Design, synthesis and structure-activity relationship study of piperazinone-containing thieno[3,2-d]pyrimidine derivatives as new PI3Kδ inhibitors.

Two classes of piperazinone-containing thieno[3,2-d]pyrimidines were designed and synthesized as new PI3Kδ inhibitors in this study. Detailed SAR study with respect to the piperazinone substituents at the 6-position of thieno[3,2-d]pyrimidine core demonstrated that piperazinone-containing thieno[3,2-d]pyrimidines would be more potent and selective for PI3Kδ than their piperazine counterparts, which led to the discovery of several potent PI3Kδ inhibitors with comparable or better antiproliferative activity against a panel of non-Hodgkin lymphoma (NHL) cell lines as compared with idelalisib. Our study will promote the development of new PI3Kδ inhibitors based on piperazinone-containing thieno[3,2-d]pyrimidine scaffold.

Identification of a potent and selective phosphatidylinositol 3-kinase δ inhibitor for the treatment of non-Hodgkin's lymphoma.

PI3Kδ has proved to be an effective target for anti-lymphoma drugs. However, the application of current approved PI3Kδ inhibitors has been greatly limited due to their specific immune-mediated toxicity and increased risk of infection, it is necessary to develop more PI3Kδ inhibitors with new scaffold. In this study, SAR study with respect to piperazinone-containing purine derivatives led to the discovery of a potent and selective PI3Kδ inhibitor, 4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (WNY1613). WNY1613 exhibits good antiproliferative activity against a panel of non-Hodgkin's lymphoma (NHL) cell lines by inducing cancer cell apoptosis and inhibiting the phosphorylation of PI3K and MAPK downstream components. In addition, it can also prevent the tumor growth in both SU-DHL-6 and JEKO-1 xenograft models without observable toxicity. WNY1613 thus could be developed as a promising candidate for the treatment of NHL after subsequent extensive pharmacodynamics and pharmacokinetics investigation.

Discovery and Structure-Resistance Relationship Study of New Thieno[2,3-b] Pyridine HCV NS4B Inhibitors.

The non-structural protein 4B (NS4B) of hepatitis C virus (HCV) has emerged as a promising target for chronic hepatitis C treatment. The thieno[2,3-b]pyridine HCV inhibitor 2 has demonstrated properties as a NS4B inhibitor. Subsequent hybridization of 2 with our recently published imidazo[2,1-b]thiazole NS4B inhibitor 3 resulted in the discovery of several more potent compounds with sub-micromolar EC50 against HCV genotype 1b replicon. More importantly, the resistant profile study of the new synthesized HCV inhibitors illustrated that the bicyclic scaffold would mediate the resistance of H3R and Q26R mutations, while the piperazinone motif would mediate the resistance of H94R, F98C and V105M mutations, and the C3- amino group would disrupt the interaction between piperazinone motif and NS4B. This structure-resistance relationship detail could help us to develop new NS4B inhibitors with higher resistant barrier in the future.

Synthesis and biological evaluation of N-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)benzenesulfonamide derivatives as new BET bromodomain inhibitors for anti-hematologic malignancies activities.

The bromodomain and extra-terminal proteins (BETs), in particular BRD4, has been reported to play important roles in cancer, inflammation, obesity, cardiovascular disease, and neurological disorders. In this paper, a series of benzomorpholinone derivatives were synthesized and biologically evaluated as BETs inhibitors. Detailed structure-activity relationship studies led to the discovery of several new potent compounds, of which 15h and 15i displayed [Formula: see text] values of 2.8 and 4.5 [Formula: see text] against BRD4 (D1), respectively, and showed good anti-proliferation activities against four hematologic malignancies cell lines at low-micromolar concentrations, including MV4-11, OCI-LY10, Pfeifer, and Su-DHL-6 cells. This chemotype could be further optimized with respect to its potency and drug-like properties in the future.

Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives.

