Thionated aminofluorophthalimides reduce classical markers of cellular inflammation in LPS-challenged RAW 264.7 cells.

Herein, we present the synthesis of several fluorinated pomalidomide derivatives and their thionated counterparts with subsequent biological evaluation against classical markers of cellular inflammation. Treatment in LPS-challenged cells effected varying reductions in levels of secreted TNF-α and nitrite relative to basal amounts. While arene fluorination and thioamidation had marginal and sporadic effects on TNF-α production, specific 7-position fluorination combined with subsequent increases in carbonyl thionation produced compounds 11, 14, and 15 which demonstrated corresponding and escalating anti-nitrite activities concurrent with minimal cellular toxicity. In this regard, compound 15 displayed roughly 96 % cell viability combined with a 65 % drop in nitrite production when supplied to RAW cells challenged with 60 ng/mL LPS. When a focused family of fluorinated isomers were directly compared, the analogous 5-fluorinated isomer 17 displayed comparable minimal toxicity but markedly less anti-nitrite activity versus 15 in RAW cells challenged with 70 ng/mL LPS. Compound 15 was subsequently screened in human liver microsomes for preliminary Phase 1 analysis where it demonstrated heightened stability relative to its non-fluorinated counterpart 3,6'-dithiopomalidomide 4, a result in line with the expected metabolic fortitude provided by fluorination at the sensitive pomalidomide 7-position.

A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Synthesis and pharmacological evaluation of pomalidomide derivatives useful for sickle cell disease treatment.

Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide-nitric oxide (NO) donor derivatives (3a-f) and evaluated their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging 0.3-30.3%. Compound 3d was the most effective HbF inducer for CD34+ cells, exhibiting an effect similar to that of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-α levels in human mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer prototype with the potential to treat SCD symptoms.

Discovery of KRas G12C-IN-3 and Pomalidomide-based PROTACs as degraders of endogenous KRAS G12C with potent anticancer activity.

A series of KRAS G12C-targeting PROTACs (PROteolysis TArgeting Chimeras) were designed and synthesized based on KRas G12C-IN-3 (a KRAS G12C inhibitor) and pomalidomide as degraders of KRAS G12C with a molecular weight of < 900. Among them, compound KP-14 (m.w. = 852.16; tPSA = 174.53) showed the highest KRAS G12C-degrading capability in NCI-H358 cancer cells (DC50≈1.25 µM). KP-14 bound to KRAS G12C through the acrylamide warhead and recruited the E3 ligase CRBN, causing rapid and sustained KRAS G12C degradation which led to suppression of MAPK signaling pathway in NCI-H358 cells. In addition, KP-14 selectively induced the degradation of KRAS G12C but not other KRAS isoforms such as G13D via PROTAC mechanism. Furthermore, KP-14 exhibited potent antiproliferative activity against NCI-H358 cancer cells and was able to suppress the formation of NCI-H358 tumor colonies. Collectively, this work suggests that KP-14 may serve as a tool compound for exploring the degradation of KRAS G12C by PROTAC strategy and deserve further investigation as a potential anticancer agent.

Discovery of a new class of PROTAC BRD4 degraders based on a dihydroquinazolinone derivative and lenalidomide/pomalidomide.

BRD4 has emerged as an attractive target for anticancer therapy. However, BRD4 inhibitors treatment leads to BRD4 protein accumulation, together with the reversible nature of inhibitors binding to BRD4, which may limit the efficacy of BRD4 inhibitors. To address these problems, a protein degradation strategy based on the proteolysis targeting chimera (PROTAC) technology has been developed to target BRD4 recently. Herein, we present our design, synthesis and biological evaluation of a new class of PROTAC BRD4 degraders, which were based on a potent dihydroquinazolinone-based BRD4 inhibitor compound 6 and lenalidomide/pomalidomide as ligand for E3 ligase cereblon. Gratifyingly, several compounds showed excellent inhibitory activity against BRD4, and high anti-proliferative potency against human monocyte lymphoma cell line THP-1. Especially, compound 21 (BRD4 BD1, IC50 = 41.8 nM) achieved a submicromolar IC50 value of 0.81 µM in inhibiting the growth of THP-1 cell line, and was 4 times more potent than compound 6. Moreover, the mechanism study established that 21 could effectively induce the degradation of BRD4 protein and suppression of c-Myc. All of these results suggested that 21 was an efficacious BRD4 degrader for further investigation.

