Novel 6-amino-1,3,5-triazine derivatives as potent BTK inhibitors: structure-activity relationship (SAR) analysis and preliminary mechanism investigation.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of B-cell related malignancies. Irreversible inhibition of BTK by a covalent inhibitor has been proved to be a clinically effective therapy. However, most irreversible BTK inhibitors also inhibit other kinases including JAK3 and EGFR, leading to some adverse events. Herein, we reported the structure-based design and optimization of a series of irreversible BTK inhibitors bearing the 6-amino-1,3,5-triazine scaffold. Most of the synthesized compounds demonstrated considerable BTK inhibition and improved anti-proliferative activity against Raji and Ramos cells. Among them, compound C11 exhibited potent BTK inhibition (BTK IC50 = 17.0 nM) and a desirable selectivity profile especially over EGFR. Moreover, C11 effectively blocked activation of BTK and downstream signaling, arrested the cell cycle in G0/G1 phase and induced apoptosis in Raji cells. Its irreversible binding mode was further investigated by both molecular modeling and a washout experiment. Collectively, C11 is a novel selective irreversible BTK inhibitor worthy of further in-depth research.

X-ray crystallography study and optimization of novel benzothiophene analogs as potent selective estrogen receptor covalent antagonists (SERCAs) with improved potency and safety profiles.

Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.

Structural optimization of tetrahydroisoquinoline-hydroxamate hybrids as potent dual ERα degraders and HDAC inhibitors.

Both estrogen receptor α (ERα) and histone deacetylases (HDACs) are valid therapeutic targets for anticancer drug development. Combination therapies using diverse ERα antagonists or degraders and HDAC inhibitors have been proven effective in endocrine-resistant ER + breast cancers based on the crosstalk between ERα and HDAC pathway. In this study, we reported the optimization of a series of methoxyphenyl- or pyridinyl- substituted tetrahydroisoquinoline-hydroxamates, which were optimized from 31, a dual ERα degrader/HDAC inhibitor previously reported by our group. Most of the synthesized compounds displayed potent ERα degradation efficacy and antiproliferative activity. Among them, A04 demonstrated the best anti-proliferation activity (MCF-7 IC50 = 1.96 µM) and HDAC6 inhibitory activity (HDAC6 IC50 = 25.96 nM), which is slightly more potent than the lead compound 31 (MCF-7 IC50 = 4.38 µM, HDAC6 IC50 = 63.03 nM). In addition, compound A04 exerted ERα-independent HDAC6-inhibiting effect without agonistic activity in endometrial cells. These results demonstrated that A04 is a novel and promising dual ERα degrader/HDAC inhibitor worthy of further development.

Benzothiophene derivatives as selective estrogen receptor covalent antagonists: Design, synthesis and anti-ERα activities.

Estrogen receptor α emerged as a well validated therapeutic target of breast cancer for decades. However, approximately 50% of patients who initially responding to standard-of-care (SoC), such as undergo therapy of Tamoxifen, generally inevitably progress to an endocrine-resistance ER+ phenotype. Recently, selective estrogen receptor covalent antagonists (SERCAs) targeted to ERα have been demonstrated as a therapeutic alternative. In the present study, series of novel 6-OH-benzothiophene (BT) derivatives targeting ERα and deriving from Raloxifene were designed, synthesized, and biologically evaluated as covalent antagonists. Driven by the antiproliferative efficacy in ER+ breast cancer cells, our chemical optimization finally led to compound 19d that with potent antagonistic activity in ER+ tumor cells while without agonistic activity in endometrial cells. Moreover, the docking simulation was carried out to elucidate the binding mode, revealing 19d as an antagonist and covalently binding to the cysteine residue at the 530 position of ER helix H11.

Synthesis and evaluation of novel thiosemicarbazone and semicarbazone analogs with both anti-proliferative and anti-metastatic activities against triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive cancer with high mortality and recurrence rates. Hecogenin, a steroidal sapogenin, is reported as a potential anti-tumor agent against breast cancer. However, the moderate activity limits its further application in clinical. With the aim to identify novel analogues that are especially efficacious in therapy of TNBC, a series of novel hecogenin thiosemicarbazone and semicarbazone derivatives were designed, synthesized and biologically evaluated. Screening of cytotoxicity revealed that 4c could potently inhibit the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cells), lung cancer cells (A549) and colon cancer cells (HT-29) at low µM level. Importantly, further mechanism studies indicated the ability of 4c in inducing apoptosis of MDA-MB-231 cells by arresting the cell cycle. Moreover, 4c notably suppressed the migration and invasion of MDA-MB-231 cells compared to its parent hecogenin at the equal concentration.

