and followed by 50 mg/kg BrdU ip
injection. In 2 h, the mice were perfused and brain sections were immunostained with anti-BrdU and DAPI. The positive cells in dentate gyrus
were highlighted by arrow (left panels). Quantitative analysis of the BrdU positive cells in dentate gyrus (right panel). (B) 7,8-DHF and its
derivative upregulate TrkB activation in dentate gyrus. Paraffin section were deparaffinized in xylene and rehydrated gradient ethanol solution.
Samples were boiled in 10 mM sodium citrate buffer for 20 min for antigen retrieval purpose. Brain sections were incubated with anti-TrkB
(BD biosciences, San Jose, CA) 1:50, and anti-p-TrkB Y816 was used at 1:300 dilution. Secondary antibody were applied using antirabbit-
Alexa 594 (red), antimouse-FITC (green). DAPI (blue) was used for nuclear staining.
antiproliferative effect, whereas 40-DMA-7,8-DHF has no
effect (Supplemental Figure 5A,B). Neither of the compounds
provokes cell death in HEK293 cells (Supporting Information
Figure 5C,D). Neuronal cell death assay reveals that both
compounds are nontoxic for primary cortical neurons up to
5 μM (Supporting Information Figure 5E,F).
Emerging evidence supports thatBDNF and TrkB receptor
are implicated in both the development of mood disorders and
the action of antidepressants.31-33 BDNF-mediated TrkB
signaling is both sufficient and necessary for antidepressant-
like behaviors.21,34 The antidepressanteffect by imipramine or
fluoxetine are abolished in transgenic mice with reduced

8282 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Liu et al.
Figure 6. 40-Dimethylamino-7,8-dihydroxyflavone and 7,8-dihydroxyflavone demonstrate antidepressant effect in a TrkB-dependent manner.
(A) Forced swim test with 40-DMA-7,8-DHF and 7,8-DHF compounds. Male C57BL/6J mice (8 mice/group) were orally administrated by
gavage with 5 mg/kg 40-DMA-7,8-DHF and 7,8-DHF and vehicle solvent saline for 21 days and subjected to a forced swim test (6 min,
immobility recorded in the last 4 min). Data are presented as mean ( SEM. Analysis of variance (ANOVA) revealed significant difference
between vehicle and either 7,8-DHF or 40-DMA-7,8-DHF (n = 6, ***P < 0.0001 vs vehicle). (B) TrkB but not TrkA is activated by 40-DMA-
7,8-DHF and 7,8-DHF in mouse brain. The brain lysates from above chronically treated mice were analyzed by immunoblotting with anti-p-TrkA
794 and p-TrkB 817. (C) Forced swim test with TrkB F616A knockin mice. Male TrkB knockin mice were given the regular drinking water or
1NMPP1 (25 μM) containing drinking water one day before we started to inject the drugs and sustained throughout the whole experiment. The
indicated control (saline) and drugs were administrated for 5 days. Data are presented as mean ( SEM; analysis of variance (ANOVA) revealed
significant effect between vehicle and either 7,8-DHF (n = 7 mice, **P < 0.001) or 40-DMA-7,8-DHF (**P < 0.001) in TrkB KI mice. None of
the drugs produced a significant change in 1NMPP1 treated TrkB KI mice (n = 6 to 7 mice) as compared to control.
BDNF or TrkB signaling.9,35 All these findings indicate that
TrkB signaling is crucial in the antidepressant effect. Here, we
provide further evidence supporting that TrkB signaling is
critical for this process. We show that both 40-DMA-7,8-DHF
and 7,8-DHF, which mimic BDNF and potently activate the
TrkB receptor, exhibit evident antidepressant effects in both
wild-type mice and TrkB F616A knockin mice. However, the
antidepressant activity by 7,8-DHF and 40-DMA-7,8-DHF are
blunted in TrkB F616A knockin mice, when the TrkB recep-
tor is inhibited by 1NMPP1 (Figure 6). These data demon-
strate that these compounds render the antidepressant effect
through activating the TrkB receptor signaling cascade. In
wild-type mice, both compounds reveal comparable antidepres-
sant effects at 5 mg/kg. At a dose of 1 mg/kg, 4,-DMA-7,8-DHF
displays a more potent effect than 7,8-DHF (Figure 3C).
