Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy.

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5% of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop "potentiators" for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of "potentiator" for colorectal cancer immunotherapy.

Quinazoline-based hydroxamic acid derivatives as dual histone methylation and deacetylation inhibitors for potential anticancer agents.

Cancer is a common malignant disease with complex signaling networks, which means it is unmanageable to cancer therapy by using single classical targeted drug. Recently, dual- or multitarget drugs have emerged as a promising option for cancer therapies. Although many multifunctional compounds targeting HDAC have been validated, as far as we know, there is no molecule targeting GLP and HDAC synchronously. In the present work, we designed and synthesized a series of quinazoline-based hydroxamic acid derivatives as dual GLP and HDAC inhibitors. These hybrid compounds showed potent enzymatic inhibitory activities against GLP and HDAC1/6 with IC50 values in the nanomolar range of less than 190 nM. Furthermore, most of our compounds displayed significant broad spectrum cytotoxic activities apart from D3 and D8 against all the tested cancer cells with IC50 values less than 50 µM. D1, D6 and D7 showed more potent cytotoxic activities than D2, D4 and D5 in those cancer cells. Especially, compound D7 showed potent inhibitory potency activity against both GLP and HDAC1/6 with IC50 values of 1.3, 89, 13 nM. Besides, D7 exhibited the most potent antiproliferative activity against all the tested cancer cells. Further evaluations indicated that D7 could inhibit the methylation and deacetylation of H3K9 on protein level. Moreover, D7 could induce cancer cell apoptosis, G0/G1 cell cycle arrest, and partly block migration and invasion. All these thorough evaluations warranted D7 as a promising lead compound worth further optimization and development for cancer therapy.

Discovery of the PARP (poly ADP-ribose polymerase) inhibitor 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide for the treatment of cancer.

In this work, two series of cyclic amine-containing benzimidazole carboxamide derivatives were designed and synthesized as potent anticancer agents. PARP1/2 inhibitory activity assays indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was investigated against four human cancer cell lines (MDA-MB-436, MDA-MB-231, MCF-7 and CAPAN-1), and several compounds exhibited strong cytotoxicity to tumor cells. Among them, 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17d) was found to be effective PARP1/2 inhibitors (IC50 = 4.30 and 1.58 nM, respectively). In addition, 17d possessed obvious selective antineoplastic activity and noteworthy microsomal metabolic stability. What's more, further studies revealed that 17d was endowed with an excellent ADME profile. These combined results indicated that 17d could be a promising candidate for the treatment of cancer.

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors.

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.

Development of a versatile DNMT and HDAC inhibitor C02S modulating multiple cancer hallmarks for breast cancer therapy.

DNMT and HDAC are closely related to each other and involved in various human diseases especially cancer. These two enzymes have been widely recognized as antitumor targets for drug discovery. Besides, research has indicated that combination therapy consisting of DNMT and HDAC inhibitors exhibited therapeutic advantages. We have reported a DNMT and HDAC dual inhibitor 15a of which the DNMT enzymatic inhibitory potency needs to be improved. Herein we reported the development of a novel dual DNMT and HDAC inhibitor C02S which showed potent enzymatic inhibitory activities against DNMT1, DNMT3A, DNMT3B and HDAC1 with IC50 values of 2.05, 0.93, 1.32, and 4.16 µM, respectively. Further evaluations indicated that C02S could inhibit DNMT and HDAC at cellular levels, thereby inversing mutated methylation and acetylation and increasing expression of tumor suppressor proteins. Moreover, C02S regulated multiple biological processes including inducing apoptosis and G0/G1 cell cycle arrest, inhibiting angiogenesis, blocking migration and invasion, and finally suppressing tumor cells proliferation in vitro and tumor growth in vivo.

Discovery of 2-(1-(3-(4-Chloroxyphenyl)-3-oxo- propyl)pyrrolidine-3-yl)-1H-benzo[d]imidazole-4-carboxamide: A Potent Poly(ADP-ribose) Polymerase (PARP) Inhibitor for Treatment of Cancer.

A series of benzimidazole carboxamide derivatives have been synthesized and characterized by 1H-NMR, 13C-NMR and HRMS. PARP inhibition assays and cellular proliferation assays have also been carried out. Compounds 5cj and 5cp exhibited potential anticancer activities with IC50 values of about 4 nM against both PARP-1 and PARP-2, similar to the reference drug veliparib. The two compounds also displayed slightly better in vitro cytotoxicities against MDA-MB-436 and CAPAN-1 cell lines than veliparib and olaparib, with values of 17.4 µM and 11.4 µM, 19.8 µM and 15.5 µM, respectively. The structure-activity relationship based on molecular docking was discussed as well.

