Optimization of a small tropomyosin-related kinase B (TrkB) agonist 7,8-dihydroxyflavone active in mouse models of depression

Article abstract of DOI:10.1021/jm301099x

Structure-activity relationship study shows that the catechol group in 7,8-dihdyroxyflavone, a selective small TrkB receptor agonist, is critical for agonistic activity. To improve the poor pharmacokinetic profiles intrinsic to catechol-containing molecules and to elevate the agonistic effect of the lead compound, we initiated the lead optimization campaign by synthesizing various bioisosteric derivatives. Here we show that the optimized 2-methyl-8-(4′- (pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one derivative possesses enhanced TrkB stimulatory activity. Chronic oral administration of this compound significantly reduces the immobility in forced swim test and tail suspension test, two classical antidepressant behavioral animal models, which is accompanied by robust TrkB activation in hippocampus of mouse brain. Further, in vitro ADMET studies demonstrate that this compound possesses the improved features compared to the previous lead compound. Hence, this optimized compound may act as a promising lead candidate for in-depth drug development for treating various neurological disorders including depression.

Full text of DOI:10.1021/jm301099x

Article
pubs.acs.org/jmc
Optimization of a Small Tropomyosin-Related Kinase B (TrkB)
Agonist 7,8-Dihydroxyflavone Active in Mouse Models of Depression
Xia Liu,† Chi-Bun Chan,† Qi Qi,† Ge Xiao,‡ Hongbo R. Luo,§ Xiaolin He,∥ and Keqiang Ye*,†
†Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 615 Michael Street, Atlanta, Georgia
30322, United States
‡Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, Georgia 30341, United States
§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
∥Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, 303 E.
Chicago Avenue, Searle 8-417, Chicago, Illinois 60611, United States
S
* Supporting Information
ABSTRACT: Structure−activity relationship study shows that the catechol group in 7,8-
dihdyroxyflavone, a selective small TrkB receptor agonist, is critical for agonistic activity. To
improve the poor pharmacokinetic profiles intrinsic to catechol-containing molecules and to
elevate the agonistic effect of the lead compound, we initiated the lead optimization campaign by
synthesizing various bioisosteric derivatives. Here we show that the optimized 2-methyl-8-(4′-
(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one derivative possesses enhanced
TrkB stimulatory activity. Chronic oral administration of this compound significantly reduces
the immobility in forced swim test and tail suspension test, two classical antidepressant behavioral
animal models, which is accompanied by robust TrkB activation in hippocampus of mouse brain.
Further, in vitro ADMET studies demonstrate that this compound possesses the improved features compared to the previous
lead compound. Hence, this optimized compound may act as a promising lead candidate for in-depth drug development for
treating various neurological disorders including depression.
with different structural backbones.10,11 Our studies demon-
strate that these small molecules exert potent neuroprotective
INTRODUCTION
■
Brain-derived neurotrophic factor (BDNF) is a member of the
neurotrophin family, which includes nerve growth factor
(NGF), NT-3, and NT-4/5. BDNF binding to its cognate
receptor, TrkB, triggers its dimerization through conforma-
effects in stroke and PD and possess robust antidepressant
effect.10,12 Both 7,8-dihydroxyflavone (7,8-DHF)10,12 and
deoxygedunin induce neurogenesis and exhibit antidepressant-
like profile in a TrkB-dependent manner.11,12 Most recently, it
tional changes and autophosphorylation of tyrosine residues,
has been shown that 7,8-DHF mimics BDNF and improves the
resulting in activation of the three major signaling pathways,
cognitive defect in AD.13 Further, 7,8-DHF dampens the
MAPK, PI3K, and PLC-γ.1 Accumulating evidence indicates
that these molecules are also critical for antidepressant drug
development of the “depressive” phenotype.14 Like BDNF, 7,8-
DHF rescues synaptic plasticity in aged animals.15 Notably, 7,8-
efficacy. Antidepressants induce BDNF mRNA expression, as
well as autophosphorylation and activation of TrkB,1,2 and the
DHF exhibits therapeutic efficacy in a mouse model of Rett
syndrome, caused by mutations in the MeCP2 gene; MeCP2
behavioral effects of antidepressants in the forced swim test are
attenuated in mice with only one active BDNF allele (BDNF
mutant mice have reduced levels of BDNF.16 Hence, these
studies demonstrate that our small TrkB agonists mimic BDNF
and exert neuroprotective roles in various neurological diseases
and display an antidepressant-like profile.
mice), completely lacking BDNF specifically in forebrain or
expressing a dominant negative form of TrkB (trkB.T1).2,3
Ablation of TrkB, specifically in hippocampal neuronal
progenitor cells renders the mice behaviorally nonresponsive
Our preliminary structure−activity relationship (SAR) study
to chronic antidepressant treatment.4 Hence, these results
shows that the 7,8-dihydroxy groups are essential for the
agonistic effect.12 To improve the lead compound’s agonistic
suggest a model whereby chronic antidepressant treatment
activity, we have conducted an extensive SAR study and
induces BDNF expression and long-term activation of TrkB,
leading to an antidepressant effect.5−7
synthesized numerous derivatives. We have successfully
identified 4′-dimethylamino-7,8-dihydroxyflavone (4′-DMA-
The preclinical evidence strongly supports the idea that
BDNF might be useful as a therapeutic agent for a variety of
neurological disorders.8,9 To search for small molecules that
mimic the biological functions of BDNF, we invented a cell-
based assay and identified two small molecular TrkB agonists
7,8-DHF)12 that displays higher TrkB agonistic activity than
Received: July 26, 2012
© XXXX American Chemical Society
A
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a
Table 1. In Vivo Pharmacokinetic Studies
route of
administration
dose
t1/2
CL
Vz
Vss
AUClast
AUCINF
(min·ng/mL)
compd
(mg/kg) subject (min) (mL min−1 kg−1
)
(mL/kg) (mL/kg) (min·ng/mL)
4′-dimethylamino-7,8-dihydroxflavone HBr
13
iv
iv
1
mice
mice
9
168
156
2117
803
5930
5943
3
103
23085
10011
18746
19229
route of
administration
dose
bioavailability
(%)
Tmax
(min)
Cmax
(ng/mL)
t1/2
(min)
AUClast
(min·ng/mL)
AUCINF
(min·ng/mL)
compd
(mg/kg) subject
4′-dimethylamino-7,8-dihydroxflavone HBr
13
po
po
5
mice
mice
NC
2
NC
120
NC
7
NC
58
NC
NC
10
1144
1293
a
Pharmacokinetic parameters were derived from a noncompartmental model using WinNonlin 5.2. t1/2 = elimination half-life. CL = total body
clearance. Vz = volume of distribution. Vss = an estimate of the volume of distribution at steady state. AUClast = area under the concentration−time
curve from the time of dosing to the time of last observation that is greater than the limit of quantitation. AUCINF = area under the concentration−
time curve from the time of dosing, extrapolated to infinity. Linear/log trapezoidal method was used for AUClast and AUCINF calculations. Tmax
=
time of maximum observed concentration. Cmax = concentration corresponding to Tmax. NC = not calculable because plasma concentrations are too
low.
Figure 1. Organic synthesis of various flavonoids. (A) Schematic diagram of synthetic routes for 4′-dimethylamino-7,8-imidazoleflavone (compounds
11, 12, and 13). R represents dimethylamino group or 4-pyrrolidin-1-yl-group. (B) Schematic diagram of synthetic route for compound 23.
the original hit 7,8-DHF. 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 4′-DMA-7,8-DHF and the original hit 7,8-DHF strongly
promotes neurogenesis in dentate gyrus and demonstrates
B
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Figure 2. 8-(4-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-6(1H)-one exhibits elevated TrkB stimulatory activity. (A) Chemical structures of
various synthetic flavonoids. (B) 8-(4-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-6(1H)-one displays stronger TrkB stimulatory activity
than the lead compound 24. Primary cortical cultures from E17 rat embryos were treated with 500 nM various synthetic flavone derivatives for 15
min. The cell lysates (20 μg) were analyzed by p-TrkB immunoblotting (top panel) and p-Akt ELISA (bottom panel). The data were from two sets
of replicated experiments (mean SEM). (C) 8-(4-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-6(1H)-one strongly activates TrkB receptor
in mouse brain. Various indicated compounds at 1 mg/kg were orally administrated into C57 BL/6J mice, and TrkB phosphorylation and its
downstream signaling cascades including Akt and MAPK in the hippocampus of mouse brain were analyzed by immunoblotting at 4 h. Compounds
11 and 24 displayed the strongest TrkB stimulatory effect (first panel). The downstream p-Akt and p-MAPK activity coupled to the TrkB activation
patterns (third and fifth panels). p-Akt 473 ELISA results of drug treated mouse brain were analyzed (seventh panel) (∗, P < 0.05 vs control; one-
way ANOVA). The data were from two sets of replicated experiments (mean SEM).
