Structure-guided discovery of novel potent and efficacious proteolysis targeting chimera (PROTAC) degrader of BRD4.

Bromodomain-containing protein 4 (BRD4) has been identified as a potential target in the treatment of many cancers and several BRD4 inhibitors have entered clinical studies. Previous studies have shown that BRD4 degraders have potential to overcome resistance to BRD4 inhibitors. However, most of the BRD4 degraders have poor solubility and bioavailability, one of which the reason is large molecular weight. Here, we describe the design, synthesis, and evaluation studies of a BRD4 degrader based on the proteolysis targeting chimeras (PROTAC) concept. Our efforts have led to the discovery of compound 15, which is a weak inhibitor and potent BRD4 degrader with a molecular weight of 821.8. In vitro, 15 can completely degrade BRD4 at nanomolar concentration, with DC50 = 0.25 and 3.15 nM in MV4-11 and RS4-11 cell lines, respectively. Further optimization of compound 15 may reduce its molecular weight and improve druggabillity, and provide a new choice for the treatment of cancer.

Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone derivatives as BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family, has been recognized as an attractive candidate target for the treatment targeting gene transcription in several types of cancers. In this study, two types of novel compounds were designed, synthesized and evaluated as BRD4 inhibitors. Therein, pyridone derivatives were more effective against BRD4 protein and human leukemia cell lines MV4-11. Among them, compounds 11d, 11e and 11f were the most potential ones with IC50 values of 0.55 µM, 0.86 µM and 0.80 µM against BRD4, and exhibited remarkable antiproliferative activities against MV4-11 cells with IC50 values of 0.19 µM, 0.32 µM and 0.12 µM, respectively. Moreover, in western blot assay, compound 11e induced down-regulation of C-Myc, which is a significant downstream gene of BRD4. Cell cycle analysis assay also showed that compound 11e could block MV4-11 cells at G0/G1 phase. Taken together, our results suggested that compound 11e and its derivatives were a class of novel structural potential BRD4 inhibitors and could serve as lead compounds for further exploration.

Metabolism of SKLB-TB1001, a Potent Antituberculosis Agent, in Animals.

Tuberculosis is a major global health problem, and the emergence of multidrug-resistant and extensively drug-resistant strains has increased the difficulty of treating this disease. Among the novel antituberculosis drugs in the pipeline, decaprenylphosphoryl-beta-d-ribose-2-epimerase (DprE1) inhibitors such as BTZ043 and pBTZ169 exhibited extraordinary antituberculosis potency. Here, the metabolites of the new DprE1 inhibitor SKLB-TB1001 in vivo and its inhibition of cytochrome P450 isoforms and plasma protein binding (PPB) in vitro were studied. The results showed that rapid transformation and high PPB resulted in inadequate exposure in vivo and thus led to the moderate potency of SKLB-TB1001 in vivo This study provided explanations for the discrepant potency of this scaffold in vivo and in vitro Meanwhile, it also provides a rationale for lead optimization of this very promising scaffold of antituberculosis agents to prevent them from being metabolized, thus improving their exposure in vivo.

Identification of novel 2-aminothiazole conjugated nitrofuran as antitubercular and antibacterial agents.

The emergence of antibiotic resistant pathogens is an ongoing main problem in the therapy of bacterial infections. In order to develop promising antitubercular and antibacterial lead compounds, we designed and synthesized a new series of derivatives of 2-aminothiazole conjugated nitrofuran with activities against both Mycobacterium tuberculosis and Staphylococcus aureus. Eight compounds 12e, 12k, 12l, 12m, 18a, 18d, 18e, and 18j emerged as promising antitubercular agents. Structure-activity relationships (SARs) were discussed and showed that the derivatives substituted at the position-3 of benzene of 5-nitro-N-(4-phenylthiazol-2-yl)furan-2-carboxamide exhibited superior potency. The most potent compound 18e, substituted with benzamide at this position, displayed minimum inhibitory concentrations (MICs) of 0.27µg/mL against Mtb H37Ra and 1.36µg/mL against S. aureus. Furthermore, compound 18e had no obvious cytotoxicity to normal Vero cells (IC50=50.2µM). The results suggest that the novel scaffolds of aminothiazole conjugated nitrofuran would be a promising class of potent antitubercular and antimicrobial agents.