Synthesis and biological research of novel azaacridine derivatives as potent DNA-binding ligands and topoisomerase II inhibitors.

DNA and DNA-related enzymes are one of the most effective and common used intracellular anticancer targets in clinic and laboratory studies, however, most of DNA-targeting drugs suffered from toxic side effects. Development of new molecules with good antitumor activity and low side effects is important. Based on computer aided design and our previous studies, a series of novel azaacridine derivatives were synthesized as DNA and topoisomerases binding agents, among which compound 9 displayed the best antiproliferative activity with an IC50 value of 0.57µM against U937 cells, which was slightly better than m-AMSA. In addition, compound 9 displayed low cytotoxicity against human normal liver cells (QSG-7701), the IC50 of which was more than 3 times lower than m-AMSA. Later study indicated that all the compounds displayed topoisomerases II inhibition activity at 50µM. The representative compound 9 could bind with DNA and induce U937 apoptosis through the exogenous pathway.

Synthesis and investigation of novel 6-(1,2,3-triazol-4-yl)-4-aminoquinazolin derivatives possessing hydroxamic acid moiety for cancer therapy.

By merging the critical pharmacophore of EGFR/HER2 and HDAC inhibitors into one compound, a novel series of EGFR, HER-2, and HDAC multitarget inhibitors were synthesized. Compounds 9a-l contained 4-anilinoquinazolines with C-6 triazole-linked long alkyl chains of hydroxamic acid and displayed excellent inhibition against these enzymes (compound 9d exhibited the best inhibitory potency on wild-type EGFR, HDAC1, and HDAC6 with IC50 values 0.12nM, 0.72nM and 3.2nM individually). Furthermore, compounds 9b and 9d potently inhibited proliferation of five human cancer cell lines (with IC50 values between 0.49 and 8.76µM). Further mechanistic study revealed that compound 9d also regulated the phosphorylation of EGFR and HER2 and histone H3 hyperacetylation on the cellular level and induced remarkable apoptosis in BT-474 cells. Therefore, our study suggested that a system network-based multi-target drug design strategy might provided an alternate drug design method, by taking into account the synergy effect of EGFR, HER-2 and HDAC.

Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment.

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 µM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC(50) at 8.3 µM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.

2'-chloro-4'-aminoflavone derivatives selectively targeting hepatocarcinoma cells: convenient synthetic process, G(2)/M cell cycle arrest and apoptosis triggers.

A series of 2'-chloro-4'-nitroflavone and 2'-chloro-4'-aminoflavone derivatives were synthesized by a convenient synthetic process. The in vitro anti-proliferation ability of these compounds was evaluated against hepatocarcinoma cells (HepG2), breast adenocarcinoma cells (MCF-7), and human chronic myelogenous leukemia cells (K562). Most of synthetic compounds possessed notable anti-proliferation activity against HepG2 cells and little activity against MCF-7 cells and K562 cells. In particular, compounds 4c and 4e exhibited high anti-proliferation activity against HepG2 cells with IC(50) at about 2.0 µM. Further toxicity screening toward normal human hepatocytes indicated that some compounds had low toxicity against normal liver cells, among which 4e displayed very weak effects on QSG7701 and HL7702 cells, with IC(50) values >100 and 50 µM, respectively. Compound 4c, with the best anti-proliferation activity in amino-substituted flavones (IC(50) = 2.0 µM), was selected for further evaluation of its effects on apoptosis and the cell cycle. HepG2 cells were exposed to this compound at 10 µM, which induced nuclear disassembly and DNA fragmentation. Flow cytometry analysis suggested that the population of apoptotic cells greatly increased in the 4c-treated HepG2 cells, and the cell cycle was arrested at the G(2)/M phase.

Synthesis and cytotoxic activity of some novel N-pyridinyl-2-(6-phenylimidazo[2,1-b]thiazol-3-yl)acetamide derivatives.

A series of novel compounds bearing imidazo[2,1-b]thiazole scaffolds were designed and synthesized based on the optimization of the virtual screening hit compound N-(6-morpholinopyridin-3-yl)-2-(6-phenylimidazo[2,1-b]thiazol-3-yl)acetamide (5a), and tested for their cytotoxicity against human cancer cell lines, including HepG2 and MDA-MB-231. The results indicated that the compound 2-(6-(4-chlorophenyl)imidazo[2,1-b]thiazol-3-yl)-N-(6-(4-(4-methoxybenzyl)piperazin-1-yl)pyridin-3-yl)acetamide (5l), with slightly higher inhibition on VEGFR2 than 5a (5.72% and 3.76% inhibitory rate at 20 µM, respectively), was a potential inhibitor against MDA-MB-231 (IC(50) = 1.4 µM) compared with sorafenib (IC(50) = 5.2 µM), and showed more selectivity against MDA-MB-231 than HepG2 cell line (IC(50) = 22.6 µM).

