Research progress of PROTACs for neurodegenerative diseases therapy.

Neurodegenerative diseases (NDD) are characterized by the gradual deterioration of neuronal function and integrity, resulting in an overall decline in brain function. The existing therapeutic options for NDD, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, fall short of meeting the clinical demand. A prominent pathological hallmark observed in numerous neurodegenerative disorders is the aggregation and misfolding of proteins both within and outside neurons. These abnormal proteins play a pivotal role in the pathogenesis of neurodegenerative diseases. Targeted degradation of irregular proteins offers a promising avenue for NDD treatment. Proteolysis-targeting chimeras (PROTACs) function via the ubiquitin-proteasome system and have emerged as a novel and efficacious approach in drug discovery. PROTACs can catalytically degrade "undruggable" proteins even at exceptionally low concentrations, allowing for precise quantitative control of aberrant protein levels. In this review, we present a compilation of reported PROTAC structures and their corresponding biological activities aimed at addressing NDD. Spanning from 2016 to present, this review provides an up-to-date overview of PROTAC-based therapeutic interventions. Currently, most protein degraders intended for NDD treatment remain in the preclinical research phase. Overcoming several challenges is imperative, including enhancing oral bioavailability and permeability across the blood-brain barrier, before these compounds can progress to clinical research or eventually reach the market. However, armed with an enhanced comprehension of the underlying pathological mechanisms and the emergence of innovative scaffolds for protein degraders, along with further structural optimization, we are confident that PROTAC possesses the potential to make substantial breakthroughs in the field of neurodegenerative diseases.

Novel 2,6-disubstituted benzofuran-3-one analogues improve cerebral ischemia/reperfusion injury via neuroprotective and antioxidative effects.

There are no highly effective and safe medicines for clinical treatment of ischemic stroke, although the natural product 3-n-butylphthalide (NBP) has been approved in China for mild and moderate ischemic stroke. To discover more potent anti-cerebral ischemic agents and overcome the low stability by phthalide derivatives, benzofuran-3-one was selected as a core moiety and two types of nitric oxide (NO)-donating groups were incorporated into the structure. In this work, a series of 2,6-disubstituted benzofuran-3-one derivatives were designed and synthesised as NBP analogues, and tested as neuroprotective and antioxidative agents. Compounds 5 (without an NO donor) and 16 (with an NO donor) displayed more potent neuroprotective effects than the established clinical drugs Edaravone and NBP. More importantly, 5 and 16 also exhibited good antioxidative activity without cytotoxicity in rat primary neuronal and PC12 cells. Most active compounds showed good blood-brain barrier permeability in a parallel artificial membrane permeability assay. Furthermore, compound 5 reduced the ischemic infarct area significantly in rats subjected to ischemia/reperfusion injury, downregulated ionised calcium-binding adaptor molecule 1 and glial fibrillary acidic protein in inflammatory cells, and upregulated nerve growth factor.

Design, synthesis and evaluation of anti-proliferative activity of 2-aryl-4-aminoquinazoline derivatives as EGFR inhibitors.

A class of 2-aryl-4-aminoquinazoline derivatives (7a-7j, 8a-8h, 9a-9h and 10a-10k) were designed, synthesized and evaluated as EGFR inhibitors. The anti-proliferative activity of compounds in vitro showed that compound 9e was considered to be a promising derivative. Compared with the lead compound Angew2017-7634-1, 9e exhibited excellent inhibitory activity against A549, NCI-H460 and H1975 cell lines, with IC50 values of 14.33 ± 1.16 µM, 17.81 ± 1.25 µM and 13.41 ± 1.14 µM, respectively. Moreover, 9e could effectively inhibit against Ba/F3-EGFRDel19/T790M/C797S cell lines. In the kinase experiment, the most promising compound 9e exhibited excellent enzymatic inhibitory activity and selectivity for EGFRL858R/T790M, with an IC50 value of 0.74 µM. Further activity studies showed that 9e could not only induce remarkable cell-apoptosis of A549, but also block A549 cell lines in S-phase in a concentration-dependent manner. Furthermore, molecular docking study revealed the binding mode of 9e. All in all, we analyzed the structure-activity relationship of the target compounds, and explored their mechanism of action.

Synthesis of Halogenated 1,5-Diarylimidazoles and Their Inhibitory Effects on LPS-Induced PGE2 Production in RAW 264.7 Cells.