Conceivably, we might be able to distinguish these two com-
pounds’ antidepressant effects at this low dose. Interestingly,
40-DMA-7,8-DHF exhibits a much more potent antidepres-
sant effect than the parental compound 7,8-DHF in TrkB
F616A knockin mice, and the antidepressant effect is com-
pletely inhibited by 1NMPP1, supporting that this antide-
pressant effect is exerted through TrkB receptor. Collectively,
these findings demonstrate that 40-DMA-7,8-DHF possesses
the stronger agonistic effect on TrkB than the lead.
Accumulating evidence supports that hippocampal neurogen-
esis is required for the efficacy of antidepressant drugs.36-38
Ablation of TrkB specifically in hippocampal neural progenitor
cells prevents chronic antidepressant-induced neurogenesis and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8283
renders the mice behaviorally nonresponsive to chronic anti-
depressant treatment.10 Our neurogenesis assay on the animal
with chronic drug treatment supports that both 40-DMA-7,
8-DHF and the parental lead evidently provoke neurogenesis
inthedenta gyrus of drug-treatedmice (Figure5). This finding
fits with the previous report that exogenous BDNF promotes
proliferation of hippocampal NPCs.39 The robust antidepres-
sant effects of these two TrkB agonists support an important
role for BDNF in mediating the biological response to chronic
ADtreatments.40,41 Together, our datasupportthat40-DMA-
7,8-DHF is a more potent synthetic TrkB agonist than 7,
8-DHF. The SAR study points a clear direction for the future
optimization of a 7,8-DHF TrkB agonist.
solution of gallacetophenone 30,40-dimethyl ether (5.83 g,
29.72 mmol) in dry pyridine (25 mL) was added (4-dimethy-
lamino)benzoyl chloride (8.19 g, 44.58 mmol) in three portions
over 15 min. The mixture was stirred at room temperature for 2 h.
The reaction was acidified with 2 M HCl and extracted with
ethyl acetate. The combined organics were washed with brine,
dried with MgSO4, and concentrated under reduced pressure.
The crude product is purified by flash chromatography
(10% EtOAc/hexanes) to afford 8.45 g (83%) of 3 as a white
crystalline solid.
Experimental Section
Cells, Reagents, and Mice. NGF and BDNF were from
Roche. Anti-p-TrkB 817 was from Epitomics. Anti-TrkB anti-
body was from Biovision. Anti-TrkA was from Cell Signaling.
TrkB F616A mice have been described previously.18 TrkB F616A
mice and wild-type C57BL/6 mice were bred in a pathogen-free
environment in accordance with Emory Medical School guide-
lines All chemicals not included above were purchased from Sigma.
7,8-Dihydroxyflavone was purchased from TCI. The flavanoids
were from Indofine (Hillsborough, NJ 08833, USA). NMR
spectrum (Bruker AV300K, 300 MHz), MS spectrum (Shimadzu
LCMS), HPLC (PE, dual pumper, SPD detector, ODS-C18
reverse phase, 254 nm, CH3CN-H2O-0.1%TFA). Phospho-
TrkB Y816 antibody was raised against [H]-CKLQNLA-
KASPV-pY-LDILG-[OH] (aa 806-822) (EM437 and EM438)
as rabbit polyclonal antibody in Covance. The antiserum was
purified by affinity columns. This phospho-TrkB was utilized
for immunostaining the brain sections. Anti-TrkB (Cell Signal-
ing, which recognizes both full-length and truncated TrkB) and
anti-TrkB (Biovision, which only recognizes full-length TrkB)
were used for immunoblotting. Mouse Monoclonal anti-TrkB
(BD Bioscience) was used for immunostaining. P-Akt 473 Sand-
wich ELISA was from Cell Signaling. BDNF was from Roche.
Antiphospho-TrkA 785, anti-TrkA, Phospho-Akt-473, anti-Akt,
and Antiphospho-Erk1/2 antibodies were from Cell Signaling.
Anti-p-TrkB Y817 antibody were from Epitomics.
General Synthesis of 1,3-Diketones (4a-c): 1-(4-(Dimethyl-
amino)phenyl)-3-(2-hydroxy-3,4-dimethoxyphenyl)propane-1,
3-dione (4a). A solution containing 6-acetyl-2,3-dimethoxy-
phenyl 4-(dimethylamino)benzoate 3 (8.45 g, 24.6 mmol),
anhydrous powdered potassium hydroxide (2.08 g, 36.9
mmol), and pyridine (50 mL) was heated at 50 °C for 2 h.
