Discovery of Novel Diphenyl Acrylonitrile Derivatives That Promote Adult Rats’ Hippocampal Neurogenesis

We previously discovered WS-6 as a new antidepressant in correlation to its function of stimulating neurogenesis. Herein, several different scaffolds (stilbene, 1,3-diphenyl 1-propene, 1,3-diphenyl 2-propene, 1,2-diphenyl acrylo-1-nitrile, 1,2-diphenyl acrylo-2-nitrile, 1,3-diphenyl trimethylamine), further varied through substitutions of twelve amide substituents plus the addition of a methylene unit and an inverted amide, were examined to elucidate the SARs for promoting adult rat neurogenesis. Most of the compounds could stimulate proliferation of progenitors, but just a few chemicals possessing a specific structural profile, exemplified by diphenyl acrylonitrile 29b, 32a, and 32b, showed better activity than the clinical drug NSI-189 in promoting newborn cells differentiation into mature neurons. The most potent diphenyl acrylonitrile 32b had an excellent brain AUC to plasma AUC ratio (B/P = 1.6), suggesting its potential for further development as a new lead.

Considering the presence of the blood-brain barrier (BBB) and the cell type complexity required for neurogenesis in the hippocampus, devising simple, predictive assays has proven difficult, and little consensus exists regarding which receptors or enzymes represent ideal targets [13].For this reason, conducting in vivo phenotype screening is essential to examine their potential in promoting neurogenesis.As a result, P7C3 and NSI-189 (Figure 1) were demonstrated to be two of the most promising molecules.P7C3 showed a great potential in treating a variety of neurodegenerative diseases, while NSI-189 is in Phase II as a monotherapy in major depressive disorder [14][15][16][17][18][19].It is noted that P7C3 and NSI-189 have different modes of action.P7C3 is a neuroprotective agent (by lowering the death rate of newborn cells), although the researchers initially intended to search for molecules with a capacity to stimulate proliferation [13,20].In other words, a significant increase in newborn cells in the hippocampus exposed to NSI-189 was observed after a short time, while the significance when exposed to P7C3 was observed after a long time [15].
molecules.P7C3 showed a great potential in treating a variety of neurodegenerative diseases, while NSI-189 is in Phase II as a monotherapy in major depressive disorder [14][15][16][17][18][19].It is noted that P7C3 and NSI-189 have different modes of action.P7C3 is a neuroprotective agent (by lowering the death rate of newborn cells), although the researchers initially intended to search for molecules with a capacity to stimulate proliferation [13,20].In other words, a significant increase in newborn cells in the hippocampus exposed to NSI-189 was observed after a short time, while the significance when exposed to P7C3 was observed after a long time [15].To produce new mature neurons in the hippocampal dentate gyrus (DG) region, both the proliferation of progenitors and differentiation into neurons are key steps for neurogenesis.Unfortunately, the structure-activity relationships (SARs) of the stimulators are less investigated at the stage of differentiation [4].It is unclear which pharmacophore could determine the fate of the newborn progenitors in an adult mammalian brain.We previously discovered N-trans-3′,4′-methylenedioxystilben-4-yl acetamide (coded WS-6, Figure 1) based on natural pterostilbene [21].WS-6 showed an excellent capacity in promoting the proliferation of progenitors and differentiation into neurons in rodents' hippocampi, which possessed the same mode of action as that of NSI-189 [21].Later, WS-6 proved to be a new antidepressant in correlation to its function of stimulating neurogenesis [22].
We herein report SARs of stilbene analogs both at the proliferation stage and the differentiation stage.Thus, we noted that only a few of the chemicals with the specific structural profile have a further capacity to promote newborn cell differentiation into mature neurons.