Design, synthesis, and biological evaluation of new challenging thalidomide analogs as potential anticancer immunomodulatory agents.

Thalidomide and its analogs are immunomodulatory drugs that inhibit the production of certain inflammatory mediators associated with cancer. In the present work, a new series of thalidomide analogs was designed and synthesized to obtain new effective antitumor immunomodulatory agents. The synthesized compounds were evaluated for their cytotoxic activities against a panel of four cancer cell lines (HepG-2, HCT-116, PC3 and MCF-7). Compounds 33h, 33i, 42f and 42h showed strong potencies against all tested cell lines with IC50 values ranging from 14.63 to 49.90 µM comparable to that of thalidomide (IC50 values ranging from 32.12 to 76.91 µM). The most active compounds were further evaluated for their in vitro immunomodulatory activities via estimation of human tumor necrosis factor alpha (TNF-α), human caspase-8 (CASP8), human vascular endothelial growth factor (VEGF), and nuclear factor kappa-B P65 (NF-κB P65) in HCT-116 cells. Thalidomide was used as a positive control. Compounds 33h and 42f showed a significant reduction in TNF-α. Furthermore, compounds 33i and 42f exhibited significant elevation in CASP8 levels. Compounds 33i and 42f inhibited VEGF. In addition, compound 42f showed significant decrease in levels of NF-κB p65. Moreover, apoptosis and cell cycle tests of the most active compound 42f, were performed. The results indicated that compound 42f significantly induce apoptosis in HCT-116 cells and arrest cell cycle at the G2/M phase.

Pomalidomide hybrids act as proteolysis targeting chimeras: Synthesis, anticancer activity and B-Raf degradation.

As the first intracellular signaling molecule and the most frequently mutated oncogene, B-Raf represents an important target in cancer therapy. Here we report several pomalidomide hybrids acting as proteolysis targeting chimeras (PROTACs) for the degradation of B-Raf. Due to its high expression of B-Raf, MCF-7 cells are sensitive to these compounds. Among them, compound 2 can effectively kill cancer cells via inducing cells apoptosis. As a B-Raf degrader, compound 2 can accelerate the degradation of B-Raf by recruiting ubiquitin-proteasome system, and further affects the expression of Mcl-1, a downstream protein of B-Raf. The anticancer mechanism of compound 2 is quite different from its mother compound and cancer cells seem to be more sensitive to the degrader, hinting that degradation of B-Raf by PROTAC is a potential way for cancer treatment.

Development of the first small molecule histone deacetylase 6 (HDAC6) degraders.

Histone deacetylases (HDACs) decrease the acetylation level of histones and other non-histone proteins. Over expression of HDACs have been observed in cancers and other diseases. Targeted protein degradation by "hijacking" the natural ubiquitin-proteasome-system (UPS) recently emerged as a novel technology to "knock-out" endogenous disease-causing proteins. We applied this strategy to the development of the first small molecule degraders for zinc-dependent HDACs by conjugating non-selective HDAC inhibitors with E3 ubiquitin ligase ligands. Through cell-based assays, we discovered novel bifunctional molecules (dHDAC6) that could selectively degrade HDAC6. Further mechanistic studies indicated that HDAC6 was selectively removed by the UPS.

Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology.

Design, synthesis and biological evaluation of Lenalidomide derivatives as tumor angiogenesis inhibitor.