C
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Figure 3. 8-(4-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-6(1H)-one strongly activates TrkB and reduces the immobility in forced swim
test. (A) Time course assay of 8-(4-(dimethylamino)phenyl)chromeno[7,8-d]imidazol-6(1H)-one. Compound (32) and compound (11) at 1 mg/
kg were orally administrated into C57 BL/6J mice, and TrkB phosphorylation and its downstream signaling cascades including Akt in mouse brain
were analyzed by immunoblotting at various time points. TrkB activation by compound 11 peaked at 4 h, whereas the maximal TrkB activation by
compound 32 in mouse brain occurred at 1−2 h. Arrows indicate the p-TrkB in mature glycosylated or unglycosylated forms (first panels). The
downstream Akt activation pattern tightly correlated with the upstream TrkB activation (third panels). (B) p-Akt S473 in drug-treated mouse brain
was by ELISA using 20 μg of brain lysates (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 vs control; one-way ANOVA). The data were from two sets of
replicated experiments (mean SEM). (C) Forced swim test with compounds 11 and 32. The test (6 min, immobility recorded in the last 4 min)
was performed with male C57BL/6J mice that have been orally administrated 5 mg/kg compound 11, compound 32, or vehicle solvent saline for 21
days. Data are presented as the mean SEM (n = 6, ∗∗, P < 0.01 vs vehicle, Student t test). (D) Locomotor activity assay. Drug-treated mice as
stated in (C) were tested for locomotor activity at day 22. Compound 11 but not 32 significantly increased the locomotor activity compared to
vehicle control. Data are presented as the mean SEM (n = 6; ∗, P < 0.05; ∗∗∗, P < 0.001; two-way ANOVA). (E) TrkB but not TrkA is activated
by compounds 11 and 32 in mouse brain. The brain lysates from chronic drug-treated mice were analyzed by immunoblotting with anti-p-TrkA 794
and p-TrkB 816.
D
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marked antidepressant-like profile.12 However, catechol con-
taining compounds usually have short in vivo half-lives and are
prone to be cleared in the circulatory system following
oxidation, glucuronidation, sulfation, or methylation.17,18 The
potential metabolic pathways implicated in catechol-containing
7,8-DHF clearance in the circulatory system may remain
similar. To overcome the intrinsic pharmacokinetics (PK)
shortcomings that catechol groups are associated with, we
replaced 7,8-dihydroxy groups with an imidazole ring. In this
report, we expand on our previous findings to include the
synthesis of numerous imidazole-substituted flavonoids with
improved in vitro ADMET profiles, efficacy in activating the
TrkB receptor in mouse brain, and elevated antidepressant
effects.
step in the synthesis is indicated in the Experimental
Procedures.
8-(4′-(Dimethylamino)phenyl)chromeno[7,8-d]-
imidazol-6(1H)-one Displays Increased TrkB Stimulatory
Effect. Our previous study shows that the electron donor
dimethylamino group at the 4′ position of the B ring
significantly elevates the agonistic effect.12 Hence, we wanted
to test the 3′-dimethylamino or the 4′-morpholino group’s
effect on 7,8-DHF’s TrkB agonistic activity. Further, we
wondered about the effect of the electron-withdrawing group,
fluoro, on 7,8-DHF or 4′-DMA-7,8-DHF’s agonistic activity
(Figure 2A). To compare the TrkB activation by these
compounds, we prepared primary cortical cultures and treated
them with 500 nM various compounds for 15 min and
collected the cell lysates. Compound 11 exhibited a stronger
effect in triggering TrkB activation than the lead compound 4′-
DMA-7,8-DHF (24 in Figure 2A). 3′-Dimethylamino-7,8-DHF
(28) or 4′-morpholino-7,8-DHF (29) exhibited activity
comparable to that of the lead compound 24. Fluoride
substitution at position 3 or 5 (32, 33, 34, and 35) did not
significantly affect 7,8-DHF’s TrkB agonistic activity. Fluoride
substitution at the 4′ position on the B ring (30) inhibited its
activity, which might be due to its electron-withdrawing effect.
As we showed before,12 replacing an O atom with an N atom in
the C ring (compounds 25 and 26) diminished agonistic
activity (Figure 2B, upper panel). The p-Akt ELISA results
were similar to the TrkB activation pattern (Figure 2B, lower
panel). To further explore these compounds’ effects on TrkB
activation in mouse brain, we orally administrated 1 mg/kg
each compound and monitored TrkB’s activity at 4 h. As
expected, the lead compound 24 clearly activated the TrkB
receptor; 4′-dimethylamino-7,8-imidazoleflavone (11) also
robustly activated TrkB. The rest of the compounds displayed
a similar effect, as was observed by the in vitro assay (Figure
2C, top panel). Accordingly, the downstream p-Akt and p-
MAPK signalings were activated by both compounds 24 and
11. p-Akt ELISA analysis also correlated with the observations
of p-TrkB immunoblotting (Figure 2C, bottom panel).
