Design, synthesis and biological evaluation of MNK-PROTACs.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) can regulate cellular mRNA translation by controlling the phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E), which plays an important role in tumor initiation, development, and metastasis. Although small-molecule MNK inhibitors have made significant breakthroughs in the treatment of various malignancies, their clinical application can be limited by drug resistance, target selectivity and other factors. The strategy of MNK-PROTACs which selectively degrades MNK kinases provides a new approach for developing small-molecule drugs for related diseases. In this study, DS33059, a small-molecule compound modified based on the ongoing clinical trials drug ETC-206, was chosen as the target protein ligand. A series of novel MNK-PROTACs were designed, synthesized and evaluated biological activity. Several compounds showed good inhibitory activities against MNK1/2. Besides, compounds exhibited moderate to excellent anti-proliferative activity in A549 and TMD-8 cells in vitro. In particular, compound II-5 significantly inhibited A549 (IC50 = 1.79 µM) and TMD-8 (IC50 = 1.07 µM) cells. The protein degradation assay showed that compound II-5 had good capability to degrade MNK1. The MNK-PROTACs strategy represents a new direction in treating tumors and deserves further exploration.

Design, synthesis and biological evaluation of KRASG12C-PROTACs.

Several small-molecule covalent inhibitors of KRASG12C have made breakthrough progress in the treatment of KRAS mutant cancer. However, the clinical application of KRASG12C small-molecule inhibitors may be limited by adaptive resistance. Emerging PROTAC strategy can achieve complementary advantages with small molecule inhibitors and improve anti-tumor efficacy. Based on AMG-510, a series of novel KRASG12C-PROTACs were designed and synthesized. The protein degradation assay showed that PROTACs I-1, II-1, III-2 and IV-1 had binding and degradation ability to KRASG12C. III-2 and IV-1 showed potent inhibitory effect on downstream p-ERK and were more potent than AMG-510. Mechanistic studies demonstrated that PROTACs exerted degradation effects through the ubiquitin-proteasome pathway. Using cell lines sensitive to KRASG12C, anti-proliferative activities of compounds were assessed. PROTACs tested showed overall anti-proliferative activities. Besides,the structure-activity relationships (SARs) of KRASG12C-PROTACs were summarized. These results supported the use of the PROTAC strategy to degrade oncogene KRASG12C and provided clues for structural optimization of KRASG12C-PROTACs.

Design, synthesis and biological evaluation of isoxazole-containing biphenyl derivatives as small-molecule inhibitors targeting the programmed cell death-1/ programmed cell death-ligand 1 immune checkpoint.

Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a number of unavoidable deficiencies such as poor pharmacokinetic properties and immunogenicity. Small-molecule PD-1/PD-L1 inhibitors offer the superiority of complementarity with monoclonal antibodies and represent an appealing alternative. A novel series of isoxazole-containing biphenyl compounds were designed, synthesized and evaluated as PD-1/PD-L1 inhibitors in this paper. The structure-activity relationship of the novel synthesized compounds indicated that the ring-closure strategy of introducing isoxazole could be employed and the 3-cyanobenzyl group was significant for the inhibitory activity against the PD-1/PD-L1 protein-protein interactions. Molecular docking studies were performed to help understand the binding mode of the small-molecule inhibitor with the PD-L1 dimer. In particular, compound II-12 was a promising anti-PD-1/PD-L1 inhibitor with the IC50 value of 23.0 nM, providing valuable information for future drug development.

Design, synthesis and biological evaluation of macrocyclic derivatives as TRK inhibitors.

Tropomyosin receptor kinases (TRKA, TRKB, TRKC) are transmembrane receptor tyrosine kinases, which are respectively encoded by NTRK1, NTRK2, and NTRK3 genes. Herein, we reported the design, synthesis and Structure-Activity Relationship (SAR) investigation of a series of macrocyclic derivatives as new TRK inhibitors. Among these compounds, compound 9e exhibited strong kinase inhibitory activity (TRKG595R IC50 = 13.1 nM) and significant antiproliferative activity in the Ba/F3-LMNA-NTRK1 cell line (IC50 = 0.080 µM) and compound 9e has shown a better inhibitory effect (IC50 = 0.646 µM) than control drug LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cell line. These results indicate that compound 9e is a potential TRK inhibitor for further investigation.

Discovery of 3,5-dimethylisoxazole derivatives as novel, potent inhibitors for bromodomain and extraterminal domain (BET) family.

