Synthesis of novel thiazolidinic-phthalimide derivatives evaluated as new multi-target antiepileptic agents.

Epilepsy is a disease that affects millions of people around the globe and has a multifactorial cause. Inflammation is a process that can be involved in the development of seizures. Thus, the present study proposed the design and synthesis of new candidates for antiepileptic drugs that would also control the inflammatory process. Nine new derivatives of the substituted thiazophthalimide hybrid core were obtained with satisfactory purity ≥99% and yields between 27% and 87%. All compounds showed cell viability values greater than 90% in the culture of PBMC cells from healthy volunteers and, therefore, were not considered cytotoxic. These compounds modulated proinflammatory cytokines IFN-y and IL-17A and can mitigate inflammation. Acute toxicity studies of compound 7i in an animal model indicated that the compound has low toxicity and an LD50 greater than 2 g/kg in healthy adult rats. The same compound did not show positive results for anticonvulsant activity through the PTZ test. However, 7i demonstrates the interaction with the target GABA-A receptor in silico, indicating a possible activity as an agonist of that receptor. Thus, further studies are needed to investigate the anticonvulsant activity, in particular, using models in which the inflammatory process triggers epileptic seizures.

Pd-Catalyzed C(sp2)-H olefination: synthesis of N-alkylated isoindolinone scaffolds from aryl amides of amino acid esters.

Isoindolinone is a constituent of various natural products and synthetic biologically active compounds. The classical multi-step synthetic methods used to prepare various indolinone derivatives are tedious and challenging. One-pot synthetic methods are attractive and economical. Transition-metal-catalyzed C-H activation is an emerging tool for synthesizing natural products and small organic molecules via reducing the number of synthetic steps necessary. This paper describes the synthesis of N-alkyl-3-methenyl chiral isoindolinone derivatives from aryl amides of L-amino acids and non-activated alkene via Pd-catalyzed C(sp2)-H olefination. Herein, the amino acid residue acts as a directing group for olefination at the aryl ring, and then cyclization occurs at the amide NH. Hence, this methodology could be helpful to transform standard amino acids into respective chiral isoindolinone derivatives.

Discovery of ARD-2585 as an Exceptionally Potent and Orally Active PROTAC Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report herein the discovery of exceptionally potent and orally bioavailable PROTAC AR degraders with ARD-2585 being the most promising compound. ARD-2585 achieves DC50 values of ≤0.1 nM in the VCaP cell line with AR gene amplification and in the LNCaP cell line carrying an AR mutation. It potently inhibits cell growth with IC50 values of 1.5 and 16.2 nM in the VCaP and LNCaP cell lines, respectively, and achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. It is more efficacious than enzalutamide in inhibition of VCaP tumor growth and does not cause any sign of toxicity in mice. ARD-2585 is a promising AR degrader for extensive investigations for the treatment of advanced prostate cancer.

De novo Assembly of the Benzenoid Ring as a Core Strategy for Synthesis of the Isoindolinone Natural Products Isohericerin, Erinacerin A, and Sterenin A.

Here we describe the use of the hexadehydro-Diels-Alder (HDDA) reaction for the de novo construction of the isoindolinone scaffold and its application to the synthesis of the title natural products. The key isoindolinone-forming HDDA reaction involved an unprecedented substrate motif in which an amide carbonyl group was conjugated to the 4π 1,3-diyne component. In addition, a dimethylsilyl (-SiMe2H) substituent was exploited to trigger a Fleming-Tamao-Kumada oxidation for the installation of an essential phenolic hydroxyl group.

Biotransformations of anthranilic acid and phthalimide to potent antihyperlipidemic alkaloids by the marine-derived fungus Scedosporium apiospermum F41-1.

