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.

Design, synthesis, and evaluation of thiazolecarboxamide derivatives as stimulator of interferon gene inhibitors.

Stimulator of interferon gene (STING) plays critical roles in the cytoplasmic DNA-sensing pathway and in the induction of inflammatory response. Aberrant cytoplasmic DNA accumulation and STING activation are implicated in numerous inflammatory and autoimmune diseases. Here, we reported the discovery of a series of thiazolecarboxamide-based STING inhibitors through a molecular planarity/symmetry disruption strategy. The privileged compound 15b significantly inhibited STING signaling and suppressed immune-inflammatory cytokine levels in both human and murine cells. In vivo experiments demonstrated 15b effectively ameliorated immune-inflammatory cytokines upregulation in MSA-2-stimulated and Trex1-D18N mice. Furthermore, compound 15b exhibited enhanced efficacy in suppressing interferon-stimulated gene 15 (ISG15), a critical positive feedback regulator of STING. Overall, compound 15b deserves further development for the treatment of STING-associated inflammatory and autoimmune diseases.

Discovery and optimization of 3-(indolin-5-yloxy)pyridin-2-amine derivatives as potent necroptosis inhibitors.

Necroptosis is a form of regulated necrotic cell death and has been confirmed to play pivotal roles in the pathogenesis of multiple autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. The development of necroptosis inhibitors may offer a promising therapeutic strategy for the treatment of these autoimmune diseases. Herein, starting from the in-house hit compound 1, we systematically performed structural optimization to discover potent necroptosis inhibitors with good pharmacokinetic profiles. The resulting compound 33 was a potent necroptosis inhibitor for both human I2.1 cells (IC50 < 0.2 nM) and murine Hepa1-6 cells (IC50 < 5 nM). Further target identification revealed that compound 33 was an inhibitor of receptor interacting protein kinase 1 (RIPK1) with favorable selectivity. In addition, compound 33 also exhibited favorable pharmacokinetic profiles (T1/2 = 1.32 h, AUC = 1157 ng·h/mL) in Sprague-Dawley rats. Molecular docking and molecular dynamics simulations confirmed that compound 33 could bind to RIPK1 with high affinity. In silico ADMET analysis demonstrated that compound 33 possesses good drug-likeness profiles. Collectively, compound 33 is a promising candidate for antinecroptotic drug discovery.

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.

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 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.

DW14383 is an irreversible pan-FGFR inhibitor that suppresses FGFR-dependent tumor growth in vitro and in vivo.

Fibroblast growth factor receptor (FGFR) is a promising anticancer target. Currently, most FGFR inhibitors lack sufficient selectivity and have nonnegligible activity against kinase insert domain receptor (KDR), limiting their feasibility due to the serious side effects. Notably, compensatory activation occurs among FGFR1-4, suggesting the urgent need to develop selective pan-FGFR1-4 inhibitors. Here, we explored the antitumor activity of DW14383, a novel irreversible FGFR1-4 inhibitor. DW14383 exhibited equivalently high potent inhibition against FGFR1, 2, 3 and 4, with IC50 values of less than 0.3, 1.1, less than 0.3, and 0.5 nmol/L, respectively. It is a selective FGFR inhibitor, exhibiting more than 1100-fold selectivity for FGFR1 over recombinant KDR, making it one of the most selective FGFR inhibitors over KDR described to date. Furthermore, DW14383 significantly inhibited cellular FGFR1-4 signaling, inducing G1/S cell cycle arrest, which in turn antagonized FGFR-dependent tumor cell proliferation. In contrast, DW14383 had no obvious antiproliferative effect against cancer cell lines without FGFR aberration, further confirming its selectivity against FGFR. In representative FGFR-dependent xenograft models, DW14383 oral administration substantially suppressed tumor growth by simultaneously inhibiting tumor proliferation and angiogenesis via inhibiting FGFR signaling. In summary, DW14383 is a promising selective irreversible pan-FGFR inhibitor with pan-tumor spectrum potential in FGFR1-4 aberrant cancers, which has the potential to overcome compensatory activation among FGFR1-4.

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.

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.

Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity.

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC50 values of 0.72 and 2.6 µM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

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.

Design, synthesis and biological evaluation of pyrazolylaminoquinazoline derivatives as highly potent pan-fibroblast growth factor receptor inhibitors.

Fibroblast growth factor receptors (FGFRs) are important oncology targets due to the dysregulation of this signaling pathway in a wide variety of human cancers. We identified a series of pyrazolylaminoquinazoline derivatives as potent FGFR inhibitors with low nanomolar potency. The representative compound 29 strongly inhibited FGFR1-3 kinase activity and suppressed FGFR signaling transduction in FGFR-addicted cancer cells; FGFRs-driven cell proliferation was also strongly inhibited regardless of mechanistic complexity implicated in FGFR activation, which further confirmed that 29 was a potent pan-FGFR inhibitor. The flexibility of our structure offered the potential to preserve good affinity for mutant FGFR, which is important for developing TKIs with long-term efficacy.

DW10075, a novel selective and small-molecule inhibitor of VEGFR, exhibits antitumor activities both in vitro and in vivo.