Bromodomain and extra-terminal (BET) is a promising therapeutic target for various hematologic cancers. We used the BRD4 inhibitor compound 13 as a lead compound to develop a variety of compounds, and we introduced diverse groups into the position of the compound 13 orienting toward the ZA channel. A series of compounds (14-23, 38-41, 43, 47-49) bearing triazolopyridazine motif exhibited remarkable BRD4 protein inhibitory activities. Among them, compound 39 inhibited BRD4(BD1) protein with an IC50 of 0.003 µM was superior to lead compound 13. Meanwhile, compound 39 possess activity, IC50 = 2.1 µM, in antiproliferation activity against U266 cancer cells. On the other hand, compound 39 could arrest tumor cells into the G0/G1 phase and induce apoptosis, which was consistent with its results in inhibiting cell proliferation. Biological and biochemical data suggest that BRD4 protein might be a therapeutic target and that compound 39 is an excellent lead compound for further development.

Design, synthesis and biological evaluation of imidazopyridazine derivatives containing isoquinoline group as potent MNK1/2 inhibitors.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are located at the meeting-point of ERK and p38 MAPK signaling pathways, which can phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at the conserved serine 209 exclusively. MNKs modulate the translation of mRNA involved in tumor-associated signaling pathways. Consequently, selective inhibitors of MNK1/2 could reduce the level of phosphorylated eIF4E. Series of imidazopyrazines, imidazopyridazines and imidazopyridines derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against diffuse large B-cell lymphoma (DLBCL) cell lines. In particular, compound II-5 (MNK1 IC50 = 2.3 nM; MNK2 IC50 = 3.4 nM) exhibited excellent enzymatic inhibitory potency and proved to be the most potent compound against TMD-8 and DOHH-2 cell lines with IC50 value of 0.3896 µM and 0.4092 µM respectively. These results demonstrated that compound II-5 could be considered as a potential MNK1/2 inhibitor for further investigation.

Discovery of 1,3,4-oxadiazole derivatives as potential antitumor agents inhibiting the programmed cell death-1/programmed cell death-ligand 1 interaction.

Inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction by small-molecule inhibitors is emerging cancer immunotherapy. A series of novel 1,3,4-oxadiazole derivatives were designed, synthesized, and evaluated for their activities in vitro and vivo to find potent inhibitors of the PD-1/PD-L1 interaction. Among them, compoundⅡ-14exhibited outstanding biochemical activity, with an IC50of 0.0380 µM. Importantly, compound II-14, with a TGI value of 35.74 %, had more potent efficacy in a mouse tumor model compared to that in the control group. Surprisingly, when compound II-14 combined with 5-FU in a mouse tumor model having a TGI value of 64.59 %, which showed potential anti-tumor synergistic effects. Furthermore, immunohistochemistry analysis demonstrated thatcompound II-14 activated the immune microenvironment by promoting the infiltration of CD4+ T cells into tumor tissues. These results indicate that compound II-14 is a promising lead compound for further development of small-molecule PD-1/PD-L1 inhibitors for cancer therapy.

Design, synthesis and biological evaluation of imidazolopyridone derivatives as novel BRD4 inhibitors.

Bromodomain containing protein 4 (BRD4) has been demonstrated to play critical roles in cellular proliferation and cell cycle progression. In this study, using the BRD4 inhibitor Fragment 9 as a lead compound, a series of imidazolopyridone derivatives were designed and tested for their inhibitory activity against BRD4 protein in vitro. Among them, HB100-A7 showed excellent BRD4(1) inhibitory activities with an IC50 value of 0.035 µM in amplified luminescent proximity homogeneous assay (Alphascreen). The result of MTT assay showed that HB100-A7 could suppress the proliferation of pancreatic cancer cells. In addition, flow cytometry further illustrated that HB100-A7 treatment resulted in G0/G1 phase arrest and promoted apoptosis of BxPc3 cells. Furthermore, the in vivo study found that HB100-A7 displayed significant tumor growth inhibition in a pancreatic mouse tumor model (Panc-02). Moreover, IHC staining suggested that HB100-A7 induce cell apoptosis in pancreatic cancer tumor tissue. Together, this study revealed, for the first time, HB100-A7 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the BRD4 protein.

Discovery and structure - activity relationship exploration of pyrazolo[1,5-a]pyrimidine derivatives as potent FLT3-ITD inhibitors.

