Discovery of efficient stimulators for adult hippocampal neurogenesis based on scaffolds in dragon's blood

Article abstract of DOI:10.1016/j.ejmech.2017.05.025

Reduction of hippocampal neurogenesis caused by aging and neurological disorders would impair neural circuits and result in memory loss. A new lead compound (N-trans-3′,4'-methylenedioxystilben-4-yl acetamide 27) has been discovered to efficiently stimulate adult rats' neurogenesis. In-depth structure-activity relationship studies proved the necessity of a stilbene scaffold that is absent in highly cytotoxic analogs such as chalcones and heteroaryl rings and inactive analogs such as diphenyl acetylene and diphenyl ethane, and validated the importance of an NH in the carboxamide and a methylenedioxy substituent on the benzene ring. Immunohistochemical staining and biochemical analysis indicate, in contrast to previously reported neuroprotective chemicals, N-stilbenyl carboxamides have extra capacity for neuroproliferation-type neurogenesis, thereby providing a foundation for improving the plasticity of the adult mammalian brain.

Full text of DOI:10.1016/j.ejmech.2017.05.025

European Journal of Medicinal Chemistry 136 (2017) 382e392
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of efficient stimulators for adult hippocampal neurogenesis
based on scaffolds in dragon's blood
,
,
,
Jian-Hua Liang a *, Liang Yang a 1, Si Wu a 1, Si-Si Liu a, Mark Cushman b, Jing Tian c,
Nuo-Min Li a, Qing-Hu Yang a, He-Ao Zhang a, Yun-Jie Qiu a, Lin Xiang a, Cong-Xuan Ma a,
Xue-Meng Li a, Hong Qing a
,
**
a School of Life Science, Beijing Institute of Technology, Beijing 100081, China
b Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer Research, Purdue University
47907 USA
c Biomedical School, Beijing City University, Beijing 100094, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Reduction of hippocampal neurogenesis caused by aging and neurological disorders would impair neural
circuits and result in memory loss. A new lead compound (N-trans-30,4'-methylenedioxystilben-4-yl
acetamide 27) has been discovered to efficiently stimulate adult rats' neurogenesis. In-depth struc-
ture-activity relationship studies proved the necessity of a stilbene scaffold that is absent in highly
cytotoxic analogs such as chalcones and heteroaryl rings and inactive analogs such as diphenyl acetylene
and diphenyl ethane, and validated the importance of an NH in the carboxamide and a methylenedioxy
substituent on the benzene ring. Immunohistochemical staining and biochemical analysis indicate, in
contrast to previously reported neuroprotective chemicals, N-stilbenyl carboxamides have extra capacity
for neuroproliferation-type neurogenesis, thereby providing a foundation for improving the plasticity of
the adult mammalian brain.
Received 3 February 2017
Received in revised form
8 May 2017
Accepted 9 May 2017
Available online 10 May 2017
Keywords:
Neurogenesis
Stilbene
SAR
© 2017 Elsevier Masson SAS. All rights reserved.
Dragon's blood
BrdU
NeuN
1. Introduction
progressive loss of neurons. Promoting adult neurogenesis in situ
doesn't have to require invasive transplantation and suppression of
Adult neurogenesis in mammals was documented in the 1960s,
which overturned the long-held dogma that no new neurons are
added in the adult mammalian brain [1]. The reservoirs of neural
stem cells or neural progenitor cells (NPCs) normally exist within
two regions: the subgranular zone (SGZ) of the hippocampal den-
tate gyrus (DG) and the subventricular zone (SVZ) of the lateral
ventricles. Neurogenesis in the hippocampus plays an important
role in improving learning and memory [2]. Unfortunately, adult
neurogenesis continuously and severely declines with age and
during neurodegenerative disease progress [3e5], especially in AD
(Alzheimer's disease) patients [6]. Currently, no medical treatments
can halt or reverse AD progression, which is characterized by
prominent amyloid plaques, neurofibrillary tangles, and the
host rejection [7], so it is a highly attractive strategy to reverse the
loss of functional neurons that occurs in aging and AD progress,
especially in view of the background of several failures in clinical
trials targeted at hot spot b-amyloid.
Unlike traditional targets such as enzymes and receptors,
stemistry, namely controlling the fate (proliferation and differen-
tiation) of stem cells in situ by chemicals, lacks an efficient strategy
to discover new leads [8]. Despite great efforts on the discovery of
the promoters [9], only a short list of chemicals have survived
in vivo neurogenesis screening, including donepezil [10], galant-
amine, memantine [11] curcumin [12], nodakenin [13], asarone
[14], allantoin [15], tetrahydrohyperforin [16], 4'-dimethylamino-
flavone [17], isoxazole [18,19], spinosin [20], isoquinoline-dione
[21] and aminopropyl carbazole [22,23]. Due to the presence of
the Blood-Brain Barrier (BBB), the promotion of neurogenesis by
small molecules has not been established to occur at low doses of
sub-milligram per kg. Most of the reported promotion of neuro-
genesis is heavily dependent on the neuroprotective mechanism. In
* Corresponding author.
** Corresponding author.
E-mail addresses: ljhbit@bit.edu.cn (J.-H. Liang), hqing@bit.edu.cn (H. Qing).
These authors contributed equally to this work with the first author.
1
http://dx.doi.org/10.1016/j.ejmech.2017.05.025
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
383
other words, the promoters exert their neurogenesis effect by
protecting newborn neurons from month-long apoptosis instead of
directly stimulating proliferation of NPCs [24]. Therefore, the search
for direct stimulators, instead of neuroprotective agents, with the
capacity to promote efficient neuroproliferation-type neurogenesis
is still an open challenge.
agent P7C3-A20 detected in the brain tissue (0.9
m
M, after an oral
dose of 20 mg/kg) [22]. A strategy of in vitro cytotoxicity evaluation
prior to SAR-based in vivo screening was adopted to pinpoint a lead.
Compounds with very low cytotoxicity were selected for further
in vivo evaluation. However, some moderately to highly cytotoxic
compounds were also selected in order to comprehensively un-
derstand the SAR. Next, 11-week old rats were treated intraperi-
toneally for 28 consecutive days with compounds followed by two
BrdU injections on the next day. The newborn cells were immu-
nohistochemically labeled by the BrdU marker.
Recently, we found that an extract of Dragon's Blood (a bright
red resin derived from Dracaena cochinchinensis) is capable of
stimulating adult neurogenesis and has beneficial effects on
improvement of learning ability (Unpublished data). Dragon's
Blood contains a mixture of phenolic compounds of the stilbene
and chalcone series such as resveratrol, pterostilbene, loureirin A,
loureirin B and the like. It would be interesting to define the
effective promoters in the extract. Thus far, no attempts have been
made on stilbene and analogous scaffolds because of negative
neurogenesis results from resveratrol. Resveratrol has been proved
to reduce adult neurogenesis in a dose-dependent manner and it
significantly impaired learning and memory in healthy adult mice
[25]. Instead of searching for neuroprotective polyphenol radical
scavengers that have limited potential because they are PAINS
compounds (pan-assay interference compounds) [26], the present
study looked for novel scaffolds that could efficiently promote adult
neurogenesis.