Design, synthesis and antitumor evaluation of novel 1H-indole-2-carboxylic acid derivatives targeting 14-3-3η protein.

In this work, a series of novel 1H-indole-2-carboxylic acid derivatives targeting 14-3-3η protein were designed and synthesized for treatment of liver cancer. After structural optimization for several rounds, C11 displayed a relatively better affinity with 14-3-3η, as well as the best inhibitory activities against several typical human liver cancer cell lines, including Bel-7402, SMMC-7721, SNU-387, Hep G2 and Hep 3B cells. Compound C11 also displayed best inhibitory activity against chemotherapy-resistant Bel-7402/5-Fu cells. Besides, C11 was rather safe against hERG and possessed moderate T1/2 and CL values in liver microsomes. In anti-proliferation, trans-well and cell apoptosis assays, C11 also showed its huge potential as a potent antitumor agent. Then, Western blot assay was conducted, following analyzed by molecular docking, the anti-proliferative mechanisms of this small-molecule inhibitor were revealed. Moreover, C11 was demonstrated to induce G1-S phase cell cycle arrest in liver cancer cells.

Discovery of benzimidazole derivatives as novel multi-target EGFR, VEGFR-2 and PDGFR kinase inhibitors.

Multi-target EGFR, VEGFR-2 and PDGFR inhibitors are highly useful anticancer agents with improved therapeutic efficacies. In this work, we used two virtual screening methods, support vector machines (SVM) and molecular docking, to identify a novel series of benzimidazole derivatives, 2-aryl benzimidazole compounds, as multi-target EGFR, VEGFR-2 and PDGFR inhibitors. 2-Aryl benzimidazole compounds were synthesized and their biological activities against a tumor cell line HepG-2 and specific kinases were evaluated. Among these compounds, compounds 5a and 5e exhibited high cytotoxicity against HepG-2 cells with IC50 values at ∼2 µM. Further kinase assay study showed that compound 5a have good EGFR inhibitory activity and moderate VEGFR-2 and PDGFR inhibitory activities, while 5e have moderate EGFR inhibitory activity and slightly weaker VEGFR-2 and PDGFR inhibitory activities. Molecular docking analysis suggested that compound 5a more tightly interacts with EGFR and PDGFR than compound 5e. Our study discovered a novel series of benzimidazole derivatives as multi-target EGFR, VEGFR-2 and PDGFR kinases inhibitors.

2-Chloro-1-[4-(2-fluoro-benz-yl)piperazin-1-yl]ethanone.

In the title compound, C(13)H(16)ClFN(2)O, the piperazine ring is flanked by 1-(2-fluoro-benz-yl)piperazine and adopts a chair conformation. The dihedral angle between the fluoro-phenyl ring and the four planar C atoms (r.m.s. = 0.0055 Å) of the piperazine chair is 78.27 (7)°, whereas the dihedral angle between the four planar C atoms of the piperazine chair and the ethanone plane is 55.21 (9) Å; the Cl atom displaced by1.589 (2) Šout of the plane.

Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines.

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI-H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFRT790M resistance mutation harboring NCI-H1975 cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI-H1975 cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring tumor cell lines (NCI-H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Synthesis and Anti-Influenza Virus Effects of Novel Substituted Polycyclic Pyridone Derivatives Modified from Baloxavir.

In this work, a series of novel substituted polycyclic pyridone derivatives were designed and synthesized as potent anti-influenza agents. The cytopathic effect (CPE) assay and cytotoxicity assay indicated that all of the compounds possessed potent anti-influenza virus activity and relatively low cytotoxicity; some of them inhibited the replication of influenza A virus (IAV) at picomolar concentrations. Further studies revealed that, at a concentration of 3 nM, three compounds (10a, 10d, and 10g) could significantly reduce the M2 RNA amounts and M2 protein expression of IAV and inhibit the activity of RNA-dependent RNA polymerase (RdRp). Among them, (R)-12-(5H-dibenzo[a,d][7]annulen-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (10a) was found to be a promising anti-influenza drug candidate with good human liver microsomal stability, as well as with better selectivity index and oral bioavailability than Baloxavir.