8-(4′-(Dimethylamino)phenyl)chromeno[7,8-d]-
imidazol-6(1H)-one Is Active in Mouse Models of
Depression with Increased Locomoter Activity. To gain
insight into TrkB activation kinetics, 8-(4′-(dimethylamino)-
phenyl)chromeno[7,8-d]imidazol-6(1H)-one (11) was admin-
istered to C57BL6 mice at 1 mg/kg via oral gavage. For
comparison, we employed compound 32 in the same
procedure. The mouse brains were collected, and TrkB
activation and its downstream Akt signaling were analyzed by
immunoblotting. Compound 11 activated TrkB receptor in a
time-dependent manner, peaking at 4 h and fading away at 16
h. The p-Akt signal was in alignment with the upstream p-TrkB
activity. Compound 32 displayed less effect on TrkB activation
than compound 11 (Figure 3A, top and third panels). P-Akt
ELISA correlated with p-Akt immunoblotting results for both
compounds. Compound 11 gradually activated Akt and
climaxed at 8 h, where the p-Akt signal was elevated by
about 250% compared to the control. Compound 32 also
elicited p-Akt activation peaking at 1 h, declining at 4 h, and
returning to the baseline at 8 h. The peak magnitude of Akt
activation by compound 32 was significantly less than that by
compound 11 (Figure 3B). Forced swim test (FST) is broadly
used for screening potential antidepressant drugs and is widely
used to measure antidepressant activity. The FST is a good
screening tool with good reliability and predictive validity.19,20
RESULTS
■
Synthesis of Imidazole Flavonoid and Indole Flavo-
noid Derivatives. Previous study shows that the catechol
group plays a critical role in regulating 7,8-DHF and its
derivatives’ TrkB agonistic effect. Since catechol-containing
compounds usually possess relatively poor PK profiles, we
synthesized numerous analogues of this compound to enhance
the desired biological and physical properties of the lead
compound 4′-DMA-7,8-DHF. The in vivo PK data for this
compound are included in Table 1. To synthesize the
intermediate 4-dimethylaminobenzoyl chloride or 4-pyrroli-
din-1-ylbenzoyl chloride, we conducted the reactions described
in the top of Figure 1A. 4-(Dimethylamino)benzoyl chloride
(7, R = dimethylamino) was coupled to N-(4-acetyl-3-hydroxy-
2-nitrophenyl)acetamide (6) in the presence of triethylamine to
yield 3-acetamido-6-acetyl-2-nitrophenyl 4-(dimethylamino)-
benzoate (8), which was subsequently cyclized to afford 7-
amino-2-(4-(dimethylamino)phenyl)-8-nitro-4H-chromen-4-
one (9). This compound was reduced to generate 7,8-diamino-
2-(4-(dimethylamino)phenyl)-4H-chromen-4-one hydrochlor-
ide (10). A solution of this compound was refluxed in
HCO2H for 1 h to produce a yellow solid, which was
recrystallized to yield pure 8-(4′-(dimethylamino)phenyl)-
chromeno[7,8-d]imidazol-6(1H)-one (compound 11). The
4′-pyrrolidinoimidazole derivatives were synthesized with
similar routes, producing a mixture of imidazole flavonoid
(compound 12) and methylimidazole flavonoid (compound
13) with 60:40 ratio (Figure 1A), which was separated with
pre-HPLC to give compound 12 as an orange solid and a dark-
yellow solid compound 13. The yield for the 2-methylimidazole
side product in 4′-dimethylamino derivative was very low
compared to that for compound 11. For compound 12, 16
hydrogen atoms were present in the 1H NMR, while for
compound 13, 18 protons appeared. We reasoned that the
methyl group was on the imidazole ring because the proton
between the two nitrogen atoms of the imidazole ring was
missing (8.81 ppm in compound 12) and the chemical shift of
methyl group was consistent with the structure. To prepare the
intermediate 7-methoxy-6-indolecarboxylic chloride (21), we
employed compound 14 as a starting material, via the reactions
described in the top of Figure 1B. The obtained intermediate
compound 21 was then coupled to 4′-pyrrolidinophenyl methyl
ketone in the presence of LiHMDS, and the reaction mixture
was quenched with NH4Cl water solution to give compound 22
(1-(4′-pyrrolidinophenyl)-3-(7′-methoxy-6-indolyl)propane-
1,3-dione), which was subsequently cyclized in the presence of
HBr to afford compound 23 (Figure 1B). The yield for each
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Figure 4. 2-Methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in primary neurons
and mouse brain. (A) Chemical structures of various synthetic 4′-pyrrolidinoflavone derivatives. (B) 2-Methyl-8-(4-(pyrrolidin-1-yl)phenyl)-
chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in primary neurons. Rat primary neurons were treated with 500 nM
various compounds for 15 min. Cell lysates (20 mg) were analyzed with various antibodies as indicated. (C) 2-Methyl-8-(4-(pyrrolidin-1-
yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in mouse brain. Various compounds at 1 mg/kg were
orally administrated into C57 BL/6J mice, and TrkB phosphorylation (first panel) and its downstream signaling cascades including Akt and MAPK
in the hippocampus of mouse brain were analyzed by immunoblotting at 2 h. The downstream p-Akt and p-MAPK activity were coupled to the TrkB
activation patterns (third and fourth panels).
To explore whether these compounds possess any anti-
depressant effect, we chronically treated C57/BL6 J mice
with 5 mg/kg compound, once a day for 3 weeks. At the end of
the treatment, we conducted the locomoter activity assay,
followed by a forced swim test. We found that both compounds
significantly reduced the immobility and the effect of
compound 11 was more robust than that of compound 32
(Figure 3C). Nonetheless, compound 11 substantially
augmented locomoter activity compared to compound 32
and vehicle control (Figure 3D). Immunoblotting with brain
tissues from both cortex and hippocampus demonstrated that
both compounds 11 and 32 clearly escalated TrkB phosphor-
ylation after 3 weeks of drug treatment compared to vehicle
control but compound 11 displayed a stronger effect than
compound 32. The TrkA receptor was not activated by any of
these compounds (Figure 3E), demonstrating that they are
TrkB receptor-specific agonists.
2-Methyl-8-(4′-(pyrrolidin-1-yl)phenyl)chromeno[7,8-
d]imidazol-6(1H)-one Demonstrates Greater Potency
than 8-(4′-(Dimethylamino)phenyl)chromeno[7,8-d]-
imidazol-6(1H)-one. Though compound 11 possessed robust
TrkB stimulatory effect and reduces immobility in forced swim
test, it escalated locomotor activity after 3 weeks of
administration. To alleviate this concern, we synthesized
several imidazole or indole-substituted flavonoid compounds
(Figure 4A). Immunoblotting and p-Akt ELISA analysis
demonstrated that both compounds 13 and 23 displayed
higher activity than compound 11 in triggering TrkB and Akt
activation in primary neurons (Figure 4B). We made similar
observations about TrkB receptor and Akt activation in mouse
brain 2 h after oral administration of 1 mg/kg compounds
(Figure 4C). Hence, we chose to focus on compounds 13 and
23 to examine their antidepressant effect in both forced swim
test and tail suspension test assays.
2-Methyl-8-(4′-(pyrrolidin-1-yl)phenyl)chromeno[7,8-
d]imidazol-6(1H)-one Demonstrates Antidepressant-like
Profile without Altering Locomoter Activity. Next, we
treated the mice with compounds 13 and 23 at a dosage of 2.5
mg/kg via oral gavage once a day for 3 weeks and to explore
their effect on locomotor activity. FST showed that compound
13 significantly reduced the immobility by 45% compared to
vehicle control; by contrast, compound 23 had no effect
(Figure 5A). The tail suspension test (TST) has become one of
the most widely used models for assessing antidepressant-like
activity in mice. The test is based on the fact that animals
subjected to the short-term, inescapable stress of being
suspended by their tail will develop an immobile posture.
Various antidepressant medications reverse the immobility and
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Figure 5. 2-Methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in mouse brain and
exhibits antidepressant effect. (A) Forced swim test (6 min, immobility recorded in the last 4 min) was performed with male C57BL/6J mice that
have been orally administrated 2.5 mg/kg 13, 23, or vehicle solvent saline for 21. Compound 13 but not compound 23 significantly decreased the
immobility. Data are presented as the mean SEM (n = 8; ∗, P < 0.05; Student’s t test). (B) Tail suspension test. The drug-treated mice were
subjected to tail suspension assay. Compound 13 but not 23 reduced the immobility. Data are presented as the mean SEM (n = 8; ∗, P < 0.05;
Student’s t test). (C) Locomotor activity assay. None of the tested compounds significantly altered the locomotor activity. (D) Both compounds 13
and 23 activate TrkB and its downstream signaling cascades. Various compounds at 2.5 mg/kg were orally administered into C57 BL/6J mice, and
TrkB phosphorylation and its downstream effector Akt activation in the hippocampus were analyzed by immunoblotting after behavioral tests. Both
compounds 13 and 23 elevated TrkB phosphorylation (first panel). The downstream p-Akt activity was also up-regulated by compounds 13 and 23
(fourth panel). The ratio P-TrkB/total TrkB in drug-treated mouse brain was analyzed (sixth panel). The data were from two sets of replicated
experiments and are expressed as the mean SEM (∗, P < 0.05 vs control; Student’s t test). (E) Both compounds 13 and 23 elevated TrkB
phosphorylation in the hippocampus. The drugs in chronic drug-treated mice were perfused, and the brain sections were stained with anti-p-TrkB
816 and anti-TrkB antibodies. The p-TrkB activated neurons were labeled with white arrows.
G
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Figure 6. In vitro cytotoxicity and genotoxicity assay. (A) Cytotoxicity assay in hepatocyte HepG2 cells. HepG2 cells were treated with various
concentrations of flavonoids for 24 h. The drug-treated cells were subjected LDH assay. Data are presented as the mean SEM (n = 3). (B)
Micronuclei assay in hepatocyte HepG2 cells. HepG2 cells were treated with 50 μM various compounds for 24 h. The nuclei were stained with DAPI
and analyzed under a fluorescent microscope. Data are presented as the mean SEM (n = 3; ∗∗∗, P < 0.001 vs vehicle; one-way ANOVA). (C)
Comet assays. HepG2 cells were treated with 100 μM various compounds for 24 h. The percentage of lesion DNA in tail was used as a parameter for
measurement of DNA damage. Data are presented as the mean SEM (n = 3; ∗∗∗, P < 0.001 vs vehicle; one-way ANOVA).
promote an escape-related behavior.21 We made similar
observations of compound 13 significantly reducing the
immobility versus vehicle control, whereas compound 23
lacked efficacy (Figure 5B). Further, locomotor activity analysis
revealed that neither compound 13 nor 23 altered the motion
activity compared to vehicle control (Figure 5C). Immunoblot-
ting using both anti-p-TrkB Y816 and anti-p-TrkB Y706
antibodies with brain tissues demonstrated that both
compounds 13 and 23 markedly activated TrkB. P-Akt
immunoblotting also correlated with the upstream TrkB
activation (Figure 5D, top, second and fourth panels).