Design and synthesis of the first PARP-1 and proteasome dual inhibitors to treat breast cancer.

PARP-1 is a crucial factor in repairing DNA single strand damage and maintaining genomic stability. However, the use of PARP-1 inhibitors is limited to combination with chemotherapy or radiotherapy, or as a single agent for indications carrying HRR defects. The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. Proteasome inhibitors can cooperate with PARP-1 inhibitors to inhibit DNA homologous recombination repair function. In this study, we designed and synthesized the first dual PARP-1 and proteasome inhibitor based on Olaparib and Ixazomib. Both compounds 42d and 42i exhibited excellent proliferation inhibition and dual-target synergistic effects on cells that were insensitive to PARP-1 inhibitors. Further mechanistic evaluations revealed that 42d and 42i could inhibit homologous recombination repair function by down-regulating the expression of BRCA1 and RAD51. Additionally, 42i induced more significant apoptosis and showed better inhibitory effect on cell proliferation in clonal formation experiments in breast cancer cells than 42d. In summary, our study presented a new class of dual PARP-1/proteasome inhibitors with significant synergistic effects for the treatment of breast cancer.

Synthesis and biological evaluation of 1,2,4-triazole and 1,3,4-thiadiazole derivatives as potential cytotoxic agents.

A series of new 3-amino-5-sulfanyl-1,2,4-triazole and 2-amino-5-sulfanyl-1,3,4-thiadiazole derivatives have been synthesized and their cytotoxicities were evaluated on a panel of human cancer cell lines (BxPC-3, H1975, SKOV-3, A875, HCT116, etc.). The best one (compound 5m) exhibited activities with IC50 values ranging from 0.04 to 23.6 µM against nine human cancer cell lines. Further biological evaluation indicated that DNA replication was blocked by treatment with compound 5m in HCT116 cells.

(E)-3-Dimethyl-amino-1-(1,3-thia-zol-2-yl)prop-2-en-1-one.

In the title compound, C8H10N2OS, the 3-(dimethyl-amino)-prop-2-en-1-one unit is approximately planar [give r.m.s. deviation] and the mean plane through the seven non-H atoms makes a dihedral angle of 8.88 (3)° with the thia-zole ring. The carbonyl and ring C=N double bonds adjacent to the carbonyl group are trans [N-C-C-O = 172.31 (15) °], while the conformation of the carbonyl and propene double bonds is cis [O-C-C-C = 2.2 (2)°]. In the crystal, short C-H⋯N and C-H⋯O inter-actions together with C-H⋯π inter-actions generate a three-dimensional network.

Application and Study of ROCK Inhibitors in Pulmonary Fibrosis: Recent Developments and Future Perspectives.

Rho-associated coiled-coil-containing kinases (ROCKs), serine/threonine protein kinases, were initially identified as downstream targets of the small GTP-binding protein Rho. Pulmonary fibrosis (PF) is a lethal disease with limited therapeutic options and a particularly poor prognosis. Interestingly, ROCK activation has been demonstrated in PF patients and in animal PF models, making it a promising target for PF treatment. Many ROCK inhibitors have been discovered, and four of these have been approved for clinical use; however, no ROCK inhibitors are approved for the treatment of PF patients. In this article, we describe ROCK signaling pathways and the structure-activity relationship, potency, selectivity, binding modes, pharmacokinetics (PKs), biological functions, and recently reported inhibitors of ROCKs in the context of PF. We will also focus our attention on the challenges to be addressed when targeting ROCKs and discuss the strategy of ROCK inhibitor use in the treatment of PF.