Exploration of SARs Based on WS-6
WS-6 [21] was used as the lead compound for further structural modification and optimization.We initially examined the impact of substituents such as various amides, a fluorine atom, a methylenedioxy group, and a 3,5-dimethoxy group on the activity (Scheme 1).To evaluate the influence of linker length on the activity of the scaffold, we designed two diphenyl propene skeletons (Scheme 2).We incorporated a cyano group or a tertiary nitrogen linker (Schemes 3 and 4) to adjust the lipid-water partition coefficient, which was a crucial factor in determining the ability of small molecules to penetrate the blood-brain barrier.To further develop new substituents, we designed twelve different amides or sulfonamides (illustrated in Figure 2).Finally, we introduced an inverted amide to the stilbene and inserted an additional methylene unit between the amide and the stilbene (Scheme 5).To produce new mature neurons in the hippocampal dentate gyrus (DG) region, both the proliferation of progenitors and differentiation into neurons are key steps for neurogenesis.Unfortunately, the structure-activity relationships (SARs) of the stimulators are less investigated at the stage of differentiation [4].It is unclear which pharmacophore could determine the fate of the newborn progenitors in an adult mammalian brain.We previously discovered N-trans-3 ′ ,4 ′ -methylenedioxystilben-4-yl acetamide (coded WS-6, Figure 1) based on natural pterostilbene [21].WS-6 showed an excellent capacity in promoting the proliferation of progenitors and differentiation into neurons in rodents' hippocampi, which possessed the same mode of action as that of NSI-189 [21].Later, WS-6 proved to be a new antidepressant in correlation to its function of stimulating neurogenesis [22].
We herein report SARs of stilbene analogs both at the proliferation stage and the differentiation stage.Thus, we noted that only a few of the chemicals with the specific structural profile have a further capacity to promote newborn cell differentiation into mature neurons.

Exploration of SARs Based on WS-6
WS-6 [21] was used as the lead compound for further structural modification and optimization.We initially examined the impact of substituents such as various amides, a fluorine atom, a methylenedioxy group, and a 3,5-dimethoxy group on the activity (Scheme 1).To evaluate the influence of linker length on the activity of the scaffold, we designed two diphenyl propene skeletons (Scheme 2).We incorporated a cyano group or a tertiary nitrogen linker (Schemes 3 and 4) to adjust the lipid-water partition coefficient, which was a crucial factor in determining the ability of small molecules to penetrate the blood-brain barrier.To further develop new substituents, we designed twelve different amides or sulfonamides (illustrated in Figure 2).Finally, we introduced an inverted amide to the stilbene and inserted an additional methylene unit between the amide and the stilbene (Scheme 5).

Chemistry
The procedures to synthesize the stilbene 8, 9, and 12 are outlined in Scheme Commercially The procedures to synthesize the diphenyl propene 18, 23, and 24 are outlined i Scheme 2. The preparation of chalcone (15) began with the aldol condensation reaction o commercially available phenyl ethanone (13) with benzaldehyde ( 14), followed b reduction with NaBH4 and then dehydration to obtain diphenyl propylene (17).Region isomerization of olefin (22) can be performed in a similar way using benzaldehyde (1 o 2) and phenylethyl ketone (19).The diphenyl propene 18b, 23b, 23i, and 24b wer prepared through the acylation reaction of 17 or 22 with methanesulfonyl chloride o isobutyric anhydride.
The procedures to synthesize the diphenyl acrylonitrile 29 and 32 are outlined i Scheme 3. A condensation reaction of commercially available benzaldehyde (1) an phenylacetonitrile (25) followed by the reduction of nitro to amino groups yielded 2
The procedures to synthesize the diphenyl acrylonitrile 29 and 32 are outlined Scheme 3. A condensation reaction of commercially available benzaldehyde (1) a phenylacetonitrile (25) followed by the reduction of nitro to amino groups yielded
The procedures to synthesize the diphenyl propene 18, 23, and 24 are outlined in Scheme 2. The preparation of chalcone (15) began with the aldol condensation reaction of commercially available phenyl ethanone (13) with benzaldehyde ( 14), followed by reduction with NaBH 4 and then dehydration to obtain diphenyl propylene (17).Regional isomerization of olefin (22) can be performed in a similar way using benzaldehyde (1 or 2) and phenylethyl ketone (19).The diphenyl propene 18b, 23b, 23i, and 24b were prepared through the acylation reaction of 17 or 22 with methanesulfonyl chloride or isobutyric anhydride.
The procedures to synthesize the diphenyl acrylonitrile 29 and 32 are outlined in Scheme 3. A condensation reaction of commercially available benzaldehyde (1) and phenylacetonitrile (25) followed by the reduction of nitro to amino groups yielded 28.Similarly, 31 was achieved from 14 and 26.The diphenyl acrylonitrile 29a-29b and 32a-32b were obtained through acylation.
The procedure to synthesize the diphenyl trimethylamine 36 is outlined in Scheme 4. Condensation and reduction of commercially available N-(4-formylphenyl)acetamide (33) and 3,4-methylenedioxymethanamine (34) yielded the intermediate 35.Compound 36a was obtained through methylation on the nitrogen atom.
The procedures to synthesize the target compounds 39b and 42b are outlined in Scheme 5.The Heck reaction of 3 with commercially available 37 led to the acid 38, and acylation of 38 with an amine produced the inverted amide 39b.The amide installed onto the additional methylene group 42b was obtained through a Heck reaction of 40 followed by acylation of 41.