Quantitative p-Akt ELISA assay supported that both com-
pounds notably activated Akt, fitting with the observations by
Western blotting (Figure 5D, bottom panel). Immunohisto-
chemistry staining with anti-p-TrkB demonstrated that TrkB
was activated by these two chemicals in dentate gyrus after 3
weeks of treatment (Figure 5E, white arrows). Therefore,
chronic treatment with compound 13 promotes TrkB
activation in the hippocampus of mice and produces potent
antidepressant-like profile.
In Vitro ADMET Profiles of the Imidazole Derivatives.
7,8-DHF and other flavonoids have recently been shown to
possess impressive antigenotoxic effects against DNA lesions
and micronuclei induced in human hepatocellular carcinoma
cells, HepG2, by a potent mutagen and carcinogen benzo[a]-
pyrene (B(a)P).22 In order to gain insight into the liabilities of
H
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the drug candidates, we examined their in vitro toxicity. An
LDH cytotoxicity assay, using HepG2 cells treated with various
compounds for 24 h, demonstrated that none of the tested
compounds induced noticeable cytotoxicity even up to 100 μM,
suggesting that these compounds do not trigger cell death even
at very high concentrations (Figure 6A). Micronuclei formation
is an important end point in genotoxicity study. To assess
whether these synthetic compounds possess any potential
carcinogenicity, we conducted the micronucleus assay by
treating HepG2 cells with 50 μM various compounds for 24
h, followed by DAPI staining. Quantitative analysis revealed
that none of the tested compounds exhibited significant effect,
whereas the positive control B(a)P robustly induced micro-
nuclei (Figure 6B). Next, we performed a Comet assay with
100 μM drug-treated HepG2 cells. As a positive control, we
chose etoposide, a topoisomerase inhibitor, which causes DNA
strand breaks. Interestingly, compound 11 but not other
synthetic derivatives induced DNA lesions (Figure 6C). Hence,
these toxicity experiments support that compounds 12, 13, and
23 possess negligible cytotoxicity or genotoxicity.
To explore these compounds’ in vitro ADMET profiles, we
conducted numerous in vitro assays. Human liver microsomal
stability assay showed that after 1 h of incubation, compound
23 had 1.2% remaining whereas compound 13 had 25.4%
unchanged, supporting that compound 13 is more metabol-
ically stable than compound 23. Reactive metabolic screening
assays support that compound 13 is quite stable in vitro
whereas compound 23 formed numerous adducts (Tables 2
13. The hERG inhibition assay revealed that compound 13 did
not possess any concentration-dependent inhibition activity of
hERG. A human plasma protein binding experiment showed
that both compounds 13 and 23 displayed approximately 99.9%
of protein binding (Table 5). Hence, compound 13 possesses a
much more preferable in vitro ADMET profile than compound
23, fitting with its remarkable in vivo antidepressant efficacy.
Table 5. Summary of Plasma Protein Binding
mean
plasma
test
concn
compd (μM)
fraction
bound
(%)
test
species
Fuplasma
(%)
recovery
(%)
binding
classification
13
5
human
0.03
99.9
86.3
high
binding
23
5
human
0.07
99.9
97
high
binding
To investigate whether compound 13 possesses an improved
in vivo PK profiles compared to the lead compound 24, we
conducted in vivo pharmacokinetic studies in mice. The PK
parameters are summarized in Table 1. For the lead compound
24, we employed the dosage of 1 mg/kg via iv injection and 5
mg/kg for the po route. At 1 mg/kg, the in vivo half-life, t1/2, of
the lead compound 24 in circulation was around 9 min and
AUClast was about 5930 (min·ng/mL), but the bioavailability of
the lead compound 24 was not measurable at 5 mg/kg (Table
1). Thus, we increased the doses of the tested compound 13.
The t1/2 for compound 13 was about 103 min with AUClast of
approximately 18 746 (min·ng/mL) at a dose of 3 mg/kg.
Moreover, the bioavailability was around 2% at a 10 mg/kg
dosage. Hence, these data demonstrate that compound 13 may
possess an elevated PK profile compared to the lead compound
24.
Table 2. Summary of Microsomal Stability Screening
test
concn
(μM)
mean remaining
parent with NADH
(%)
mean remaining
parent NADPH-free
(%)
test
species
compd
13
23
1
1
human
human
25.4
1.2
92.2
79.6
DISCUSSION
■
In the current report, we show that the synthetic 2-methyl-8-(4-
(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one
(compound 13) possesses improved in vitro ADMET features
and is active in mouse models of depression. Moreover, in the
mouse brain hippocampus, this compound activates the TrkB
receptor and exerts robust antidepressant effects in both FST
and TST assays. Therefore, our study demonstrates that this
compound is a potential candidate for in-depth drug develop-
ment. Molecular modeling also supports that 7,8-DHF and
compound 13 might bind to the LRR motif on TrkB ECD
(Supporting Information Figure 1), fitting with our previous
results found with in vitro binding assay.10,11 This approach
may shed light on our future drug design for further improving
the lead compound, if the cocrystal (agonist/TrkB ECD)
structure is experimentally resolved.
Table 3. Summary of Reactive Metabolite Identification
potential reactive
metabolites identified
compd
scan
m/z
comment
13
precursor
no
no adduct
detected
neutral loss
precursor
no
23
yes
yes
627, 643
neutral loss
605, 629,
659, 675
and 3). CYP screen inhibition demonstrated that at 3 μM,
CYP1A2 was inhibited about 27.3% by compound 13 whereas
compound 23 inhibited 37.4%. At 30 μM, the inhibition
patterns contained similar trends. The detailed inhibition data
are summarized in Table 4. To test the possible cardiovascular
toxicity, we examined hERG inhibition triggered by compound
Our previous structure−activity relationship (SAR) study
demonstrates that the 7,8-dihydroxy groups on the A ring and
the middle heteroatomic chromen-4-one C ring are essential for
Table 4. Summary of CYP Screening
compd
test concn (μM)
CYP3A4 modazolam (%)
CYP3A4 testoterone (%)
CYP2C9 (%)
CYP2C19 (%)
CYP2D6 (%)
CYP1A2 (%)
13
30
3
40.5
10.8
48.1
3.9
43.3
4.5
73.6
12.4
52.9
11.9
57.5
11.1
90.0
16.2
32.0
8.7
66.1
27.3
75.9
37.4
23
30
3
18.7
3.4
81.6
7.2
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the TrkB stimulatory effect. Additionally, the 4′-position on the
B ring is also critical for the agonistic effect. An electron-
withdrawing group, such as F, or an electron-donating OH at
this position suppresses the activity. Nonetheless, replacement
with a dimethylamino or pyrrolidino group yields the desired
activity. On the basis of the lead compound 4′-DMA-7,8-DHF,
we initiated a lead optimization campaign by synthesizing a
series of new compounds. Since the lead compound exhibits
robust agonistic effect and potent antidepressant efficacy, we
employed the bioisosteric strategy to enhance the desired
biological or physical properties of a compound without making
significant changes in chemical structure. We added a fluoride
group at different positions and replaced the 7,8-dihydroxy
groups with an imidazole ring or changed the dimethylamino
group into a pyrrolidino ring. From compounds 31 and 32, we
found that addition of a fluoride group barely affects TrkB
stimulatory activity (Figure 2). Replacing the 7,8-dihydroxy
groups with an imidazole ring in compound 11 elevated its
agonistic activity compared to the lead compound 24, though
the in vivo TrkB stimulatory activity remained comparable for
these two compounds (Figure 2B,C). Although compound 11
strongly activated TrkB in mouse brain and decreased the
immobility in FST, this compound highly augmented the
locomotor activity as well (Figure 3), suggesting that it might
reduce the immobility in FST by increasing the locomotor
activity. Since the dimethylamino group may be prone to
metabolic demethylation, we replaced the dimethylamino
group with a pyrrolidino group. Remarkably, compound 13
not only displayed higher agonistic activity than compound 11
but also attenuated the locomotor enhancement effect by
compound 11 (Figure 5). As expected, compound 13 was
active in both FST and TST depression behavioral assays. It
remains unclear why compound 23 exhibits potent TrkB
stimulatory activity after oral administration but fails to show
significant immobility reduction activity in either antidepressant
behavioral assay. Conceivably, its high reactivity and poor
microsomal stability account for its lack of an efficacious
antidepressant effect (Tables 2 and 3).