A new diphenylamine derivative, scediphenylamine A (1), together with six phthalimide derivatives (2-7) and ten other known compounds (8-17) were obtained from the marine-derived fungus Scedosporium apiospermum F41-1 fed with synthetically prepared anthranilic acid and phthalimide. The structure and absolute configuration of the new compound were determined by HRMS, NMR, and X-ray crystallography. Evaluation of their lipid-lowering effect in 3T3-L1 adipocytes showed that scediphenylamine A (1), N-phthaloyl-tryptophan-methyl ester (4), 5-(1,3-dioxoisoindolin-2-yl) pentanamide (5), perlolyrine (10) and flazine (11) significantly reduced triglyceride level in 3T3-L1 cells by inhibiting adipogenic differentiation and synthesis with the EC50 values of 4.39, 2.79, 3.76, 0.09, and 4.52 µM, respectively. Among them, perlolyrine (10) showed the most potent activity, making it a candidate for further development as a potential agent to treat hyperlipidemia.

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC50 around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.

Discovery of a new series of PI3K-δ inhibitors from Virtual Screening.

PI3K-δ mediates key immune cell signaling pathways and is a target of interest for treatment of oncological and immunological disorders. Here we describe the discovery and optimization of a novel series of PI3K-δ selective inhibitors. We first identified hits containing an isoindolinone scaffold using a combined ligand- and receptor-based virtual screening workflow, and then improved potency and selectivity guided by structural data and modeling. Careful optimization of molecular properties led to compounds with improved permeability and pharmacokinetic profile, and high potency in a whole blood assay.

A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Design and Synthesis of EZH2-Based PROTACs to Degrade the PRC2 Complex for Targeting the Noncatalytic Activity of EZH2.

EZH2 mediates both PRC2-dependent gene silencing via catalyzing H3K27me3 and PRC2-independent transcriptional activation in various cancers. Given its oncogenic role in cancers, EZH2 has constituted a compelling target for anticancer therapy. However, current EZH2 inhibitors only target its methyltransferase activity to downregulate H3K27me3 levels and show limited efficacy because of inadequate suppression of the EZH2 oncogenic activity. Therefore, therapeutic strategies to completely block the oncogenic activity of EZH2 are urgently needed. Herein, we report a series of EZH2-targeted proteolysis targeting chimeras (PROTACs) that induce proteasomal degradation of PRC2 components, including EZH2, EED, SUZ12, and RbAp48. Preliminary assessment identified E7 as the most active PROTAC molecule, which decreased PRC2 subunits and H3K27me2/3 levels in various cancer cells. Furthermore, E7 strongly inhibited transcriptional silencing mediated by EZH2 dependent on PRC2 and transcriptional activation mediated by EZH2 independent of PRC2, showing significant antiproliferative activities against cancer cell lines dependent on the enzymatic and nonenzymatic activities of EZH2.

Discovery of Proteolysis-Targeting Chimera Molecules that Selectively Degrade the IRAK3 Pseudokinase.

We report the first disclosure of IRAK3 degraders in the scientific literature. Taking advantage of an opportune byproduct obtained during our efforts to identify IRAK4 inhibitors, we identified ready-to-use, selective IRAK3 ligands in our compound collection with the required properties for conversion into proteolysis-targeting chimera (PROTAC) degraders. This work culminated with the discovery of PROTAC 23, which we demonstrated to be a potent and selective degrader of IRAK3 after 16 h in THP1 cells. 23 induced proteasome-dependent degradation of IRAK3 and required both CRBN and IRAK3 binding for activity. We conclude that PROTAC 23 constitutes an excellent in vitro tool with which to interrogate the biology of IRAK3.

Dual Parasiticidal Activities of Phthalimides: Synthesis and Biological Profile against Trypanosoma cruzi and Plasmodium falciparum.