Reaction was cooled to room temperature, acidified with
2 M HCl, extracted with ethyl acetate, washed with brine,
dried with MgSO4, and evaporated under reduced pressure
to yield 7.59 g (90%) of crude propanedione 4. The crude
reaction mixture was carried forward without further
purification.
General Synthesis of Dimethoxy Chromen-4-ones (5a-c): 2-
(4-(Dimethylamino)phenyl)-7,8-dimethoxy-4H-chromen-4-one
(5a). A solution of 1-(4-(dimethylamino)phenyl)-3-(2-hydroxy-
3,4-dimethoxyphenyl)propane-1,3-dione (4) (11.5 g, 33.4 mmol)
in glacial acetic acid (100 mL) and concentrated sulfuric acid
(1 mL) was refluxed for 1 h. The reaction mixture was then
poured into ice and extracted with ethyl acetate. The combined
organics were washed with brine, dried with MgSO4, and
concentrated under reduced pressure to yield a dark solid. Flash
column chromatography (30% EtOAc/hexane) yielded 6.41 g
(60%) as a yellow-brown solid.
Synthesis of 7,8-Dihydroxyflavone Derivatives. 1H and 13C
NMR spectra were recorded with a Varian 300 spectrometer.
Chemical shifts are reported as δ values (parts per million, ppm).
Infrared spectra were recorded on a Bruker Equinox 55 FTIR
spectrophotometer. Peaks are reported in cm-1. Melting points
were obtained with a Barnstead Electrothermal MelTemp ap-
paratus. ESI MS spectra were recorded on an Applied Biosys-
tems 4000 QTrap spectrometer. Gallacetophenone, Gallaceto-
phenone 30,40-dimethyl ether (1), and all acid chlorides were
purchased from Sigma Aldrich (acid chlorides were also pre-
pared directly from the corresponding carboxylic acids). All
other reagents and solvents were purchased from commercial
sources and purified by standard procedures. All reactions were
performed under a dry N2 atmosphere in flame-dried glassware.
The purity of the compounds (>95%) is confirmed by HPLC.
General Procedures for the Synthesis of 7,8-DHF Derivatives
According to Figure 2.
2-(4-(Dimethylamino)phenyl)-7,8-dihydroxy-4H-chromen-4-
one HBr (40-DMA-7,8-DHF HBr, 6). A solution of 2-(4-(di-
3
3
methylamino)phenyl)-7,8-dimethoxy-4H-chromen-4-one (v)
(0.462 g, 1.42 mmol) in aqueous hydrobromic acid (48%, 10 mL)
was refluxed overnight. After cooling, the reaction mixture was
diluted with water, neutralized with saturated NaHCO3, and
extracted with 1-butanol. The organic phase was washed with water,
dried with MgSO4, and evaporated under reduced pressure.
Recrytallization from 50% methanol/dichloromethane provided
0.221 g (52%) of 6.
General Synthesis of Aryl Benzoates (3a-c): 6-Acetyl-2,
3-dimethoxyphenyl 4-(dimethylamino)benzoate (3a). To a

8284 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Liu et al.
1H NMR (300 MHz, DMSO-d6) 7.96(d, J = 8.80,2H), 7.35
(d, J = 8.56, 1H), 6.91 (d, J = 8.64, 1H), 6.85 (d, J = 8.76,2H),
6.64 (s,1H), 3.02 (s, 6H). MS-ESI: cald 297; found: 298 (M þ H)þ.
purified by flash chromatography to afford (E )-ethyl 3-(2,
3-dimethoxyphenylamino)-3-(4-fluorophenyl)acrylate (11).
Anal. for C17H15NO4 1.06HBr: Calcd: C, 53.30; H, 4.23; N,
3
3.66; Br, 22.11. Found: C, 53.08; H, 4.23; N, 3.42; Br, 21.81; mp:
267.8-270.4 °C; HPLC: 100%.
2-(4-Fluorophenyl)-7,8-dimethoxyquinolin-4(1H )-one (12). A
solution of (E)-ethyl 3-(2,3-dimethoxyphenylamino)-3-(4-fluor-
ophenyl)acrylate (11) in Ph2O was heated to 250 °C for 10 min
concentrated. The residue was purified by flash chromatogra-
phy (CH2Cl2 -MeOH, 40:1) to afford 2-(4-fluorophenyl)-7,
8-dimethoxyquinolin-4(1H)-one (12).