Based on the scaffolds identified in Dragon's Blood, a pool of
analogs was rationally designed and screened that consisted of
stilbene, diphenyl acetylene, diphenylethane, chalcone, and other
modified scaffolds that included heteroatomic linkers and hetero-
aryl rings. We iteratively designed compounds according to a set of
rules for CNS-permeation [27,28] and determined the efficacy to
illuminate SAR by employing a cell proliferation marker (BrdU) and
a mature neuron marker (NeuN), which finally led to discovery of a
novel series of efficient stimulators N-stilbenyl carboxamides that
featured a crucial methylenedioxy group on the benzene ring not
containing the carboxamide. None of the series of compounds
presented in Fig. 1 has been disclosed (accessed by SCIfinder on Oct.
1, 2016). The advantages of the scaffold include achirality and low-
cost synthetic accessibility. This report documents significant effi-
cacy (P < 0.001) with potency at sub-milligram per kg.
2.1. In vitro cytotoxicity screening and structure-cytotoxicity
relationships
The cytotoxicity data of all the compounds are listed in Table S1
(Supporting Information). Human neuroblastoma cells were used
for evaluating the cytotoxicity. Due to low solubility, 62 was not
tested. Generally, the introduction of a hydroxyl (12e21) or an
amino group (23, 24 and 25) on the stilbene resulted in higher
cytotoxicity, particularly at the medium and the high dose.
Methylation of the hydroxyl groups (2 vs. 15; 7 vs. 12) and acety-
lation of the amino groups (27 vs. 24; 30 vs. 23) minimized the
unfavorable effects. In contrast, some hydrophobic groups such as
methyl and trifluromethyl groups also have negative effects on
survival of the cells (6, 16, 20, 50, 51 and 54). Substitution of the
methyl groups with hydrophilic methoxy groups (6 vs.10; 16 vs. 22;
54 vs. 48) and shrinkage of long alkyl groups to shorter ones (34, 28
vs. 27; 56, 55 vs. 48; 52 vs. 53) are beneficial for improving the
survival ratio. Therefore, it is clear that low stilbene analog toxicity
requires a balance of hydrophilicity and hydrophobicity.
Replacement of the substituted benzene rings with heteroaryl
groups led to high toxicity (71, 72, and 73). The position of substi-
tution on the phenyl ring of the stilbene is also an important factor.
The para position is much more favorable than the meta position
(27 vs. 33; 32 vs. 31; 7 vs. 44) or the ortho position (7 vs. 9). But this
trend was not observed in the framework of diphenyl acetylene (58
vs. 57). The variation of the linkers between the two phenyl groups
generally has a slight impact on the toxicity (27 vs. 60; 48 vs. 7; 22
vs. 59), but sometimes it has great influence (27 vs. 58; 57 vs. 33).
Compared to the stilbene analogs, the insertion of a carbonyl group
into the stilbene, resulting in a chalcone, appeared to dramatically
increase the toxicity (43 vs. 66; 27 vs. 67; 22 vs. 65). Generally,
chalcones (63, 64, 65, 66, 67 and 68) are highly toxic with the
exception of 69. A direct ring closure of 69 to the dihydroflavone 70
resulted in low survival of the cells. The presence of an unsub-
stituted amide is necessary for the stilbene to maintain low toxicity
in contrast to an acetyl group (27 vs. 41) or an N-methylacetamide
(27 vs. 40).
2. Results
We designed a variety of stilbenes (Fig. 2, compounds 1e47),
diphenyl acetylenes (Fig. 3, compounds 48e58), diphenyl ethanes
(Fig. 4, compounds 59e60), and a variety of hetero linkers and
heteroaryl substitutions in the stilbene scaffold (Fig. 5, compounds
61e73). For details see the Supporting Information (the known
compounds are labeled with CAS codes). About 37 out of 73 com-
pounds for in vitro tests are unknown, and 21 out of 32 compounds
for in vivo assays are novel.
Neuroblastoma cell viability data for stilbenes that stimulate
neurogenesis are listed in Fig. 6. Most of the stilbenes produce
dose-dependent cytotoxicity with the exception of 22 (3,5-O-
dimethylresveratrol; known as pterostilbene) and 10 (4-O-ace-
tylpterostilbene). In contrast, structure-cytotoxicity relationships
show that the compounds sharing the common 3,4-
Long-term preventive treatment of AD definitely requires a
strict demand for low cytotoxicities of the candidates. We exam-
ined the survival of cells exposed to three concentrations (100, 500
and 1000
mM) of the stilbene analogs. The lowest concentration
(100 M) is far higher than the level of the potent neuroprotective
m
Fig. 1. Structures and retrosynthetic analysis of novel N-stilbenyl carboxamides.

384
J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
1 R1=Cl, R2, R3=H, R4, R5=OCH3
2 R1= OCH3, R2, R3, R4=H, R5=CN
3 R1= OCH3, R2, R3, R4=H, R5=OCF3
4 R1= OCH3, R2, R3=H, R4=NH2, R5=F
5 R1=AcO, R2, R3=H, R4 R5=OCH2O
6 R1=AcO, R2, R4=H, R3, R5=CH3
7 R1= OCH3, R2, R3=H, R4 R5=OCH2O
8 R1=CN, R2, R3=H, R4 R5=OCH2O
9 R1, R3=H, R2= OCH3, R4 R5=OCH2O
11
10 R1=AcO, R2, R4=H, R3, R5=OCH3
12 R1= H, R2 R3= OCH2O
23 R1, R2= OCH3
24 R1 R2= OCH2O
25 R1= H, R2= OCF3
27 R1= NHCOCH3, R2=H, R3 R4=OCH2O
13 R1, R2= H, R3= OCF3
14 R1, R2, R3= OCH3
28 R1= NHCOCH2CH3, R2=H, R3 R4=OCH2O
29 R1= NHCOCF3, R2=H, R3 R4=OCH2O
30 R1=NHCOCH3, R2=H, R3, R4=OCH3
31 R1=NHCOCH3, R2, R3=H, R4=OCH3
32 R1=NHCOCH3, R2, R4=H, R3=OCH3
33 R1=H, R2=NHCOCH3, R3 R4=OCH2O
34 R1=NHCO(CH2)2CH3, R2=H, R3 R4=OCH2O
35 R1=NHCOCH(CH3)2, R2=H, R3 R4=OCH2O
36 R1= NHOSOCH3, R2=H, R3 R4=OCH2O
37 R1=NHCOPh, R2=H, R3 R4=OCH2O
38 R1=NHCO(2-thienyl), R2=H, R3 R4=OCH2O
39 R1=NHCO(cyclo-Pr), R2=H, R3 R4=OCH2O
15 R1, R2= H, R3=CN
16 R1, R3= CH3, R2= H
17 R1= H, R2, R3= OCH3
18 R1= H, R2 R3= OCH2CH2O
19 R1, R3=F, R2= H
20 R1, R3=CF3, R2= H
21 R1, R3= H, R2= COCH3
22 R2= H, R1, R3= OCH3
26
41 R1 R2=OCH2O, R3, R5=H, R4 = COCH3
42 R1 R2=OCH2O, R3, R5=H, R4 =OBn
43 R1, R3=H, R2=N(CH3)2, R4 R5=OCH2O
44 R1 R2=OCH2O, R3, R4=H, R5 =OCH3
45 R1, R3=OCH3, R2 =H, R4 R5 =OCH2O
40
46
47
Fig. 2. Structures of a variety of stilbenes (compounds 1e47; the synthetic procedures are illustrated in Schemes S1eS10).