Structure-Activity Relationships
Previous in vitro or in vivo studies mainly focused on stimulating the proliferation effect of the small molecules [21].In this study, a two-step protocol was employed.First, the stimulating function of the compounds on progenitor cells in the rodents' hippocampi was examined with using bromodeoxyuridine (BrdU) labeling.Next, the potent compounds were further screened to ascertain their ability to promote the differentiation of neural progenitor cells, as confirmed by dual labeling of BrdU and NeuN (neuronal nuclear antigen).

Structure-Activity Relationships for Stimulating Proliferation
Adult rats were administered intraperitoneally with the prepared compounds at a dose of 4.0 mg/kg/day for a period of 28 days, followed by a BrdU pulse.After 24 h, the rats were sacrificed, and their brain tissues were isolated and cryosectioned.Immunohistochemical staining was performed and eight brain tissue slices were randomly selected for statistical analyzing [21].
To investigate the impact of amide pharmacophores on proliferative activity, a series of stilbenes, namely, 8c (N,N-dimethylaminoformamide), 8d (cyclobutylformamide), 8g (benzylformamide), 8h (pyridi-2-ylformamide), 8j (N,N-dimethylsulfonamide), 8k (cyclopropylsulfonamide), 8l (benzenesulfonamide), 9b (isobutyramide), and 12f (benzamide) were designed.The data in Figures 3 and 4 shows that both sulfonamides and amides can enhance the proliferation of brain progenitor cells.However, irrespective of an amide or a sulfonamide, the introduction of (hetero) aryl substituents (8g, 8h, 8l, Figure 3; 12f, Figure 4) adversely affected the proliferative activity.Conversely, cyclic alkyl groups and aza-alkyl groups (8d, 8j, 8k, Figure 3) demonstrated a greater propensity for enhancing activity.In contrast to stilbenes, diphenylpropene 18b and 23b maintained the same proliferative activity as 9b (Figure 4).To gain a deep understanding of the impact of diphenyl propene on proliferative activity, we substituted 3,4-methylenedioxy with 3,5dimethoxy (23b vs. 24b) or replaced amide with sulfonamide (23b vs. 23i).The activity of 24b remained the same, while the activity of methanesulfonamide 23i decreased, which underscored the different structure-activity relationships in the diphenyl propene and the stilbene frameworks (Figure 5).In contrast to stilbenes, diphenylpropene 18b and 23b maintained the same proliferative activity as 9b (Figure 4).To gain a deep understanding of the impact of diphenyl propene on proliferative activity, we substituted 3,4-methylenedioxy with 3,5-dimethoxy (23b vs. 24b) or replaced amide with sulfonamide (23b vs. 23i).The activity of 24b remained the same, while the activity of methanesulfonamide 23i decreased, which underscored the different structure-activity relationships in the diphenyl propene and the stilbene frameworks (Figure 5).An attempt to bridge two phenyl rings using a trimethylamine linker (36a) instead of alkene linkers abolished the activity (Figure 5).The diphenyl acrylonitrile 29b exhibited satisfactory activity.However, compound 8e with a cyano group shifted from the linker to the amide substituent did not display significant activity.These findings corroborated that stilbene was the preferred scaffold with proliferative activity and was amenable to certain structural modifications.
The amide inversion (39b) displayed activity comparable to that found in amides 8c, An attempt to bridge two phenyl rings using a trimethylamine linker (36a) instead of alkene linkers abolished the activity (Figure 5).The diphenyl acrylonitrile 29b exhibited satisfactory activity.However, compound 8e with a cyano group shifted from the linker to the amide substituent did not display significant activity.These findings corroborated that stilbene was the preferred scaffold with proliferative activity and was amenable to certain structural modifications.
The amide inversion (39b) displayed activity comparable to that found in amides 8c, 8d, and 29b (Figure 6) [21].Unfortunately, the introduction of an additional methylene group between the amide and the stilbene led to a loss of activity (42b).These experimental findings emphasized the diverse SARs exhibited by amide substituents.
BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle group, and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01; * p < 0.05).
An attempt to bridge two phenyl rings using a trimethylamine linker (36a) instead of alkene linkers abolished the activity (Figure 5).The diphenyl acrylonitrile 29b exhibited satisfactory activity.However, compound 8e with a cyano group shifted from the linker to the amide substituent did not display significant activity.These findings corroborated that stilbene was the preferred scaffold with proliferative activity and was amenable to certain structural modifications.
The amide inversion (39b) displayed activity comparable to that found in amides 8c, 8d, and 29b (Figure 6) [21].Unfortunately, the introduction of an additional methylene group between the amide and the stilbene led to a loss of activity (42b).These experimental findings emphasized the diverse SARs exhibited by amide substituents.Compounds 8c, 8d, 29b, and 39b (but not 42b) showed significant proliferation-promoting activity (The stilbene 8d and the diphenyl acrylonitrile 29b were repeated to check reproducibility).Elevenweek-old rats (n = 2) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle Figure 6.SARs of compounds 8c, 8d, 29b, 39b, and 42b in the proliferation stage of neurogenesis.Compounds 8c, 8d, 29b, and 39b (but not 42b) showed significant proliferation-promoting activity (The stilbene 8d and the diphenyl acrylonitrile 29b were repeated to check reproducibility).Eleven-week-old rats (n = 2) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle group, and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; *** p < 0.001; * p < 0.05).