Design, synthesis and biological evaluation of 7-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-2,3-dihydro-1H-inden-1-one derivatives as potent FAK inhibitors for the treatment of ovarian cancer.

Focal adhesion kinase (FAK) promotes tumor progression by intracellular signal transduction and regulation of gene expression and protein turnover, which is a compelling therapeutic target for various cancer types, including ovarian cancer. However, the clinical responses of FAK inhibitors remain unsatisfactory. Here, we describe the discovery of FAK inhibitors using a scaffold hopping strategy. Structure-activity relationship (SAR) exploration identified 36 as a potent FAK inhibitor, which exhibited inhibitory activities against FAK signaling in vitro. Treatment with 36 not only decreased migration and invasion of PA-1 cells, but also reduced expression of MMP-2 and MMP-9. Moreover, 36 inhibited tumor growth and metastasis, and no obvious adverse effects were observed during the in vivo study. These results revealed the potential of FAK inhibitor 36 for treatment of ovarian cancer.

N-Methyl-4-(4-pivalamido-phenyl-sulfan-yl)picolinamide hemihydrate.

In the title compound, C(18)H(21)N(3)O(2)S·0.5H(2)O, the benzene ring makes dihedral angles of 88.59 (6) and 40.74 (8)° with the pyridine ring and the amide group, respectively. The water O atom lies on a twofold axis. In the crystal, the organic mol-ecules and the water mol-ecules are linked via O-H⋯O hydrogen bonds, while the organic mol-ecules are connected to each other via N-H⋯O hydrogen bonds, forming a three-dimensional network.

Matrix metalloproteinases inhibitors in idiopathic pulmonary fibrosis: Medicinal chemistry perspectives.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with limited therapeutic options and a particularly poor prognosis. Matrix metalloproteinases (MMPs), promising targets for the treatment of IPF, have been identified as playing a pivotal role in IPF. Although the pathological processes of MMPs and IPF have been verified, there are no MMP inhibitors for the treatment of IPF in the clinic. In this review, we will present the latest developments in MMP inhibitors, including pharmacophores, binding modes, selectivity and optimization strategies. In addition, we will also discuss the future development direction of MMP inhibitors based on emerging tools and techniques.

Design, synthesis, and biological evaluation of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as novel dual-PLK1/BRD4 inhibitors.

Protein kinase inhibitors and epigenetic regulatory molecules are two main kinds of anticancer drugs developed in recent years. Both kinds of drugs harbor their own advantages and disadvantages in the treatment of cancer, and the development of small molecules which could target at kinases and epigenetic targets simultaneously can avoid the defects of drugs which only targets at kinases or epigenetic proteins. In this study, a series of 4,5-dihydro-[1,2,4]triazolo [4,3-f]pteridine derivatives were designed and synthesized based on the structure of PLK1 inhibitor BI-2536. Subsequent targets affinity screen and antiproliferative activity test led to the discovery of the most potent dual PLK1/BRD4 inhibitor 9b with good potency for both PLK1 (IC50 = 22 nM) and BRD4 (IC50 = 109 nM) as well as favorable antiproliferative activity against a panel of cancer cell lines. 9b could induce cell cycle arrest and apoptosis in acute myeloid leukemia cell line MV 4-11 in a concentration dependent manner. It could also downregulate the transcription of several proliferation-related oncogenes, including c-MYC, MYCN and BCL-2. Finally, in a MV4-11 mouse xenograft model, 9b exhibited favorable in vivo antitumor activity with 66% tumor growth inhibition (TGI) at a dose of 60 mg/kg while without obvious toxicity. This study thus provided us a start point for the development of new dual PLK1/BRD4 inhibitors as anticancer agents.

Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-one derivatives as anti-tumor agents targeting TOPK.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a serine-threonine mitogen-activated protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of 1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK inhibitors. Some of them displayed very potent anti-cancer activity with IC50s less than 100 nM, superior than reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy compared to OTS964. Pharmacokinetic studies reveal its good oral bioavailability. Our findings therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic agent against colorectal cancer.

Synthesis and biological evaluation of (1,2,4)triazole[4,3-a]pyridine derivatives as potential therapeutic agents for concanavalin A-induced hepatitis.

A series of (1,2,4)triazole[4,3-a]pyridine (TZP) derivatives have been designed and synthesized. Compound 8d was identified as having the most potent inhibitory activity on NO release in response to lipopolysaccharide (LPS) stimulation and inhibition of the migration induced by MCP-1 protein on RAW264.7 macrophages. Based on the screening data, an immunofluorescence assay and a real-time qPCR assay were conducted, indicating that compound 8d suppressed NF-κB p65 translocation and expression of inflammatory genes by concanavalin A (Con A)-induced RAW264.7 macrophages. More importantly, 8d also exhibited potent efficacy, alleviating Con A-induced hepatitis by downregulating the levels of plasma alanine transaminase (ALT), aspartate transaminase (AST) and inflammatory infiltration in a mouse autoimmune hepatitis (AIH) model. In addition, the flow cytometry (FCM) data showed that compound 8d inhibited the accumulation of MDSCs in the liver of Con A-induced mice. These findings raise the possibility that compound 8d might serve as a potential agent for the treatment of AIH.

Palladium-catalyzed intramolecular C-H arylation of 2-halo-N-Boc-N-arylbenzamides for the synthesis of N-H phenanthridinones.

A palladium catalyzed synthesis of N-H phenanthridinones was developed via C-H arylation. The protocol gives phenanthridinones regioselectively by one-pot reaction without deprotection. It exhibits broad substrate scope and affords targets in up to 95% yields. Importantly, it could be applied for the less reactive o-chlorobenzamides.

Identification of a new series of benzothiazinone derivatives with excellent antitubercular activity and improved pharmacokinetic profiles.

Nitrobenzothiazinone (BTZ) is a promising scaffold with potent activity against M. tuberculosis by inhibiting decaprenylphosphoryl-beta-d-ribose 2'-oxidase (DprE1). But unfavorable durability poses a challenge to further development of this class of agents. Herein, a series of BTZs bearing a variety of different substituents at the C-2 position were designed and synthesized. Compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37Ra and were profiled for metabolic stability, plasma protein-binding capacity and pharmacokinetics in vivo. In general, these new BTZs containing N-piperazine, N-piperidine or N-piperidone moiety have excellent antitubercular activity and low cytotoxicity. Several of the compounds showed improved microsomal stability and lower plasma protein-binding, opening a new direction for further lead optimization. And we obtained compound 3o, which maintained good anti-tuberculosis activity (MIC = 8 nM) and presented better in vitro ADME/T and in vivo pharmacokinetic profiles than reported BTZ compound PBTZ169, which may serve as a candidate for the treatment of tuberculosis.

A novel benzothiazinethione analogue SKLB-TB1001 displays potent antimycobacterial activities in a series of murine models.

New chemotherapeutic compounds and regimens are needed to combat multidrug-resistant Mycobacterium tuberculosis. Here, we used a series of murine models to assess an antitubercular lead compound SKLB-TB1001. In the Mycobacterium bovis bacillus Calmette-Guérin and the acute M. tuberculosis H37Rv infection mouse models, SKLB-TB1001 significantly attenuated the mycobacterial load in lungs and spleens. The colony forming unit counts and histological examination of lungs from H37Rv infected mice revealed that the benzothiazinethione analogue SKLB-TB1001 as a higher dose level was as effective as isoniazid. Moreover, in a multidrug-resistant (MDR)-TB mouse model, SKLB-TB1001 showed significant activity in a dose-dependent manner and was more effective than streptomycin. These results suggested that SKLB-TB1001 could be an antitubercular drug candidate worth further investigation.