Discovery of novel 2H-chromene-3-carbonyl derivatives as selective estrogen receptor degraders (SERDs): Design, synthesis and biological evaluation.

Selective estrogen receptor degraders (SERDs) not only block ERα activity but degrade this receptor at the same time and are effective in relapsed ERα positive breast cancer patients who have accepted other endocrine therapies. Herein, through scaffold hopping of coumarin skeleton, a series of 2H-chromene-3-carbonyl-based SERDs with phenyl acrylic acid group as the side chain were designed and synthesized. Compound XH04 containing 7-hydroxy-2H-chromene-3-carbonyl skeleton exhibited the most potent activities in 2D (IC50 = 0.8 µM) and 3D cells culture models (MCF-7) and had the best ERα binding affinity as well. Furthermore, the significant antiestrogen property of compound XH04 was confirmed by inhibiting the expression of progesterone receptor (PgR) mRNA in MCF-7 cells. On the other hand, the outgoing ERα degradation property of compound XH04 was qualitatively and quantificationally verified by immunofluorescence analysis and Western blot assay in MCF-7 cells. Besides, compound XH04 repressed the expression level of Ki67 in MCF-7 cells and induced the apoptosis increase of this tumor cells in a dose-dependent manner like approved-SERD fulvestrant (2), while compound XH04 exhibited better preliminary pharmacokinetics in human and rat liver microsomes in vitro and a lower LogD7.4 value than fulvestrant. And further molecular docking study revealed that compound XH04 possessed a proverbial and typical binding model with ERα like other reported SERD. All these results confirmed that 7-hydroxy-2H-chromene-3-carbonyl structure could be a feasible skeleton for design of ERα antagonists including SERDs and compound XH04 is a promising candidate for further development of ERα + breast cancer therapy agents.

Discovery of novel steroidal-chalcone hybrids with potent and selective activity against triple-negative breast cancer.

A series of novel steroidal-chalcone derivates were designed and synthesized based on the molecular hybridization strategy and further evaluated for their growth inhibitory activity against three human cancer cell lines. The MTT results indicated that most compounds were apparently more sensitive to human breast cancer cells MDA-MB-231. Compounds 8 and 18 exerted the best cytotoxic activity against triple-negative MDA-MB-231 cells with the IC50 values of 0.42 µM and 0.52 µM respectively, which were 23-fold increase or more compared with 5-Fu. Further mechanism studies demonstrated that compound 8 could induce cells apoptosis through regulating Bcl-2/Bax proteins and activating caspase-3 signaling pathway. Moreover, compound 8 could upregulate the cellular ROS levels which accelerated the apoptosis of MDA-MB-231 cells. In addition, interestingly, cell cycle assay showed that compound 8 could arrest MDA-MB-231 cells at S phase but not commonly anticipated G2/M phase. These evidences fully confirmed that compound 8 could be a potential candidate that deserves further development as an antitumor agent against triple-negative breast cancer.

A new class of 1,3,5-triazine-based selective estrogen receptor degraders (SERDs): Lead optimization, molecular docking and dynamic simulation.