48 R1= OCH3, R2=H, R3 R4=OCH2O
49 R1= OCH3, R2=H, R3, R4=OCH3
50 R1= OCH3, R2, R4=CH3, R3=H
51 R1= OCH3, R2, R4=CF3, R3=H
52 R1= (CH2)4CH3, R2, R4=H, R3 =OBn
57 R1= H, R2= NHCOCH3
58 R1= NHCOCH3, R2= H
53 R1= CH3, R2, R4=H, R3 =OBn
54 R1= CH3, R2=H, R3 R4=OCH2O
55 R1= OCH2CH3, R2=H, R3 R4=OCH2O
56 R1=O(CH2)4CH3, R2=H, R3 R4=OCH2O
Fig. 3. Structures of a variety of diphenyl acetylenes (compounds 48e58; the synthetic procedures are illustrated in Scheme S11 -S12).
methylenedioxy stilbene substructure such as 27 (acetamide), 36
(methanesulfonamide),
37
(benzamide),
39
(cyclo-
propanecarboxamide) and 46 (acetamide plus additional fluorina-
tion on the benzene ring) generally have low cytotoxicities. In
particular, the cell survival rate exceeded 80% for all three doses of
27, 37 and 39.
59 R1= OH, R2, R4= OCH3, R3= H
2.2. The discovery of a novel lead compound stimulating
neurogenesis
60 R1= NHCOCH3, R2=H, R3 R4= OCH2O
Fig. 4. Structures of a variety of diphenyl ethanes (compounds 59e60; the synthetic
procedures are illustrated in Scheme S13).
To differentiate neuroproliferation-type neurogenesis from

J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
385
70
61 R1= OCH3, R2 = H
63 R1= OCH3, R2= OH, R3, R6 = H, R4 R5=OCH2O
64 R1, R5= OCH3, R2= OH, R3, R4, R6 = H
62 R1= H, R2 = OCH3
65 R1, R3= OCH3, R2, R4, R6 = H, R5= OH
66 R1, R3, R6 = H, R2=N(CH3)2, R4 R5=OCH2O
67 R1, R3, R6 = H, R2= NHCOCH3, R4 R5=OCH2O
68 R1= OH, R2= OCH3, R3, R6 = H, R4 R5=OCH2O
69 R1 R2=OCH2O, R3, R4 = H, R5=OCH3, R6 = OH
72 3-thienyl
73 2-thienyl
71
Fig. 5. Structures of a variety of hetero linkers and heteroaryl substitutions in the stilbene scaffold (compounds 61e73; the synthetic procedures are illustrated in Scheme S14 -S18).
neuroprotection-type neurogenesis, rats were treated intraperito-
neally for 28 consecutive days followed by the BrdU injections and
were then sacrificed 24 h after a pulse of the BrdU. None of the
chalcones survived cytotoxicity screening due to extremely high
cytotoxicity. In the first round, 11 compounds 4, 5, 11, 12, 17, 27, 48,
49, 53, 57, and 60 with various skeletons of stilbene, diphenyl
acetylene, diphenylethane and a variety of substituents were sub-
jected to an in vivo examination. In contrast to the vehicle control
group, only the 27-treated group (an example of R ¼ methyl and
X ¼ H in Fig. 1) appeared BrdU positive in the SGZ-GCL (granule cell
Fig. 7. Proliferation of NPCs promoted by the analogs of the lead compound 27. Eleven-
week old rats were given vehicle control and solutions of the compounds intraperi-
toneally at a dose of 2.2 mg/kg/day for 28 days followed by a BrdU pulse. The rats were
layer). In other words, 27 directly stimulated the proliferation of
NPCs. Meanwhile, preliminary SAR indicated that replacement of
the acetamide of 27 with a hydroxyl (compound 12) or an acetoxy
sacrificed 24 h after the BrdU injections. Data are expressed as mean SEM (n ¼ 8). (V:
(compound 5) group, or hydrogenation of 27 to 60, abolished the
efficacy. Compound 57 with an acetamide at a meta position of the
diphenyl acetylene is also inactive. The substituted stilbene 27 is
associated with a high degree of structural specificity and is a novel
compound for directly promoting adult neurogenesis.
vehicle control; ***P < 0.001; *P < 0.05).
significant efficacy relative to the vehicle-treated group (Fig. 7).
Despite the inactivity of 48 that was revealed in the first round, the
analog 61 was constructed having an elongated linker connecting
the two substituted benzene rings. This resulted in moderate effi-
cacy. Clearly, the relative potencies conferred by the substituents on
neurogenesis at the cell proliferation stage ranked as follows: 3,4-
methylenedioxy > 3, 4-dimethoxy > 4-methoxy.
Next,
a
homolog of 27, compound 28 possessing
a
4-
propionamide substituent in combination with
a
3,4-
methylenedioxy moiety, was also prepared. The rats treated with
28 were also BrdU-labeling positive with a decreasing statistical
significance (Fig. 7).
To further ascertain the impacts of the lead 27 on the new-born
cell fate (at cell differentiation stage), rats were sacrificed 28 days
instead of 24 h after the pulse of BrdU. The group that received a
dose of 2.0 mg/kg had augmented BrdU þ cells to an extent as high
as 4.2 times versus the control group (Fig. 8). Moreover, the number
of BrdU þ cells counted at day 28 after the pulse of BrdU is far
To investigate the importance of the methylenedioxy moiety,
compound 30 was produced with methoxy groups in the para and
meta positions, and 32 with a single methoxy group at the para
position was also prepared. Compound 31 with a methoxy group at
the meta position was not evaluated in vivo due to its high toxicity.