Chagas disease and malaria are two neglected tropical diseases (NTDs) that prevail in tropical and subtropical regions in 149 countries. Chagas is also present in Europe, the US and Australia due to immigration of asymptomatic infected individuals. In the absence of an effective vaccine, the control of both diseases relies on chemotherapy. However, the emergence of parasite drug resistance is rendering currently available drugs obsolete. Hence, it is crucial to develop new molecules. Phthalimides, thiosemicarbazones, and 1,3-thiazoles have been used as scaffolds to obtain antiplasmodial and anti-Trypanosoma cruzi agents. Herein we present the synthesis of 24 phthalimido-thiosemicarbazones (3 a-x) and 14 phthalimido-thiazoles (4 a-n) and the corresponding biological activity against T. cruzi, Plasmodium falciparum, and cytotoxicity against mammalian cell lines. Some of these compounds showed potent inhibition of T. cruzi at low cytotoxic concentrations in RAW 264.7 cells. The most active compounds, 3 t (IC50 =3.60 µM), 3 h (IC50 =3.75 µM), and 4 j (IC50 =4.48 µM), were more active than the control drug benznidazole (IC50 =14.6 µM). Overall, the phthalimido-thiosemicarbazone derivatives were more potent than phthalimido-thiazole derivatives against T. cruzi. Flow cytometry assay data showed that compound 4 j was able to induce necrosis and apoptosis in trypomastigotes. Analysis by scanning electron microscopy showed that T. cruzi trypomastigote cells treated with compounds 3 h, 3 t, and 4 j at IC50 concentrations promoted changes in the shape, flagella, and surface of the parasite body similar to those observed in benznidazole-treated cells. The compounds with the highest antimalarial activity were the phthalimido-thiazoles 4 l (IC50 =1.2 µM), 4 m (IC50 =1.7 µM), and 4 n (IC50 =2.4 µM). Together, these data revealed that phthalimido derivatives possess a dual antiparasitic profile with potential effects against T. cruzi and lead-like characteristics.

Discovery of phthalimide derivatives as novel inhibitors of a soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) inhibitors are effective in reducing blood pressure, inflammation, and pain in a number of mammalian disease models. As most classical urea-based sEH inhibitors suffer from poor solubility and pharmacokinetic properties, the development of novel sEH inhibitors with an improved pharmacokinetic specification has received a great deal of attention. In this study, a series of amide-based sEH inhibitors bearing a phthalimide ring as the novel secondary pharmacophore (P2 ) was designed, synthesized, and evaluated. Docking results illustrated that the amide group as the primary pharmacophore (P1 ) was placed at a suitable distance from the three key amino acids (Tyr383, Tyr466, and Asp335) for an effective hydrogen bonding. In agreement with these findings, most of the newly synthesized compounds demonstrated moderate to high sEH inhibitory activities, relative to 12-(3-adamantan-1-yl-ureido)dodecanoic acid as the reference standard. Compound 12e with a 4-methoxybenzoyl substituent exhibited the highest sEH inhibitory activity, with an IC50 value of 1.06 nM. Moreover, the ADME properties of the compounds were evaluated in silico, and the results revealed appropriate predictions.

Chiral Phase Transfer Catalysis in the Asymmetric Synthesis of a 3,3-Disubstituted Isoindolinone and Determination of Its Absolute Configuration by VCD Spectroscopy.

In this work we report our endeavors toward the development of an asymmetric synthesis of a 3,3-disubstituted isoindolinone, dimethyl 2-(1-methyl-3-oxoisoindolin-1-yl)malonate, via asymmetric cascade reaction of 2-acetylbenzonitrile with dimethylmalonate and the determination of its absolute configuration (AC) by vibrational circular dichroism (VCD). Bifunctional ammonium salts, derived from trans-1,2-cyclohexanediamine in combination with inorganic bases under phase transfer conditions, were the most effective catalytic systems, leading to the target in high yields and moderate enantioselectivity. An efficient process of heterochiral crystallization allowed the increase of the enantiopurity up to 96% ee and in an acceptable overall yield. An important aim of the present work is the comparison of different VCD methodologies for AC determination of the target compound.

Substituted adamantylphthalimides: Synthesis, antiviral and antiproliferative activity.