Selective estrogen receptor degrader (SERD) that acts as not only ER antagonist, but also ER degrader, would be useful for the treatment for drug-resistance ER+ breast cancer. However, most of currently available SERD candidates involve very limited molecular scaffolds and are still in clinical trials. In this study, we introduced a 1,3,5-triazine ring into a homobibenzyl motif extracted from amounts of ER ligands and synthesized sixteen SERDs bearing acrylic acid or acrylic amide side chains that possess both ERα antagonism and degradation properties. And all compounds were screened for their anti-proliferative activity against ER+ MCF-7 and Ishikawa cell lines. Among them, compound XHA1614 displayed potent growth inhibition activity against MCF-7 and Ishikawa cells with IC50 values of 3.15 µM and 3.11 µM, respectively. Moreover, XHA1614 could dramatically degrade ER level at 1 nM in a Western blotting assay and afforded an outstanding antagonistic activity via suppressing the expression of progesterone receptor messenger RNA in MCF-7 cells in a RT-PCR assay. Further molecular docking and dynamic simulation on properly selected derivative furnished insights into its binding profile within ERα. Our findings suggest that the 1,3,5-triazine core was a feasible alternative to currently reported SERD scaffold, and provide information that will be useful for further development of promising SERDs candidates for breast cancer therapies.

3, 9-di-O-substituted coumestrols incorporating basic amine side chains act as novel apoptosis inducers with improved pharmacological selectivity.

There is much interest in the use of phytoestrogens such as coumestrol in breast cancer intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However, the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of coumestrol led to the most promising compound 7e with improved antiproliferative activity (1.7-fold increase) against MCF-7 cells, decreased estrogen activity (>20 times weaker ERα binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with distinct pharmacological properties, providing an excellent starting point to future development of novel tumor-vascular disrupting agents targeting tubulin.

Lipid accumulation inhibitory activities of novel isoxazole-based chenodeoxycholic acids: Design, synthesis and preliminary mechanism study.

In continuation of our drug discovery program on hyperlipidemia, a series of novel isoxazole-chenodeoxycholic acid hybrids were designed, synthesized and evaluated for their lipid-lowering effects. Preliminary screening of all the synthesized compounds was done by using a 3T3-L1 adipocyte model, in which the most active compound 16b could significantly reduce the lipid accumulation up to 30.5% at a nontoxic concentration 10 µM. Further mechanism studies revealed that 16b blocked lipid accumulation via activating FXR-SHP signaling pathway, efficiently down-regulated the expression of key lipogenesis regulator SREBP-1c.

Structure-guided design and synthesis of isoflavone analogs of GW4064 with potent lipid accumulation inhibitory activities.

Our group has previously reported a series of isoflavone derivatives with antidyslipidemic activity. With this background, a series of isoflavone analogs of GW4064 were designed, synthesized and evaluated the lipid-lowering activity of analogs. As a result, most of compounds significantly reduced the lipid accumulation in 3T3-L1 adipocytes and four of them (10a, 11, 15c and 15d) showed stronger inhibitory than GW4064. The most potent compound 15d exhibited promising agonistic activity for FXR in a cell-based luciferase reporter assay. Meanwhile, 15d up-regulated FXR, SHP and BSEP gene expression and down-regulated the mRNA expression of lipogenesis gene SREBP-1c. Besides, an improved safety profile of 15d was also observed in a HepG2 cytotoxicity assay compared with GW4064. The obtained biological results were further confirmed by a molecular docking study showing that 15d fitted well in the binding pocket of FXR and interacted with some key residues simultaneously.

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ.

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC50 = 139 nM; PI3Kδ: IC50 = 275 nM) and showed some selectivity against PI3Kδ compared to PI3Kß/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.

Lipid reducing activity of novel cholic acid (CA) analogs: Design, synthesis and preliminary mechanism study.

Bile acids, initially discovered as endogenous ligands of farnesoid X receptor (FXR), play a central role in the regulation of triglyceride and cholesterol metabolism and have recently emerged as a privileged structure for interacting with nuclear receptors relevant to a large array of metabolic processes. In this paper, phenoxy containing cholic acid derivatives with excellent drug-likeness have been designed, synthesized, and assayed as agents against cholesterol accumulation in Raw264.7 macrophages. The most active compound 14b reduced total cholesterol accumulation in Raw264.7 cells up to 30.5% at non-toxic 10 µM and dosage-dependently attenuated oxLDL-induced foam cell formation. Western blotting and qPCR results demonstrate that 14b reduced both cholesterol and lipid in Raw264.7 cells through (1) increasing the expression of cholesterol transporters ABCA1 and ABCG1, (2) accelerating ApoA1-mediated cholesterol efflux. Through a cell-based luciferase reporter assay and molecular docking analysis, LXR was identified as the potential target for 14b. Interestingly, unlike conventional LXR agonist, 14b did not increase lipogenesis gene SREBP-1c expression. Overall, these diverse properties disclosed herein highlight the potential of 14b as a promising lead for further development of multifunctional agents in the therapy of cardiovascular disease.