A series of halogenated 1,5-diarylimidazole compounds were synthesized and their inhibitory effects on LPS-induced PGE2 production in RAW 264.7 cells were evaluated. A wide variety of 2,4-, 4-, and 2-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles were synthesized for SAR study via two different pathways. Overall, 4-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles, regardless of the species of halogen, exhibited very strong inhibitory activities of PGE2 production. Among them, 4-chloro-5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole (3, IC50 3.3 nM ± 2.93), and 4-chloro-5-(4-chlorophenyl)-1-(4-methylsulfonylphenyl)imidazole (13, IC50 5.3 nM ± 0.23) showed the best results.

Discovery of thiapyran-pyrimidine derivatives as potential EGFR inhibitors.

A series of novel thiapyran-pyrimidine derivatives (10a-10h, 11a-11g, 12a-12f, 13a-13f, 14a-14f) were synthesized and their antiproliferative activities were tested. Most of the target compounds showed good activity on one or more cancer cell lines but low activity on human normal cell LO2. The most promising compound 13a exhibited the similar IC50 values on A549 and H1975 cell lines to the lead drug Olmutinib, and exhibited excellent activity and selectivity on EGFRT790M/L858R in the kinase experiment. AO and Hoechst33258 staining indicated that 13a could effectively induce H1975 cells apoptosis. Cell cycle and apoptosis analysis suggested that 13a could block cancer cells in G2/M phase and induce into late apoptosis in a manner of concentration-dependent. The structure-activity relationship of 13a was analyzed to explore its mechanism. All the results showed that 13a was a promising EGFR inhibitor.

Design, synthesis and biological evaluation of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as potential dual PI3Kα/mTOR inhibitors.

The phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) have been regarded as promising targets for the treatment of cancer. Herein, we synthesized a new series of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as novel PI3Kα/mTOR dual inhibitors for cancer therapy. All compounds were evaluated for the IC50 values against three cancer cell lines (A549, MCF-7 and Hela). Most of the target compounds exhibited moderate to excellent anti-tumor activities against these three tested cancer cell lines especially against A549 and Hela cancer cell lines. Among them, the most promising compound 13g showed excellent anti-tumor potency for A549, MCF-7 and Hela cell lines with IC50 values of 0.20 ± 0.05 µM, 1.25 ± 0.11 µM and 1.03 ± 0.24 µM, respectively. Notably, according to the result of enzymatic activity assay, compound 13g was identified as a novel PI3Kα/mTOR dual inhibitor, which had an approximately 10-fold improvement in mTOR inhibition, compared to the class I PI3K inhibitor 1 (pictilisib, GDC-0941), with IC50 values of 525 nM to 48 nM. And western blot analysis indicated compound 13g could efficiently suppress the phosphorylation of AKT at the dose of 0.1 µM, which further demonstrated compound 13g had significant inhibitory effect on the PI3K/Akt/mTOR pathway. Furthermore, compound 13g could stimulate A549 cells arrest at G0/G1 phase in a dose-dependent manner, and induced apoptosis at a low concentration.

Design and synthesis of tetrahydropyridopyrimidine derivatives as dual GPR119 and DPP-4 modulators.

Based on the approach of merged pharmacophores of GPR119 agonists and DPP-4 inhibitors, a series of tetrahydropyridopyrimidine compounds were designed as dual GPR119 and DPP-4 modulators with hypoglycemic activity. Seven fragments extracted from DPP-4 inhibitors were hybridized with the scaffold of tetrahydropyridopyrimidine. Among them, compound 51 displayed most potent GPR119 agonistic activity (EC50 = 8.7 nM) and good inhibition rate of 74.5% against DPP-4 at 10 µM. Furthermore, the blood glucose AUC0-2h of 51 was reduced to 19.5% in the oral glucose tolerance test (oGTT) at the dose of 30 mg/kg in C57BL/6N mice, which was more potent than that of vildagliptin (16.4%) at the same dose. The docking study of compound 51 with DPP-4 indicated GPR119 agonists could inhibit DPP-4 to serve as dual GPR119 and DPP-4 modulators.

Optimisation of novel 4, 8-disubstituted dihydropyrimido[5,4-b][1,4]oxazine derivatives as potent GPR 119 agonists.

GPR119 is a promising target for discovery of anti-type 2 diabetes mellitus agents. We described the optimisation of a novel series of pyrimido[5,4-b][1,4]oxazine derivatives as GPR119 agonists. Most designed compounds exhibited good agonistic activities. Among them, compound 10 and 15 demonstrated the potent EC50 values (13 and 12 nM, respectively) and strong inherent activities. Moreover, significant hypoglycaemic effect of compound 15 was observed by reducing the blood glucose AUC0-2h at the dose of 30 mg/kg, which is stronger than Vildagliptin (23.4% reduction vs. 17.9% reduction).

Effects of novel pyrrolomycin MP1 in MYCN amplified chemoresistant neuroblastoma cell lines alone and combined with temsirolimus.

