Design, synthesis and evaluation of 2-pyrimidinylindole derivatives as anti-obesity agents by regulating lipid metabolism.

Obesity, a global pandemic posing a growing threat to human health, necessitates the development of effective and safe anti-obesity agents. Our previous studies highlighted the lipid-lowering effects of indolylquinazoline Bouchardatine and its derivatives. In this study, we employed scaffold hopping and simplification strategies to design and synthesize two new series derivatives by modifying the D ring. Extensive discussions have been conducted regarding the structure-activity relationship between lipid-lowering activity and the new compounds. These discussions have resulted in the discovery of 2-pyrimidinylindole derivatives as a promising scaffold for anti-obesity treatment. The new 2-pyrimidinylindole derivatives exhibited comparable lipid-lowering activity to the previously reported indolylquinazoline derivatives, including SYSU-3d and R17, with reduced toxicity. The most potent compound, 5a, demonstrated a larger therapeutic index, improved aqueous solubility and oral bioavailability compared to the previous lead compounds. In vivo evaluation indicated that 5a effectively reduced lipid accumulation in adipose tissue, improved glucose tolerance, and mitigated insulin resistance and liver function damage caused by a high-fat and high-cholesterol diet. Mechanism studies indicated that 5a may regulate lipid metabolism through the modulation of the PPARγ signaling pathway. Overall, our study has identified a highly active compound 5a, and provided the basis for further development of 2-pyrimidinylindole as a promising scaffold for obesity treatment.

Design and Synthesis of Bouchardatine Derivatives as a Novel AMP-Activated Protein Kinase Activator for the Treatment of Colorectal Cancer.

Metabolic reprogramming is a crucial hallmark of tumorigenesis. Modulating the reprogrammed energy metabolism is an attractive anticancer therapeutic strategy. We previously found a natural product, bouchardatine, modulated aerobic metabolism and inhibited proliferation in the colorectal cancer cell (CRC). Herein, we designed and synthesized a new series of bouchardatine derivatives to discover more potential modulators. We applied the dual-parametric high-content screening (HCS) to evaluate their AMP-activated protein kinase (AMPK) modulation and CRC proliferation inhibition effect simultaneously. And we found their antiproliferation activities were highly correlated to AMPK activation. Among them, 18a was identified with nanomole-level antiproliferation activities against several CRCs. Interestingly, the evaluation found that 18a selectively upregulated oxidative phosphorylation (OXPHOS) and inhibited proliferation by modulating energy metabolism. Additionally, this compound effectively inhibited the RKO xenograft growth along with AMPK activation. In conclusion, our study identified 18a as a promising candidate for CRC treatment and suggested a novel anti-CRC strategy by AMPK activating and OXPHOS upregulating.

Matrine counteracts obesity in mice via inducing adipose thermogenesis by activating HSF1/PGC-1α axis.

Promoting energy expenditure is known to curb obesity and can be exploited for its treatment. Our previous study has demonstrated that activation of HSF1/PGC-1α axis efficiently induced mitochondrial biogenesis and adaptive oxidation and thus ameliorating lipid accumulation, however, whether it can be a therapeutic approach for metabolic disorders treatment needs explored. Here, a high-efficient and specific HSF1/PGC-1α activator screening system was established and the natural clinical liver-protecting agent matrine was identified as a robust HSF1/PGC-1α activator. Matrine treatment efficiently induced mitogenesis and thermogenic program in primary mouse adipose stem cell derived adipocytes by enriching HSF1 to the promoter of Pgc-1α. Deficiency of PGC-1α in adipocytes diminished the browning induction ability of matrine. Oral administration of matrine to the obese mice induced by high fat and high cholesterol diet increased energy expenditure and corrected the degeneration of thermogenesis in brown adipose tissue (BAT). Also, matrine treatment markedly induced the transformation of brown-like adipocytes in subcutaneous white adipose tissue (sWAT) via a mechanism of HSF1/PGC-1α, thereby attenuating obesity and myriads of metabolic disorders. This led to an improvement in adaptive thermogenesis to cold stimuli. These findings are of great significance in understanding the regulation mechanisms of the HSF1/PGC-1α axis in thermogenesis and providing a novel therapeutic approach for obesity treatment. Matrine may have potential therapeutic implications for the treatment of obesity in clinics.

A novel HSF1 activator ameliorates non-alcoholic steatohepatitis by stimulating mitochondrial adaptive oxidation.

