Lupenone-Rich Fraction Derived from Cissus quadrangularis L. Suppresses Lipid Accumulation in 3T3-L1 Adipocytes.

Cissus quadrangularis L. (CQ) has potential as a therapeutic for managing obesity and balancing metabolic activity, but the main bioactive compound and regulatory mechanism remain unknown. Herein, the CQ hexane extract was fractionated into 30 fractions (CQ-H) using flash column chromatography and analyzed using thin-layer chromatography. The direct antiadipogenesis effect of CQ-H fractions was tested on 3T3-L1 preadipocytes. Lupenone-rich fractions 2H and 3H were identified as containing potent antiadipogenesis agents that reduced differentiated cell numbers and intracellular lipid droplet size. Although the overall mitochondrial density remained unchanged, differentiated cells exhibited a higher mitochondrial density than that in non-differentiated cells. Additionally, 2H increased mitochondrial activity in both cell types as shown by their differentiation and lipid formation stages. Lupenone was isolated from 2H (Lu-CQ) and shown to dose-dependently inhibit adipogenesis, with 2H being more potent than Lu-CQ. Lu-CQ and 2H downregulated the expression of Pparg2 mRNA and upregulated that of glucose transporter genes, Slc2a1 and Slc2a4. Lu-CQ and 2H induced increased glucose uptake by 3T3-L1 cells. These findings suggest that lupenone-rich fractions in CQ contribute to balancing metabolic activity and reducing adipose tissue formation. Further exploration of CQ and its components may prompt innovative strategies for managing obesity and metabolic disorders.

meta-Ureidophenoxy-1,2,3-triazole hybrid as a novel scaffold for promising HepG2 hepatocellular carcinoma inhibitors: Synthesis, biological evaluation and molecular docking studies.

Thirty-one meta-ureidophenoxymethyl-1,2,3-triazole derivatives were designed and synthesized via nucleophilic addition, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition (CuAAC). The evaluation of their cytotoxicity using MTT assay indicated that almost all derivatives exhibited significantly superior inhibitory activity against hepatocellular carcinoma cell line HepG2 compared to the parental molecule sorafenib (1). Among the series, 5r was the most potent anti-HepG2 agent with IC50 = 1.04 µM, which was almost 5-fold more active than sorafenib (IC50 = 5.06 µM), while the cytotoxic activity against human embryonal lung fibroblast cell line MRC-5 remained comparable to sorafenib. The synthetic derivative 5r, thus, possessed 5.2-time higher selectivity index (SI) than that of sorafenib. Molecular docking studies revealed an efficient interaction of 5r at the same sorafenib's binding region in both B-Raf and VEGFR-2 with lower binding energies than those of sorafenib, consistent with its cytotoxic effect. Furthermore, 5r was proven to induce apoptosis in a dose-dependent manner similar to sorafenib. In addition, the prediction using SwissADME suggested that 5r possessed appropriate drug properties conforming to Veber's studies. These findings revealed that the newly designed meta-ureidophenoxy-1,2,3-triazole hybrid scaffold was a promising structural feature for an efficient inhibition of HepG2. Moreover, derivative 5r emerged as a promising candidate for further development as a targeted anti-cancer agent for hepatocellular carcinoma (HCC).

The Design and Synthesis of a New Series of 1,2,3-Triazole-Cored Structures Tethering Aryl Urea and Their Highly Selective Cytotoxicity toward HepG2.