Structure-Activity Relationships for Promoting Differentiation
In an effort to investigate the effects of various compounds on the differentiation fate of neural progenitor cells following proliferation, we selected a set of nine active compounds (8c, 8d, 8j, 8k, 18b, 23b, 24b, 29b, and 39b) and an inactive compound, 42b.The study employed a rat model, wherein nine-week-old rats were administered intraperitoneally with the compound at a dose of 4.0 mg/kg/day for 28 days and then treated with a pulse of BrdU.Instead of sacrificing the rats 24 h after the BrdU injections, the rats were sacrificed after another 28 days.The brains were then isolated and cryosectioned.Eight randomly selected slices were subjected to immunohistochemical staining.This staining enabled the co-localization of the BrdU-incorporated cells with the NeuN marker, enabling the definition of the compounds that could promote the differentiation of newborn neural progenitor cells into mature functional neurons.
However, after a 28 day waiting period, only five compounds (8c, 8d, 8k, 29b and 39b) could promote the proliferation of progenitors (Figure 7A).Compounds 18b, 23b, and 24b, featuring a diphenyl propylene scaffold, did not exhibit significant proliferative activity over the longer 28 day period.Regarding the amide and sulfonamide substructures, cycloalkyl substituents were more favorable than the aza-alkyl groups to enhance the proliferative activity (8k vs. 8j, 8d vs. 8c).Compound 39b, characterized by amide inversion, exhibiting proliferative activity within both the short and long durations, whereas compound 42b, with the insertion of a methylene group between the amide and the benzene ring, remained devoid of proliferative activity.Unexpectedly, the analysis of BrdU and NeuN double staining revealed that only compound 29b significantly increased the number

Structure-Activity Relationships of 29b Analogs
Inspired by the success of 29b, we synthesized 29a, an acetamide analog of compound 29b.The corresponding isomers of 29a and 29b, 32a and 32b, varying the position of the cyano group, were also prepared.Because NSI-189 was a stimulator for neurogenesis [17], we utilized NSI-189 as a control.The results depicted in Figure 8 showed that both 29a and NSI-189 were inactive at a low dose of 4 mg/kg, while the other three compounds, 29b, 32a, and 32b, exhibited proliferative activity after a day.

