Optimization of a series of novel, potent and selective Macrocyclic SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

Identification of a novel series of azabenzimidazole-derived inhibitors of spleen tyrosine kinase.

Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC50 = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI50 = 210 nM.

Novel potent and selective pyrazolylpyrimidine-based SYK inhibitors.

Hybridisation of amino-pyrimidine based SYK inhibitors (e.g. 1a) with previously reported diamine-based SYK inhibitors (e.g. TAK-659) led to the identification and optimisation of a novel pyrimidine-based series of potent and selective SYK inhibitors, where the original aminomethylene group was replaced by a 3,4-diaminotetrahydropyran group. The initial compound 5 achieved excellent SYK potency. However, it suffered from poor permeability and modest kinase selectivity. Further modifications of the 3,4-diaminotetrahydropyran group were identified and the interactions of those groups with Asp512 were characterised by protein X-ray crystallography. Further optimisation of this series saw mixed results where permeability and kinase selectivity were increased and oral bioavailability was achieved in the series, but at the expense of potent hERG inhibition.

Optimization of a series of potent, selective and orally bioavailable SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254).

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.

Correlation between serum prealbumin and prognosis of patients with hepatocellular carcinoma after hepatectomy.

OBJECTIVE: To determine whether serum prealbumin levels are associated with long-term survival after hepatectomy in patients with primary hepatocellular carcinoma(HCC). METHODS: A consecutive sample of 526 patients with HCC who underwent potentially curative hepatectomy from August 2007 to August 2010 was retrospectively analyzed. Patients were classified as having normal or reduced serum prealbumin based on cut-off values of 200 or 182 mg/L. RESULTS: Multivariate analysis identified the preoperative level of serum prealbumin as an independent prognostic factor of long-term survival (P < 0.05): Survival was significantly better for those with normal levels than for those with reduced levels, based on either cut-off value. Similar results were observed in subgroup analyses based on the degree of cirrhosis, level of ɑ-fetoprotein and Barcelona Clinic Liver Cancer stage. CONCLUSIONS: Preoperative level of serum prealbumin may be useful for predicting long-term survival in patients with HCC after hepatectomy.

Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain.

Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

Identification of azabenzimidazoles as potent JAK1 selective inhibitors.

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

Discovery of heterocyclic sulfonamides as sphingosine 1-phosphate receptor 1 (S1P1) antagonists.

We have discovered a novel class of heterocyclic sulfonamides that act as antagonists of the S1P1 receptor. While members of this series identified from a high-throughput screen showed promising levels of potency in a cell-based assay measuring the inhibition of receptor internalization, most compounds were excessively lipophilic and contained an oxidation-prone thioether moiety. As a result, such compounds suffered from poor physical properties and metabolic stability, limiting their utility as in vivo probes. By removing the thioether group and systematically developing an understanding of structure-activity relationships and the effects of lipophilicity on potency within this series, we have been able to identify potent compounds with vastly improved physical properties. A representative enantiopure triazole sulfonamide (33) has measurable bioavailability following a low (3mg/kg) oral dose in rat, highlighting an achievement of the early hit-to-lead efforts for this series.

Factors influencing recurrence and model development for recurrence of minimally invasive percutaneous transhepatic lithotripsy: a single-center retrospective study.