Internal tandem duplications of FLT3 (FLT3-ITD) occur in approximately 25% of all acute myeloid leukemia (AML) cases and confer a poor prognosis. Optimization of the screening hit 1 from our in-house compound library led to the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives as potent and selective FLT3-ITD inhibitors. Compounds 17 and 19 displayed potent FLT3-ITD activities both with IC50 values of 0.4 nM and excellent antiproliferative activities against AML cell lines. Especially, compounds 17 and 19 inhibited the quizartinib resistance- conferring mutations, FLT3D835Y, both with IC50 values of 0.3 nM. Moreover, western blot analysis indicated that compounds 17 and 19 potently inhibited the phosphorylation of FLT3 and attenuated downstream signaling in AML cells. These results indicated that pyrazolo[1,5-a]pyrimidine derivatives could be promising FLT3-ITD inhibitors for the treatment AML.

Insight into the dichotomous regulation of STING activation in immunotherapy.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway (cGAS-STING) is a hub linking innate immunity and adaptive immunity against pathogen infection by inducing the production of type I interferon (IFN-I). It also plays pivotal roles in modulating tumorigenesis by ensuring the antigen presentation, T cell priming, activation, and tumor regression. Given its antitumor immune properties, cGAS-STING has attracted intense focus and several STING agonists have entered into clinical trials. However, some problems still exist when activating STING for use in oncological indications. It is remarkable that multiple downstream cytokines such as TNF-α, IL-6 may lead to inflammatory disease and even tumor metastasis in practical trials. Besides, there is a synergistic effect when STING agonists are combined with other immunotherapies. In this review, we discussed the advanced understanding between STING and anti-tumor immunity, as well as a variety of promising clinical treatment strategies.

DW14006 as a direct AMPKα1 activator improves pathology of AD model mice by regulating microglial phagocytosis and neuroinflammation.

Alzheimer's disease (AD) is a progressively neurodegenerative disease with typical hallmarks of amyloid ß (Aß) plaque accumulation, neurofibrillary tangle (NFT) formation and neuronal death extension. In AD brain, activated microglia phagocytose Aß and neuronal debris, but also aggravate inflammation stress by releasing inflammatory factors and cytotoxins. Improving microglia on Aß catabolism and neuroinflammatory intervention is thus believed to be a promising therapeutic strategy for AD. AMP-activated protein kinase (AMPK) is highly expressed in microglia with AMPKα1 being tightly implicated in neuroinflammatory events. Since indirect AMPKα1 activators may cause side effects with undesired intracellular AMP/ATP ratio, we focused on direct AMPKα1 activator study by exploring its potential function in ameliorating AD-like pathology of AD model mice. Here, we reported that direct AMPKα1 activator DW14006 (2-(3-(7-chloro-6-(2'-hydroxy-[1,1'-biphenyl]-4-yl)-2-oxo-1,2-dihydroquinolin-3-yl)phenyl)acetic acid) effectively improved learning and memory impairments of APP/PS1 mice, and the underlying mechanisms have been intensively investigated. DW14006 reduced amyloid plaque deposition by promoting microglial o-Aß42 phagocytosis and ameliorated innate immune response by polarizing microglia to an anti-inflammatory phenotype. It selectively enhanced microglial phagocytosis of o-Aß42 by upgrading scavenger receptor CD36 through AMPKα1/PPARγ/CD36 signaling and suppressed inflammation by AMPKα1/IκB/NFκB signaling. Together, our work has detailed the crosstalk between AMPKα1 and microglia in AD model mice, and highlighted the potential of DW14006 in the treatment of AD.

Design, synthesis and biological activity of bicyclic carboxamide derivatives as TRK inhibitors.

'precision medicine' is characterized by the selection of targeted drugs based on genetic characteristics of tumor from patients, and no longer selected basis on the type of cancer tissue. Among them, clinical trials on neurotrophin receptor tyrosine kinase genes (NTRK) have proven that great anti-cancer effects can be achieved in different cancer patients. In this paper, a novel total of twenty compounds in two categories have been designed and synthesized. Results of Kinase activity tests showed that I-9 (TRKA IC50 = 1.3 nM, TRKAG595R IC50 = 6.1 nM), and I-10 (TRKA IC50 = 1.1 nM, TRKAG595R IC50 = 5.3 nM) have significant inhibitory activity, and results of cell viability tests showed that I-9 and I-10 can maintain a great inhibitory effect in the Ba/F3-LMNA-NTRK1 cell line(IC50 = 81.1 nM and 41.7 nM, respectively), and in Ba/F3-LMNA-NTRK1-G595R cell line, I-9 and I-10 have better cell activity (IC50 was 495.3 nM, 336.6 nM, respectively) compared with the positive control drug LOXO-101. These results indicate that I-9 and I-10 are potential TRK inhibitors that can overcome drug resistance for further investigation.