AIM: Targeting the VEGF/VEGF receptor (VEGFR) pathway has proved to be an effective antiangiogenic approach for cancer treatment. Here, we identified 6-((2-((3-acetamidophenyl)amino)pyrimidin-4-yl)oxy)-N-phenyl-1-naphthamide (designated herein as DW10075) as a novel and highly selective inhibitor of VEGFRs. METHODS: In vitro tyrosine kinase activity was measured using ELISA, and intracellular signaling pathway proteins were detected by Western blot analysis. Endothelial cell proliferation was examined with CCK-8 assays, and tumor cell proliferation was determined with SRB assays. Cell migration, tube formation and rat aortic ring assays were used to detect antiangiogenic activity. Antitumor efficacy was further evaluated in U87-MG human glioblastoma xenograft tumors in nude mice receiving DW10075 (500 mg · kg(-1) · d(-1), po) for two weeks. RESULTS: Among a panel of 21 kinases tested, DW10075 selectively inhibited VEGFR-1, VEGFR-2 and VEGFR-3 (the IC50 values were 6.4, 0.69 and 5.5 nmol/L, respectively), but did not affect 18 other kinases including FGFR and PDGFR at 10 µmol/L. DW10075 significantly blocked VEGF-induced activation of VEGFR and its downstream signaling transduction in primary human umbilical vein endothelial cells (HUVECs), thus inhibited VEGF-induced HUVEC proliferation. DW10075 (1-100 nmol/L) dose-dependently inhibited VEGF-induced HUVEC migration and tube formation and suppressed angiogenesis in both the rat aortic ring model and the chicken chorioallantoic membrane model. Furthermore, DW10075 exhibited anti-proliferative activity against 22 different human cancer cell lines with IC50 values ranging from 2.2 µmol/L (for U87-MG human glioblastoma cells) to 22.2 µmol/L (for A375 melanoma cells). In U87-MG xenograft tumors in nude mice, oral administration of DW10075 significantly suppressed tumor growth, and reduced the expression of CD31 and Ki67 in the tumor tissues. CONCLUSION: DW10075 is a potent and highly selective inhibitor of VEGFR that deserves further development.

DW09849, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, prevents PI3K signaling and preferentially inhibits proliferation of cells containing the oncogenic mutation p110α (H1047R).

Phosphatidylinositol 3-kinase, α isoform (PI3Kα) plays essential roles in cell metabolism, growth, and proliferation and has been validated as a promising anticancer target. In an effort to search for new PI3Kα-selective inhibitors, DW series compounds were designed and synthesized aiming to reduce the off-target effects of their parent compound PIK-75 [2-methyl-5-nitro-1-benzenesulfonic acid 2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide], which was reported to selectively target PI3Kα. A series of compounds named DW series potently inhibited the kinase activity of PI3Kα with little activity against PI3K-related protein kinases and a panel of 15 tyrosine kinases. Similar to PIK-75, DW series compounds were more potent to inhibit PI3Kα among four class I PI3K isoforms, whereas a representative compound DW09849 [(E)-N'-((6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N-ethyl-2-methyl-5-nitrobenzohydrazide] displayed distinct binding mode compared with PIK-75. Although DW series compounds inhibited proliferation of rhabdomyosarcoma RH30 cells at elevated 50% inhibitory concentrations (IC50) in comparison with PIK-75, they were more selective than PIK-75 to inhibit PI3K signaling in the cellular context. In particular, DW09849 significantly and persistently blocked PI3K/protein kinase B signaling in RH30 cells, which consequently arrested RH30 cells in the G1 phase. Moreover, DW09849 selectively suppressed the proliferation and clonogenesis of transformed RK3E/HR cells harboring oncogenic mutation of p110α H1047R, as well as a panel of human breast cancer cells containing mutated PI3Kα, which is consistent with the finding that DW09849 demonstrated preference against H1047R mutated PI3Kα in molecular docking stimulation. These results suggest that DW series compounds, especially DW09849, selectively targeting PI3Kα with less off-target effects than PIK-75, provide new clues for the design and discovery of new specific PI3Kα inhibitors for cancer therapy.

Identification of DW532 as a novel anti-tumor agent targeting both kinases and tubulin.

AIM: 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities. METHODS: In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy. RESULTS: DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC50 values were 4.9 and 5.5 µmol/L, respectively), and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10 µmol/L) induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo. CONCLUSION: DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.

Discovery of hematopoietic progenitor kinase 1 inhibitors using machine learning-based screening and free energy perturbation.

Hematopoietic progenitor kinase 1 (HPK1) is a key negative regulator of T-cell receptor (TCR) signaling and a promising target for cancer immunotherapy. The development of novel HPK1 inhibitors is challenging yet promising. In this study, we used a combination of machine learning (ML)-based virtual screening and free energy perturbation (FEP) calculations to identify novel HPK1 inhibitors. ML-based screening yielded 10 potent HPK1 inhibitors (IC50 < 1 µM). The FEP-guided modification of the in-house false-positive hit, DW21302, revealed that a single key atom change could trigger activity cliffs. The resulting DW21302-A was a potent HPK1 inhibitor (IC50 = 2.1 nM) and potently inhibited cellular HPK1 signaling and enhanced T-cell function. Molecular dynamics (MD) simulations and ADME predictions confirmed DW21302-A as candidate compound. This study provides new strategies and chemical scaffolds for HPK1 inhibitor development.Communicated by Ramaswamy H. Sarma.