Lenalidomide is a type of immunomodulatory agent with anti-tumor activity by mainly expressed in the anti-angiogenesis. In order to enhance the pharmacological activity of Lenalidomide, a series of Lenalidomide derivatives were designed as tumor angiogenesis inhibitors. The potential anti-angiogenesis targets of Lenalidomide derivatives were virtual screened on Auto-Dock 4.0 by using reverse docking method. The six target proteins, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, BCR-ABL tyrosine kinase, p38 mitogen activated protein kinase and metal protein kinase, were chosen as the targets. The Lenalidomide derivatives were synthesized by alkylated, acylated or sulfonylated Lenalidomide and verified by the 1H NMR, 13C NMR and LC-MS. Their anti-cancer activities were detected by using CCK-8 in the esophageal carcinoma cell line EC9706. The results indicate that the inhibitory activities of Lenalidomide derivatives were higher than that of Lenalidomide.

Stereoselective Synthesis of (R)-3-Methylthalidomide by Piperidin-2-one Ring Assembly Approach.

A simple and stereoselective synthesis of 3-methylthalidomide, a configurationally stable thalidomide analog, is presented. Herein we describe the synthesis of (R)-3-methylthalidomide starting from (S)-alanine by piperidin-2-one ring assembly approach in high yield and enantiomeric purity without using a chiral auxiliary or reagent. Starting from (R)-alanine, the corresponding (S)-3-methylthalidomide can be prepared using the same methodology.

Efficient palladium-catalyzed double carbonylation of o-dibromobenzenes: synthesis of thalidomide.

We describe here a convenient and mild procedure for double carbonylation of o-dibromobenzenes with various 2-amino pyridines and naturally occurring amines, thus providing in good to excellent yields N-substituted phthalimides by using this palladium-catalyzed carbonylation procedure. Furthermore, for the first time we have applied the developed synthetic protocol for the synthesis of biologically active molecule thalidomide via a single step carbonylative cyclization reaction in excellent yield.

Synthesis and evaluation of thalidomide and phthalimide esters as antitumor agents.

A series of thalidomide and phthalimide ester analogs were efficiently synthesized from N-chloromethylthalidomide, N-chloromethylphthalimide, and N-(2-bromoethyl)phthalimide derivatives with various biologically important carboxylic acids. The synthesized compounds were purified and characterized by various chromatographic and spectroscopic techniques. The antitumor activity of all the synthesized compounds was screened against human liver and breast cancer cells, which showed that phthalimide ester 6a was the best cytotoxic compound against MCF7 cells, while all of the tested compounds showed a non-cytotoxic effect against HepG2 cells. Compounds 5a, 6a, and 7a possess immunosuppressant effect, while compounds 5c, 5d, 6c, 6d, 7c, and 7d showed an immunostimmulatory effect. Meanwhile, estimation of the binding affinity for all the synthesized compounds toward the vascular endothelial growth factor receptor (VEGFR) showed that compounds 5a, 5b, and 7d were the most potent inhibitors.

Structure-activity relationship studies of thalidomide analogs with a taxol-like mode of action.

Anti-microtubule agents such as paclitaxel and docetaxel have played an important role in the treatment of cancer for many years. Recently, a small molecule that has a taxol-like mode of action (5HPP-33) was reported. Herein, the detailed structure-activity relationship (SAR) studies of 5HPP-33 analogs that are substituted at the isoindole and phenyl rings are described. Bulky substitutions (such as di-isopropyl groups) on the phenyl ring result in the isoindole and phenyl rings being perpendicular to each other. It was found that this conformation is critical for anti-microtubule activity. These studies have provided valuable information, which will be helpful in the design of more potent analogs.

Preparation, characterization, and in vitro intestinal permeability evaluation of thalidomide-hydroxypropyl-ß-cyclodextrin complexes.

Thalidomide is emerging as a therapeutic agent with renewed clinical importance, presenting anti-inflammatory, immunomodulatory, and antineoplasic properties. In this work, we studied the complexation of thalidomide with cyclodextrins as a strategy to circumvent the poor aqueous solubility of the drug. Thalidomide-hydroxypropyl-ß-cyclodextrin complexes were obtained by kneading method and were characterized by differential scanning calorimetry, powder X-ray diffractometry, and scanning electronic microscopy. The aqueous solubility and in vitro dissolution of thalidomide were significantly improved through the complexation. Physicochemical analysis of the complexes in solid state revealed a decreased crystallinity of the complexed drug in comparison with free thalidomide. Thalidomide was able to dissociate from the complexes and permeates across intestinal epithelial Caco-2 cells with a favorable high permeability profile equivalent to that of the free drug. In summary, the present results suggest that thalidomide-hydroxypropyl-ß-cyclodextrin complexes could be regarded as a promising strategy for improving the gastrointestinal absorption of thalidomide.

