Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

Preclinical and early clinical studies of a novel compound SYHA1813 that efficiently crosses the blood-brain barrier and exhibits potent activity against glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and is poorly controlled. Previous studies have shown that both macrophages and angiogenesis play significant roles in GBM progression, and co-targeting of CSF1R and VEGFR is likely to be an effective strategy for GBM treatment. Therefore, this study developed a novel and selective inhibitor of CSF1R and VEGFR, SYHA1813, possessing potent antitumor activity against GBM. SYHA1813 inhibited VEGFR and CSF1R kinase activities with high potency and selectivity and thus blocked the cell viability of HUVECs and macrophages and exhibited anti-angiogenetic effects both in vitro and in vivo. SYHA1813 also displayed potent in vivo antitumor activity against GBM in immune-competent and immune-deficient mouse models, including temozolomide (TMZ) insensitive tumors. Notably, SYHA1813 could penetrate the blood-brain barrier (BBB) and prolong the survival time of mice bearing intracranial GBM xenografts. Moreover, SYHA1813 treatment resulted in a synergistic antitumor efficacy in combination with the PD-1 antibody. As a clinical proof of concept, SYHA1813 achieved confirmed responses in patients with recurrent GBM in an ongoing first-in-human phase I trial. The data of this study support the rationale for an ongoing phase I clinical study (ChiCTR2100045380).

Design, synthesis, and STING-agonistic activity of benzo[b]thiophene-2-carboxamide derivatives.

STING is an important immune-associated protein that localizes in the endoplasmic reticulum membrane. Upon being activated by its agonists, STING triggers the IRF and NF-κB pathways, which generates type I interferons and proinflammatory cytokines, and ultimately primes the innate immune responses to achieve valid antitumor efficacy. We designed and synthesized a series of benzo[b]thiophene-2-carboxamide derivatives. Through STING-agonistic activity evaluation, compounds 12d and 12e exhibited marginal human STING-activating activities. Western blot analysis demonstrated that both 12d and 12e treatment increased the phosphorylation of the downstream signaling molecules (TBK1 and IRF3) of STING. The proposed binding mode of 12d/12e and STING protein displayed that two canonical hydrogen bonds, a π-π stacking interaction, as well as a π-cation interaction formed between the agonist and the CDN-binding domain of STING protein.

Discovery of 10H-Benzo[b]pyrido[2,3-e][1,4]oxazine AXL Inhibitors via Structure-Based Drug Design Targeting c-Met Kinase.

Receptor tyrosine kinase AXL exerts pivotal roles in cancer cell survival, metastasis, and drug resistance. Pharmacologic or genetic targeting of the aberrant AXL signaling has proven preferable antitumor efficacies in both preclinical and clinical studies, which highlights AXL as an attractive antitumor drug target. By conformational restriction of the anilinopyrimidine 10e and systematic structure-activity relationship (SAR) exploration, we discovered 10H-benzo[b]pyrido[2,3-e][1,4]oxazine 16j as a potent and orally bioavailable AXL inhibitor. As a type II AXL inhibitor, compound 16j displayed about 15-fold selectivity for AXL over its highly homologous kinase c-Met. And it significantly blocked cellular AXL signaling, inhibited AXL-mediated cell proliferation, and impaired growth arrest-specific protein 6 (Gas6)/AXL-stimulated cell migration and invasion. Moreover, 16j exhibited significant antitumor efficacy in AXL-driven xenograft model at a well-tolerant dosage, causing tumor stasis or regression.

Structural insights into a shared mechanism of human STING activation by a potent agonist and an autoimmune disease-associated mutation.

Stimulator of interferon gene (STING) is increasingly exploited for the potential in cancer immunotherapy, yet its mechanism of activation remains not fully understood. Herein, we designed a novel STING agonist, designated as HB3089 that exhibits robust and durable anti-tumor activity in tumor models across various cancer types. Cryo-EM analysis reveals that HB3089-bound human STING has structural changes similar to that of the STING mutant V147L, a constitutively activated mutant identified in patients with STING-associated vasculopathy with onset in infancy (SAVI). Both structures highlight the conformational changes of the transmembrane domain (TMD), but without the 180°-rotation of the ligand binding domain (LBD) previously shown to be required for STING activation. Further structure-based functional analysis confirmed a new STING activation mode shared by the agonist and the SAVI-related mutation, in which the connector linking the LBD and the TMD senses the activation signal and controls the conformational changes of the LBD and the TMD for STING activation. Together, our findings lead to a new working model for STING activation and open a new avenue for the rationale design of STING-targeted therapies either for cancer or autoimmune disorders.

Discovery of pyrrolo[2,3-d]pyrimidine derivatives as potent Axl inhibitors: Design, synthesis and biological evaluation.

Axl has emerged as an attractive target for cancer therapy due to its strong correlation with tumor growth, metastasis, poor survival, and drug resistance. Herein, we report the design, synthesis and structure-activity relationship (SAR) investigation of a series of pyrrolo[2,3-d]pyrimidine derivatives as new Axl inhibitors. Among them, the most promising compound 13b showed high enzymatic and cellular Axl potencies. Furthermore, 13b possessed preferable pharmacokinetic properties and displayed promising therapeutic effect in BaF3/TEL-Axl xenograft tumor model. Compound 13b may serve as a lead compound for new antitumor drug discovery.

Synthesis of triazolotriazine derivatives as c-Met inhibitors.

Receptor tyrosine kinase c-Met is an important antitumor drug target. Triazolotriazine analogues 2-10 were prepared efficiently and evaluated the enzymatic and cellular c-Met activities. Brief structure-activity relationships of triazolotriazine core and CF2-quinoline part were investigated, leading to the discovery of compound 8 with nanomolar enzymatic c-Met activity, and subnanomolar MKN45 and EBC-1 cellular potencies. The proposed binding model of 8 and c-Met unraveled that two canonical hydrogen bonds and a π-π stacking interaction formed between the inhibitor and the ATP binding site of c-Met kinase domain, which accounted for its potent c-Met activities.