Target cancer drug therapy is an alternative treatment for advanced hepatocellular carcinoma (HCC) patients. However, the treatment using approved targeted drugs has encountered a number of limitations, including the poor pharmacological properties of drugs, therapy efficiency, adverse effects, and drug resistance. As a consequence, the discovery and development of anti-HCC drug structures are therefore still in high demand. Herein, we designed and synthesized a new series of 1,2,3-triazole-cored structures incorporating aryl urea as anti-HepG2 agents. Forty-nine analogs were prepared via nucleophilic addition and copper-catalyzed azide-alkyne cycloaddition (CuAAC) with excellent yields. Significantly, almost all triazole-cored analogs exhibited less cytotoxicity toward normal cells, human embryonal lung fibroblast cell MRC-5, compared to Sorafenib and Doxorubicin. Among them, 2m' and 2e exhibited the highest selectivity indexes (SI = 14.7 and 12.2), which were ca. 4.4- and 3.7-fold superior to that of Sorafenib (SI = 3.30) and ca. 3.8- and 3.2-fold superior to that of Doxorubicin (SI = 3.83), respectively. Additionally, excellent inhibitory activity against hepatocellular carcinoma HepG2, comparable to Sorafenib, was still maintained. A cell-cycle analysis and apoptosis induction study suggested that 2m' and 2e likely share a similar mechanism of action to Sorafenib. Furthermore, compounds 2m' and 2e exhibit appropriate drug-likeness, analyzed by SwissADME. With their excellent anti-HepG2 activity, improved selectivity indexes, and appropriate druggability, the triazole-cored analogs 2m' and 2e are suggested to be promising candidates for development as targeted cancer agents and drugs used in combination therapy for the treatment of HCC.

Synthesis of sorafenib analogues incorporating a 1,2,3-triazole ring and cytotoxicity towards hepatocellular carcinoma cell lines.

A series of 1,2,3-triazole-containing Sorafenib analogues, in which the aryl urea moiety of Sorafenib (1) was replaced with a 1,2,3-triazole ring linking a substituted phenoxy fragment, were prepared successfully via Huisgen 1,3-dipolar cycloaddition and nucleophilic aromatic substitution. The studies of cytotoxicity towards human hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh7, indicated that p-tert-butylphenoxy analogue 2m showed significant inhibitory activity against Huh7 with IC50 = 5.67 ± 0.57 µM. More importantly, 2m showed low cytotoxicity against human embryonal lung fibroblast cell line, MRC-5, with IC50 > 100 µM, suggesting its highly selective cytotoxic activity (SI > 17.6) towards Huh7 which is much superior to that of Sorafenib (SI = 6.73). The molecular docking studies revealed that the analogue 2m bound B-RAF near the binding position of Sorafenib, while it interacted VEGFR2 efficiently at the same binding position of Sorafenib. However, 2m exhibited moderate inhibitory activity toward B-RAF, implying that its anti-Huh7 effect might not strictly relate to inhibition of B-RAF. Wound healing and BrdU cell proliferation assays confirmed anti-cell migration and anti-cell proliferative activities towards Huh7. With its inhibitory efficiency and high safety profile, 2m has been identified as a promising candidate for the treatment of HCC.

Isatin sulfonamides: potent caspases-3 and -7 inhibitors, and promising PET and SPECT radiotracers for apoptosis imaging.

Caspases-3 and -7 play an essential role in apoptosis. Isatin sulfonamides have been identified as potent inhibitors of these executing caspases. Besides pharmacological application, these compounds can also serve as recognition units to target caspases using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) when labeled with a positron or a gamma emitter. Fluorinated, alkylated, arylated isatin derivatives, in addition to derivatives modified with heterocycles, have been prepared in order to improve their binding potency, selectivity and metabolic stability. Structural optimization has led to stable, highly active inhibitors, which after labeling have been applied in PET studies in tumor mouse models and for first preclinical and clinical investigations with healthy human volunteers. The results support further development of such radiotracers for clinical apoptosis imaging.

Synthesis of 7-halogenated isatin sulfonamides: nonradioactive counterparts of caspase-3/-7 inhibitor-based potential radiopharmaceuticals for molecular imaging of apoptosis.

N-Alkylated (S)-7-halogen-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins were developed as a new group of nonradioactive reference compounds for future radiotracers. Inhibitor potency studies of these compounds suggest that the binding pockets readily accommodate both the 7-halogen substituents and aliphatic side chains (methyl to n-butyl) as well as some ω-fluorinated analogues (3-fluoropropyl and 4-fluorobutyl) at the isatin nitrogen. Indeed, compared to the halogen free parent compounds, some 7-halogenated derivatives exhibited slightly improved inhibitory potencies with IC50 values up to 2.6 nM (caspase-3) and 3.3 nM (caspase-7), respectively. Moreover, the 7-position of isatin, a potential cytochrome P450 hydroxylation site, was substituted by I, Br, Cl, and F to potentially enhance the metabolic stability of isatin sulfonamides. As an example, the radiotracer [(18)F]39 that was produced by (19)F/(18)F isotope exchange was shown to be stable in human blood serum after incubation at 37 °C for at least 90 min.