Compounds 30 (3,4-dimethoxy) and 32 (4-methoxy) failed to show
Fig. 6. Cytotoxicities of the compounds with elevated efficacy in stimulating neurogenesis. L, M, and H represent low concentration, medium concentration and high concentration
(100, 500 and 1000 M), respectively. (Cell type: SH-SY5Y).
m

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J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
Fig. 8. Compound 27 significantly promotes adult hippocampal neurogenesis (differentiation and maturation of newborn NPCs). The rats were sacrificed 28 days instead of 24 h
after the BrdU injections. A-D represent BrdU/NeuN co-staining of vehicle control, low dose (2.0 mg/kg), medium dose (4.0 mg/kg), and high dose (20.0 mg/kg) with scale bars of
250
m
m, respectively. E and F are images of high magnification (40 ꢀ ) of the inset in A (control group) and B (compound 27, low dose) with scale bars of 75
mm, respectively. G and H
represent BrdU positive cells in E and F, respectively. I) the numbers of BrdU þ cells; J) proportion of BrdU þ NeuNþ in BrdU þ cells (V, vehicle control group; 27 L, 27 M and 27H
represent low dose (2.0 mg/kg), medium dose (4.0 mg/kg), and high dose (20.0 mg/kg)). Quantifications are presented as mean SEM (n ¼ 8). ***P < 0.001; *P < 0.05.
higher than that at day 1. In other words, the NPCs continued to
proliferate after termination of administration of 27. This is strik-
ingly different from the reported neuroprotective function, where
the newborn NPCs died relatively slowly and finally survived more
than the untreated ones [24].
novel lead that promotes efficient and sustained neurogenesis at
lower doses.
2.3. In-depth structure-activity relationships of the lead 27
The BrdU-incorporated cells were co-localized with the NeuN
marker to determine whether compound 27 could promote the
differentiation of the newborn NPCs into mature functional neu-
rons. Dose-response analysis (Fig. 8) indicated that the number of
newborn NPCs was inversely proportional to the daily dose while
the percentage of BrdU/NeuN double positive BrdU þ cells slightly
increased with an elevation of the dose. As a result, 2.3-, 3.1- and
4.6- fold net neurogenesis, which was co-expressed with BrdUþ
and NeuNþ, was observed relative to the control group at 20.0, 4.0
and 2.0 mg/kg, respectively. This emphasizes that compound 27 is a
According to the dose-response analysis of 27, we chose 4.0 mg/
kg as the top dose in the subsequent in-depth SAR studies. Next, the
3,4-methylenedioxy moiety of the stilbene was held constant while
other modifications were made. Compared to 27, fluorination on
the 3-postion adjacent to the amide (see 46) retained the potency,
but methylation of the nitrogen of the amide (see 40) diminished
the activity in addition to producing high toxicity. As substitution at
the para position generally confers lower cytotoxicity, a series of
3,4-methylenedioxystilbenes were designed that were substituted
at the para position. For example, 7 (methoxy), 8 (cyano), 43

J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
387
(dimethylamino), 29 (trifluoroacetamide), 34 (butyramide), 35
(isobutyramide), 36 (methanesulfonamide), 37 (benzamide), 38 (2-
thienylformamide) and 39 (cyclopropanecarboxamide) were
investigated, and the cell proliferation results are illustrated in
Fig. 9. Compounds with cyano (8) and the amides (34, 35, 36, 37 and
39) were found to be more efficient than the vehicle with statistical
significance. Interestingly, the cyano-substituted version of 27 (i.e.
8) proved to be much superior to the methoxy version (7) and the
dimethylamino version (43). Compound 8 has considerable po-
tency compared to the vehicle control group (P < 0.0001). It is
noted that, among the variety of amides, the electron-withdrawing
groups present in 29 and 38 confer activity that is inferior to the
others. In particular, 29 is totally inactive, while the efficacy of 38 is
moderate.
In addition, compounds 10, 22 and 47, sharing the common 3,5-
dimethoxystilbene substitution pattern, have similar potencies that
are relatively high (Fig. 9). The high activities of 10 and 22
contradict the SAR previously drawn on the 3,4-
methylenedioxystilbenes. The basic 3,5-dimethoxystilbene struc-
ture tolerates the presence of a hydroxyl (22) or an acetoxyl (10)
group as well as an acetamide (47). Nevertheless, it should be
pointed out that 22 and 10 were inferior to 47, showed an abnormal
dose-toxicity relationship, and possessed high toxicity at low con-
centration. Structural modifications of the linker of 27 led to
compounds 55 and 58 having an acetylene bridge between the two
aromatic rings. The diminished efficacy of 55 and 58 (Fig. 9) was
observed. Thus, maximal promotion of neurogenesis requires a
stilbene scaffold substituted with 3,4-methylenedioxy (alterna-
tively 3,5-dimethoxy) and amide moieties with unsubstituted NH.
As a result, ten compounds were found to be capable of
enhancing BrdU incorporation 2.2e3.5 times as high as the control
(Fig. 9). Among the six novel compounds, compounds 35, 36, 37 (all
P < 0.0001) and 39 (P < 0.01) were selected for further studies
because of their structural novelty, and compounds 34 and 46 were
discarded. Of the four known compounds, namely 8, 10, 22 and 47,
compound 10 is the most potent. Compound 10 is an acetylated
derivative of pterostilbene 22. It is reported that pterostilbene, not
resveratrol, has positive effects on improving cognitive perfor-
mance at an oral dose of 120 mg/kg for 8 weeks [29]. Given the
likelihood of 10 being the acetylated prodrug of pterostilbene, it
was selected as a positive control (instead of resveratrol and pter-
ostilbene) for further studies because 10 may more readily pene-
trate the BBB compared to pterostilbene 22 and resveratrol.
Dose-response assays (Fig. 10) revealed that the neurogenesis
efficacy of 10 (4-O-acetylpterostilbene) dropped off sharply with a
decrease in the administered dose, and there was no longer a sta-
tistically significant response at the dose of 0.5 mg/kg relative to the
vehicle group. Although 36 (a methanesulfonamide version of 27)
has a similar dose-response curve to 10, it is potent at all three dose
levels (4.0, 1.0 and 0.5 mg/kg; all P < 0.001). Surprisingly, 39
reversed the trend, with the efficacy of 39 increasing with a decline
in the daily dose. In contrast, 35 and 37 shared a V-shaped dose-
response curve. Encouragingly, the candidates presented in Fig. 1
(35, R ¼ isopropyl; 37, R ¼ phenyl; 39, R ¼ cyclopropyl; X ¼ H)
retained significant potency relative to vehicle group, which was
even documented at the low dose of 0.5 mg/kg (P < 0.001). In a
word, N-stilbenyl carboxamides 35, 36, 37 and 39 showed advan-
tages of stimulating effects over 4-O-acetylpterostilbene 10, a
resveratrol derivative, at sub-milligram per kg doses.
To further ascertain the effects of lower doses of N-stilbenyl
carboxamides on the fate of newborn NPCs at sub-milligram per kg,
the rats were dosed with a compound 37 that has extremely low
toxicity at 0.5 mg/kg and 0.05 mg/kg for 28 consecutive days fol-
lowed by a pulse of BrdU. After waiting another 28 days, the rats
were sacrificed and the level of neurogenesis was assayed by BrdU
and NeuN double immunostaining. The immunohistochemical re-
sults confirmed that adult neurogenesis mediated by 37 was
observed even at 0.05 mg/kg, as illustrated in Fig. 11. The group that
received a dose of 0.5 mg/kg and 0.05 mg/kg had augmented
BrdU þ cells co-localized with BrdU þ to an extent as high as 3.2-
and 1.6- fold versus the control group, respectively.