Discovery of a new class of PROTAC BRD4 degraders based on a dihydroquinazolinone derivative and lenalidomide/pomalidomide.

BRD4 has emerged as an attractive target for anticancer therapy. However, BRD4 inhibitors treatment leads to BRD4 protein accumulation, together with the reversible nature of inhibitors binding to BRD4, which may limit the efficacy of BRD4 inhibitors. To address these problems, a protein degradation strategy based on the proteolysis targeting chimera (PROTAC) technology has been developed to target BRD4 recently. Herein, we present our design, synthesis and biological evaluation of a new class of PROTAC BRD4 degraders, which were based on a potent dihydroquinazolinone-based BRD4 inhibitor compound 6 and lenalidomide/pomalidomide as ligand for E3 ligase cereblon. Gratifyingly, several compounds showed excellent inhibitory activity against BRD4, and high anti-proliferative potency against human monocyte lymphoma cell line THP-1. Especially, compound 21 (BRD4 BD1, IC50 = 41.8 nM) achieved a submicromolar IC50 value of 0.81 µM in inhibiting the growth of THP-1 cell line, and was 4 times more potent than compound 6. Moreover, the mechanism study established that 21 could effectively induce the degradation of BRD4 protein and suppression of c-Myc. All of these results suggested that 21 was an efficacious BRD4 degrader for further investigation.

Design, synthesis and biological evaluation of novel chroman derivatives as non-selective acetyl-CoA carboxylase inhibitors.

Acetyl-CoA carboxylases (ACCs) are the rate-limiting enzymes in the de no lipogenesis, which play an important role in the synthesis and oxidation of fatty acid. Recent research reveals that ACCs are tightly relevant to many kinds of metabolic diseases and cancers. In this study, we synthesized a series of chroman derivatives and evaluated their ACCs inhibitory activities, obtaining compound 4s with IC50 value of 98.06 nM and 29.43 nM of binding activities in ACC1 and ACC2, respectively. Compound 4s exhibited the most potent anti-proliferation activity against A549, H1975, HCT116 and H7901 cell lines (values of IC50: 0.578 µΜ, 1.005 µΜ, 0.680 µΜ and 1.406 µΜ, respectively). Docking studies were performed to explain the structure-activity relationships. These results indicate that compound 4s is a promising ACC1/2 inhibitor for the potent treatment of cancer.

Design, synthesis and biological evaluation of bicyclic carboxylic acid derivatives as IDO1 inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a vital role in tumor immune escape and has emerged as a promising target for cancer immunotherapy. In this study, a novel series of bicyclic carboxylic acid derivatives were designed, synthesized and evaluated for inhibitory activities against IDO1. Among these, compound 9c exhibited strong IDO1 inhibitory activity (HeLa cellular IC50 = 2.6 nM, THP-1 cellular IC50 = 11.2 nM). Further anti-tumor studies in vivo have shown that compound 9c has a great inhibitory effect on tumor growth in mice CT26 model as a single agent or in combination with 5-fluorouracil (inhibition rate was 53.9% and 92.7%, respectively). These results indicate that compound 9c is a effective IDO1 inhibitor for further investigation.

Development of small-molecule BRD4 degraders based on pyrrolopyridone derivative.

Bromodomain-containing protein 4 (BRD4) plays a crucial role in the epigenetic regulation of gene transcription and some BRD4 inhibitors have been advanced to clinical trials. Nevertheless, the clinical application of BRD4 inhibitors could be limited by drug resistance. As an alternative strategy, the emerging Proteolysis Targeting Chimeras (PROTACs) technology has the potential to overcome the drug resistance of traditional small-molecule drugs. Based on PROTACs approaches, several BRD4 degraders were developed and have been proved to degrade BRD4 protein and inhibit tumor growth. Herein, we present the design, synthesis, and biological evaluation of pyrrolopyridone derivative-based BRD4 degraders. Four synthesized compounds displayed comparative potence against BRD4 BD1 with IC50 at low nanomolar concentrations. Anti-proliferative activity of 32a against BxPC3 cell line (IC50 = 0.165 µM) was improved by about 7-fold as compared to the BRD4 inhibitor ABBV-075. Furthermore, degrader 32a potently induced the degradation of BRD4 and inhibited the expression of c-Myc in BxPC3 cell line in a time-dependent manner. The exploration of intracellular antitumor mechanism showed 32a induced cell cycle arrest and apoptosis effectively. All the results demonstrated that compound 32a could be considered as a potential BRD4 degrader for further investigation.