Catechol-related compounds usually possess poor pharma-
cokinetic profiles because of oxidation, glucuronidation,
sulfation, or methylation. For instance, catechol-containing
apomorphine is a non-narcotic morphine derivative that acts as
a potent dopaminergic agonist. Apomorphine metabolism
occurs through several enzymatic pathways, including N-
demethylation, sulfation, glucuronidation, and catechol O-
methylation as well as nonenzymatic oxidation.17 L-DOPA is
the primary component of Parkinson’s disease (PD) therapy;
this drug is usually administered orally, but it is extensively
metabolized in the gastrointestinal tract, so relatively little
circulates in the bloodstream as intact L-DOPA. To minimize
the conversion to dopamine outside the central nervous system,
L-DOPA is usually given in combination with peripheral
inhibitors of aromatic L-amino acid decarboxylase and COMT
(catechol methyltransferase) inhibitor.18 Our preliminary in
vivo metabolism study shows that 7,8-DHF is also subjected to
oxidation, glucuronidation, sulfation, and methylation (Sup-
porting Information Figure 2). Among the modifications,
glucuronidation and sulfation are mainly responsible for the in
vivo clearance of the flavonoids. In addition, we have detected
the O-methylated metabolites in both the plasma and brain
samples after oral administration. Conceivably, these mod-
ification pathways may explain the relatively short half-lives of
7,8-DHF and its synthetic derivatives. Alteration of this labile
group into the bioisosteric imidazole derivatives escalates the
PK profile of compound 13 (Table 1). Though compound 13
displays an improved ADMET profile than the lead compound
24, its oral bioavailability remains poor and the in vivo half-life
is relatively short. Additional medicinal chemistry is needed to
further optimize the lead compound and to improve the PK
parameters.
Among the leading causes of drug candidate failure (∼60%)
are poor PK/ADME, toxicological properties, and adverse
effects, which contribute more significantly than “lack of
efficacy” (∼30%). In order to optimize the ADME properties
and also foresee liabilities of drug candidates, in vitro and in
vivo ADMET (ADME plus toxicity) filters are being developed
and implemented in various stages of the drug discovery and
development process to alert to potential ADMET issues in the
clinic.23 Toxicology- and pharmacology-related safety has
become a leading cause for compound failure during preclinical
development and clinical trials. Most withdrawals of marketed
drugs were associated with hERG inhibition24,25 and
hepatotoxicity.26 Accordingly, we have examined our com-
pounds’ in vitro ADMET profiles. Remarkably, compounds 12,
13, and 23 exhibited negligible cytotoxicity or genotoxicity
(Figure 6). The hERG inhibition index is also trifling for
compound 13. Moreover, the microsomal stability and reactive
metabolic screening assays support that compound 13 is more
stable than compound 23 in vitro. Though compound 13’s in
vivo half-life is about 103 min in mice, its oral bioavailability is
only 2%. Nonetheless, it exhibited a much improved in vivo PK
profile than the lead compound 24 (Table 1), demonstrating
our progress in the optimization mission. It is worth noting that
there is significant discrepancy between the remarkable
therapeutic efficacy in the mouse models of antidepression
and poor in vivo oral bioavailability. The potential explanation
for this difference might be the compound’s extreme potency so
that a low concentration in the circulation system is sufficient
for provoking the physiological effects. It is also possible that
the metabolites of the compound are active as well. For
instance, the B ring in compound 13 can be metabolized via
hydroxylation in the lead compound 24 and compound 13. Our
previous study shows that hydroxylation on 2′ or 3′ position
escalates 7,8-DHF’s agonistic activity.12 On the other hand, it
remains unclear how 4′-DMA-7,8-DHF, which has a short half-
life, exerts its physiological activities in animals. Conceivably, its
O-methylated or B-ring hydroxylated metabolites in mouse
brain might contribute to these actions. Clearly, further
medicinal chemistry is necessary to continue developing
compound 13 to meet the preclinical standards for a promising
candidate. Specifically, we will determine the potential
metabolites, which may shed light on how to metabolically
stabilize this compound. Further, we will reduce the candidate’s
plasma protein binding affinity so that we can improve its brain
bioavailability. Plausibly, the resynthesized novel compounds
will possess longer t1/2 and much-improved oral bioavailability
with low toxicity. Together, our data support that 7,8-imidazole
and 4′-pyrrolidone-substituted flavones are excellent lead
compounds justifying further medicinal modification. These
compounds not only provide a novel tool to dissect the
biological functions of BDNF/TrkB signaling but also act as
good lead compounds with great potential for future drug
development for various neurological diseases, including
depression.
J
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Journal of Medicinal Chemistry
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(100 mL) and washed with 1 N HCl (100 mL) and water (50 mL).
The organic phase was separated, dried with sodium sulfate, filtered,
and concentrated to afford a gray solid, which was purified (PE/EA =
1/1) to afford compound 8 (1.2 g, yield of 75% with purity of >95% by
HPLC).
7-Amino-2-(4′-(dimethylamino)phenyl)-8-nitro-4H-chro-
men-4-one (9). A mixture of 3-acetamido-6-acetyl-2-nitrophenyl 4-
(dimethylamino)benzoate (compound 8, 2 g) and potassium
hydroxide (8 g) in pyridine (20 mL) was heated to 60 °C for 1 h
and then poured into icy 1 N HCl (100 mL). The yellow solid was
collected and dissolved in acetic acid (20 mL) and concentrated
sulfuric acid. The resulting mixture was heated to 110 °C for 30 min.
The mixture was cooled to room temperature and then poured into
saturated sodium carbonate. The yellow solid was filtered and dried in
vacuo to afford 7-amino-2-(4-(dimethylamino)phenyl)-8-nitro-4H-
chromen-4-one (compound 9 with R = dimethylamino) (1.5 g, yield
of 89% with purity of >95% by HPLC).
EXPERIMENTAL PROCEDURES
■
Cells, Reagents, and Mice. Anti-p-TrkB 817 was from Epitomics.
Anti-p-TrkB 706 was from Santa Cruz. Anti-TrkB antibody was from
Cell Signaling. The wild-type C57BL/6 mice were bred in a pathogen-
free environment in accordance with Emory Medical School
guidelines. All chemicals not included above were purchased from
Sigma. 7,8-DHF was purchased from TCI. The syntheses of
compounds 24−27 were reported in our previous study.12 The
fluoro-substituted flavonoids in Figure 2A (compounds 25, 30, 31, 32,
33, 34, and 35) were from Sundia (Shanghai, China). Compounds 28
and 29 in Figure 2A were provided by NIMH, Chemical Synthesis and
Drug Supply Program at RTI. Experiments included NMR (Bruker
AV300K, 300 MHz), MS (Shimadzu LCMS), and HPLC (PE, dual
pumper, SPD detector, ODS-C18 reverse phase, 254 nm, CH3CN−
H2O−0.1%TFA). Phospho-TrkB Y816 antibody was described
before.12 This phospho-TrkB was utilized for immunostaining the
brain sections. Anti-TrkB (Cell Signaling, which recognizes both full-
length and truncated TrkB) was used for immunoblotting. p-Akt 473
Sandwich ELISA was from Cell Signaling. BDNF was from Peprotech.