BACKGROUND: The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM). METHODS: Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated. RESULTS: The IC50 of MP1 was 0.096 µM in BE-2c cells compared to 0.89 µM in IMR, and >50 µM in SKN-AS. The IC50 of MP1 plus TEM in BE-2c cells was 0.023 µM. MP1 inhibited metabolism leading to quiescence and produced a decline in cell cycle S-phase. Electron microscopy showed cristae loss and rounding up of mitochondria. Gene and protein expression for MYCN and MCL-1 declined while LCII and cleaved PARP increased. Protein expression of BAX, bcl-2, and BRD-4 were not significantly changed after MP1 treatment. The in-vivo concentrations of MP1 in blood and tumor were sufficient to produce the biologic effects seen in-vitro. MP1 plus TEM produced a complete response in 3 out of 5 tumor bearing mice. In a second mouse study, the combination of MP1 and TEM slowed tumor growth compared to control. CONCLUSIONS: MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.

Synthesis and evaluation of novel fused pyrimidine derivatives as GPR119 agonists.

A novel series of fused pyrimidine derivatives were designed, synthesized and evaluated as GPR119 agonists. Among them, cyclohexene fused compounds (tetrahydroquinazolines) showed greater GPR119 agonistic activities than did dihydrocyclopentapyrimidine and tetrahydropyridopyrimidine scaffolds. Analogues (16, 19, 26, 28, 42) bearing endo-N-Boc-nortropane amine and fluoro-substituted aniline exhibited better EC50 values (0.27-1.2 µM) though they appeared to be partial agonists.

Design, Synthesis, and Biological Evaluation of Novel Thienopyrimidine Derivatives as PI3Kα Inhibitors.

Three series of novel thienopyrimidine derivatives 9a-l, 15a-l, and 18a-h were designed and synthesized, and their IC50 values against four cancer cell lines HepG-2, A549, PC-3, and MCF-7 were evaluated. Most compounds show moderate cytotoxicity against the tested cancer cell lines. The most promising compound 9a showed moderate activity with IC50 values of 12.32 ± 0.96, 11.30 ± 1.19, 14.69 ± 1.32, and 9.80 ± 0.93 µM, respectively. The inhibitory activities of compounds 9a and 15a against PI3Kα and mTOR kinase were further evaluated. Compound 9a exhibited PI3Kα kinase inhibitory activity with IC50 of 9.47 ± 0.63 µM. In addition, docking studies of compounds 9a and 15a were also investigated.

Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2'-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

Design and synthesis of novel senkyunolide analogues as neuroprotective agents.

A class of senkyunolide analogues bearing benzofuranone fragment were designed, synthesized and evaluated for their neuroprotective effect in models of oxygen glucose deprivation (OGD) and oxidative stress. All tested compounds showed neuroprotection profile based on the cell viability assay. In particular, derivatives 1f-1i possessing furoxan-based nitric oxide releasing functionality exhibited significant biological activities in OGD models. More importantly, compound 1g containing short linker with furoxan displayed the most potent neuroprotection at the concentration of 100 µM (cell survival up to 145.2%). Besides, 1g also showed the middle level neuroprotective effect in model of oxidative stress.

Design and synthesis of novel pyrimido[5,4-d]pyrimidine derivatives as GPR119 agonist for treatment of type 2 diabetes.

We described the discovery and optimization of a novel series of pyrimidopyrimidine derivatives as G-protein coupled receptor 119 (GPR119) agonists against type 2 diabetes. Most designed compounds displayed significant GPR119 agonistic activities. Optimized analogues 15a and 21e exhibited highly potent agonistic activities with single digit EC50 values (2.2 nM and 8.1 nM, respectively). Therefore, 15a and 21e were evaluated for their oral glucose tolerance test (oGTT) in C57BL/6N mice. Compound 15a reduced the blood glucose area of under curve from 0 to 2 h (AUC0-2h) to 13.5% at the dose of 15 mg/kg comparing with Metformin reduced 18% of AUC0-2h at the dose of 300 mg/kg.

Coumarin-dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against Alzheimer's disease: Design, synthesis and biological evaluation.