BACKGROUND AND PURPOSE: Non-alcoholic steatohepatitis (NASH) is the more severe form of metabolic associated fatty liver disease (MAFLD) and no pharmacological treatment as yet been approved. Identification of novel therapeutic targets and their agents is critical to overcome the current inadequacy of drug treatment for NASH. EXPERIMENTAL APPROACH: The correlation between heat shock factor 1 (HSF1) levels and the development of NASH and the target genes of HSF1 in hepatocyte were investigated by chromatin-immunoprecipitation sequencing. The effects and mechanisms of SYSU-3d in alleviating NASH were examined in relevant cell models and mouse models (the Ob/Ob mice, high-fat and high-cholesterol diet and the methionine-choline deficient diet-fed mice). The actions of SYSU-3d in vivo were evaluated. KEY RESULTS: HSF1 is progressively reduced with mitochondrial dysfunction in NASH pathogenesis and activation of this transcription factor by its newly identified activator SYSU-3d effectively inhibited all manifestations of NASH in mice. When activated, the phosphorylated HSF1 (Ser326) translocated to nucleus and bound to the promoter of PPARγ coactivator-1α (PGC-1α) to induce mitochondrial biogenesis. Thus, increasing mitochondrial adaptive oxidation and inhibiting oxidative stress. The deletion of HSF1 and PGC-1α or recovery of HSF1 in HSF1-deficiency cells showed the HSF1/PGC-1α pathway was mainly responsible for the anti-NASH effects of SYSU-3d independent of AMP-activated protein kinase (AMPK). CONCLUSION AND IMPLICATIONS: Activation of HSF1 is a practical therapeutic approach for NASH treatment via the HSF1/PGC-1α/mitochondrial pathway and SYSU-3d can be considered as a potential candidate for the treatment of NASH.

Palladium oxidative addition complex-enabled synthesis of amino-substituted indolyl-4(3H)-quinazolinones and their antitumor activity evaluation.

The indolyl-4(3H)-quinazolinone core is an important structural motif in functional molecules. However, few methods exist for its direct modification, which limits its potential application. Reported herein is a palladium-mediated amination of halogen-containing indolyl-4(3H)-quinazolinones with a variety of primary and secondary amines via the corresponding palladium oxidative addition complexes. The protocol allows the facile synthesis of indolyl-4(3H)-quinazolinone derivatives with amino groups at all the positions of the benzene ring in moderate to good yields with mild reaction conditions and good functional group tolerance. Furthermore, the antitumor activity of these products was evaluated.

CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae).

In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD-/+ and NlCSAD-/-) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.

Discovery of a promising agent IQZ23 for the treatment of obesity and related metabolic disorders.

Discovery of novel anti-obesity agents is a challenging and promising research area. Based on our previous works, we synthesized 40 novel ß-indoloquinazoline analogues by altering the skeleton and introducing preferential side chains, evaluated their lipid-lowering activity and summarized the structure-activity relationships. In combination with an evaluation of the lipid-lowering efficacies, AMP-dependent activated protein kinase (AMPK) activating ability and liver microsomal stability, compound 23 (named as IQZ23) was selected for further studies. IQZ23 exerted a high efficacy in decreasing the triglyceride level (EC50 = 0.033 µM) in 3T3-L1 adipocytes. Mechanistic studies revealed the lipid-lowering activity of IQZ23 was dependent on the AMPK pathway by modulating ATP synthase activity. This activation was accompanied by mitochondrial biogenesis and oxidation capacity increased, and insulin sensitivity enhanced in pertinent cell models by various interventions. Correspondingly, IQZ23 (20 mg/kg, i.p.) treatment significantly reversed high fat and cholesterol diet (HFC)- induced body weight increases and accompanying clinical symptoms of obesity in mice but without indicative toxicity. These results indicate that IQZ23 could be a useful candidate for the treatment of obesity and related metabolic disorders.

Bouchardatine analogue alleviates non-alcoholic hepatic fatty liver disease/non-alcoholic steatohepatitis in high-fat fed mice by inhibiting ATP synthase activity.

BACKGROUND AND PURPOSE: Non-alcoholic hepatic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome in the liver and non-alcoholic steatohepatitis (NASH) represents its advanced stage. R17 derived from bouchardatine, shows benefits in the metabolic syndrome, but has not been tested in the liver. The present study examined the pharmacological effects of R17 in a model of NAFLD/NASH and its mode of action. EXPERIMENTAL APPROACH: The effects of R17 were examined in mice fed a high-fat (HF) diet to induce the pathological characteristics of NAFLD/NASH and in cultures of HuH7 cells. We used histological and immunohistochemical techniques along with western blotting and siRNA. Generation of ROS and apoptosis were measured. KEY RESULTS: Administration of R17 (20 mg·kg-1 , i.p. every other day) for 5 weeks reversed HF-induced hepatic triglyceride content, inflammation (inflammatory cytokines and macrophage numbers), injury (hepatocyte ballooning and apoptosis, plasma levels of alanine aminotransferase and aspartate aminotransferase), and fibrogenesis (collagen deposition and mRNA expression of fibrosis markers). In cultured cells, R17 reduced cell steatosis from both lipogenesis and fatty acid influx. The attenuated inflammation and cell injury were associated with inhibition of both endoplasmic reticulum (ER) stress and oxidative stress. Notably, R17 activated the liver kinase B1-AMP-activated protein kinase (AMPK) pathway by inhibiting activity of ATP synthase, rather than direct stimulation of AMPK. CONCLUSION AND IMPLICATIONS: R17 has therapeutic potential for NAFLD/NASH. Its mode of action involves the elimination of ER and oxidative stresses, possibly via activating the LKB1-AMPK axis by inhibiting the activity of ATP synthase.