WZ66, a novel acetyl-CoA carboxylase inhibitor, alleviates nonalcoholic steatohepatitis (NASH) in mice.

The global prevalence of nonalcoholic steatohepatitis (NASH) increases incredibly. NASH ends up to advanced liver disease, which is highly threatening to human health. Currently, treatment of NASH is very limited. Acetyl-CoA carboxylases (ACC1/ACC2) are proved as effective drug targets for NASH. We aimed to develop novel ACC inhibitors and evaluate their therapeutic value for NASH prevention. ACC inhibitors were obtained through structure-based drug design, synthesized, screened from ACC enzymatic measurement platform and elucidated in cell culture-based assays and animal models. The lipidome and microbiome analysis were integrated to assess the effects of WZ66 on lipids profiles in liver and plasma as well as gut microbiota in the intestine. WZ66 was identified as a novel ACC1/2 inhibitor. It entered systemic circulation rapidly and could accumulate in liver. WZ66 alleviated NASH-related liver features including steatosis, Kupffer cells and hepatic stellate cells activation in diet-induced obese mice. The triglycerides (TGs) and other lipids including diglycerides (DGs), phosphatidylcholine (PC) and sphingomyelin (SM) were decreased in WZ66-treated mice as evidenced by lipidome analysis in livers. The lipids profiles in plasma were also altered with WZ66 treatment. Plasma TG were moderately increased, while the activation of SREBP1c was not detected. WZ66 also downregulated the abundance of Allobaculum, Mucispirillum and Prevotella genera as well as Mucispirillum schaedleri species in gut microbiota. WZ66 is an ideal lead compound and a potential drug candidate deserving further investigation in the therapeutics of NASH.

A near-infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells.

Using the characteristics of hydrogen peroxide that are able to cleave phenyl-boric acid selectively and efficiently, we here report a dicyanoisophorone-boric acid (DCP-BA)-based near-infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn-on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.

Design, synthesis and biological evaluation of imidazo[1,5-a]pyrazin-8(7H)-one derivatives as BRD9 inhibitors.

BRD9 is the subunit of mammalian SWI/SNF chromatin remodeling complex (BAF). SWI/SNF complex mutations were found in nearly 20% of human cancers. The biological role played by BRD9 bromodomain remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. In this paper, we synthesized a series of imidazo[1,5-a]pyrazin-8(7H)-one derivatives as potent BRD9 inhibitors and evaluated their BRD9 inhibitory activity in vitro and anti-proliferation effects against tumor cells. Gratifyingly, compound 27 and 29 exhibited robust potency of BRD9 inhibition with IC50 values of 35 and 103 nM respectively. Docking studies were performed to explain the structure-activity relationship. Furthermore, compound 27 potently inhibited cell proliferation in cell lines A549 and EOL-1 with an IC50 value of 6.12 µM and 1.76 µM respectively. The chemical probe, compound 27, was identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Design, synthesis and biological evaluation of pyridone-aminal derivatives as MNK1/2 inhibitors.

Excessive phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) plays a major role in the dysregulation of mRNA translation and the activation of tumor cell signaling. eIF4E is exclusively phosphorylated by mitogen-activated protein kinase interacting kinases 1 and 2 (MNK1/2) on Ser209. So, MNK1/2 inhibitors could decrease the level of p-eIF4E and regulate tumor-associated signaling pathways. A series of pyridone-aminal derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against hematologic cancer cell lines. In particular, compound 42i (MNK1 IC50 = 7.0 nM; MNK2 IC50 = 6.1 nM) proved to be the most potent compound against TMD-8 cell line with IC50 value of 0.91 µM. Furthermore, 42i could block the phosphorylation level of eIF4E in CT-26 cell line, and 42i inhibited the tumor growth of CT-26 allograft model significantly. These results indicated that compound 42i was a promising MNK1/2 inhibitor for the potent treatment of colon cancer.