Anti-phospho-TrkA 794, anti-TrkA, phospho-Akt-473, anti-Akt, and
anti-phospho-Erk1/2 antibodies were from Cell Signaling.
7,8-Diamino-2-(4′-(dimethylamino)phenyl)-4H-chromen-4-
one Hydrochloride (10).
A solution of 7-amino-2-(4-
(dimethylamino)phenyl)-8-nitro-4H-chromen-4-one (compound 9,
900 mg, 2.77 mmol) and 10% Pd/C (450 mg) in methanol (9 mL)
and concentrated hydrochloride (aqueous, 9 mL) was stirred in an
atmosphere of hydrogen overnight. The solid was filtered and the
filtrate was evaporated at reduced pressure to afford 7,8-diamino-2-(4′-
(dimethylamino)phenyl)-4H-chromen-4-one hydrochloride (com-
pound 10) as a light yellow solid (810 mg, yield of 88% with purity
of >95% by HPLC).
Synthesis of Compounds 11, 12, 13, and 23. 4-Aminomethyl
Benzoate (2). A solution of compound 1 (6.857 g, 50 mmol) in
MeOH (120 mL) was treated with SOCl2 (7 g, 59 mmol, 1.2 equiv) at
−10 °C, and the mixture was stirred at room temperature overnight.
LC−MS and TLC showed the reaction was over and the desired
product was found. The mixture was concentrated and the residue was
washed with Et2O to give compound 2 as a pale-yellow solid (with
8-(4′-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-
6(1H)-one (11). A solution of 7,8-diamino-2-(4′-(dimethylamino)-
phenyl)-4H-chromen-4-one hydrochloride (compound 10, 500 mg) in
HCO2H (5 mL) was heated to reflux for 1 h. The volatiles were
evaporated at reduced pressure, and the residue was partitioned
between EA/isopropanol = 20/1 (50 mL) and saturated sodium
carbonate (25 mL). The organic phase was separated, dried with
sodium sulfate, filtered, and concentrated to afford a yellow solid,
which was recrystallized from EA (25 mL) to afford a light yellow solid
1
purity of ∼95% by HPLC, 6.0 g, used for the next step without H
NMR confirmed).
Methyl 4-Pyrrolidin-1-ylbenzoate (3). A mixture of compound
2 (3.02 g, 20 mmol), K2CO3 (5.52 g, 40 mmol), and 1,4-
dibromobutane (5.18 g, 24 mmol) in dioxane (30 mL) and water
(30 mL) was treated with TBAB (tert-butylamine borane), and the
mixture was refluxed for 48 h. LC−MS and TLC showed the reaction
was not over and the desired product was found. The mixture was
concentrated, and the residue was diluted with EA (ethyl acetate),
washed with water and brine, and dried over Na2SO4. The mixture was
concentrated and purified by silica gel (PE (petroleum ether) to PE/
EA = 5:1 to 3:1) to give compound 3 as a pale-yellow solid (with
1
(compound 11 with R= dimethylamino, 111 mg, yield of 24%). H
NMR (300 MHz, DMSO-d6) δ 8.46 (m,1H), 10.09 (br s, 1H), 8.01
(m, 2H), 7.82 (m, 1H), 7.61 (m, 2H), 6.84−6.88 (m, 3H). MS-ESI
calculated, 305; found, 306(M + H)+. HPLC: 99.23%
8-(4′-(Pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-
6(1H)-one (12 and 13). To a solution of compound N-(4-acetyl-3-
hydroxy-2-nitrophenyl)acetamide (compound 6, 700 mg, 2.94 mmol)
in dry DCM (dichloromethane,10 mL) was added DIPEA (N,N-
diisopropylethylamine, 0.8 mL) followed by compound 7 4-
(pyrrolidin-1-yl)benzoyl chloride (950 mg) at 0 °C, and the resulting
mixture was stirred at room temperature overnight. The mixture was
quenched with water and extracted with dichloromethane. The
combined extracts were washed with water and brine and dried over
Na2SO4. The mixture was concentrated and purified by silica gel (PE
to PE/EA = 10:1 to 7:1 to 5:1 to 3:1) to give the coupled ester
compound as an orange solid (750 mg), which was then followed by
similar procedures as described for compound 11. After cyclization
with KOH/pyridine, followed by H2SO4/AcOH reflux, the nitro group
in the intermediate was reduced with Fe (300 mg, 5.36 mmol) and
NH4Cl (154 mg, 2.85 mmol) to yield the reduced 7,8-diamino
compound 10 with R = pyrrolidin-1-yl. A mixture of compound 7,8-
diamino-2-(4′-(N-pyrrolidino)phenyl)-4H-chromen-4-one hydro-
chloride (1.1 g) in HCOOH (10 mL) was refluxed for 2 h. TLC
showed the reaction was over. Combined with other batches, the
mixture was basified to pH ≈ 8 by 1 N NaOH. The mixture was then
concentrated and purified by silica gel (dichloromethane to dichloro-
methane/MeOH = 100:1 to 50:1 to 20:1) to give a mixture of
compound 12 and 2-methylated compound 13 (80 mg), which was
purified by pre-HPLC to give compound 12 as an orange solid (50 mg,
1
1
purity of >95% by HPLC, 1.12 g, confirmed by H NMR). H NMR
(400 MHz, DMSO-d6): δ 7.82 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.8 Hz,
2H), 3.81 (s, 3H), 3.36 (m, 4H), 2.02 (m, 4H).
4-(Pyrrolidin-1-yl)benzoic Acid (4). A mixture of compound 3
(1.12 g, 5.46 mmol) and NaOH (1.1 g, 27.3 mmol) in MeOH (15
mL) and water (15 mL) was refluxed overnight. LC−MS and TLC
showed the reaction was over and the desired product was found. The
mixture was concentrated and the residue was acidified with 6 N HCl
to pH ≈ 3 and the precipitate was collected by filtration and dried with
vacuum to give compound 4 as a white solid (with purity of >95% by
HPLC, 600 mg, confirmed by 1H NMR). 1H NMR (400 MHz,
DMSO-d6): δ 12.03 (br s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 6.54 (d, J =
8.4 Hz, 2H), 3.30 (m, 4H), 1.97 (m, 4H).
4-(Pyrrolidin-1-yl)benzoyl Chloride (5). To a suspension of
compound 4 (115 mg, 0.6 mmol) in DCM (2 mL) at 0 °C was added
DMF followed by (COCl)2 (91 mg, 0.72 mmol). The mixture was
stirred at room temperature for 2 h, and a clear solution was obtained.
TLC (quenched by MeOH) showed the reaction was over. The
mixture was concentrated, and to the residue was added toluene. Then
the mixture was concentrated twice to give compound 5 as a pale-
yellow solid, which was used for the next step without further
purification.
8-(4′-(Dimethylamino)phenyl)chromeno[7,8-d]imidazol-
6(1H)-one (11). 3-Acetamido-6-acetyl-2-nitrophenyl 4-
(Dimethylamino)benzoate (8). To a mixture of N-(4-acetyl-3-
hydroxy-2-nitrophenyl)acetamide (compound 6, 1 g, 4.1 mmol, 1.0
equiv) and triethylamine (1.5 mL) was added 4-(dimethylamino)-
benzoyl chloride (compound 7 with R = dimethylamino, 6.3 mmol) in
three portions at 0 °C. Then the mixture was stirred at room
temperature for 3 h. The mixture was diluted with dichloromethane
1
confirmed by H NMR) and compound 13 as dark-orange solid (25
mg, confirmed by 1H NMR). For 12: 1H NMR (400 MHz, CD3OD) δ
8.81 (s, 1H), 8.04 (m, 3H), 7.68 (d, J = 8.8 Hz, 1H), 6.78 (s, 1H), 6.66
(d, J = 8.8 Hz, 2H), 3.37 (m, 4H), 2.07 (m, 4H). HPLC: 96%. MS-ESI
calculated, 331.4; found, 332.1 (M + 1)+. For 13: 1H NMR (400 MHz,
K
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Journal of Medicinal Chemistry
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CD3OD): δ 8.09 (d, J = 8.4 Hz, 1H), 8.01(d, J = 8.4 Hz, 2H), 7.64 (d,
J=8.4 Hz, 1H), 6.78 (s, 1H), 6.69(d, J = 8.8 Hz, 2H), 3.41 (m, 4H),
2.92 (s, 3H), 2.10 (m, 4H); HPLC: 94%; MS-ESI: calculated: 345.4;
found: 345.9 (M + 1)+.