Synthesis, 18F-radiolabeling, and in vivo biodistribution studies of N-fluorohydroxybutyl isatin sulfonamides using positron emission tomography.

The effector caspases-3 and -7 play a central role in programmed type I cell death (apoptosis). Molecular imaging using positron emission tomography (PET) by tracking the activity of executing caspases might allow the detection of the early onset as well as therapy monitoring of various diseases induced by dysregulated apoptosis. Herein, four new fluorinated diastereo- and enantiopure isatin sulfonamide-based potent and selective caspase-3 and -7 inhibitors were prepared by cyclic sulfate ring-opening with fluoride. All fluorohydrins exhibited excellent in vitro affinities (up to IC50 = 11.8 and 0.951 nM for caspase-3 and -7, respectively), which makes them appropriate PET radiotracer candidates. Therefore, N-(4-[(18)F]fluoro-3(R)-hydroxybutyl)- and N-(3(S)-[(18)F]fluoro-4-hydroxybutyl)-5-[1-(2(S)-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin were synthesized in 140 min with 24% and 10% overall radiochemical yields and specific activities of 10-127 GBq/µmol using [(18)F]fluoride in the presence of Kryptofix and subsequent acidic hydrolysis. In vivo biodistribution studies in wild-type mice using PET/computed tomography imaging proved fast clearance of the tracer after tail vein injection.

Influence of 4- or 5-substituents on the pyrrolidine ring of 5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin derivatives on their inhibitory activities towards caspases-3 and -7.

A series of new 4- or 5-substituted pyrrolidine derivatives of 5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin bearing additional n-butyl or 4-fluorobutyl groups at the isatin nitrogen were prepared and their inhibitory activities have been tested against caspases-3 and -7, which are known to participate in the execution of the programmed cell death, called apoptosis. Several analogues fluorinated at the 4-position of the pyrrolidine ring were also synthesized since such inhibitors might be developed as (18)F-radiotracers for molecular imaging of activated caspases in vivo by PET. Enantiomerically pure diastereomeric 4-fluoropyrrolidinyl derivatives inhibited the enzymes in the nanomolar scale, i.e.100-1000 times more efficient than the corresponding 4-methoxy analogues. The 4,4-difluorinated compound showed the best result with IC50 = 362 nM and 178 nM for the aforementioned caspases. In contrast, the 4-methoxy and 4-trifluoromethyl analogues exhibited less inhibition potencies for the enzymes in the µM scale, whereas all 4-OPEG4 (PEG4 = tetraethyleneglycol) and 5-methoxymethyl derivatives were inactive.

Synthesis of new fluorinated, 2-substituted 5-pyrrolidinylsulfonyl isatin derivatives as caspase-3 and caspase-7 inhibitors: nonradioactive counterparts of putative PET-compatible apoptosis imaging agents.

Downstream caspases-3 and -7 are essential to execute the programmed type I cell death (apoptosis). In order to better understand their role, specific inhibitors of these enzymes are required, which after radiolabeling can be applied to non-invasively visualize and monitor apoptotic pathways in vivo using Positron Emission Tomography (PET). Therefore, 2-methoxyethyl-, 2-methoxypropyl-, 2-ethoxymethyl-, 2-(2-fluoroethoxymethyl)-, and 2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl analogues of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (2) were prepared and their in vitro binding affinities towards caspases-1, -3, -6 and -7 were evaluated and compared to that of the lead structure 2. While the inhibition potencies against caspases-1 and -6 were in the micromolar range, all synthesized compounds exhibited excellent and selective inhibition of caspases-3 and -7 in the nanomolar range up to IC50=4.79 nM and 7.47 nM, respectively. These highly potent 2-substituted analogues of 2 might be developed as anti-apoptosis agents and some selected fluorinated inhibitors might be useful as potential PET radiotracers for apoptosis imaging after (18)F-labeling.