Tuberculosis (TB) remains a major human health problem. New therapeutic antitubercular agents are urgent needed to control the global tuberculosis pandemic. We synthesized a new series of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives and evaluated their anti-mycobacterial activities against Mycobacterium tuberculosis H37Ra as well as their druggabilities. The results showed that most of these derivatives, especially the compounds with simple alkyl side chains, exhibited good antitubercular activities and favorable aqueous solubilities with no obvious cytotoxicity. It suggested that the 4-carbonyl piperazine substituents in benzothiazinone scaffold were well tolerated, in which the compound 8h, with an antitubercular activity of MIC 0.008 µM, exhibited an excellent aqueous solubility of 104 µg/mL, which was 100-fold better than the potent DprE1 inhibitor Comp.1 (BTZ038), also more soluble than PBTZ169.

Discovery of novel N-(5-(tert-butyl)isoxazol-3-yl)-N'-phenylurea analogs as potent FLT3 inhibitors and evaluation of their activity against acute myeloid leukemia in vitro and in vivo.

FLT3 inhibitors have been explored as a viable therapy for acute myeloid leukemia (AML). However, the clinical outcomes of these FLT3 inhibitors were underwhelming except AC220. Therefore, the development of novel FLT3 inhibitors with high potency against both FLT3-WT and FLT3-ITD mutants are strongly demanded at the present time. In this study, we designed and synthesized a series of novel N-(5-(tert-butyl)isoxazol-3-yl)-N'-phenylurea derivatives as FLT3 inhibitors. SAR studies focused on the fused rings led to the discovery of a series of compounds with high potency against FLT3-ITD-bearing MV4-11 cells and significantly inhibitory activity toward FLT3. Among these compounds, N-(5-(tert-butyl)isoxazol-3-yl)-N'-(4-(7-methoxyimidazo[1,2-a]pyridin-2-yl)phenyl)urea (16i), displayed acceptable aqueous solubility, desirable pharmacokinetic profile and high cytotoxicity selectivity against MV4-11 cells. This compound can inhibit phosphorylation of FLT3 and induce apoptosis in a concentration-dependent manner. Further in vivo antitumor studies showed that 16i led to complete tumor regression in the MV4-11 xenograft model at a dose of 60 mg/kg/d while without observable body weight loss. This study had provided us a new chemotype of FLT3 inhibitors as novel therapic candidates for AML.

Discovery and structure-activity relationships study of novel thieno[2,3-b]pyridine analogues as hepatitis C virus inhibitors.

Current treatment for hepatitis C is barely satisfactory, there is an urgent need to develop novel agents for combating hepatitis C virus infection. This study discovered a new class of thieno[2,3-b]pyridine derivatives as HCV inhibitors. First, a hit compound characterized by a thienopyridine core was identified in a cell-based screening of our privileged small molecule library. And then, structure activity relationship study of the hit compound led to the discovery of several potent compounds without obvious cytotoxicity in vitro (12c, EC50=3.3µM, SI >30.3, 12b, EC50=3.5µM, SI >28.6, 10l, EC50=3.9µM, SI >25.6, 12o, EC50=4.5µM, SI >22.2, respectively). Although the mechanism of them had not been clearly elucidated, our preliminary optimization of this class of compounds had provided us a start point to develop new anti-HCV agents.

Synthesis and structure-activity relationships evaluation of benzothiazinone derivatives as potential anti-tubercular agents.

N-Alkyl and heterocycle substituted 1,3-benzothiazin-4-one (BTZ) derivatives were synthesized. The anti-mycobacterial activities of these compounds were evaluated by determination of minimal inhibitory concentration (MIC) for Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv. It was found that an extended or branched alkyl chain analog could enhance the potency, and activities of N-alkyl substituted BTZs were not affected by either nitro or trifluoromethyl at 6-position. Trifluoromethyl plays an important role in maintaining anti-tubercular activity in the piperazine or piperidine analogs. Compound 8o, which contains an azaspirodithiolane group, showed a MIC of 0.0001 µM against M. tuberculosis H37Rv, 20-fold more potent than BTZ043 racemate. These results suggested that the volume and lipophilicity of the substituents were important in maintaining activity. In addition, compound 8o was nontoxic to Vero cells and orally bioavailable in a preliminary pharmacokinetics study.