Bouchardatine suppresses rectal cancer in mice by disrupting its metabolic pathways via activating the SIRT1-PGC-1α-UCP2 axis.

Cancer metabolism is an attractive target of the therapeutic strategy for cancer. The present study identified bouchardatine (Bou) as a potent suppressor of rectal cancer growth by cycle-arresting independent of apoptosis. In cultured HCT-116 rectal cancer cells, Bou increased glucose uptake/oxidation and capacity of mitochondrial oxidation. These effects were associated with an upregulation of uncoupling protein 2 (UCP2) and the activation of its upstream Sirtuin 1 (SIRT1)/(Liver kinase B1) LKB1- (Adenosine monophosphate-activated protein kinase) AMPK axis. The pivotal role of UCP2 in the cancer-suppressing effect was demonstrated by overexpressing UCP2 in HCT-116 cells with similar metabolic effects to those produced by Bou. Interestingly, Bou activated peroxisome proliferators activated receptor γ coactivator 1α (PGC-1α) and recruited it to the promoter of UCP2 in HCT-116 cells along with deacetylation (thus activation) by SIRT1. The requirement of SIRT1 for the cancer-suppressing effect through the PGC-1α-UCP2 was confirmed by the reciprocal responses to Bou in HCT-116 with defected and overexpressed SIRT1. Whereas knockdown, mutation or pharmacological inhibition of SIRT1 all abolished Bou-induced deacetylation/activation of PGC-1α, the opposing effects were observed after overexpressing SIRT1. In mice, administration of Bou (50 mg/kg) also suppressed the growth of rectal cancer associated with increases the UCP2 expression and mitochondria capacity in the tumor. Collectively, our findings suggest that Bou has a therapeutic potential for the treatment of rectal cancer by disrupting the metabolic path of cancer cells via activating the PGC-1α-UCP2 axis with SIRT1 as its primary target.

Design, synthesis and biological evaluation of novel bouchardatine analogs as potential inhibitors of adipogenesis/lipogenesis in 3T3-L1 adipocytes.

Inhibition of the differentiation of adipocytes and reduced lipid synthesis are efficacious approaches for treating obesity-related metabolic disorders. Bouchardatine (Bou) is a natural alkaloid that has been reported to moderately inhibit the differentiation of 3T3-L1 cells without inducing toxicity. To explore the importance of aldehyde group at 8a-position of Bou and optimize the activity, we synthesized 35 (31 novel) compounds by discarding or replacing aldehyde group with halogen and introducing different amine chains at 5-position of Bou. The lipid-lowering activity was evaluated using a cell-based screening system. The substitution of the group at the 8a-position of compounds was important for its lipid-lowering activity, and the SAR was discussed. The selective compound 6e showed a 93-fold increase in its lipid-lowering effect (EC50 = 0.24 µM) compared with Bou (EC50 ≈ 25 µM). Further mechanistic studies revealed that compound 6e activated AMP-activated protein kinase (AMPK) pathway and inhibited MCE activity to block cell proliferation and induce cell cycle arrest at the early stage of differentiation, thus decreasing the expression of adipogenic factors and fatty acid synthesis-related proteins.

A rapid UPLC-MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics.

A reliable high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid-phase extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R(2) > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra- and inter-day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats.

In vitro evaluation of tectoridin, tectorigenin and tectorigenin sodium sulfonate on antioxidant properties.

Tectoridin (4',5,7-thrihydroxy-6-methoxyisoflavone-7-O-ß-d-glucopyranoside) isolated from the flowers of Pueraria thunbergiana is reported to have less hepatoprotective, hypoglycemic, antiallergic and anaphylaxis inhibitory activity than its aglycone form tectorigenin. To obtain tectorigenin, tectoridin was hydrolyzed in the current study. However, practical limitations of tectorigenin do exist due to its poor water-solubility. To increase its water-solubility, tectorigenin was sulfonated with sulfuric acid (98wt.%) and mixed with saturated salt water to produce tectorigenin sodium sulfonate. Tectoridin and the two transfer products were identified by UV, IR, HPLC-MS, (1)H NMR and (13)C NMR, and the solubility of tectorigenin sodium sulfonate was increased about 9-fold than tectorigenin. Antioxidant experiments of tectoridin, tectorigenin and modified tectorigenin in vitro including reducing power, superoxide anion radical scavenging activity, hydroxyl radical scavenging activity, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity and anti-lipid peroxidation were carried on comparing with ascorbic acid (Vc) or butylated hydroxytoluene (BHT). The results suggested that the antioxidant activity in all the experimental systems exhibited the same order as follows: tectorigenin sodium sulfonate>tectorigenin>tectoridin. Due to the high water-solubility and good antioxidant properties with tectorigenin sodium sulfonate, appropriate chemical modifications could greatly improve the biological activities of the naturally occurring products.