Design, synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents.

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC50 value of 4.52±2.47µM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure-activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.

Design, synthesis and biological evaluation of novel 3-substituted 4-anilino-coumarin derivatives as antitumor agents.

Various 3-substituted 4-anilino-coumarin derivatives have been designed, synthesized and their anti-proliferative properties have been studied. The in vitro cytotoxicity screening was performed against MCF-7, HepG2, HCT116 and Panc-1 cancer cell lines by MTT assay. Most of the synthesized compounds exhibited comparable anti-proliferative activity to the positive control 5-Fluorouracil against these four tested cancer cell lines. Among the different substituents at C-3 position of coumarin scaffold, 3-trifluoroacetyl group showed the most promising results. Especially, compounds 33d (IC50=16.57, 5.45, 4.42 and 5.16µM) and 33e (IC50=20.14, 6.71, 4.62 and 5.62µM) showed excellent anti-proliferative activities on MCF-7, HepG2, HCT116 and Panc-1 cell lines respectively. In addition, cell cycle analysis and apoptosis activation revealed that 33d induced G2/M phase arrest and apoptosis in MCF-7 cells in a dose-dependent manner. Low toxicity of compounds 33d and 33e was observed against human umbilical vein endothelial cells (HUVECs), suggesting their acceptable safety profiles in normal cells. Furthermore, the results of in silico ADME studies indicated that both 33d and 33e exhibited good pharmacokinetic properties.

Targeting focal adhesion kinase (FAK) for cancer therapy: FAK inhibitors, FAK-based dual-target inhibitors and PROTAC degraders.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays an essential role in regulating cell proliferation, migration and invasion through both kinase-dependent enzymatic function and kinase-independent scaffolding function. The overexpression and activation of FAK is commonly observed in various cancers and some drug-resistant settings. Therefore, targeted disruption of FAK has been identified as an attractive strategy for cancer treatment. To date, numerous structurally diverse inhibitors targeting distinct domains of FAK have been developed, encompassing kinase domain inhibitors, FERM domain inhibitors, and FAT domain inhibitors, with several FAK inhibitors advanced to clinical trials. Moreover, given the critical role of FAK scaffolding function in signal transduction, FAK-targeted PROTACs have also been developed. Although no current FAK-targeted therapeutics have been approved for the market, the combination of FAK inhibitors with other anticancer drugs has shown considerable promise in the clinic. This review provides an overview of current drug discovery strategies targeting FAK, including the development of FAK inhibitors, FAK-based dual-target inhibitors and proteolysis-targeting chimeras (PROTACs) in both literature and patent applications. Accordingly, their design and optimization process, mechanisms of action and biological activities are discussed to offer insights into future directions of FAK-targeting drug discovery in cancer therapy.

Dual-target inhibitors based on ERα: Novel therapeutic approaches for endocrine resistant breast cancer.

Estrogen receptor alpha (ERα), a nuclear transcription factor, is a well-validated therapeutic target for more than 70% of all breast cancers (BCs). Antagonizing ERα either by selective estrogen receptor modulators (SERMs) or selective estrogen receptor degraders (SERDs) forms the foundation of endocrine therapy and has achieved great success in the treatment of ERα positive (ERα+) BCs. Unfortunately, despite initial effectiveness, endocrine resistance eventually emerges in up to 30% of ERα+ BC patients and remains a significant medical challenge. Several mechanisms implicated in endocrine resistance have been extensively studied, including aberrantly activated growth factor receptors and downstream signaling pathways. Hence, the crosstalk between ERα and another oncogenic signaling has led to surge of interest to develop combination therapies and dual-target single agents. This review briefly introduces the synergisms between ERα and another anticancer target and summarizes the recent advances of ERα-based dual-targeting inhibitors from a medicinal chemistry perspective. Accordingly, their rational design strategies, structure-activity relationships (SARs) and biological activities are also dissected to provide some perspectives on future directions for ERα-based dual target drug discovery in BC therapy.