OBJECTIVE: To identify factors influencing recurrence after percutaneous transhepatic choledochoscopic lithotripsy (PTCSL) and to develop a predictive model. METHODS: We retrospectively analyzed clinical data from 354 patients with intrahepatic and extrahepatic bile duct stones treated with PTCSL at Qinzhou First People's Hospital between February 2018 and January 2020. Patients were followed for three years and categorized into non-recurrence and recurrence groups based on postoperative outcome. Univariate analysis identified possible predictors of stone recurrence. Data were split using the gradient boosting machine (GBM) algorithm, assigning 70% as the training set and 30% as the test set. The predictive performance of the GBM model was assessed using the receiver operating characteristic (ROC) curve and calibration curve, and compared with a logistic regression model. RESULTS: Six factors were identified as significant predictors of recurrence: age, diabetes, total bilirubin, biliary stricture, number of stones, and stone diameter. The GBM model, developed based on these factors, showed high predictive accuracy. The area under the ROC curve (AUC) was 0.763 (95% CI: 0.695-0.830) for the training set and 0.709 (95% CI: 0.596-0.822) for the test set. Optimal cutoff values were 0.286 and 0.264, with sensitivities of 62.30% and 66.70%, and specificities of 77.20% and 68.50%, respectively. Calibration curves indicated good agreement between predicted probabilities and observed recurrence rates in both sets. DeLong's test revealed no significant differences between the GBM and logistic regression models in predictive performance (training set: D = 0.003, P = 0.997 > 0.05; test set: D = 0.075, P = 0.940 > 0.05). CONCLUSION: Biliary stricture, stone diameter, diabetes, stone number, age, and total bilirubin significantly influence stone recurrence after PTCSL. The GBM model, based on these factors, demonstrates robust accuracy and discrimination. Both GBM and logistic regression models effectively predicted stone recurrence post-PTCSL.

Identification and Evaluation of Reversible Covalent Binders to Cys55 of Bfl-1 from a DNA-Encoded Chemical Library Screen.

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

Adjuvant immune checkpoint inhibitors associated with higher recurrence-free survival in postoperative hepatocellular carcinoma (PREVENT): a prospective, multicentric cohort study.

BACKGROUND: Adjuvant therapy may improve survival of patients with hepatocellular carcinoma (HCC) after curative resection. This study compared safety and efficacy outcomes between patients at high risk of recurrence who received different types of adjuvant therapy or no such therapy after hepatic resection for HCC. METHODS: Recurrence-free survival (RFS), overall survival, and adverse events were compared among patients who received adjuvant immune checkpoint inhibitors (ICIs) alone, ICIs with tyrosine kinase inhibitors (TKIs), or no adjuvant therapy between 13 March 2019 and 19 March 2022. This study was registered on ClinicalTrials.gov (NCT05221398). RESULTS: Of the 517 patients in final analysis, 432 (83.6%) received no adjuvant therapy, 53 (10.2%) received ICIs alone, and 32 (6.2%) received adjuvant ICIs and TKIs. During median follow-up of 34.0 months (IQR 27.8 to 41.6 months), RFS was significantly longer among patients who received either type of adjuvant therapy (25.2 months, 95%CI 16.4-34.0) than among those who received none (16.1 months, 95%CI 12.9-19.4), and this difference remained significant after propensity score matching (HR 0.52, 95%CI 0.35-0.76, P = 0.004). Overall survival was unaffected by either type of adjuvant therapy, while significant difference was observed between patients who received adjuvant therapy or not after propensity score matching (HR 0.31, 95%CI 0.17-0.59, P = 0.005). The rate of grade 3 or 4 adverse events was similar between the two types of adjuvant therapy. CONCLUSIONS: ICIs alone or with TKIs may improve RFS of patients at high risk of HCC recurrence after curative resection.

Discovery of the Potent and Selective Inhaled Janus Kinase 1 Inhibitor AZD4604 and Its Preclinical Characterization.

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

Discovery of novel hedgehog antagonists from cell-based screening: Isosteric modification of p38 bisamides as potent inhibitors of SMO.

Cell-based subset screening of compounds using a Gli transcription factor reporter cell assay and shh stimulated cell differentiation assay identified a series of bisamide compounds as hedgehog pathway inhibitors with good potency. Using a ligand-based optimization strategy, heteroaryl groups were utilized as conformationally restricted amide isosteres replacing one of the amides which significantly increased their potency against SMO and the hedgehog pathway while decreasing activity against p38α kinase. We report herein the identification of advanced lead compounds such as imidazole 11c and 11f encompassing good p38α selectivity, low nanomolar potency in both cell assays, excellent physiochemical properties and in vivo pharmacokinetics.

Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKε kinases.

The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.

Discovery of (2R)-N-[3-[2-[(3-Methoxy-1-methyl-pyrazol-4-yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide (AZD4205) as a Potent and Selective Janus Kinase 1 Inhibitor.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 2.

INTRODUCTION: Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 1.

INTRODUCTION: Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 1 covering A through to I. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.