A novel small-molecule YLT256 inhibits proliferation and induces apoptosis both in vitro and in vivo in solid tumors.

Pancreatic carcinoma is a still unsolved health problem all over the world with poor prognosis and high mortality rate. YLT256, a novel synthesized chemical small inhibitor, displays potent antineoplastic activities via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT256 showed growth inhibition against a broad spectrum of human cancer cell lines and pancreatic cancer cell line BxPc-3 was the most sensitive with an IC50 of 0.42µM. We also found YLT256 could induce apoptosis of BxPc-3 cells in a dose-dependent manner. Western blot analysis revealed that the occurrence of its apoptosis was associated with activation of caspases-3 and -9, up-regulation of pro-apoptotic Bak, and down-regulation of anti-apoptotic Bcl-2. Moreover, YLT256-treated resulted in changes of mitochondrial membrane potential (Δψm), and generation of reactive oxygen species (ROS). Furthermore, our data also revealed that YLT256 suppressed the growth of established tumor-bearing xenograft models without obvious side effects. Immunohistochemical analyses and TUNEL assay revealed an increase in cleaved caspase-3-positive cells and TUNEL-positive cells, a decrease in Ki67-positive cells upon YLT256. Together, all the results of present study provided evidence demonstrating that YLT256 could be a promising potential drug candidate for pancreatic cancer therapy.

Benzothiazinethione is a potent preclinical candidate for the treatment of drug-resistant tuberculosis.

New chemotherapeutic compounds are needed to combat multidrug-resistant Mycobacterium tuberculosis (Mtb), which remains a serious public-health challenge. Decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1 enzyme) has been characterized as an attractive therapeutic target to address this urgent demand. Herein, we have identified a new class of DprE1 inhibitors benzothiazinethiones as antitubercular agents. Benzothiazinethione analogue SKLB-TB1001 exhibited excellent activity against Mtb in the Microplate Alamar blue assay and intracellular model, meanwhile SKLB-TB1001 was also highly potent against multi-drug resistant extensively and drug resistant clinical isolates. Importantly, no antagonism interaction was found with any two-drug combinations tested in the present study and the combination of SKLB-TB1001 with rifampicin (RMP) was proved to be synergistic. Furthermore, benzothiazinethione showed superb in vivo antitubercular efficacy in an acute Mtb infection mouse model, significantly better than that of BTZ043. These data combined with the bioavailability and safety profiles of benzothiazinethione indicates SKLB-TB1001 is a promising preclinical candidate for the treatment of drug-resistant tuberculosis.

Selective inhibition of EZH2 by ZLD1039 blocks H3K27 methylation and leads to potent anti-tumor activity in breast cancer.

Enhancer of zeste homolog 2 (EZH2) is a candidate oncogenic driver due to its prevalent overexpression and aberrant repression of tumor suppressor genes in diverse cancers. Therefore, blocking EZH2 enzyme activity may present a valid therapeutic strategy for the treatment of cancers with EZH2 overexpression including breast cancers. Here, we described ZLD1039 a potent, highly selective, and orally bioavailable small molecule inhibitor of EZH2, which inhibited breast tumor growth and metastasis. ZLD1039 considerably inhibited EZH2 methyltransferase activity with nanomolar potency, decreased global histone-3 lysine-27 (H3K27) methylation, and reactivated silenced tumor suppressors connected to increased survival of patients with breast cancer. Comparable to conditional silencing of EZH2, its inhibition by ZLD1039 decreased cell proliferation, cell cycle arrest, and induced apoptosis. Comparably, treatment of xenograft-bearing mice with ZLD1039 led to tumor growth regression and metastasis inhibition. These data confirmed the dependency of breast cancer progression on EZH2 activity and the usefulness of ZLD1039 as a promising treatment for breast cancer.