2-(4-Fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one HBr (40-
3
F-7,8-DHF HBr, 7). 1H NMR (300 MHz, DMSO-d6) δ 8.20-
3
8.25 (dd, J = 8.80, 2H), 7.35 (d, J = 8.56, 1H), 6.91 (d, J = 8.64,
1H), 6.85 (d, J=8.76,2H), 6.64 (s,1H), 3.02 (s, 6H). MS-ESI:
calcd 272.0478; found 272.0485.
2-(4-Fluorophenyl)-7,8-dihydroxyquinolin-4(1H)-one HBr (13).
3
To a solution of 2-(4-fluorophenyl)-7,8-dimethoxyquinolin-
4(1H)-one (500 mg) in CH2Cl2 was added slowly BBr3 at 0 °C
under N2, and then the reaction was stirred at rt for 3 h. The
reaction was cooled down to 0 °C and quenched by adding
slowly MeOH. Concentrated and the residue was purified by
recrystallization from methyl tert-butyl ether to afford 2-(4-
fluorophenyl)-7,8-dihydroxyquinolin-4(1H)-one (13) as a red
solid (200 mg). 1H NMR (300 MHz, DMSO-d6) δ 10.83 (br s,
1H), 10.09 (br s,1H), 7.92 (q, J1 = 5.1, J2 = 8.1 2H), 7.70 (d, J =
8.7, 1H), 7.46 (t, J =8.7,2H), 7.30 (d, J =9, 1H), 6.87 (s,1H). MS-
7,8-Dihydroxy-2-(pyrimidin-5-yl)-4H-chromen-4-one HBr (8).
3
1H NMR (300 MHz, CDCl3) δ 10.27 (brs, 1H), 9.65 (brs, 1H),
9.52 (s, 2H), 9.32 (s, 1H), 7.39 (d, J = 8.7, 1H), 7.10 (s, 1H),
6.94 (d, J=8.7,1H); calcd 256; found: 257(M þ H)þ. Anal. for
C13H8N2O4 0.45H2O 0.3HBr: Calcd: C, 54.10; H, 3.21; N, 9.71;
3
3
Br, 8.31. Found: C: 54.30 ; H, 3.39; N, 9.41; Br, 8.37; mp > 300 °C.
HPLC: 89.3%.
ESI: calcd 271; found 272 (M þ H)þ. Anal. For C15H10FNO3
3
0.87HBr 2.4H2O: Calcd: C, 46.81; H, 4.10; N, 3.64; Br, 18.06.
Found: C, 46.75; H, 3.80; N, 3.26; Br, 17.46; mp>300. HPLC:
98.9%.
3
2-(4-(Dimethylamino)phenyl)-7-hydroxy-8-methoxy-4H-chro-
men-4-one HBr (40-DMA-8-H-7M-F HBr). 1H (CDCl3) δ 7.95
(d, J = 9 Hz, 1H), 7.75 (d, J = 9 Hz, 2H), 7.08 (d, J = 9 Hz, 1H),
3
3
7,8-Dihydroxy-2-phenylquinolin-4(1H)-one HBr (14). 1H
6.78 (d, J = 9 Hz, 2H), 6.65 (s, 1H), 3.99 (s, 3H), 3.08 (s, 6H). 13
C
3
NMR (300 MHz, DMSO-d6) δ 10.71 (br s, 1H), 7.81 (d, J =
7.2, 2H), 7.65 (d, J = 9.3, 1H), 7.57-7.66 (m, 3H), 7.23 (d, J =
9.3, 1H), 6.81 (s, 1H). MS-ESI (negative): calcd 253; found 252
(M - 1). Anal. For C15H11NO3 0.8HBr 0.96H2O: Calcd: C,
(CDCl3) δ 177.1, 163.2, 153.0, 128.1, 121.0, 111.9, 109.8, 103.6,
61.7, 57.2.
Representative Procedures for the Synthesis of 7,8-Dihydroxy
Quinolin-4(1H )-one Derivatives According to Figure 2B.
3
3
53.74; H, 4.12; N, 4.18; Br, 18.90. Found: C, 53.45; H, 3.96; N,
4.43; Br, 19.11; mp: 269.8-274.2 °C. HPLC: 100%.
