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

Abstract

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

Figure 1. Organic synthesis of various flavonoids

A, Schematic diagram of synthetic routes for 4′-dimethylamino-7,8-imidazole flavone (compounds 11, 12 and 13). R represents dimethylamino group or 4-pyrrolidin-1-yl-group. B, Schematic diagram of synthetic route for compound 23.

Figure 2. 8-(4-(dimethylamino)phenyl)chromeno [7,8-d]imidazol-6(1H)-one exhibits elevated TrkB stimulatory activity

A, The chemical structures of various synthetic flavonoids. B, 8-(4- (dimethylamino)phenyl)chromeno [7,8-d]imidazol-6(1H)-one displays stronger TrkB stimulatory activity than the lead compound 24. Primary cortical cultures from E17 rat embryos were treated with 500 nM of various synthetic flavone derivatives for 15 min. The cell lysates (20 μg) were analyzed by p-TrkB immunoblotting (top panel) and p-Akt ELISA (bottom panel). The data were from two sets of replicated experiments (mean ± SEM). C, 8-(4-(dimethylamino) phenyl)chromeno [7,8-d]imidazol-6(1H)-one strongly activates TrkB receptor in mouse brain. One mg/kg of various indicated compounds were orally administrated into C57 BL/6J mice and TrkB phosphorylation and its downstream signaling cascades including Akt and MAPK in the hippocampus of mouse brain were analyzed by immunoblotting at 4 h. Compounds 11 and 24 displayed the strongest TrkB stimulatory effect (1^st panel). The downstream p-Akt and p-MAPK activity coupled to the TrkB activation patterns (3^rd and 5^th panels). P-Akt 473 ELISA in drug treated mouse brain was analyzed (7^th panel) (*: P<0.05 vs control; one-way ANOVA). The data were from two sets of replicated experiments (mean ± SEM).

Figure 3. 8-(4-(dimethylamino)phenyl)chromeno [7,8-d]imidazol-6(1H)-one strongly activates TrkB and reduces the immobility in forced swim test

A, Time course assay of 8-(4-(dimethylamino)phenyl)chromeno [7,8-d]imidazol-6(1H)-one. One mg/kg of compound (32) and compound (11) were orally administrated into C57 BL/6J mice and TrkB phosphorylation and its downstream signaling cascades including Akt in mouse brain were analyzed by immunoblotting at various time points. TrkB activation by compound 11 peaked at 4 h, whereas the maximal TrkB activation by compound 32 in mouse brain occurred at 1–2 h. Arrows indicate the p-TrkB in mature glycosylated or unglycosylated forms (1^st panels). The downstream Akt activation pattern tightly correlated with the upstream TrkB activation (3^rd panels). B, P-Akt S473 in drug-treated mouse brain was by ELISA using 20 μg brain lysates. (*: P<0.05, **: P<0.01, ***, P<0.001 vs control; one-way ANOVA). The data were from two sets of replicated experiments (mean ± SEM). C, Forced swim test with compound 11 and 32. The test (6 min, immobility recorded in the last 4 min) were performed in male C57BL/6J mice that have been orally administrated with 5 mg/kg compound 11, compound 32 or vehicle solvent saline for 21 days. Data are presented as mean ± SEM (n=6, **P<0.01 vs vehicle, Student t-test). D. Locomotor activity assay. Drug-treated mice as stated in C were subjected locomotor activity at day 22. Compound 11 but not 32 significantly increased the locomotor activity compared to vehicle control. Data are presented as mean ± SEM (n=6, *: P<0.05, ***P<0.001, two-way ANOVA). E. TrkB but not TrkA is activated by compound 11 and 32 in mouse brain. The brain lysates from chronic drug-treated mice were analyzed by immunoblotting with anti-p-TrkA 794 and p-TrkB 816.

Figure 4. 2-Methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in primary neurons and mouse brain

A, Chemical structures of various synthetic 4′-pyrrolidino-flavone derivatives. B, 2-methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in primary neurons. Rat primary neurons were treated with 500 nM various compounds for 15 min. Cell lysates (20 mg) were analyzed with various antibodies as indicated. C, 2-methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in mouse brain. One mg/kg of various compounds were orally administrated into C57 BL/6J mice and TrkB phosphorylation (1^st panel) and its downstream signaling cascades including Akt and MAPK in the hippocampus of mouse brain were analyzed by immunoblotting at 2 h. The downstream p-Akt and p-MAPK activity coupled to the TrkB activation patterns (3^rd and 4^th panels).

Figure 5. 2-methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-d]imidazol-6(1H)-one (compound 13) triggers TrkB activation in mouse brain and exhibits antidepressant effect

A, Forced swim test (6 min, immobility recorded in the last 4 min) was performed in male C57BL/6J mice that have been orally administrated with 2.5 mg/kg compound 13, 23 or vehicle solvent saline for 21. Compound 13 but not compound 23 significantly decreased the immobility. Data are presented as mean ± SEM (n=8, *: P<0.05, Student’s t-test). B, Tail suspension test. The drug-treated mice were subjected tail suspension assay. Compound 13 but not 23 reduced the immobility. Data are presented as mean ± SEM (n=8, *: P<0.05, Student’s t-test). C. Locomotor activity assay. None of the tested compounds significantly altered the locomotor activity. D. Both compounds 13 and 23 activate TrkB and its downstream signaling cascades. 2.5 mg/kg of various compounds were orally administrated into C57 BL/6J mice and TrkB phosphorylation and its downstream effector Akt activation in the hippocampus were analyzed by immunoblotting after behavioral tests. Both compound 13 and 23 evidently elevated TrkB phosphorylation (1^st panel). The downstream p-Akt activity was also upregulated by compounds 13 and 23 (4^th panel). The ratio of P-TrkB/total TrkB in drug-treated mouse brain was analyzed (6^th panel). The data were from two sets of replicated experiments and are expressed as mean ± SEM (*, P<0.05 vs control, Student’s t-test). E, Both compound 13 and 23 elevated TrkB phosphorylation in hippocampus. The chronic drug-treated mice were perfused and the brain sections were stained with anti-p-TrkB 816 and anti-TrkB antibodies. The p-TrkB activated neurons were labeled with white arrows.

Figure 6. In vitro cytotoxicity and genotoxicity assay

A, Cytotoxicity assay in hepatocyte HepG2 cells. HepG2 cells were treated with various concentrations of flavonoids for 24 h. The drug-treated cells were subjected LDH assay. Data are presented as mean ± SEM. (n=3). B, Micronuclei assay in hepatocyte HepG2 cells. HepG2 cells were treated with 50 μM of various compounds for 24 h. The nuclei were stained with DAPI and analyzed under a fluorescent microscope. Data are presented as mean ± SEM. (n=3, ***P<0.001 vs vehicle, One-way ANOVA). C, Comet assays. HepG2 cells were treated with 100 μM of various compounds for 24 h. The percentage of lesion DNA in tail was used as a parameter for measurement of DNA damage. Data are presented as mean ± SEM. (n=3, ***P<0.001 vs vehicle, One-way ANOVA).