Structure-Activity Relationships of 29b Analogs
Inspired by the success of 29b, we synthesized 29a, an acetamide analog of compound 29b.The corresponding isomers of 29a and 29b, 32a and 32b, varying the position of the cyano group, were also prepared.Because NSI-189 was a stimulator for neurogenesis [17], we utilized NSI-189 as a control.The results depicted in Figure 8 showed that both 29a and NSI-189 were inactive at a low dose of 4 mg/kg, while the other three compounds, 29b, 32a, and 32b, exhibited proliferative activity after a day.Next, we further examined the effects of 29a, 29b, 32a, 32b, and NSI-189 on differentiation fate of newborn progenitors (Figure 9).The trend in the differentia stage was observed to be consistent with that in the proliferation stage.Among compounds, isobutyramide was more favorable than acetamide in enhancing activity vs. 29a, 32b vs. 32a).The position of the cyano group played a crucial role in defin activity, because 32a and 32b, with a cyano substitution distal to the amide-substitu phenyl ring, are more active than 29a and 29b, with a cyano substitution adjacent to amide-substituted phenyl ring.Compound 32b showed the highest activity, while 29a NSI-189 had no significant neurogenesis-promoting activity at the dose of 4.0 mg/kg.Next, we further examined the effects of 29a, 29b, 32a, 32b, and NSI-189 on the differentiation fate of newborn progenitors (Figure 9).The trend in the differentiation stage was observed to be consistent with that in the proliferation stage.Among the compounds, isobutyramide was more favorable than acetamide in enhancing activity (29b vs. 29a, 32b vs. 32a).The position of the cyano group played a crucial role in defining activity, because 32a and 32b, with a cyano substitution distal to the amide-substituted phenyl ring, are more active than 29a and 29b, with a cyano substitution adjacent to the amide-substituted phenyl ring.Compound 32b showed the highest activity, while 29a and NSI-189 had no significant neurogenesis-promoting activity at the dose of 4.0 mg/kg.

Pharmacokinetic Studies
We conducted pharmacokinetic experiments (n = 3 rats per group) on the most promising compound, 32b, to examine its pharmacokinetic parameters and distribution (Table 1).The maximum drug concentrations observed in the plasma and brain tissue of rats were 132 ng/mL and 168 ng/g, respectively.The exposure of the drug in brain tissue in terms of AUC (area under the curve) (i.e. the brain to plasma ratio, B/P) was 1.59 times higher than that in plasma (383 h*ng/mL in plasma and 608 h*ng/g in brain tissue).The half-life of the drug in brain tissue was found to be better than in plasma.Taken together, compound 32b could effectively traverse the BBB and exhibited great overall uptake in the brain.Finally, we performed cytotoxicity tests on compounds 29b, 32a, and 32b.Among them, the IC50 values of compounds 29b and 32b were greater than 200 μM, and only compound 32a had a weak inhibitory effect (IC50 = 109.29 ± 12.91 μM) on hCMEC/D3 cell lines (Figure S1).

Pharmacokinetic Studies
We conducted pharmacokinetic experiments (n = 3 rats per group) on the most promising compound, 32b, to examine its pharmacokinetic parameters and distribution (Table 1).The maximum drug concentrations observed in the plasma and brain tissue of rats were 132 ng/mL and 168 ng/g, respectively.The exposure of the drug in brain tissue in terms of AUC (area under the curve) (i.e. the brain to plasma ratio, B/P) was 1.59 times higher than that in plasma (383 h*ng/mL in plasma and 608 h*ng/g in brain tissue).The half-life of the drug in brain tissue was found to be better than in plasma.Taken together, compound 32b could effectively traverse the BBB and exhibited great overall uptake in the brain.Calculating the logP (lipid-water partition coefficients) indicated 3.82 for acetamide WS-6 and 4.66 for an isobutyramide analog of WS-6.However, ClogP of the isobutyamides 29b and 32b was comparable to WS-6 (Clog P: 3.28 for 29b and 32b; 2.44 for acetamide 29a and 32a).Thus, the introduction of the cyano group played a role in regulating the lipid-water partition coefficient, which may account for the improved ability of 32b to penetrate the BBB.