Design, synthesis, and structure-activity relationship studies of 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazole derivatives as necroptosis inhibitors.

The development of necroptosis inhibitors has emerged as a promising strategy to effectively mitigate necroptosis-related inflammatory diseases, neurodegenerative diseases, and cancers. In this paper, we reported a series of 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazole derivatives as potent necroptosis inhibitors. The representative compound 26 displayed potent anti-necroptotic activity in both human and mouse cellular assays and exhibited potent inhibitory activity against receptor-interacting protein kinase 1 (RIPK1). In vivo pharmacokinetic studies were performed to determine the oral exposure of compound 26. Finally, molecular docking elucidated that compound 26 could effectively bind to the allosteric pocket of RIPK1 and serve as a type III inhibitor. Taken together, our findings highlighted that compound 26 represented a promising lead compound for future necroptosis inhibitor development.

Discovery of a 1,6-naphthyridin-4-one-based AXL inhibitor with improved pharmacokinetics and enhanced in vivo antitumor efficacy.

The receptor tyrosine kinase AXL has emerged as an attractive target in anticancer drug discovery. Herein, we described the discovery of a new series of 1,6-naphthyridin-4-one derivatives as potent AXL inhibitors. Starting from a low in vivo potency compound 9 which was previously reported by our group, we utilized structure-based drug design and scaffold hopping strategies to discover potent AXL inhibitors. The privileged compound 13c was a highly potent and orally bioavailable AXL inhibitor with an IC50 value of 3.2 ± 0.3 nM. Compound 13c exhibited significantly improved in vivo antitumor efficacy in AXL-driven tumor xenograft mice, causing tumor regression at well-tolerated dose, and demonstrated favorable pharmacokinetic properties (MRT = 16.5 h, AUC0-∞ = 59,815 ng h/mL) in Sprague-Dawley rats. These results suggest that 13c is a promising therapeutic candidate for AXL-targeting cancer treatment.

Mycophenolic acid derivative 118 improves outcome of skin grafts by suppressing IL-17 production.

AIM: To investigate the effects and underlying mechanisms of 118, a novel derivative of mycophenolic acid, in a murine allogeneic skin graft model. METHODS: Skin grafts were conducted by grafting BALB/c donor tail skin into C57BL/6 skin beds (allograft) or by grafting female C57BL/6 donor tail skin into female C57BL/6 skin beds (syngraft). The mice were treated with the derivative 118 (40 mg·kg(-1)·d(-1), po) for 13 d (3 d before and 10 d after transplantation). Skin grafts, splenocytes and graft-infiltrated lymphocytes were isolated and examined ex vivo. The effects of the derivative 118 on naive CD4(+) T cell differentiation were examined in vitro. RESULTS: Treatment with the derivative 118 dramatically increased the survival rate of murine allogeneic skin grafts. Flow cytometric analysis and H&E staining showed that the derivative significantly decreased inflammatory cell infiltration into the grafts. The levels of the chemokines CXCL1, CXCL2, CCL7, and CCL2 were reduced in the derivative 118-treated grafts. Additionally, the derivative 118 significantly suppressed the IL-17 levels in the grafts but did not affect the differentiation of systemic helper T cells in the murine allogeneic skin graft model. Furthermore, IL-23p19 expression was suppressed in the grafts from the derivative 118-treated group, which might be due to decreases in TLR4 and MyD88 expression. Finally, the derivative 118 did not exert direct influences on helper T cell differentiation in vitro. CONCLUSION: Treatment with the mycophenolic acid derivative 118 improves murine allogeneic skin grafts by decreasing IL-23 expression and suppressing local IL-17 secretion in the grafts, rather than directly inhibiting Th17 differentiation.

Design, synthesis and pharmacological evaluation of 2,3-dihydrobenzofuran IRAK4 inhibitors for the treatment of diffuse large B-cell lymphoma.

Interleukin-1 receptor associated kinase 4 (IRAK4) is a critical mediator of MYD88 L265P-induced NF-κB activation, indicating it is a promising therapeutic target for diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of 2,3-dihydrobenzofuran IRAK4 inhibitors through structure-based drug design. The representative compound 22 exhibited strong IRAK4 inhibitory potency (IRAK4 IC50 = 8.7 nM), favorable kinase selectivity and high antiproliferative activity against the MYD88 L265P DLBCL cell line (OCI-LY10 IC50 = 0.248 µM). Compound 22 also exhibited the ability to inhibit the activation of IRAK4 signaling pathway and induce apoptosis in MYD88 L265P DLBCL cell line. In combination with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, 22 showed enhanced apoptosis-inducing effect and antiproliferative potency. The most advanced compound 22 in this inhibitor series holds promise for further development into efficacious and selective IRAK4 inhibitors for the treatment of DLBCL.