A series of new coumarin-dithiocarbamate hybrids were designed and synthesized as multitarget agents for the treatment of Alzheimer's disease. Most of them showed potent and clearly selective inhibition towards AChE and MAO-B. Among these compounds, compound 8f demonstrated the most potent inhibition to AChE with IC50 values of 0.0068 µM and 0.0089 µM for eeAChE and hAChE, respectively. Compound 8g was identified as the most potent inhibitor to hMAO-B, and it is also a good and balanced inhibitor to both hAChE and hMAO-B (0.114 µM for hAChE; 0.101 µM for hMAO-B). Kinetic and molecular modeling studies revealed that 8g was a dual binding site inhibitor for AChE and a competitive inhibitor for MAO-B. Further studies indicated that 8g could penetrate the BBB and exhibit no toxicity on SH-SY5Y neuroblastoma cells. More importantly, 8g did not display any acute toxicity in mice at doses up to 2500 mg/kg and could reverse the cognitive dysfunction of scopolamine-induced AD mice. Overall, these results highlighted 8g as a potential multitarget agent for AD treatment and offered a starting point for design of new multitarget AChE/MAO-B inhibitors based on dithiocarbamate scaffold.

Design, Synthesis and Biological Evaluation of Novel Phenylsulfonylurea Derivatives as PI3K/mTOR Dual Inhibitors.

Five series of novel phenylsulfonylurea derivatives, 19a⁻d, 20a⁻d, 21a⁻d, 22a⁻d and 23a⁻d, bearing 4-phenylaminoquinoline scaffold were designed, synthesized and their IC50 values against four cancer cell lines (HepG-2, A549, PC-3 and MCF-7) were evaluated. Most compounds showed moderate cytotoxicity activity against the cancer cell lines. Structure⁻activity relationships (SARs) and pharmacological results indicated that introduction of 4-aminoquinoline scaffold and phenylsulfonylurea scaffold were beneficial for anti-tumor activity. Moreover, para-methoxyl substitution of 4-anilino moiety and para-halogen substitution of phenylsulfonylurea have different impacts on different series of compounds. Furthermore, the micromolecule group substitution in the 6-position of the quinoline ring have a slight impact on the cellular activity of the target compounds.

Synthesis and biological evaluation of pyrimidine derivatives with diverse azabicyclic ether/amine as novel GPR119 agonist.

A class of novel pyrimidine derivatives bearing diverse conformationally restricted azabicyclic ether/amine were designed, synthesized and evaluated for their GPR119 agonist activities against type 2 diabetes. Most compounds exhibited superior hEC50 values to endogenous lipid oleoylethanolamide (OEA). Analogs with 2-fluoro substitution in the aryl ring showed more potent GPR119 activation than those without fluorine. Especially compound 27m synthesized from endo-azabicyclic alcohol was observed to have the best EC50 value (1.2nM) and quite good agonistic activity (112.2% max) as a full agonist.

Nitric oxide-donating derivatives of hederacolchiside A1: Synthesis and biological evaluation in vitro and in vivo as potential anticancer agents.

A series of nitric oxide (NO) donating derivatives of hederacolchiside A1 bearing triterpenoid saponin motif were designed, synthesized and evaluated for their anticancer activity. All of the tested furoxan-based NO releasing compounds showed significant proliferation inhibitory activities. Especially compound 6a exhibited strong cytotoxicity (IC50=1.6-6.5µM) against four human tumor cell lines (SMMC-7721, NCI-H460, U251, HCT-116) in vitro and the highest level of NO releasing. Furthermore, compound 6a was revealed low acute toxicity to mice and weak haemolytic activity with potent tumor growth inhibition against mice H22 hepatocellular cells in vivo (51.5%).

Novel 5-nitropyrimidine derivatives bearing endo-azabicyclic alcohols/amines as potent GPR119 agonists.

A series of GPR119 agonists based on a 5-nitropyrimidine scaffold bearing endo-azabicyclic substituents were synthesized and evaluated for their GPR119 agonistic activities. Most compounds exhibited much stronger EC50 values than that of oleoylethanolamide (OEA). Among them, derivatives from endo-azabicyclic alcohols displayed more potent GPR119 agonistic activities than compounds with endo-azabicyclic amines. Especially the optimized compounds (6, 7, 8, 12, 17) were shown to have potent biological activities and were identified as full agonists. Isopropyl carbamate compound 8 synthesized from endo-azabicyclic alcohol was observed to have the best EC50 value (0.6nM). Generally 2-fluoro substitution of the aryl group at the C4 position of 5-nitropyrimidine scaffold resulted in the increase of biological activity.

Synthesis and biological evaluation of Hederacolchiside A1 derivatives as anticancer agents.

Modification of Hederacolchiside A1 (HA1) on 28-COOH gave a series of novel triterpenoid saponin compounds containing ester or amide group. Comparing with natural product HA1, several derivatives showed decreased toxicity in the mice acute toxicity trial and increased the anticancer activity in vitro. Especially compound 1 exhibited the strongest antiproliferative activities against human cancer cell lines tested (IC50=1.1-4.6µM) and potent tumor inhibition rate in vivo (46.8%).