In this study, three groups of adamantylphthalimides, bearing different substituents at the phthalimide moiety, N-(4'-R2 )phthalimidoadamantanes (1-7), 3-[N-(4'-R2 )phthalimido]-1-adamantanols (8-10), and 3-[N-(4'-R2 )phthalimido]adamantane-1-carboxylic acids (11-15), were synthesized and screened against tumor cells and viruses. The most potent compounds are not substituted at the adamantane and bear an OH or NH2 substituent at the phthalimide (compounds 3 and 5). The antiproliferative activities of compounds 3 and 5 are in the micromolar range, much higher than the one of thalidomide. A minor antiviral activity against cytomegalovirus and varicella-zoster virus was found for compounds 3 and 5, but these compounds lacked selectivity. The results presented are important for the rational design of the next-generation compounds with anticancer and antiviral activities.

Advances in Synthesis and Medicinal Applications of Compounds Derived from Phthalimide.

Phthalimide derivatives have been presenting several promising biological activities in the literature, such as anti-inflammatory, analgesic, antitumor, antimicrobial and anticonvulsant. The most well-known and studied phthalimide derivative (isoindoline-1,3-dione) is thalidomide: this compound initially presented important sedative effects, but it is now known that thalidomide has effectiveness against a wide variety of diseases, including inflammation and cancer. This review approaches some of the recent and efficient chemical synthesis pathways to obtain phthalimide analogues and also presents a summary of the main biological activities of these derivatives found in the literature. Therefore, this review describes the chemical and therapeutic aspects of phthalimide derivatives.

Design, Synthesis, and Herbicidal Activity of Novel Diphenyl Ether Derivatives Containing Fast Degrading Tetrahydrophthalimide.

To seek new protoporphyrinogen oxidase (PPO) inhibitors with better biological activity, a series of novel diphenyl ether derivatives containing tetrahydrophthalimide were designed based on the principle of substructure splicing and bioisomerization. PPO inhibition experiments exhibited that 6c is the most potential compound, with the half-maximal inhibitory concentration (IC50) value of 0.00667 mg/L, showing 7 times higher activity than Oxyfluorfen (IC50 = 0.0426 mg/L) against maize PPO and similar herbicidal activities to Oxyfluorfen in weeding experiments in greenhouses and field weeding experiments. In view of the inspected bioactivities, the structure-activity relationship (SAR) of this series of compounds was also discussed. Crop selection experiments demonstrate that compound 6c is safe for soybeans, maize, rice, peanuts, and cotton at a dose of 300 g ai/ha. Accumulation analysis experiments showed that the accumulation of 6c in some crops (soybeans, peanuts, and cotton) was significantly lower than Oxyfluorfen. Current work suggests that compound 6c may be developed as a new herbicide candidate in fields.

Benzo[4,5]thieno[2,3-d]pyrimidine phthalimide derivative, one of the rare noncompetitive inhibitors of dipeptidyl peptidase-4.

A small library of benzo[4,5]thieno[2,3-d]pyrimidine phthalimide and amine derivatives was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4). The phthalimide derivatives exhibited better activity than the amine precursors, with 2-(2-(3-chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)isoindoline-1,3-dione (compound 14) as the most effective inhibitor (IC50 = 34.17 ± 5.11 µM). The five most potent selected inhibitors did not show cytotoxicity to a greater extent on Caco-2 cells, even at a concentration of 250 µM. Compound 14 is considered as a novel representative of the rare noncompetitive DPP-4 inhibitors. Molecular docking and dynamics simulation indicated the importance of the Tyr547, Lys554, and Trp629 residues of DPP-4 in the formation of the enzyme-inhibitor complex. These observations could be potentially utilized for the rational design and optimization of novel (structurally similar, with phthalimide moiety, or different) noncompetitive DPP-4 inhibitors, which are anyway rare, but favorable in terms of the saturation of substrate competition.

Design and Synthesis of N-phenyl Phthalimides as Potent Protoporphyrinogen Oxidase Inhibitors.