Evaluation of Deuterium-Labeled JNJ38877605: Pharmacokinetic, Metabolic, and in Vivo Antitumor Profiles.

c-Met inhibitor JNJ38877605 has proven curative as an antitumor agent, while its clinical study was terminated due to renal toxicity. It was reported that the renal toxicity was caused by the poor solubility of its aldehyde oxidase (AO) metabolites. Therefore, blocking AO oxidation of JNJ38877605 might diminish the toxic metabolites and overcome the renal toxicity. Compound 3, the AO metabolic site deuterated JNJ38877605, was then synthesized as the target molecule. In vitro monkey liver S9 fraction incubation of 3 manifested that the renal toxic metabolite M2-2 was significantly reduced, which connoted that this deuteration has partly blocked AO oxidation. After po. nasal gavage to cynomolgus monkeys, compound 3 revealed decreased AO metabolites M2-2 and M3-2 in the plasma as well as 1.88-fold AUC and 1.56-fold Cmax compared with JNJ38877605, indicating that deuterium replacement significantly blocked AO metabolism in vivo. Besides, metabolic profiles of 3 were investigated by analysis of the plasma and the urine of the po. administrated cynomolgus monkeys. Moreover, after oral administration to the EBC-1 tumor-bearing nude mice, compound 3 exhibited a better antitumor efficacy than JNJ38877605. In conclusion, deuteration on the AO metabolic site of JNJ38877605 improved its AO metabolism, oral exposure, as well as in vivo antitumor efficacy.

Simm530, a novel and highly selective c-Met inhibitor, blocks c-Met-stimulated signaling and neoplastic activities.

The aberrant c-Met activation has been implicated in a variety of human cancers for its critical role in tumor growth, metastasis and tumor angiogenesis. Thus, c-Met axis presents as an attractive therapeutic target. Notably, most of these c-Met inhibitors currently being evaluated in clinical trials lack selectivity and target multiple kinases, often accounting for the undesirable toxicities. Here we described Simm530 as a potent and selective c-Met inhibitor. Simm530 demonstrated >2,000 fold selectivity for c-Met compared with other 282 kinases, making it one of the most selective c-Met inhibitors described to date. This inhibitor significantly blocked c-Met signaling pathways regardless of mechanistic complexity implicated in c-Met activation. As a result, Simm530 led to substantial inhibition of c-Met-promoted cell proliferation, migration, invasion, ECM degradation, cell scattering and invasive growth. In addition, Simm530 inhibited primary human umbilical vascular endothelial cell (HUVEC) proliferation, decreased intratumoral CD31 expression and plasma pro-angiogenic factor interleukin-8 secretion, suggesting its significant anti-angiogenic properties. Simm530 resulted in dose-dependent inhibition of c-Met phosphorylation and tumor growth in c-Met-driven lung and gastric cancer xenografts. And, the inhibitor is well tolerated even at doses that achieve complete tumor regression. Together, Simm530 is a potent and highly selective c-Met kinase inhibitor that may have promising therapeutic potential in c-Met-driven cancer treatment.

Discovery of 6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-yl)methyl)quinoline as a highly potent and selective c-Met inhibitor.

c-Met/HGF overexpression has been detected in many human malignancies including tumors which are resistant to anticancer therapy. Disrupting the aberrant c-Met/HGF axis has enjoyed significant progress in both preclinical and clinical antitumor campaign. To eliminate the OCH2-related metabolic deficiency of our previously reported triazolotriazine 2, we synthesized a series of CH2-/CF2-linked triazolotriazines and assessed their c-Met activities, leading to the highly potent compound 23 with IC50 values of 0.24 nM of enzymatic activity in c-Met and 0.85 nM of cellular activity in EBC-1 cancer cell line, as well as with complete tumor regression in EBC-1 xenograft mice model at dose of 25 mg/kg via oral administration. Based on its potent anti-proliferative activities and favorable pharmacokinetic properties, 23 has been selected as a drug candidate for preclinical investigation.

Discovery of Anilinopyrimidines as Dual Inhibitors of c-Met and VEGFR-2: Synthesis, SAR, and Cellular Activity.

Both c-Met and VEGFR-2 are important targets for cancer therapies. Here we report a series of potent dual c-Met and VEGFR-2 inhibitors bearing an anilinopyrimidine scaffold. Two novel synthetic protocols were employed for rapid analoguing of the designed molecules for structure-activity relationship (SAR) exploration. Some analogues displayed nanomolar potency against c-Met and VEGFR-2 at enzymatic level. Privileged compounds 3a, 3b, 3g, 3h, and 18a exhibited potent antiproliferative effect against c-Met addictive cell lines with IC50 values ranged from 0.33 to 1.7 µM. In addition, a cocrystal structure of c-Met in complex with 3h has been determined, which reveals the binding mode of c-Met to its inhibitor and helps to interpret the SAR of the analogues.

O-linked triazolotriazines: potent and selective c-Met inhibitors.

The HGF/c-Met signaling pathway mediates a variety of important biological activities, but dysregulation of the pathway is also closely associated with poor prognosis in a wide range of human cancers. c-Met is considered to be among the most promising therapeutic targets for anticancer drug discovery. Herein we report the discovery of a series of O-linked triazolotriazines that show sub-nanomolar inhibition of c-Met activity. Among these new compounds, 6 a exhibits high c-Met inhibitory potency in both enzymatic and cellular assays with great selectivity.