2-(4-(Pyrrolidin-1-yl)phenyl)pyrano[3,2-g]indol-4(9H)-one
(23). To a solution of compound 4′-N-pyrrolidinobenzoyl methyl
ketone (154 mg, 0.813 mmol) in dry THF(10 mL) at −20 °C was
added LiHMDS (2 mL, 2 mmol), and the mixture was stirred at −20
°C for 1 h. Compound 21 7-methoxy-6-indoloyl chloride (200 mg,
0.976 mmol) with (COCl)2) in dry THF was added, and the mixture
was stirred at −20 °C for 1 h and then at room temperature overnight.
TLC and LC−MS showed the desired product was found. The
mixture was quenched with aqueous NH4Cl and extracted with EA,
and the combined extracts were washed with water and brine and
dried over Na2SO4. The mixture was concentrated and purified by
silica gel (PE to PE/EA = 5:1 to 2:1) to give intermediate compound
22, which was refluxed in HBr (48%, 6 mL) for 3 h. TLC showed the
reaction was over. The mixture was combined with other batches and
cooled to room temperature. Water was added and the mixture was
basified to pH ≈ 8 by NaOH (1 N) and concentrated and purified by
silica gel (dichloromethane to dichloromethane/MeOH = 20:1 to
10:1) to give crude compound 23 as a brown solid. This crude product
was purified by pre-HPLC to give compound 23 as a brown solid. 1H
NMR (400 MHz, DMSO-d6): δ 12.40 (br s, 1H), 8.16 (d, J = 8.8 Hz,
2H), 7.70 (s, 1H), 7.57 (s, 2H), 6.80 (s, 1H), 6.68 (m, 3H), 3.36 (m,
4H), 2.00 (m, 4H). HPLC: 99%. MS-ESI calculated, 330.4; found,
331.1 (M + 1)+.
Phospho-Akt S473 ELISA. PathScan phospho-Akt1 (Ser473)
Sandwich ELISA kit was purchased from Cell Signaling (catalog no.
7160). A 100 μL sample in sample diluent (supplied in the kit) was
added to each well and incubated overnight at 4 °C. After 4× wash
with 200 μL of wash buffer (supplied in the kit), 100 μL/well
detection antibody was added and the mixture was incubated for 1 h at
37 °C. After 4× wash, 100 μL of HRP-linked secondary antibody
(supplied in the kit) was added, and the mixture was incubated for 30
min at 37 °C. After a final wash, 100 μL of 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 a 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.
TrkB Agonists Drug Administration. Male C57BL/6 mice aged
2 months were orally administrated 4′-DMA-7,8-DHF derivatives in a
single dose of 1 mg/kg for 4 h. The control mice were injected with
saline. The mice were sacrificed, and brains were homogenized and
ultracentrifuged. The supernatant (40 μg) was employed for SDS−
PAGE and immunoblotting analysis with indicated antibodies,
respectively. Male C57BL/6 mice aged 2 months were orally
administrated compound 32 or 11 at a dose of 5 mg kg−1 day−1
each and compound 13 or 23 at a dose of 2.5 mg kg−1 day−1 each for
21 days. BrdU (100 mg/kg) was ip injected 2 h before the TrkB
agonist treated animals were sacrificed and the hippocampal section
lysates were analyzed by immunoblotting with p-TrkB and total TrkB
antibodies, p-AKT ELISA.
Immunohistochemistry Staining. Brain tissues were fixed in 4%
paraformaldehyde overnight followed by paraffin embedding. Sections
of 6 μm were cut. For immunohistochemical staining, brain sections
were deparaffinized in xylene and rehydrated 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 TrkB 816 and TrkB were
detected using specific antibodies. Paraffin sections were deparaffinized
in xylene and rehydrated in 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. Secondary
antibody was applied using anti-rabbit-Alexa 594 (red) and anti-
mouse-fluorescein isothiocyanate (FITC) (green). DAPI (blue) was
used for nuclear staining.
8-(4′-(Pyrrolidino)phenyl)chromeno[7,8-d]indoloyl-6(1H)-
one (23). Methyl 2-Methoxy-4-methylbenzoate (15). A mixture
of compound 14 (10 g, 65.73 mmol), CH3I (37.32 g, 262.9 mmol),
and K2CO3 (45.4 g, 329 mmol) in dry DMF (100 mL) was stirred at
room temperature overnight. TLC showed the reaction was over. The
mixture was filtered, and the filtrate was concentrated. The residue was
diluted with EA, washed with water and brine, and dried over Na2SO4.
The mixture was concentrated to give compound 15 as a yellow oil
1
(with purity of >95% by HPLC, 12 g, confirmed by H NMR, 100%
yield). 1H NMR (400 MHz, CDCl3): δ 7.71 (d, J = 8.4 Hz, 1H), 6.78
(m, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 2.38 (s, 3H).
Methyl 2-Methoxy-4,5-dimethylbenzoate (16). To a solution
of compound 15 (10.0 g, 55.5 mmol) in dry DMF (80 mL) was added
NCS (8.15 g, 61 mmol) in portions, and the resulting mixture was
stirred at room temperature overnight. The mixture was concentrated,
and the residue was dissolved in EA, washed with water and brine, and
dried over Na2SO4. The mixture was concentrated to give compound
16 as a yellow solid (with purity of >95% by HPLC, 11.0 g, confirmed
1
1
by H NMR). H NMR (400 MHz, CDCl3): δ 7.78 (s, 1H), 6.83 (s,
1H), 3.88 (m, 6H), 2.39 (s, 3H).
Methyl 2-Methoxy-4,5-dimethyl-3-nitrobenzoate (17). To a
solution of compound 16 (5 g, 23.3 mmol) in concentrated H2SO4
(23 mL) at 0 °C was added HNO3 (2.3 mL) in portions, and the
resulting mixture was stirred at 0 °C for 2 h. TLC showed the reaction
was over. The mixture was poured into ice and extracted with Et2O.
The combined extracts were washed with water and brine, dried, and
concentrated to give compound 17 as a yellow oil (with purity of
>95%, 4.5 g, confirmed by 1H NMR). 1H NMR (400 MHz, CDCl3): δ
7.99 (s, 1H), 3.92 (m, 6H), 2.33 (s, 3H).
(E)-Methyl 4-(2-(Dimethylamino)vinyl)-2-methoxy-5-methyl-
3-nitrobenzoate (18). A mixture of compound 17 (2.6 g, 10 mmol)
in dry DMF(10 mL) was treated with dimethylformamide−
dimethylacetal (DMF−DMA) (3.58 g, 30 mmol), and the mixture
was stirred at 120 °C overnight. TLC showed the reaction was over.
The mixture was cooled and concentrated, and the residue was
dissolved in EA, washed with water and brine, and dried over Na2SO4.
The mixture was concentrated to give a crude as a brown semisolid
(compound 18 with purity of >95%, 3 g, used for the next step without
further purification).
Methyl 7-Methoxy-1H-indole-6-carboxylate (19). A mixture of
compound 18 (3.1 g, 11.5 mmol) and Pd(OH)2 (160 mg) in MeOH
(20 mL) was stirred at room temperature under H2 overnight. TLC
showed the reaction was over. The mixture was filtered and
concentrated and purified by silica gel (PE to PE/EA = 10:1 to 5:1)
to give compound 19 as a white solid (with purity of >95%, 1.66 g,
1
1
confirmed by H NMR). H NMR (400 MHz, CDCl3): δ 8.61 (br s,
1H), 7.64 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.34 (m, 1H),
6.59 (m, 1H), 4.04 (s, 3H), 3.94 (s, 3H).