Protoporphyrinogen oxidase (PPO) has been identified as one of the most promising targets for herbicide discovery. A series of novel phthalimide derivatives were designed by molecular docking studies targeting the crystal structure of mitochondrial PPO from tobacco (mtPPO, PDB: 1SEZ) by using Flumioxazin as a lead, after which the derivatives were synthesized and characterized, and their herbicidal activities were subsequently evaluated. The herbicidal bioassay results showed that compounds such as 3a (2-(4-bromo-2,6-difluorophenyl) isoindoline-1,3-dione), 3d (methyl 2-(4-chloro-1,3-dioxoisoindolin-2-yl)-5-fluorobenzoate), 3g (4-chloro-2-(5-methylisoxazol-3-yl) isoindoline-1,3-dione), 3j (4-chloro-2-(thiophen-2-ylmethyl) isoindoline-1,3-dione) and 3r (2-(4-bromo-2,6-difluorophenyl)-4-fluoroisoindoline-1,3-dione) had good herbicidal activities; among them, 3a showed excellent herbicidal efficacy against A. retroflexus and B. campestris via the small cup method and via pre-emergence and post-emergence spray treatments. The efficacy was comparable to that of the commercial herbicides Flumioxazin, Atrazine, and Chlortoluron. Further, the enzyme activity assay results suggest that the mode of action of compound 3a involves the inhibition of the PPO enzyme, and 3a showed better inhibitory activity against PPO than did Flumioxazin. These results indicate that our molecular design strategy contributes to the development of novel promising PPO inhibitors.

[C-H Functionalization by Transition-metal-catalyst or in Situ Generated Base].

Direct functionalization of the C-H bond is a highly attractive method because it does not require any pre-functionalized starting materials. Therefore this method can be adopted to attain the atom- and step-economic synthesis of organic compounds. We recently developed novel C-H functionalization reactions using two strategies. The first strategy is transition-metal catalyzed C-H functionalization, which comprises the copper-catalyzed oxidative C(sp3)-H functionalization of 2-alkyl-N-arylbenzamides for the synthesis of N-aryl-isoindolinones. This method can be applied to various substituted substrates and the synthesis of bioactive compounds, indoprofen and DWP205190. It provides an efficient approach toward the construction of isoindolinone skeletons. The second strategy is an in situ generated base-mediated C-H functionalization comprising the deprotonative silylation of C(sp)-H and C(sp2)-H bonds by the base generated in situ from trifluoromethyltrimethylsilane (CF3SiMe3) and its activator. In the C(sp)-H silylation of terminal alkynes, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) acts as both a solvent and activator; therefore no additional activator is required. Moreover, the preferential deprotonation of terminal alkynes using this system proceeds faster than the trifluoromethylation of ketones and esters.

Aminonaphthalimide hybrids of mitoxantrone and amonafide as anticancer and fluorescent cellular imaging agents.

Novel water-soluble 4-aminonaphthalimides were synthesised and their cellular fluorescent imaging, cytotoxicity and ability to induced apoptosis evaluated. The lead compound 1 was designed from the cross-fertilisation of the basic hydrophilic amino pharmacophore of mitoxantrone, and an aminonaphthalimide scaffold of the drug candidate, amonafide. The compounds are also fluorescent pH probes based on photoinduced electron transfer (PET) and internal charge transfer (ICT). The compounds are sensitive to solvent polarity with large Stoke shifts (>90 nm) and provide emissive-coloured solutions (blue to yellow). Excited state pKas of 9.0-9.3 and fluorescence quantum yields of 0.47-0.58 were determined in water. The cytotoxicity and cellular fluorescent imaging properties of the compounds were tested on human cancer cell lines K562 and MCF-7 by the MTT assay, phase contrast and fluorescence microscopy. Compounds 1 and 3 with flexible aminoalkyl chains exhibited GI50 comparable to amonafide, while 2 and 4 with a rigid piperazine moiety and butyl chain are less cytotoxic. Fluorescence microscopy with 1 allowed for the visualization of the intracellular microenvironment exemplifying the potential utility of such hybrid molecules as anticancer and fluorescent cellular imaging agents.