Design, synthesis and biological evaluation of new thalidomide analogues as TNF-α and IL-6 production inhibitors.

Several thalidomide analogues were synthesized and compared to thalidomide and its more active analogue, lenalidomide, for their ability to inhibit the production of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and interleukin (IL)-6 by LPS-activated peripheral blood mononuclear cells (PBMCs). Among these compounds, two analogues containing sulfonyl group displayed interesting downregulation of TNF-α and IL-6 production.

Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity.

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 µM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 µM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease.

Design, synthesis and biological evaluation of thioether-containing lenalidomide and pomalidomide derivatives with anti-multiple myeloma activity.

Lenalidomide and its analogs are well-known for treating multiple myeloma. In this work, designed sulfide-modified lenalidomide and pomalidomide were synthesized and evaluated. The anti-proliferative activity against MM.1S cell line of 3ak (IC50 = 79 nM) was similar to lenalidomide (IC50 = 81 nM). Compared to benzylic thioether substituted lenalidomide 3a, the half-live (T1/2) of 4-F-phenyl-thioether analogs 3ak in human liver microsomes was promoted from 3 min to 416.7 min. The corresponding metabolic factor of 3ak was increased from 2.8% to 79.5%, which was slightly lower than lenalidomide (91.5%). Moreover, the IKZF1 degradation of 3y and 3ak was well related with corresponding IC50 values, which suggested the IKZF1 degradation efficiency is correlated to the responses of MM1. S cells. Furthermore, the oral administration of compounds 3y and 3ak at dosages of 60 mg/kg could delay tumor growth in female CB-17 SCID mice. This research helped to prompt the stability of thioether lenalidomide analogs, which paved the way for developing better molecules for treating multiple myeloma.

Folate-Guided Protein Degradation by Immunomodulatory Imide Drug-Based Molecular Glues and Proteolysis Targeting Chimeras.

Molecular glues and proteolysis targeting chimeras (PROTACs) are promising new therapeutic modalities. However, the lack of specificity for molecular glue- or PROTAC-mediated proteolysis in cancer cells versus normal cells raises potential toxicity concerns that will likely limit their clinical applications. Here, we developed a general strategy to deliver immunomodulatory imide drug (IMiD)-based molecular glues and PROTACs to folate receptor α (FOLR1)-positive cancer cells. Specifically, we designed a folate-caged pomalidomide prodrug, FA-S2-POMA, by incorporating a folate group as a caging and guiding element and validated its degradation effect on its neo-substrates in FOLR1-positive cancer cells in a FOLR1-dependent manner. We also developed a folate-caged pomalidomide-based anaplastic lymphoma kinase (ALK) PROTAC, FA-S2-MS4048, which effectively degraded ALK fusion proteins in cancer cells, again in a FOLR1-dependent manner. This novel approach provides a generalizable platform for the targeted delivery of IMiD-based molecular glues and PROTACs to FOLR1-expressing cancer cells with the potential to ameliorate toxicity.

Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders.

SHP2, a non-receptor tyrosine phosphatase, plays a pivotal role in numerous oncogenic cell-signaling cascades like RAS-ERK, PI3K-AKT and JAK-STAT. On the other hand, proteolysis targeting chimera (PROTAC) has emerged as a promising strategy for the degradation of disease-related protein of interest (POI). SHP2 degradation via the PROTAC strategy will provide an alternative startegy for SHP2-mediated cancer therapy. Herein we described the design, synthesis and evaluation of a series of thalidomide-based heterobifunctional molecules and identified 11(ZB-S-29) as the highly efficient SHP2 degrader with a DC50 of 6.02 nM. Further mechanism investigation illustrated that 11 came into function through targeted SHP2 protein degradation.