Identification of a selective BRD4 PROTAC with potent antiproliferative effects in AR-positive prostate cancer based on a dual BET/PLK1 inhibitor.

BRD4-targeted proteolysis targeting chimera (PROTAC) have exhibited promising in vitro and in vivo anticancer activity in a number of cancer models. However, the clinical development of current reported BRD4-PROTACs have stagnated, largely due to the safety risks caused by their poor degradation selectivity. In this study, we designed and synthesized a series of PROTACs based on our recently reported dual BET/PLK1 inhibitor WNY0824, which led to the discovery of an isoform-selective and potent BRD4-PROTAC 12a (WWL0245). WWL0245 exhibited excellent selective cytotoxicity in the BETi sensitive cancer cell lines, including AR-positive prostate cancer cell lines. It could also efficiently induce ubiquitin-proteasomal degradation of BRD4 in AR-positive prostate cancer cell lines, with sub-nanomolar half-maximal degrading concentration (DC50) and maximum degradation (Dmax) > 99%. Moreover, WWL0245 induced cell cycle arrest at the G0/G1 phase and apoptosis in AR-positive prostate cancer by downregulation of the protein levels of AR, PSA and c-Myc as well as transcriptionally suppressed AR-regulated genes. WWL0245 was thus expected to be developed as a promising drug candidate for AR-positive prostate cancer and a valuable tool compound to study the biological function of BRD4.

BRD4 inhibition sensitizes aggressive non-Hodgkin lymphomas to PI3Kδ inhibitors by suppressing PI3K reactivation and c-MYC expression.

Targeting phosphatidylinositol 3-kinase δ (PI3Kδ) is an important therapeutic strategy for indolent non-Hodgkin lymphomas (NHLs). However, we previously observed reactivation of phosphatidylinositol 3-kinase (PI3K) pathways in aggressive NHL cell lines following continuous exposure to PI3Kδ inhibitors (PI3Kδi), which limited their efficacy and suggests that more studies should be focused on this reactivation to improve current PI3Kδi-based treatments. Herein we conducted a drug synergy screening that combined a marketed PI3Kδi, idelalisib, with 14 well-characterized epigenetic drugs across several types of aggressive NHL cell lines. We identified BRD4 inhibitors (BRD4i) as potent partners that, in combination with idelalisib, were capable of synergistically exerting anti-proliferative activity and inducing cell apoptosis in a panel of aggressive NHL cell lines through continuous suppression of PI3K pathways. More importantly, the combination of BRD4i and PI3Kδi simultaneously inhibited transcription and translation of the oncogenic transcription factor c-MYC, downregulating the expression of c-MYC and continuously suppressing the proliferation of cancer cells in vitro, as well as the growth of tumors in vivo even after drug withdrawal. This study, thus, reveals the potential of simultaneously targeting PI3Kδ and BRD4 as a new therapeutic strategy for aggressive forms of NHL.

Penfluridol: An antipsychotic agent suppresses lung cancer cell growth and metastasis by inducing G0/G1 arrest and apoptosis.