Phospho-AktS473 ELISA. The ELISA was carried out using
96-well Nunc-Immuno MaxSorp plates (VWR Cat. no. 62409-
024). PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit
was purchased from Cell Signaling (Cat. no. 7160). The cell
lysates from primary neurons or mouse brain that were treated
with various compounds were employed in the assay. A 100 μL
sample (20-40 μg) in sample diluent buffer (supplied in the kit)
was added to each well and incubated overnight at 4 °C. After
4ꢀ wash with 200 μL of wash buffer (supplied in the kit), 100 μL/
well detection antibody was added and incubated for 1 h at
37 °C. After 4ꢀ wash, 100 μL of HRP-linked secondary antibody
(supplied in the kit) was added and incubated for 30 min at 37 °C.
After a final wash, 100 μL of 3,30,5,50-tetramethylbenzidine
(E)-Ethyl 3-(2,3-Dimethoxyphenylamino)-3-(4-fluorophenyl)-
acrylate. A mixture of ethyl 3-(4-fluorophenyl)-3-oxopropanoate
(5.00 g, 23.8 mmol), 2,3-dimethoxybenzenamine (3.64 g, 23.8
mmol), AcOH (cat.), and CaSO4(6.5 g, 47.6 mmol) in EtOH
(100 mL) was refluxed at 75 °C under N2 for 144 h. The reaction
mixture was concentrated to afford crude product, which was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8285
(TMB) substrate was added to each well and incubated for
10 min at 37 °C. The reaction was stopped by adding 100 μL/well
stop solution. The values of each well were recorded using the
microplate reader at 450 and 650 nm. The optical density was
determined by the subtraction of the reading at 650 nm from the
readings at 450 nm.
Kainic Acid/TrkB Agonists Drug Administration. Male
C57BL/6 mice aged of 60 days were administrated orally with
a single dose of 40-DMA-7,8-DHF or 7,8-DHF (1 mg/kg each).
KA (20 mg/kg) (Sigma, MO) was ip injected. Animals were
continually monitored for 2 h for the onset of seizure activity. At
0, 4, and 8 h following TrkB agonist treatment, the animals were
sacrificed and the hippocampal section lysates were analyzed by
immunoblotting with p-TrkB, active caspase-3 and total TrkB
antibodies.
TrkB Agonists Suppress KA-Induced Neuronal Cell Death in
TrkB F616A Mice. TrkB F616A knockin mice (2-3 months old)
were fed with 1NMPP1 (25 μM) in drinking water one day
before pharmacological reagent treatment. Next day, the mice
were administrated orally by gavage with 7,8-DHF or 40-DMA-
7,8-DHF (5 mg/kg) 4 h before kainic acid (20 mg/kg). The
control mice were injected with saline, 1NMPP1, kainic acid
alone, or administrated 7,8-DHF or 40-DMA-7,8-DHF 4 h
before kainic acid. In 4 days, the mice were sacrificed and brains
were homogenated and ultracentrafuged. The supernatant (40
μg) was employed for SDS-PAGE and immunoblotting analysis
with indicated antibodies, respectively.
Immunohistochemistry Staining. Brain tissues were fixed in
4% paraformaldehyde overnight followed by paraffin embed-
ding. Sections of 6 μm were cut. For immunohistochemical
staining, brain sections were deparaffinized in xylene and rehy-
drated in graded alcohols. Endogenous peroxidase activity was
blocked by 3% hydrogen peroxide for 5 min and all slides were
boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 min.
Phosphorylated Trk B816 and Trk B were detected using specific
antibodies. Paraffin section were deparaffinized in xylene and
rehydrated gradient ethanol solution. Samples were boiled in
10 mM sodium citrate buffer for 20 min for antigen retrieval
purpose. Brain sections were incubated with anti-TrkB (BD
Biosciences, San Jose, CA) 1:50, p-TrkB 1:300 dilution. Second-
ary antibody was applied using antirabbit-Alexa 594 (red),
antimouse-fluorescein isothiocyanate (FITC) (green). DAPI
(blue) was used for nuclear staining.
containing 0.01% 1,4-diazobicyclo(2,2,2)octane and examined
under a fluorescence microscope.
Acknowledgment. This work is supported by grants from
National Institutes of Health RO1 NS045627 to K. Ye. We
are thankful to Dr. David Ginty at Johns Hopkins University
for TrkB F616A knockin mice.
Supporting Information Available: CBC analysis of TrkB
agonist-treated mice; structure-activity relationship study;
2,40-DMA-7,8-DHF is more potent than 7,8-DHF in triggering
TrkB activation in primary neurons; TrkB is activated by 7,8-
DHF via oral administration in a dose dependent manner.
histology exampination of drug-treated mice and control sal-
ine-treated mice; cell growth and cytotoxicity assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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