2.4. The impacts on Akt and Erk signaling pathway involved in
neurogenesis
Akt and Erk are the two crucial kinases that regulate the
signaling pathway of neurogenesis [30e32]. Importantly, loss of
Erk1/2 would result in complete absence of neural progenitor pools
[32]. Thus, it is worthwhile to investigate the variation of the two
kinases' levels and phosphorylation levels. It appeared that com-
pounds 27 and 37 significantly affect AKT and ERK1/2 expression
levels and phosphorylation levels at the proliferation stage (day 1
after pulse of BrdU; Fig. 12) and at the differentiation stage (day 28
after pulse of BrdU; Fig. 13). The western blot analysis as presented
in Fig. 12 indicates that 27 up-regulated pAkt and pErk levels
inversely proportional to the doses; while the dose-response curve
of 37 is V-shaped. In other words, the dose-response analysis of 27
and 37 in activating pAkt and pErk is consistent with the BrdU la-
beling results at the proliferation stage. On the other hand, the
alteration of the signaling kinases expression levels remained 28
days after the termination of injection of 27 and 37 (Fig. 13), which
accounted for the reason that 27 and 37 persistently stimulated
neurogenesis. Therefore, western blot analysis consistently sup-
ports the transcription results via BrdU/NeuN immunostaining.
3. Discussion
Discovery of novel small molecules that have the potential to
stimulate adult neurogenesis in situ is challenging. Structure-
activity relationships indicate stimulation of neurogenesis re-
quires the presence of a stilbene scaffold that is absent in inactive
analogs such as diphenyl acetylene and diphenyl ethane. Encour-
agingly, the presence of various carbamates at the 4-position in
combination with a 3,4-methylenedioxy moiety not only amelio-
rates the cytotoxicity but also dramatically enhances the efficacy of
neurogenesis. The activity was retained by replacement of the 3,4-
Fig. 9. Proliferation of NPCs promoted by the analogs of the lead 27. Eleven-week old
rats were injected with compounds and the vehicle solvent control intraperitoneally
for 28 days at a dose of 4.0 mg/kg/day followed by a BrdU pulse. The rats were
sacrificed 24 h after the BrdU injections. Data are expressed as mean SEM (n ¼ 8). (V:
vehicle control; ***P < 0.001; **P < 0.01; *P < 0.05).
methylenedioxy moiety of the lead compound with
a 3,5-
dimethoxy substitution pattern, or when the carboxamide was

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J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
Fig. 10. Dose-response data for 10, 35, 36, 37 and 39 on promoting proliferation of NPCs. Eleven-week old rats were injected with compounds and the vehicle solvent control
intraperitoneally with 4.0 (H), 1.0 (M) and 0.5 (L) mg/kg/day for 28 days followed by a BrdU pulse. The rats were sacrificed 24 h after the BrdU injections. A, B, C, D, E, F represent
compounds 10, 35, 36, 37, 39 and vehicle, respectively; the numbers 1e3 represent low dose, medium dose and high dose, respectively. G represents BrdU staining images of high
magnification (20 ꢀ ) of F with a scale bar of 50
m
m. H and I represent BrdU staining images of high magnification (20 ꢀ and 40 ꢀ ) of A3 with scale bars of 50
mm and 20 mm,
respectively. J represents dose-response quantitation of BrdU positive cells. Data are expressed as mean SEM (n ¼ 8). (V: vehicle control; ***P < 0.001; **P < 0.01; *P < 0.05).
replaced by a sulfonamide or a nitrile, or when the benzene ring
attached to the carboxamide was substituted with a fluorine atom.
The failure of the compound in which a methyl group is attached to
the nitrogen of the carboxamide indicates the importance of an
active NH. Among a variety of carboxamides presented in Fig. 1, R
equal to isopropyl, cyclopropyl, and phenyl groups seem to be the
best contributors.
carboxamides can be distinguished from the optimized resveratrol
derivative 10 at lower doses of sub-milligram per kg.
Improving hippocampus-dependent capability must be based
on abundant newborn mature neurons that can modulate the
structural and functional plasticity of the hippocampus. It is
encouraging that statistically significant proliferation of NPCs and
net neurogenesis were actually validated at low doses (compound
37, 0.5 and 0.05 mg/kg) in the present study. In addition, the BrdU/
NeuN double-staining results indicate that N-stilbenyl carbox-
amides generally affected neuronal lineage commitment. Interest-
ingly, a trend was observed that increased newborn BrdU þ cells
mediated by 27 and 37 would be accompanied with decreased ratio
of BrdU þ NeuNþ/BrdUþ, as depicted in Figs. 8 and 11. A require-
ment for an appropriate differentiation ratio of NPCs into neurons
and astrocytes could be a plausible explanation that warrants
further study [35]. Western blot analysis proved the N-stilbenyl
carboxamides such as 27 and 37 could activate the pAkt and pErk
signaling pathway both at day 1 and day 28 after pulse of BrdU.
Moreover, alteration in protein expression levels essentially ac-
counts for variation of the efficacies of stimulating neurogenesis
determined by immunohistochemical BrdU/NeuN staining.
Neuroproliferation-type neurogenesis induced by the N-stilbenyl
carboxamides was observed at stage of the proliferation of NPCs (a
BrdU marker) and at stage of the differentiation into mature neuron
(BrdU/NeuN markers). Thus, the striking difference between the N-
stilbenyl carboxamides and the known neuroprotective agents may
be ascribed to a different mechanism underlying neurogenesis.
The most well-known stilbene is resveratrol (4,30,5'-trihydrox-
ystilbene), which is a polyphenol that is reported to have detri-
mental effects on adult neurogenesis [25]. It has also been reported
that a low dose of resveratrol is not effective in models of aging and
AD [29]. On the other hand, other studies have argued that
resveratrol has beneficial effects on neurogenesis in aged rats at a
very high dose of 40 mg/kg [33]. The authors attributed the con-
flicting results to studies in juvenile vs. aged brains involved sup-
pression of oxidative stress [33]. Given the four-week waiting
period and aged rats used in the resveratrol study, a neuro-
protective mechanism involving neurogenesis cannot be ruled out.
Unfortunately, the low bioavailability of resveratrol, along with its
rapid metabolism and poor brain-penetrating capability, would
limit its potential in treating neurodegenerative diseases [29,34].
The present results suggest that 4-hydroxy-30,5'-dimethox-
ystilbene (known as pterostilbene 22) and its acetylated derivative
10 (4-acetoxy-30,5'-dimethoxystilbene) are neurogenesis pro-
moters but more cytotoxic than N-stilbenyl carboxamides.