Current discovery strategies for hepatocellular carcinoma therapeutics.

Introduction: The global incidence of hepatocellular carcinoma (HCC) is not expected to decline significantly over the next 30 years. And although the latest gene sequencing studies have established its genetic map, the potentially targetable drivers of HCC are, so far, difficult to identify. To date, only seven drugs have been approved by the FDA for unresectable HCC treatment; thus, effective therapeutic breakthroughs are still needed urgently.Areas covered: In this review, the authors discuss both genetic and epigenetic alterations in HCC and introduce the current progress with some of the representative molecular targeting inhibitors, listing some of the approved drugs for the targets of HCC. The structure-activity relationship of molecules (e.g. thalidomide, bortezomil) used for HCC is also discussed.Expert opinion: Effective therapeutic targets and effective drugs for HCC treatment are an urgent unmet need. Better understanding and characterization of genetic and epigenetic alterations, which are important to hepatocarcinogenesis, may help to understand the molecular pathogenesis of HCC, as well as provide novel therapeutic lead compounds for HCC treatment.

Antiproliferative activities of the second-generation antipsychotic drug sertindole against breast cancers with a potential application for treatment of breast-to-brain metastases.

Epidemiological observations have shown that schizophrenia patients after long-term drug treatment exhibited reduced tumor incidences. The potential anticancer effects of antipsychotic drugs are subsequently demonstrated. These drugs are of great interest as agents against untreatable brain metastases because of their ability to traverse the blood-brain barrier (BBB). Most drugs tested thus far are the first-generation antipsychotics (FGAs). But their clinical application may be limited due to high risks of deaths in elderly patients. There is an urgent need to find additional BBB-traversing anticancer agents with lower risks of deaths. In this work, we investigated antitumor activities of eight second-generation-antipsychotic (SGA) drugs, since they exhibit lower mortality rates than FGAs. We discovered that sertindole showed broad antiproliferative activities against seven cancer types including 29 cell-lines and exhibited potent effects toward breast cancer cell-lines, with half maximal concentration to inhibit proliferation by 50% (IC50) as low as 800 nM. We further found that sertindole caused cell death through autophagy-associated apoptosis and its directly-binding inhibition of 5-HT6 involved in this process. In xenotransplant mice, sertindole administration approaching maximal therapeutic dose attenuated breast-tumor growth by 22.7%. Therefore, our study reveals promising anticancer potentials of sertindole against breast cancers, with probable applications for breast-to-brain metastases.

Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for hepatocellular carcinoma treatment.

Sorafenib was the only small-molecule drug approved by FDA for treatment of the advanced hepatocellular carcinoma (HCC). Recent study indicated that YM155 was a promising agent for HCC cells with high survivin expression, however, the antitumor activity needs to be further improved. Based on molecular docking and rational design method, a series of multi-substituted benzyl acridone derivatives were designed and synthesized. MTT assay indicated that some of the synthesized compounds displayed better antiproliferative activity against HepG2 cells than YM155. Later study indicated that the representive compound 8u may directly interact with survivin protein and induce HepG2 cells apoptosis, which is different from YM155. In addition, ADME property was predicted in silico, and it performed well. Moreover, in vivo preliminary experiments showed that 8u may be a good lead compound in the treatment of HCC.

Clustered distribution of natural product leads of drugs in the chemical space as influenced by the privileged target-sites.

Some natural product leads of drugs (NPLDs) have been found to congregate in the chemical space. The extent, detailed patterns, and mechanisms of this congregation phenomenon have not been fully investigated and their usefulness for NPLD discovery needs to be more extensively tested. In this work, we generated and evaluated the distribution patterns of 442 NPLDs of 749 pre-2013 approved and 263 clinical trial small molecule drugs in the chemical space represented by the molecular scaffold and fingerprint trees of 137,836 non-redundant natural products. In the molecular scaffold trees, 62.7% approved and 37.4% clinical trial NPLDs congregate in 62 drug-productive scaffolds/scaffold-branches. In the molecular fingerprint tree, 82.5% approved and 63.0% clinical trial NPLDs are clustered in 60 drug-productive clusters (DCs) partly due to their preferential binding to 45 privileged target-site classes. The distribution patterns of the NPLDs are distinguished from those of the bioactive natural products. 11.7% of the NPLDs in these DCs have remote-similarity relationship with the nearest NPLD in their own DC. The majority of the new NPLDs emerge from preexisting DCs. The usefulness of the derived knowledge for NPLD discovery was demonstrated by the recognition of the new NPLDs of 2013-2014 approved drugs.