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

Abstract

7,8-Dihydroxyflavone is a recently identified small molecular tropomyosin-receptor-kinase B (TrkB) agonist. Our preliminary structural-activity relationship (SAR) study showed that the 7,8-dihydroxy groups are essential for the agonistic effect. To improve the lead compound's agonistic activity, we have conducted an extensive SAR study and synthesized numerous derivatives. We have successfully identified 4'-dimethylamino-7,8-dihydroxyflavone that displays higher TrkB agonistic activity than that of the lead. This novel compound also exhibits a more robust and longer TrkB activation effect in animals. Consequently, this new compound reveals more potent antiapoptotic activity. Interestingly, chronic oral administration of 4'-dimethylamino-7,8-dihydroxyflavone and its lead strongly promotes neurogenesis in dentate gyrus and demonstrates marked antidepressant effects. Hence, our data support that the synthetic 4'-dimethylamino-7,8-dihydroxyflavone and its lead both are orally bioavailable TrkB agonists and possess potent antidepressant effects.

Figure 1. Structure-activity relationship study

(A) 7,8-dihydroxyflavone chemical structure with every position in each ring labeled. (B) Chemical Structures of flavonoids from Indofine, Inc. (C) Immunoblotting analysis with neuronal lysates. Primary rat cortical neurons from E17 embryos (13 DIV) were treated with 500 nM various chemicals for 15 min. The neuronal cell lysates were collected and resolved on 10% SDS-PAGE. Immunoblotting was conducted with various antibodies. p-TrkB Y817 antibody was employed at 1:20,000–40,000 dilution. (D). Phospho-Akt 473 ELISA. The cell lysates (20 μg/sample) from the neurons treated with various indicated drugs were analyzed by p-Akt ELISA. The quantitative p-Akt activity in ELISA correlated with TrkB activity. Results were expressed as mean ± SEM (*P<0.05, compared with the vehicle control group, Student t-test, n=3).

Figure 2. Organic synthesis of various 7,8-dihydroxyflavone derivatives

(A) & (B) Schematic diagram of synthetic pathways for various 7,8-DHF derivatives.

Figure 3. 4'-dimethylamino-7,8-dihydroxyflavone displays more potent TrkB stimulatory effect than parental 7,8-dihydroxyflavone

(A) Chemical structures of various 7,8-DHF derivatives. (B) Phospho-Akt ELISA assay by the synthetic compounds in cortical neurons. Primary cortical cultures from E17 rat embryos were treated with 500 nM of various 7,8-DHF derivatives. The cell lysates were analyzed by the ELISA (left panel) (*: P<0.05, ***: P<0.001 vs vehicle, Student's t-test). Different doses of 4'-DMA-7,8-DHF and 7,8-DHF were incubated with primary cortical neurons for 15 min. The cell lysates (20 μg) were analyzed with p-Akt ELISA (right panel) (*: P<0.05, **: P<0.01, ***: P<0.001 vs control, one-way ANOVA; b: P<0.01, c: P<0.001 vs 7,8-DHF at same concentration, Student's t-test). The data were from 2 sets of replicated experiments (mean ± SEM). (C) Time course assay with 4'-DMA-7,8-DHF. Rat primary neurons were treated with 500 nM 4'-DMA-7,8-DHF fro various time points. The neuronal lysates were analyzed with various antibodies. 4'-DMA-7,8-DHF rapidly activated TrkB and its downstream signaling cascades (left panels). 4'-DMA-7,8-DHF revealed longer period of TrkB activation in mouse brain. 1 mg/kg of 4'-DMA-7,8-DHF and 7,8-DHF were orally administrated into C57 BL/6J mice and TrkB phosphorylation and its downstream signaling cascades including Akt and MAPK in mouse brain were analyzed by immunoblotting at various time points. TrkB activation by 4'-DMA-7,8-DHF peaked at 4 h, whereas the maximal TrkB activation by 7,8-DHF in mouse brain occurred at 1–2 h (middle panels). P-Akt 4734 ELISA in drug treated mouse brain was analyzed (right panel) (***: P<0.001 vs control, one-way ANOVA; a: P<0.05, c: P<0.001 vs 7,8-DHF at same concentration, Student's t-test). The data were from 2 sets of replicated experiments (mean ± SEM). (D) 7,8-dihydroxy groups are essential for the flavone's agonistic effect. Different methoxy replaced derivatives were tested on primary neurons by immunoblotting assays.