Furthermore, dose-response assays as illustrated in Fig. 10 have
indicated that the neurogenesis effects of the N-stilbenyl

J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
389
Fig. 11. Compound 37 significantly stimulates adult hippocampal neurogenesis (differentiation and maturation of newborn NPCs) at low doses of 0.05 mg/kg and 0.5 mg/kg. A-C
represent BrdU/NeuN co-staining of vehicle control, low dose (0.05 mg/kg) and high dose (0.5 mg/kg), respectively. D is an image of high magnification (40 ꢀ ) of the inset in A
(control group) with scale bars of 75
with scale bars of 75
and high dose (0.5 mg/kg)). Quantifications are presented as mean SEM (n ¼ 8). ***P < 0.001; **P < 0.01; *P < 0.05.
m
m. E is the merge of BrdU staining (F) and NeuN staining (G) images of high magnification (40 ꢀ ) of the inset in C (compound 37, high dose)
m
m. H) the numbers of BrdU þ cells; I) proportion of BrdU þ NeuNþ in BrdU þ cells (V, vehicle control group; 37 L and 37H represent low dose (0.05 mg/kg)
4. Conclusion
5. Experimental
In conclusion, analysis of a library of 73 rationally designed
compounds led to discovery of a novel chemotype with low cyto-
toxicity and high potency for promoting adult hippocampal neu-
rogenesis by employing in vivo screening of a library of stilbenes
and analogous scaffolds. The effective sub-milligram dose in vivo
also showed that N-stilbenyl carboxamides, instead of the well-
known star molecule resveratrol, point to the potential for suc-
cess of future optimization studies involving rational design and
synthesis. N-Stilbenyl carboxamides have advantages over other
reported chemicals to probe underlying neuroproliferation-type
neurogenesis instead of the common neuroprotective mechanism.
Taken together, the novel N-(methylenedioxy)stilbenyl carbox-
amides disclosed here offer a significant opportunity to improve
the plasticity of the adult mammalian brain and to develop new
treatments for neurodegenerative disorders in which adult neu-
rogenesis is impaired. Further study is underway.
5.1. Chemistry
The synthetic procedures are described in Supporting Infor-
mation. The synthetic procedures of a variety of stilbenes (Fig. 2,
compounds 1e47) are illustrated in Schemes S1eS10; the synthetic
procedures of a variety of diphenyl acetylenes (Fig. 3, compounds
48e58) are illustrated in Scheme S11 -S12; the synthetic proced-
ures of a variety of diphenyl ethanes (Fig. 4, compounds 59e60) are
illustrated in Scheme S13; the synthetic procedures of a variety of
hetero linkers and heteroaryl substitutions in the scaffold of stil-
bene (Fig. 5, compounds 61e73) are illustrated in Scheme S14 -S18.
All of the compounds were identified by 1H NMR, 13C NMR, and
HRMS with purities of higher than 95% (HPLC analysis). All of the
biological assays were conducted by investigators blind to the
compounds' structures.

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J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
Fig. 12. Western blot analysis of hippocampal neurogenesis mediated by 27 (two doses; H and L) and 37 (three doses; H, M and L) 24 h after pulse of BrdU. A and B represent
immunoblots of Akt, pAkt, ERK1/2 and pERK with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as an internal control. C - F represent the densitometric analyses of
the ratios of Akt/GAPDH, pAkt/GAPDH, ERK1/2/GAPDH and pERK1/2/GAPDH, respectively. The data were normalized to the vehicle control given a value of 1. (V: vehicle; 27H:
4.0 mg/kg; 27 L: 2.2 mg/kg; 37H: 4.0 mg/kg; 37 M: 1.0 mg/kg; 37 L: 0.5 mg/kg). ***P < 0.001; **P < 0.01; *P < 0.05.
5.2. Cell viability assays
injected intraperitoneally with the compounds once a day (0.5, 1.0,
2.0, 2.2, 4.0 or 20 mg/kg/day) for 28 days. Meanwhile, the vehicle
group of animals was injected with 10% ethanol in oil during the 28
days. At Day 29, BrdU (Sigma) (10 mg/mL) solution was prepared in
normal saline. Allof the groups of rats wereinjected twice separately
withBrdU(100 mgkg)at a 2 hinterval. Ratswere sacrificed24h after
the last injection of BrdU. BrdU staining was used to observe the
proliferation of new cells. Alternatively, rats were sacrificed 28 days
after the last injection of BrdU. Double staining (NeuN/BrdU) was
used to observe the differentiation of new born NPCs.
The MTT method was used to evaluate the cell survival ratios of
the prepared compounds. The SH-SY5Y cell line (human neuro-
blastoma cells) used in this assay was seeded in 96-well plates at a
density of 5 ꢀ 104 cells/mL and incubated at 37 ꢁC. After 24 h, the
compounds were added to each well (final concentrations: 100,
500, 1000 mM, respectively). After co-incubation for 24 h, 20 mL of
5 mg/mL MTT was added to each well, and the cells were incubated
for another 4 h at 37 ꢁC. The percentage of cell viability was
determined at 570 nm by microplate reader. All assays were con-
ducted with five parallel samples.
5.5. Immunohistochemistry
5.3. Animals
Rats (two or three rats each group) were anaesthetized with
pentobarbital sodium (70 mg/kg) and perfused with normal saline.
Brain tissue blocks were fixed for one week in 4% paraformaldehyde
at 4 ꢁC. The brain tissues collected were postfixed for 1 week and
allowed to settle in a 20%e30% sucrose solution for 72 h. Brains
were cryosectioned at 30 mm and 8 pieces were randomly selected
in each of 40 consecutive slices (n ¼ 8).
SD male rats with age 10 weeks were obtained from Peking
University Health Science Center. Following arrival, the animals
were acclimatized for 7 days. The circadian rhythm of the animals
was strictly complied with. The rats were allowed to consume
water and food at will. All experimental processes were conducted
in strict compliance with the experimental animal management
regulations and animal ethics policies of Beijing Institute of
Technology.
BrdU staining: sections were blocked in 1% H2O2 for 30 min at
room temperature (RT) and then incubated with 2 M HCl, 37 ꢁC for
1 h and rinsed in 0.1 M borate buffer (pH 8.5). The sections were
incubated in blocking buffer (5% goat serum and 0.5% Triton X-100
in PBS) for 1e2 h at RT. After that, samples were incubated with
mouse primary antibody BrdU (1:500; Millipore, Billerica, MA,
USA) at 4 ꢁC overnight and then with the appropriate DAB kit for 2 h
at RT.
5.4. Compound administration and BrdU injections
The solutions of the compounds (3 mg/mL) were prepared in
Reagent grade soybean oil containing 10% ethanol. The rats were

J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
391
Fig. 13. Western blot analysis of hippocampal neurogenesis mediated by 27 (three doses; H, M and L) and 37 (two doses; H and L) 28 days after pulse of BrdU. A and B represent
immunoblots of Akt, pAkt, ERK1/2 and pERK with GAPDH serving as an internal control. C - F represent the densitometric analyses of the ratios of Akt/GAPDH, pAkt/GAPDH, ERK1/2/
GAPDH and pERK1/2/GAPDH, respectively. The data were normalized to the vehicle control given a value of 1. (V: vehicle; 37H: 0.5 mg/kg; 37 L: 0.05 mg/kg; 27H: 20.0 mg/kg; 27 M:
4.0 mg/kg; 27 L: 2.0 mg/kg). ***P < 0.001; **P < 0.01; *P < 0.05.