Chemistry
All reagents and solvents were purchased from commercial sources (J&K Scientific) and used without further purification unless otherwise noted.All non-aqueous reactions were carried out under argon using dry solvents, unless otherwise noted.Reactions were monitored through thin-layer chromatography, and 254 nm UV light was used for visualization.Column chromatography was performed on silica gel (100-200 or 200-300 mesh) purchased from Qingdao Haiyang. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on Bruker ARX 400/500 MHz with tetramethylsilane (TMS) as an internal standard.High resolution mass spectra (HRMS) were obtained with Agilent 6210 LC-MS TOF or Agilent Q-TOF 6520 LC-MS (Table S1 and Supplementary Materials).
General procedure 3: synthesis of compounds 7 and 28.A well stirred mixture of compounds with a nitro group (10 mmol, 1 eq) and stannous chloride (40.0 mmol, 4 eq) in EtOH (40 mL) was heated to reflux for 2 h.After the reaction was completed, the solvent was removed, and the residue was stirred with NaOH (50 mL, 1 M) for another 0.5 h.The aqueous solution was extracted with ethyl acetate.The organic layer was combined, washed with saturated NaCl solution and then concentrated in vacuo.Purification through column chromatography on silica gel (dichloromethane = 100%) afforded products with an amino group.
General procedure 4(b): Pyridine (0.19 mL, 2.4 mmol) and anhydride (2.3 mmol, 3 eq) were added dropwise to a solution of compounds with an amino (0.8 mmol, 1 eq) and DMAP (0.02 g, 0.1 mmol) in CH 2 Cl 2 (20 mL) below 0 • C. The mixture was stirred at 0 • C from 0.5 h to 1.5 h.The reaction solution was poured into saturated NaHCO 3 solution, the organic layer was separated, and the solvent was removed in vacuo.The residue was purified through column chromatography on silica gel to afford pure target products.

Animal Experiments
Eight-week-old (or 10-week-old) SD male rats purchased from Peking University Health Science Center were acclimatized for one week at the Beijing Institute of Technology.All experimental protocols strictly adhered to the regulations governing the management of experimental animals and the animal ethics policy of the Beijing Institute of Technology.
The compounds were dissolved in reagent-grade soybean oil containing 10% ethanol to reach a drug concentration of 2.8 mg/mL.The rats in the experimental group received intraperitoneal injections of the compounds at a dose of 4.0 mg/kg/d for 28 consecutive days.As a control, the vehicle group was administered injections of reagent-grade soybean oil containing 10% ethanol.BrdU (Sigma-Aldrich, St. louis, MA, USA) solution, prepared in physiological saline at a concentration of 10 mg/mL, was administered to all rats on day 29.Each rat received two injections of BrdU at a dosage of 50 mg/kg, with a 2 h interval between injections.The rats were euthanized 24 h (or 28 days) after the final BrdU injection.
Two or four rats per group were anesthetized using pentobarbital sodium (70 mg/kg) and subsequently perfused with physiological saline.The brain tissue was carefully dissected and fixed in 4% paraformaldehyde at 4 • C for one week.Next, the brain tissue underwent dehydration using a phosphate-buffered saline (PBS) solution containing 20% to 30% sucrose.Finally, the brain tissue was cryosectioned into slices with a thickness of 30 µm, and eight slices were randomly selected for further analysis.
The assessment of new cell proliferation was conducted using BrdU staining.To eliminate endogenous peroxides, sections were exposed to 1% H 2 O 2 for 30 min at RT. Cellular denaturation was achieved by treating the sections with 2 M HCl at 37 • C for 1 h, followed by rinsing in 0.1 M borate buffer (pH 8.5).Tissue sections were subjected to blocking in a solution consisting of 5% goat serum and 0.5% Triton X-100 in PBS at RT for 1-2 h.The specimens were then incubated overnight at 4 • C with a mouse primary antibody specific to BrdU (diluted at 1:500; Millipore, Billerica, MA, USA).Subsequently, the DAB kit was employed for 2 h of incubation at RT.
The differentiation process of NPCs was visualized through the utilization of the NeuN/BrdU double staining technique.Tissue sections were subjected to an incubation period in a blocking buffer at RT for 1 to 2 h.The specimens were exposed to a rabbit anti-NeuN antibody (diluted at 1:500, Abcam, Cambridge, MA, UK) and a primary antibody, BrdU (diluted at 1:500), overnight at 4 • C. Afterwards, the sections were treated with anti-mouse 488 and anti-rabbit TRITC secondary antibodies for 2 h at RT.
The data were presented as mean ± SEM (standard error of the mean) and visualized using GRAPHPAD PRISM 6 (Graphpad Software, La Jolla, CA, USA).Statistical analysis was conducted utilizing one-way analysis of variance (ANOVA) followed by a post hoc test.Subsequent to the statistical treatment, the observed discrepancies were determined to be statistically significant (p < 0.05).