Figure 4. 4'-dimethylamino-7,8-dihydroxyflavone prevents neurons from apoptosis in a TrkB-dependent manner

(A & B) Active caspase-3 ELISA assay. Cortical neurons were prepared from E16 rat embryonic. The neurons were pretreated with different doses of compounds as indicated for 30 min, followed by 50 μM glutamate for 16 h. The cell lysates were analyzed by active caspse-3 ELISA. (C) 4'-DMA-7,8-DHF and 7,8-DHF prevent KA-elicited neuronal cell death. C57BL/6J mice were orally administrated with 5 mg/kg of 4'-DMA-7,8-DHF and 7,8-DHF, at different time points, the mice were i.p. injected with 20 mg/kg KA for 2 h. The brain lysates were analyzed by immunoblotting with anti-p-TrkB, anti-active caspase-3 antibodies, respectively. (D) TrkB activation is indispensable for the neuroprotective effect of 4'-DMA-7,8-DHF. 4'-DMA-7,8-DHF and 7,8-DHF suppressed KA-induced caspase-3 activation in TrkB F616A mutant knockin mice, which can not be blocked by 1NMPP1 (top panel). TrkB F616A was strongly activated by 4'-DMA-7,8-DHF and 7,8-DHF, which was blocked by 1NMPP1 (middle panel).

Figure 5. 4'-dimethylamino-7,8-dihydroxyflavone and 7,8-dihydroxyflavone promote neurogenesis

A, Neurogenesis assay. Male C57BL/6J mice were orally administrated with 5 mg/kg 4'-DMA-7,8-DHF and 7,8-DHF and vehicle solvent for 21 days, and followed by 50 mg/kg BrdU i.p. injection. In 2 h, the mice were perfused and brain sections were immunostained with anti-BrdU and DAPI. The positive cells in dentate gyrus were highlighted by arrow (left panels). Quantitative analysis of the BrdU positive cells in dentate gyrus (right panel). B, 7,8-DHF and its derivative upregulate TrkB activation in dentate gyrus. Paraffin section were deparaffinized in xylene and rehydrated gradient ethanol solution. Samples were boiled in 10 mM sodium citrate buffer for 20 min for antigen retrieval purpose. Brain sections were incubated with anti-TrkB (BD biosciences, San Jose, CA) 1:50, and anti-p-TrkB Y816 was used at 1:300 dilution. Secondary antibody were applied using anti-rabbit-Alexa 594 (red), anti-mouse-FITC (green). DAPI (blue) was used for nuclear staining.

Figure 6. 4'-dimethylamino-7,8-dihydroxyflavone and 7,8-dihydroxyflavone demonstrate antidepressant effect in a TrkB-dependent manner

(A) Forced swim test with 4'-DMA-7,8-DHF and 7,8-DHF compounds. Male C57BL/6J mice (8 mice/group) were orally administrated by gavage with 5 mg/kg 4'-DMA-7,8-DHF and 7,8-DHF and vehicle solvent saline for 21 days, and subjected to a forced swim test (6 min, immobility recorded in the last 4 min). Data are presented as mean ± SEM. Analysis of variance (ANOVA) revealed significant difference between vehicle and either 7,8-DHF or 4'-DMA-7,8-DHF (n=6, ***P<0.0001 vs vehicle). (B) TrkB but not TrkA is activated by 4'-DMA-7,8-DHF and 7,8-DHF in mouse brain. The brain lysates from above chronically treated mice were analyzed by immunoblotting with anti-p-TrkA 794 and p-TrkB 817. (C) Forced swim test with TrkB F616A knockin mice. Male TrkB knockin mice were given the regular drinking water or 1NMPP1 (25 μM) containing drinking water one day before we started to inject the drugs and sustained throughout the whole experiment. The indicated control (saline) and drugs were administrated for 5 days. Data are presented as mean ± SEM; Analysis of variance (ANOVA) revealed significant effect between vehicle and either 7,8-DHF (n=7 mice, **P<0.001) or 4'-DMA-7,8-DHF (**P<0.001) in TrkB KI mice. None of the drugs produced a significant change in 1NMPP1 treated TrkB KI mice (n=6 to 7 mice) as compared to control.