Double staining: sections were incubated in blocking buffer for
1e2 h at RT. Samples were incubated with rabbit anti-NeuN (1:500,
Abcam, Cambridge, MA, UK) and primary antibody BrdU (1:500) at
4 ꢁC overnight and then with anti-mouse 488 and anti-rabbit TRITC
for 2 h at RT.
Competing interests
The authors delare no competing financial interests.
Author contributions
J.-H.L. and H.Q conceived the idea and supervised the project. J.-
H.L designed all the compounds and the synthetic routes. H.Q.
designed all biological assays. L.Y. and S.W. performed most of
in vivo and synthetic experiments, respectively. S.-S.L., J.T., N.-M.L.,
Q.-H.Y., H.-A.Z., Y.-J.Q., L.X., C.-X.M. and X.-M.L. did the remaining
biological and chemical experiments. J.-H.L., M.C., H.Q., L.Y., S.-S.L.,
S.W., Q.-H.Y. and N.-M.L. analyzed the data and drafted this
manuscript. J.-H.L., M.C. and H.Q. provided critical analysis and
revision of the manuscript.
5.6. Immunoblotting
Rats were anaesthetized with pentobarbital sodium (70 mg/kg)
and sacrificed. The brain tissues were lysed in RIPA buffer (1% Triton
X-100, 1% sodium deoxycholate, 4% SDS, 0.15 M NaCl, and 0.05 M
Tris-HCl at pH 7.2) supplemented with protease inhibitors (Com-
plete, Roche Diagnostics, Germany). The lysates were applied by
12% SDS-PAGE. Immunoblotting was performed as previously
described [36]. Rabbit anti-Akt, anti-pAkt, anti-ERK and pERK
polyclonal antibodies (Abcam, UK) were used to detect serine/
threonine-specific protein kinase and its phosphorylated prod-
ucts. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves
as a internal control.
Acknowledgement
This work is supported by a grant from the National Natural
Science Foundation of China (81673335). We are also thankful for
help from Prof. Chun Li and Si-Pin Pang.
5.7. Statistical analysis
All data are shown as mean SEM (standard error of the mean)
and represented by GRAPHPAD PRISM 6 (Graphpad Software, La
Jolla, CA, USA). One-way analysis of variance (ANOVA) with post
hoc test was used for statistical analysis. Statistical significance was
set at p < 0.05.
Appendix A. Supplementary data
The spectral data for all the compounds, experimental proced-
ures for the synthesis and cytotoxicities of all the compounds listed
in Table S1.

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J.-H. Liang et al. / European Journal of Medicinal Chemistry 136 (2017) 382e392
Neuroscience 332 (2016) 212e222.
Supplementary data related to this article can be found at http://
[20] Y. Lee, S.J. Jeon, H.E. Lee, I.H. Jung, Y.W. Jo, S. Lee, J.H. Cheong, D.S. Jang,
dx.doi.org/10.1016/j.ejmech.2017.05.025.
J.H. Ryu, Spinosin, a C-glycoside flavonoid, enhances cognitive performance
and adult hippocampal neurogenesis in mice, Pharmacol. Biochem. Behav.
145 (2016) 9e16.
References
[21] H. Lin, H. Fang, J. Wang, Q. Meng, X. Dai, S. Wu, J. Luo, D. Pu, L. Chen, D. Minick,
K. Arai, E.T. Mandeville, E. Lo, J.C. Holder, T.T. Chuang, J. Zhao, Discovery of a
novel 2,3,11,11a-tetrahydro-1H-pyrazino 1,2-b isoquinoline-1,4(6H)-dione
series promoting neurogenesis of human neural progenitor cells, Bioorg.
Med. Chem. Lett. 25 (2015) 3748e3753.
[22] K.S. MacMillan, J. Naidoo, J. Liang, L. Melito, N.S. Williams, L. Morlock,
P.J. Huntington, S.J. Estill, J. Longgood, G.L. Becker, S.L. McKnight, A.A. Pieper,
J.K. De Brabander, J.M. Ready, Development of proneurogenic, neuroprotective
small molecules, J. Am. Chem. Soc. 133 (2011) 1428e1437.
[1] C.M. Zhao, W. Deng, F.H. Gage, Mechanisms and functional implications of
adult neurogenesis, Cell 132 (2008) 645e660.
[2] W. Deng, J.B. Aimone, F.H. Gage, New neurons and new memories: how does
adult hippocampal neurogenesis affect learning and memory? Nat. Rev.
Neurosci. 11 (2010) 339e350.
[3] J. Walter, S. Keiner, O.W. Witte, C. Redecker, Age-related effects on hippo-
campal precursor cell subpopulations and neurogenesis, Neurobiol. Aging 32
(2011) 1906e1914.
[4] A.Y. Maslov, T.A. Barone, R.J. Plunkett, S.C. Pruitt, Neural stem cell detection,
characterization, and age-related changes in the subventricular zone of mice,
J. Neurosci. 24 (2004) 1726e1733.
[5] N. Taniuchi, T. Niidome, Y. Goto, A. Akaike, T. Kihara, H. Sugimoto, Decreased
proliferation of hippocampal progenitor cells in APPswe/PS1dE9 transgenic
mice, Neuroreport 18 (2007) 1801e1805.
[6] K. Boekhoorn, M. Joels, P.J. Lucassen, Increased proliferation reflects glial and
vascular-associated changes, but not neurogenesis in the presenile Alzheimer
hippocampus, Neurobiol. Dis. 24 (2006) 1e14.
[7] L.M. Tong, H. Fong, Y.D. Huang, Stem cell therapy for Alzheimer's disease and
related disorders: current status and future perspectives, Exp. Mol. Med. 47
(2015) e151.
[8] S.G. Davies, P.D. Kennewell, A.J. Russell, P.T. Seden, R. Westwood, G.M. Wynne,
Stemistry: the control of stem cells in situ using chemistry, J. Med. Chem. 58
(2015) 2863e2894.
[9] S. Wu, H. Qing, J.-H. Liang, Advance in the study of compounds with potential
activities in promotion of adult hippocampal neurogenesis, Acta Pharm. Sin.
51 (2016) 1025e1031.
[10] S. Kotani, T. Yamauchi, T. Teramoto, H. Ogura, Donepezil, an acetylcholines-
terase inhibitor, enhances adult hippocampal neurogenesis, Chem-biol.
Interact. 175 (2008) 227e230.
[11] K. Jin, L. Xie, X.O. Mao, D.A. Greenberg, Alzheimer's disease drugs promote
neurogenesis, Brain Res. 1085 (2006) 183e188.