Discovery and Proof of Concept of Potent Dual Polθ/PARP Inhibitors for Efficient Treatment of Homologous Recombination-Deficient Tumors.

DNA polymerase theta (Polθ) has recently emerged as a new attractive synthetic lethal target involved in DNA damage repair. Inactivating Polθ alone or in combination with PARP inhibitors has demonstrated substantial therapeutic potential against tumors with homologous recombination (HR) defects such as alternation of BRCA genes. Herein, we report the design and proof of concept of a highly potent dual Polθ/PARP inhibitor 25d, which exhibited low nanomolar inhibitory activities against both Polθ and PARP1. Compared to combination treatment, 25d demonstrated superior antitumor efficacy in both MDA-MB-436 cells and xenografts by inducing more DNA damage and apoptosis. Importantly, 25d retained sensitivity in PARP inhibitor-resistant MDA-MB-436 cells with 53BP1 defect. Altogether, these findings illustrate the potential advantages of 25d, a first-in-class dual Polθ/PARP inhibitor, over monotherapy in treating HR-deficient tumors, including those with acquired PARP inhibitor resistance.

Structure-Activity Relationship (SAR) Studies of Novel Monovalent AR/AR-V7 Dual Degraders with Potent Efficacy against Advanced Prostate Cancer.

Androgen receptor (AR) has been extensively established as a potential therapeutic target for nearly all stages of prostate cancer (PCa). However, acquired resistance to AR-targeted drugs inevitably develops and severely limits their clinical efficacy. Particularly, there currently exists no efficient treatment for patients expressing the constitutively active AR splice variants, such as AR-V7. Herein, we report the structure-activity relationship studies of 55 N-heterocycle-substituted hydantoins, which identified the structural motifs required for AR/AR-V7 degradation. Among them, the most potent compound 27c exhibited selective AR/AR-V7 degradation over other hormone receptors and excellent antiproliferative activities in LNCaP and 22RV1 cells. RNA sequence analysis confirmed that 27c effectively suppressed transcriptional activity of the AR signaling pathway. Importantly, 27c demonstrated potent antitumor efficacy in an enzalutamide-resistant 22RV1 xenograft model. These results highlight the potential of 27c as a promising dual AR/AR-V7 degrader for overcoming drug resistance in advanced PCa expressing AR splice variants.

Precisely Amplifying Intracellular Oxidative Storm by Metal-Organic Coordination Polymers to Augment Anticancer Immunity.

Oxidative stress accompanying the reactive oxygen species (ROS) burst governs immunocyte infiltration, activation, and differentiation in tumor microenvironments and thus can elicit robust antitumor immunity. Here, we identify a photoactive metal-organic coordination polymer (MOCP), composed of an organometallic core formed by cytotoxic mitoxantrone (MTX) acylates and photosensitive Ru(BIQ)-HDBB [BIQ = 2,2'-biquinoline, HDBB = 4,4'-di(4-benzoato)-2,2'-bipyridine] linked by Fe(II) ions via coordinate covalent bonds and an amphipathic shell encapsulating cholesterol-modified siRNA against GPX4 (siGPX4) via hydrophobic force, to precisely amplify intracellular oxidative storm. MOCPs simultaneously encapsulated MTX, Ru, and siGPX4 with efficiencies >98% and loaded Fe with efficiencies of ∼0.49%. With longer blood circulation and higher tumor accumulation, MOCPs with a 670 nm LED irradiation generate abundant ROS to induce biomembrane dysfunction and subsequently contribute to ferroptotic and immunogenic cell death, which drive tumor-associated antigen-specific immunity. MTX analogs contributed to Type I immunogenic cell death (ICD), while oxidative storm served as a damager for endo/lysosomal escape, an initiator for ferroptosis, and an inducer for type II ICD. Moreover, the blockade of CD73 that reduces extoATP catabolism unleashes immunosuppression, finally enhancing antitumor immune stimulation of MOCPs to promote orthotopic mammary cancer regression and prevent postoperative advanced cancer from recurrence and metastasis. MOCPs by exposing sufficient antigenicity thus provide a platform to synergize immune checkpoint inhibitors for the treatment of immunologically cold tumors.