Lung cancer remains the leading cause of cancer mortality because of highly malignant and metastatic potential. The current status of lung cancer treatment is limited, and more treatment options are needed. Interesting, antipsychotic drugs have been reported to show anti-cancer effects. In this present study, we investigated the anticancer potential of penfluridol (PF), an anti-schizophrenic drug, in lung cancer and its underlying mechanism in vitro and in vivo. In vitro, it could inhibit the viability of various lung cancer cells with G0/G1 phase arrest via increasing the expression level of p21/p27 and decreasing the expression levels of cyclin-CDK complex. Meanwhile, cell-cycle arrest causes DNA repair in the nucleus, which was associated with the upregulation of H2A.X and p-H2A.X. Moreover, PF could also decrease mitochondrial membrane potential and increase reactive oxygen species levels in the lung cancer cells. These results implied that PF might induce the mitochondria-mediated intrinsic apoptosis. In addition, PF inhibits the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling. In vivo, oral administration of PF at concentration of 10 mg/kg inhibited tumor growth in A549 xenograft model. Notably, PF is an approved drug and the price is exceedingly cheap, so this study demonstrates the potential of PF to treat lung cancer.

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.

Novel Dual BET and PLK1 Inhibitor WNY0824 Exerts Potent Antitumor Effects in CRPC by Inhibiting Transcription Factor Function and Inducing Mitotic Abnormality.

Castration-resistant prostate cancer (CRPC) is a lethal disease with few treatment alternatives once patients become resistant to second-generation antiandrogens. In CRPC, BET proteins are key regulators of AR- and MYC-mediated transcription, while the PLK1 inhibitor potentially downregulates AR and MYC besides influencing the cell cycle. Therefore, synchronous inhibition of BET and PLK1 would be a promising approach for CRPC therapy. This study developed a dual BET and PLK1 inhibitor WNY0824 with nanomolar and equipotent inhibition of BRD4 and PLK1. In vitro, WNY0824 exhibited excellent antiproliferation activity on AR-positive CRPC cells and induced apoptosis. These activities are attributable to its disruption of the AR-transcriptional program and the inhibition of the ETS pathway. Furthermore, WNY0824 downregulated MYC and induced mitotic abnormality. In vivo, oral WNY0824 administration suppressed tumor growth in the CRPC xenograft model of enzalutamide resistance. These findings suggest that WNY0824 is a selective dual BET and PLK1 inhibitor with potent anti-CRPC oncogenic activity and provides insights into the development of other novel dual BET- and PLK1-inhibiting drugs.

Repurposing of the anti-helminthic drug niclosamide to treat melanoma and pulmonary metastasis via the STAT3 signaling pathway.

The incidence of melanoma is increasing rapidly worldwide. Additionally, new and effective candidates for treating melanoma are needed because of the increase in drug resistance and the high metastatic potential of this cancer. The STAT3 signaling pathway plays a pivotal role in pathogenesis of melanoma, making STAT3 a promising anticancer target for melanoma therapy. Niclosamide, an FDA-approved anti-helminthic drug, has been identified as a potent STAT3 inhibitor that suppresses STAT3 phosphorylation at Tyr705 and its transcript activity. In this study, we evaluated the biological activities of niclosamide in melanoma in vitro and in vivo. Niclosamide potently inhibited the growth of four melanoma cell lines and induced the apoptosis of melanoma cells via the mitochondrial apoptotic pathway. Further, western blot analysis indicated that cell apoptosis was correlated with activation of Bax and cleaved caspase-3 and decreased expression of Bcl-2. Moreover, niclosamide markedly impaired melanoma cell migration and invasion, reduced phosphorylated STAT3Tyr705 levels, and inhibited matrix metalloproteinase-2 and -9 expression. Additionally, in a xenograft model of A375, intraperitoneal administration of niclosamide inhibited tumor growth and tumor weight in a dose-dependent manner without obvious side effects. Histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells and p-STAT3Try705-positive cells and increase in cleaved caspase-3-positive cells. Notably, niclosamide significantly inhibited pulmonary metastasis in a B16-F10 melanoma lung metastasis model, including the number of lung metastatic nodules and lung/body coefficient. Importantly, a marked reduction in myeloid-derived suppressor cells (Gr1+CD11b+) infiltration in the pulmonary metastasis tissue was observed. Taken together, these results demonstrate that niclosamide is a promising candidate for treating melanoma.

Novel selective TOPK inhibitor SKLB-C05 inhibits colorectal carcinoma growth and metastasis.