[12] S.J. Kim, T.G. Son, H.R. Park, M. Park, M.S. Kim, H.S. Kim, H.Y. Chung,
M.P. Mattson, J. Lee, Curcumin stimulates proliferation of embryonic neural
progenitor cells and neurogenesis in the adult hippocampus, J. Biol. Chem. 283
(2008) 14497e14505.
[13] Q. Gao, S.J. Jeon, H.A. Jung, H.E. Lee, S.J. Park, Y. Lee, Y. Lee, S.Y. Ko, B. Kim,
J.S. Choi, J.H. Ryu, Nodakenin enhances cognitive function and adult hippo-
campal neurogenesis in mice, Neurochem. Res. 40 (2015) 1438e1447.
[14] J. Mao, S. Huang, S. Liu, X.-L. Feng, M. Yu, J. Liu, Y.E. Sun, G. Chen, Y. Yu, J. Zhao,
G. Pei, A herbal medicine for Alzheimer's disease and its active constituents
promote neural progenitor proliferation, Aging Cell 14 (2015) 784e796.
[15] Y.J. Ahn, S.J. Park, H. Woo, H.E. Lee, H.J. Kim, G. Kwon, Q. Gao, D.S. Jang,
J.H. Ryu, Effects of allantoin on cognitive function and hippocampal neuro-
genesis, Food Chem. Toxicol. 64 (2014) 210e216.
[16] A.C. Abbott, C.C. Toledo, F.C. Aranguiz, N.C. Inestrosa, L. Varela-Nallar, Tetra-
hydrohyperforin increases adult hippocampal neurogenesis in wild-type and
APPswe/PS1 delta E9 mice, J. Alzhermers Dis. 34 (2013) 873e885.
[17] X. Liu, C.-B. Chan, S.-W. Jang, S. Pradoldej, J. Huang, K. He, L.H. Phun, S. France,
G. Xiao, Y. Jia, H.R. Luo, K. Ye, A synthetic 7,8-Dihydroxyflavone derivative
promotes neurogenesis and exhibits potent antidepressant effect, J. Med.
Chem. 53 (2010) 8274e8286.
[23] J. Naidoo, H. De Jesus-Cortes, P. Huntington, S. Estill, L.K. Morlock, R. Starwalt,
T.J. Mangano, N.S. Williams, A.A. Pieper, J.M. Ready, Discovery of a neuro-
protective
chemical,
(S)-N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-
fluoropropyl)-6-methoxypyridin -2-amine (-)-P7C3-S243, with improved
druglike properties, J. Med. Chem. 57 (2014) 3746e3754.
[24] A.A. Pieper, S. Xie, E. Capota, S.J. Estill, J. Zhong, J.M. Long, G.L. Becker,
P. Huntington, S.E. Goldman, C.-H. Shen, M. Capota, J.K. Britt, T. Kotti, K. Ure,
D.J. Brat, N.S. Williams, K.S. MacMillan, J. Naidoo, L. Melito, J. Hsieh, J. De
Brabander, J.M. Ready, S.L. McKnight, Discovery of a proneurogenic, neuro-
protective chemical, Cell 142 (2010) 39e51.
[25] H.R. Park, K.H. Kong, B.P. Yu, M.P. Mattson, J. Lee, Resveratrol inhibits the
proliferation of neural progenitor cells and hippocampal neurogenesis, J. Biol.
Chem. 287 (2012) 42588e42600.
[26] J.B. Baell, G.A. Holloway, New substructure filters for removal of pan assay
interference compounds (PAINS) from screening libraries and for their
exclusion in bioassays, J. Med. Chem. 53 (2010) 2719e2740.
[27] D.E. Clark, In silico prediction of blood-brain barrier permeation, Drug Discov.
Today 8 (2003) 927e933.
[28] W.M. Pardridge, CNS drug design based on principles of blood-brain barrier
transport, J. Neurochem. 70 (1998) 1781e1792.
[29] J. Chang, A. Rimando, M. Pallas, A. Camins, D. Porquet, J. Reeves, B. Shukitt-
Hale, M.A. Smith, J.A. Joseph, G. Casadesus, Low-dose pterostilbene, but not
resveratrol, is a potent neuromodulator in aging and Alzheimer's disease,
Neurobiol. Aging 33 (2012) 2062e2071.
[30] G. Otaegi, M.J. Yusta-Boyo, E. Vergano-Vera, H.R. Mendez-Gomez, A.C. Carrera,
J.L. Abad, M. Gonzalez, E.J. de la Rosa, C. Vicario-Abejon, F. de Pablo, Modu-
lation of the PI 3-kinase-Akt signalling pathway by IGF-I and PTEN regulates
the differentiation of neural stem/precursor cells, J. Cell Sci. 119 (2006)
2739e2748.
[31] H.S.G. Kalluri, R. Vemuganti, R.J. Dempsey, Mechanism of insulin-like growth
factor I-mediated proliferation of adult neural progenitor cells: role of Akt,
Eur. J. Neurosci. 25 (2007) 1041e1048.
[32] J. Vithayathil, J. Pucilowska, L.H. Goodnough, R.P. Atit, G.E. Landreth, Dentate
gyrus development requires ERK activity to maintain progenitor population
and MAPK pathway feedback regulation, J. Neurosci. 35 (2015) 6836e6848.
[33] M. Kodali, V.K. Parihar, B. Hattiangady, V. Mishra, B. Shuai, A.K. Shetty,
Resveratrol prevents age-related memory and mood dysfunction with
increased hippocampal neurogenesis and microvasculature, and reduced glial
activation, Sci. Rep. 5 (2015) 8075.
[34] S.M. Poulose, N. Thangthaeng, M.G. Miller, B. Shukitt-Hale, Effects of pter-
ostilbene and resveratrol on brain and behavior, Neurochem. Int. 89 (2015)
227e233.
[35] I. Matias, A.S. Buosi, F.C.A. Gomes, Functions of flavonoids in the central
nervous system: astrocytes as targets for natural compounds, Neurochem. Int.
95 (2016) 85e91.
[36] H. Qing, G.Q. He, P.T.T. Ly, C.J. Fox, M. Staufenbiel, F. Cai, Z.H. Zhang, S.C. Wei,
X.L. Sun, C.H. Chen, W.H. Zhou, K. Wang, W.H. Song, Valproic acid inhibits A
beta production, neuritic plaque formation, and behavioral deficits in Alz-
heimer's disease mouse models, J. Exp. Med. 205 (2008) 2781e2789.
[18] D. Petrik, Y.D. Jiang, S.G. Birnbaum, C.M. Powell, M.S. Kim, J. Hsieh, A.J. Eisch,
Functional and mechanistic exploration of an adult neurogenesis-promoting
small molecule, Faseb. J. 26 (2012) 3148e3162.
[19] L.E.B. Bettio, A.R. Patten, J. Gil-Mohapel, N.F. O'Rourke, R.P. Hanley, S. Kennedy,
K. Gopalakrishnan, A.L.S. Rodrigues, J. Wulff, B.R. Christie, Isx-9 can potentiate
cell proliferation and neuronal commitment in the rat dentate gyrus,