Pharmacokinetic Study
The SD rats (approximately 200 g) were randomly assigned to eight groups, with each group comprising three rats.All experimental procedures were carried out in accordance with the ethical guidelines outlined by the Ethics Committee of the Beijing Institute of Technology.The administration medium consisted of a combination of 5% dimethyl sulfoxide (DMSO) and 95% soybean oil.The drug was administered intraperitoneally at a dose of 4 mg/kg.Prior to the experiment, the experimental animals were subjected to a 12 h fasting period with free access to water.Following drug administration, blood samples were collected from the rat's orbital vein at predetermined time points.The collected blood was then placed in sodium heparin anticoagulation tubes.The rats were euthanized, and their brain tissues were carefully extracted and loaded into 5 mL EP tubes.The collection time points for plasma and brain tissue samples were as follows: 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h.Within 2 h after collection, the blood samples were centrifuged at 6000 rpm for 10 min at a temperature of 4 • C. The resulting samples were then stored in a −20 • C refrigerator.
Tissue samples were first weighed and homogenized to ensure consistent testing conditions.The determination of original compound concentrations in plasma and tissue homogenates was performed using LC-MS/MS.Specifically, 50 µL of plasma or tissue samples were mixed with 50 µL of propranolol and 100 µL of acetonitrile for protein precipitation.The resulting mixture was then centrifuged at 12,000 rpm for 5 min, and the supernatant was diluted and analyzed through LC-MS/MS.The liquid chromatography was conducted on a Thermo Scientific Q Exactive HF-X instrument (Thermo Fisher Scientific, Waltham, MA, USA), employing an ACQUITY UPLC BEH C18 Column with dimensions of 130 Å, 1.7 µm, 2.1 mm × 100 mm.Additionally, a pre-column equipped with an ACQUITY UPLC BEH C18 VanGuard Pre-Column (130 Å, 1.7 µm, 2.1 mm × 5 mm) was utilized.The liquid phase method employed gradient elution with mobile phases consisting of acetonitrile and a 0.1% formic acid aqueous solution.Mass spectrometry was conducted in positive ion mode.The quantification of drug concentration in plasma samples was performed using the internal standard method, and the obtained pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0 software.

CCK-8 Assay for Cytotoxicity
hCMEC/D3 cells were obtained from the Cell Resource Center, Peking Union Medical College (PCRC).All cells were acquired in 2023 and were maintained in culture for no more than 10 continuous passages.The cells were cultured in DMEM (11995065, Gibco, Grand Island, NY, USA) supplemented with 10% FBS (164210-50, Procell, Austin, TX, USA), 100 units of penicillin, and 100 mg/mL of streptomycin (PB180120, Procell).hCMEC/D3 cells (5 × 10 3 ) were seeded into 96-well plates in 90 µL of DMEM medium containing 10% fetal bovine serum per well.All test compounds were formulated as 10 mM stock solutions in DMSO (final concentration ≤ 0.5%).After overnight incubation, the test compound stock solution was diluted with DMEM medium containing 10% fetal bovine serum to every target concentration, and 10 µL of each was added to each well.Three parallel experiments were set up for each compound.After incubation for 48 h, 10 µL of CCK-8 reagent was added to each well in sequence according to the manufacturer's instructions.After incubating in a 37 • C incubator for 1-2 h, the absorbance was measured at a wavelength of 450 nm, using a wavelength of 620 nm as a reference, and detected with a ThermoScientific Multiskan FC (Thermo Fisher Scientific).The graphs are representative results, and this experiment was independently repeated three times with similar results.

Figure 1 .
Figure 1.The compounds promoting neurogenesis in vivo.

Figure 1 .
Figure 1.The compounds promoting neurogenesis in vivo.

Figure 3 .
Figure 3. SARs of compounds 8d, 8g, 8h, 8j, 8k, and 8l in the proliferation stage of neurogenesis.All compounds showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were administered intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the vehicle group and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(V: vehicle group; *** p < 0.001; ** p < 0.01).

Figure 3 .
Figure 3. SARs of compounds 8d, 8g, 8h, 8j, 8k, and 8l in the proliferation stage of neurogenesis.All compounds showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were administered intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the vehicle group and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(V: vehicle group; *** p < 0.001; ** p < 0.01).