The mitogen-activated protein kinase (MAPK) signaling pathway member T-LAK cell-originated protein kinase/PDZ-binding kinase (TOPK/PBK) is closely involved in tumorigenesis and progression. Its overexpression in colorectal carcinoma (CRC) exacerbates tumor malignancy, promotes metastasis and results in dismal prognosis. Therefore, targeting TOPK is a promising approach for CRC therapy. Here, we report the development of a TOPK selective inhibitor SKLB-C05, with subnanomolar inhibitory potency. In vitro, SKLB-C05 exhibited excellent cytotoxicity and anti-migration and invasion activity on TOPK high-expressing CRC cells and induced cell apoptosis. These activities could attribute to its inhibition of TOPK downstream signaling including extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase 1, 2, and 3 (JNK1/2/3), as well as downregulation of FAK/Src- MMP signaling. Furthermore, SKLB-C05 disrupted cell mitosis and blocked CRC cell cycle. In vivo, oral administration of SKLB-C05 at concentrations of 20 and 10 mg kg-1·day-1 dramatically attenuated CRC tumor xenograft growth and completely suppressed hepatic metastasis of HCT116 cells, respectively. Thus, these findings suggest that SKLB-C05 is a specific TOPK inhibitor with potent anti-CRC oncogenic activity in vitro and in vivo.

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.

Nifuroxazide exerts potent anti-tumor and anti-metastasis activity in melanoma.

Melanoma is a highly malignant neoplasm of melanocytes with considerable metastatic potential and drug resistance, explaining the need for new candidates that inhibit tumor growth and metastasis. The signal transducer and activator of the transcription 3 (Stat3) signaling pathway plays an important role in melanoma and has been validated as promising anticancer target for melanoma therapy. In this study, nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3, was evaluated for its anti-melanoma activity in vitro and in vivo. It had potent anti-proliferative activity against various melanoma cell lines and could induce G2/M phase arrest and cell apoptosis. Moreover, nifuroxazide markedly impaired melanoma cell migration and invasion by down-regulating phosphorylated-Src, phosphorylated-FAK, and expression of matrix metalloproteinase (MMP) -2, MMP-9 and vimentin. It also significantly inhibited tumor growth without obvious side effects in the A375-bearing mice model by inducing apoptosis and reducing cell proliferation and metastasis. Notably, nifuroxazide significantly inhibited pulmonary metastases, which might be associated with the decrease of myeloid-derived suppressor cells (MDSCs). These findings suggested that nifuroxazide might be a potential agent for inhibiting the growth and metastasis of melanoma.

Discovery of imidazo[2,1-b]thiazole HCV NS4B inhibitors exhibiting synergistic effect with other direct-acting antiviral agents.

The design, synthesis, and SAR studies of novel inhibitors of HCV NS4B based on the imidazo[2,1-b]thiazole scaffold were described. Optimization of potency with respect to genotype 1b resulted in the discovery of two potent leads 26f (EC50 = 16 nM) and 28g (EC50 = 31 nM). The resistance profile studies revealed that 26f and 28g targeted HCV NS4B, more precisely the second amphipathic α helix of NS4B (4BAH2). Cross-resistance between our 4BAH2 inhibitors and other direct-acting antiviral agents targeting NS3/4A, NS5A, and NS5B was not observed. For the first time, the synergism of a series of combinations based on 4BAH2 inhibitors was evaluated. The results demonstrated that our 4BAH2 inhibitor 26f was synergistic with NS3/4A inhibitor simeprevir, NS5A inhibitor daclatasvir, and NS5B inhibitor sofosbuvir, and it could also reduce the dose of these drugs at almost all effect levels. Our study suggested that favorable effects could be achieved by combining 4BAH2 inhibitors such as 26f with these approved drugs and that new all-oral antiviral combinations based on 4BAH2 inhibitors were worth developing to supplement or even replace current treatment regimens for curing HCV infection.