7-Methoxy-1H-indole-6-carboxylic Acid (20). A mixture of
compound 19 (1.66 g, 8.09 mmol) and NaOH (1.62 g, 40 0.5 mmol)
in MeOH (15 mL), THF (5 mL), and water (15 mL) was refluxed for
3 h. TLC showed the reaction was over. The mixture was cooled and
concentrated, the residue was acidified to pH ≈ 2 by 2 N HCl, and the
precipitate was collected by filtration and dried to give compound 20
as an off-white solid (with purity of >95% by HPLC, 980 mg,
confirmed by 1H NMR). 1H NMR (400 MHz, DMSO-d6): δ 12.41 (br
s, 1H), 11.59 (s, 1H), 7.48 (s, 3H), 7.40 (d, J = 8.0 Hz, 1H), 7.31 (d, J
= 8.4 Hz, 1H), 6.50 (s, 1H), 3.92 (s, 3H).
7-Methoxy-1H-indole-6-carbonyl Chloride (21). To a mixture
of compound 20 (100 mg, 0.523 mmol) in dry THF (5 mL) was
added DMF followed by (COCl)2 (77 mg, 0.61 mmol) at 0 °C, and
the resulting mixture was stirred at 0 °C for 1 h. TLC showed the
reaction was almost over. The mixture was concentrated to give
compound 21 as a yellow solid, which was used for the next step
without further purification.
Forced Swim Test. Adult male mice (2−3 months old) were
randomly submitted to a forced swim test without a preswim. Saline
and TrkB agonists were orally administrated by gavage for 21 days.
The mice were allowed to adapt to the test room for 2 days. The mice
were placed in a clear glass cylinder with a diameter of 16 cm, half-
L
dx.doi.org/10.1021/jm301099x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
filled with clear water at 24 °C (water depth of 14 cm did not allow the
mice to reach the bottom of the cylinder) for a total of 6 min, and
immobility was recorded during the last 4 min by an investigator blind
to the treatment.
Tail Suspension Test. Mice were individually suspended by the
tail to a horizontal ring stand bar 30 cm above the floor using adhesive
tape for 6 min and videotaped. Latency to immobility and time spent
immobile were scored for each mouse. Following the test, mice were
returned to their home cage. Immobility scores were compared by
unpaired t test.
Locomotor Activity. Locomotor activity was assessed using an
automated system (San Diego Instruments, La Jolla, CA, U.S.) with
photobeams that recorded ambulations (consecutive beam breaks).
Drug-treated mice were placed in the locomotor chambers, and their
activity was recorded for 2 h at 30 min intervals.
control compounds for ADMET assays are included in Supporting
Information Tables 1−4.
Homology Modeling and Docking of 7,8-DHF to TrkB ECD.
A structure-guided sequence alignment between human TrkA ECD
and human TrkB ECD was edited to accommodate the short
deletions/insertions at the tips of the connecting loops between
secondary structural elements. A monomeric structure of TrkA was
selected from the crystal structure of the TrkA-nerve growth factor
complex (PDB code 2IFG)29 as the template. Comparative modeling
was carried out with the MODELLER program by satisfaction of
spatial restraints.30 Docking of 7,8-DHF to the modeled TrkB ECD
structure was carried out with MEDock based on a maximum entropy
optimization algorithm.31 The top five probable solutions resulting
from random seed searches, with the lowest docked energies ranging
from −9.3 to −8.7 kcal/mol, were selected for graphical analysis.
Neurogenesis Analysis in TrkB-Agonists-Treated Hippo-
campus. Adult male mice (2−3 months old) were orally
administrated with saline, 13, and 23 (2.5 mg/kg) for 21 days. Then
BrdU (100 mg/kg) was ip injected. In 2 h, the mice were perfused
with 4% paraformaldehyde. Immunohistochemical staining was
performed on formalin-fixed paraffin embedded sections. Sections
from brain were cut, deparaffinized in xylene, and rehydrated in graded
alcohols. The slides were boiled in 10 mM citric acid (pH 6.0) for 10
min, followed by an incubation in 2 N HCl for 10 min in room
temperature. The slides were then permeabilized and blocked with 1%
BSA in 0.2% PBS Tween-20 (PBST). The incorporated BrdU was
stained using anti-BrdU-FITC (Abcam, U.S.) at 4 °C for 16 h. After
three times of washing in PBS, the cells were then stained with DAPI
for another 10 min at room temperature. The slides were finally
mounted with AquaMount (Lerner Laboratories, U.S.) containing
0.01% 1,4-diazobicyclo(2,2,2)octane and examined under a fluores-
cence microscope.
Micronucleus Assay. The cells treated with 7,8-DHF derivatives
at 50 μM for 24 h were washed with PBS, incubated in mild hypotonic
solution (0.075 M KCL/0.9% NaCl, 1:19) for 10 min at 37 °C, fixed
with methanol−glacial acetic acid (3:1) for 15 min at 37 °C, rinsed
with distilled water, and air-dried. Fixed cells were stained with DAPI
(2 μg/mL) for 30 min in the dark at room temperature. Cells were
rinsed with PBS and distilled water and mounted with Fluoromount-G
(Southern Biotech). Micronuclei were identified based on the criteria
specified by Miller at al.27 One-thousand cells per treatment were
analyzed using the fluorescence microscope. Data are the mean
SEM of three independent experiments.
Single Cell Gel Electrophoresis (SCGE, the Comet Assay).
The treated and control HepG2 cells embedded in 0.75% LMP (low
melting point) agarose and spread on a base layer of 1% NMP (normal
melting point) agarose in PBS buffer were placed in a lysis solution
(2.5 M NaCl, 200 mM Na2EDTA, 10 mM Tris-HCl, pH 10, and 1%
Triton X-100) at 4 °C for 2 h. Slides were transferred to an
electrophoresis box and immersed in an alkaline solution (300 mM
NaOH, 1 mM Na2EDTA, pH > 13). After a 40 min unwinding time, a
voltage of 25 V (300 mA) was applied for 30 min at 4 °C. Slides were
neutralized with 3 × 5 min washes with Tris-HCl (0.4 M, pH 7.4) and
stained with ethidium bromide (EtBr, 10 μg/mL). EtBr stained
nucleotides were examined with fluorescence microscope. Total cell
numbers in a field (>100) were counted, and the number of nucleoids
exhibiting comet tail formation was identified.22,28 Results were
quantified as the number of comet nuclei out of the total number of
nuclei observed from three independent experiments.
In Vitro ADMET and in Vivo Pharmacokinetic Studies. Mice
of 20−30 g at age of 6−8 weeks were administrated with the indicated
compounds iv or orally. At different time points (3, 10, 30, 60, 120,
240, 360, and 480 min), blood aliquots (300−400 mL) were collected
in tubes coated with lithium heparin, mixed gently, then kept on ice,
and centrifuged at 2500g for 15 min at 4 °C within 1 h of collection.
The plasma was then harvested and kept frozen at −20 °C until
further processing. For each time point, 3 mice/group were employed.
The plasma samples were analyzed by HPLC. The in vitro ADMET
assays were conducted in Apredica, Inc. The experimental conditions
for the ADMET are available at www.apredica.com. The standard and
ASSOCIATED CONTENT
■
S
* Supporting Information
Two figures showing 7,8-DHF metabolism and molecular
modeling; four tables listing the positive controls in the
ADMET profiling. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 404-712-2814. E-mail: kye@emory.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported by grant from NIDCD, National
Institutes of Health (Grant RO1 DC010204), to K.Y. The
authors thank Andrei Halavaty for the help in molecular
modeling. The authors are thankful to Dr. Obianyo at the Ye
laboratory for proofreading of the manuscript.
ABBREVIATION USED
■
7,8-DHF, 7,8-dihydroxyflavone; 4′-DMA-7,8-DHF, 4′-dimethy-
lamino-7,8-dihydroxyflavone; ECD, extracellular domain;
BDNF, brain-derived neurotrophic factor; FITC, fluorescein
isothiocyanate; NGF, nerve growth factor; DAPI, 4′,6-
diamidino-2-phenylindole; TrkB, tropomyosin-related kinase
B; NT-3, neurotrophic factor 3; MeCP2, methyl CpG binding
protein 2; FST, forced swim test; TST, tail suspension test;
COMT, catechol methyltransferase; LDH, lactate dehydrogen-
ase
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