Figure 3 .
Figure3.SARs of compounds 8d, 8g, 8h, 8j, 8k, and 8l in the proliferation stage of neurogenesis.All compounds showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were administered intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the vehicle group and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(V: vehicle group; *** p < 0.001; ** p < 0.01).

Figure 4 .
Figure 4. SARs of compounds 9b, 12f, 18b, and 23b in the proliferation stage of neurogenesis.Compounds 9b, 18b, and 23b (but not 12f) showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were exposed to the compounds intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injection.BrdU + immunoreactivity demonstrated the number of newborn cells in the vehicle group and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(V: vehicle group; *** p < 0.001; ** p < 0.01).

Figure 4 .
Figure 4. SARs of compounds 9b, 12f, 18b, and 23b in the proliferation stage of neurogenesis.Compounds 9b, 18b, and 23b (but not 12f) showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were exposed to the compounds intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injection.BrdU + immunoreactivity demonstrated the number of newborn cells in the vehicle group and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(V: vehicle group; *** p < 0.001; ** p < 0.01).

Figure 5 .
Figure 5. SARs of compounds 8e, 23i, 24b, 29b, and 36a in the proliferation stage of neurogenesis.Compounds 24b and 29b showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle group, and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01; * p < 0.05).

Figure 5 .
Figure 5. SARs of compounds 8e, 23i, 24b, 29b, and 36a in the proliferation stage of neurogenesis.Compounds 24b and 29b showed significant proliferation-promoting activity.Eleven-week-old rats (n = 2) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle group, and the drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01; * p < 0.05).

Figure 6 .
Figure 6.SARs of compounds 8c, 8d, 29b, 39b, and 42b in the proliferation stage of neurogenesis.Compounds 8c, 8d, 29b, and 39b (but not 42b) showed significant proliferation-promoting activity (The stilbene 8d and the diphenyl acrylonitrile 29b were repeated to check reproducibility).Elevenweek-old rats (n = 2) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injections.BrdU + immunoreactivity demonstrated the number of newborn cells in the control group, the vehicle

Figure 8 .
Figure 8. SARs of NSI-189, 29a, 29b, 32a, and 32b in the proliferation stage of neurogenesis.Compounds 29b, 32a, and 32b showed significant proliferation-promoting activity.Nine-week-old rats (n = 4) were administrated intraperitoneally at a dose of 4.0 mg/kg/day for 28 days, followed by a BrdU pulse.The rats were sacrificed 24 h after the BrdU injection.BrdU + immunoreactivity demonstrated the number of positive cells in the control group, vehicle group, and NSI-189 and drug treatment groups.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01; * p < 0.05).

Figure 9 .
Figure 9. SARs of compounds NSI-189, 29a, 29b, 32a, and 32b in the differentiation stage of neurogenesis.Compound 29b, 32a, and 32b showed significant neurogenesis-promoting activity.Green fluorescent BrdU + dots in the subgranular zone indicate newborn cells (white triangle), and yellow fluorescent BrdU + &NeuN + dots in the subgranular zone indicate newborn mature neurons (white triangle).Nine-week-old rats (n = 4) were administrated intraperitoneally at a dose of 4.0 mg/kg, and the rats were sacrificed 28 days instead of 24 h after the BrdU injections.(A) The number of BrdU + cells; (B) the number of NeuN + and BrdU + cells.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01).

Figure 9 .
Figure 9. SARs of compounds NSI-189, 29a, 29b, 32a, and 32b in the differentiation stage of neurogenesis.Compound 29b, 32a, and 32b showed significant neurogenesis-promoting activity.Green fluorescent BrdU + dots in the subgranular zone indicate newborn cells (white triangle), and yellow fluorescent BrdU + &NeuN + dots in the subgranular zone indicate newborn mature neurons (white triangle).Nine-week-old rats (n = 4) were administrated intraperitoneally at a dose of 4.0 mg/kg, and the rats were sacrificed 28 days instead of 24 h after the BrdU injections.(A) The number of BrdU + cells; (B) the number of NeuN + and BrdU + cells.Data are expressed as mean ± SEM (n = 8 slices).(C: control group; V: vehicle group; ** p < 0.01).