Lithocholic acid derivatives as potent modulators of the nuclear receptor RORγt.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor found in various tissues that plays a crucial role in the differentiation and proliferation of T helper 17 (Th17) cells, as well as in their generation of the pro-inflammatory cytokine IL-17A. RORγt represents a promising therapeutic target for autoimmune diseases, metabolic disorders, and multiple tumors. Despite extensive research efforts focused on the development of small molecule RORγt modulators, no drug candidates have advanced to phase 3 clinical trials owing to a lack of efficacy or safety margin. This outcome highlights the unmet need to optimize small molecule drug candidates targeting RORγt to develop effective therapies for autoimmune and inflammatory diseases. In this study, we synthesized and evaluated 3-oxo-lithocholic acid amidates as a new class of RORγt modulators. Our evaluation entailed biophysical screening, cellular screening in different platforms, molecular docking, and in vitro pharmacokinetic profiling. The top compound from our study (3-oxo-lithocholic acid amidate, A2) binds to RORγt at an equilibrium dissociation constant (KD) of 16.5 ± 1.34 nM based on microscale thermophoresis (MST). Assessment of the efficacy of A2 in the cellular RORγt reporter luciferase assay revealed a half-maximal inhibitory concentration (IC50) value of 225 ± 10.4 nM. Unlike 3-oxo-lithocholic acid, A2 demonstrated the ability to reduce the IL-17A mRNA expression levels in EL4 cells with RORγt expression using quantitative reverse transcriptase PCR (RT-PCR). Validation of the desirable physicochemical properties and stability of A2 sets the stage for the preclinical evaluation of this new class of RORγt modulators in animal models of autoimmune diseases.

Discovery of Quinolinone Hybrids as Dual Inhibitors of Acetylcholinesterase and Aß Aggregation for Alzheimer's Disease Therapy.

The development of multitargeted therapeutics has evolved as a promising strategy to identify efficient therapeutics for neurological disorders. We report herein new quinolinone hybrids as dual inhibitors of acetylcholinesterase (AChE) and Aß aggregation that function as multitargeted ligands for Alzheimer's disease. The quinoline hybrids (AM1-AM16) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. Among the tested compounds, AM5 and AM10 inhibited AChE activity by more than 80% at single-dose screening and possessed a remarkable ability to inhibit the fibrillation of Aß42 oligomers at 10 µM. In addition, dose-dependent screening of AM5 and AM10 was performed, giving half-maximal AChE inhibitory concentration (IC50) values of 1.29 ± 0.13 and 1.72 ± 0.18 µM, respectively. In addition, AM5 and AM10 demonstrated concentration-dependent inhibitory profiles for the aggregation of Aß42 oligomers with estimated IC50 values of 4.93 ± 0.8 and 1.42 ± 0.3 µM, respectively. Moreover, the neuroprotective properties of the lead compounds AM5 and AM10 were determined in SH-SY5Y cells incubated with Aß oligomers. This work would enable future research efforts aiming at the structural optimization of AM5 and AM10 to develop potent dual inhibitors of AChE and amyloid aggregation. Furthermore, the in vivo assay confirmed the antioxidant activity of compounds AM5 and AM10 through increasing GSH, CAT, and SOD activities that are responsible for scavenging the ROS and restoring its normal level. Blood investigation illustrated the protective activity of the two compounds against lead-induced neurotoxicity through retaining hematological and liver enzymes near normal levels. Finally, immunohistochemistry investigation revealed the inhibitory activity of ß-amyloid (Aß) aggregation.

Discovery of ICOS-Targeted Small Molecules Using Pharmacophore-Based Screening.

There are currently no small molecules clinically approved as immune checkpoint modulators. Besides possessing oral bioavailability, cell-penetrating capabilities and enhanced tumor penetration compared to monoclonal antibodies (mAbs), small molecules are amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may avoid immune-related adverse events associated with mAbs. The interaction of inducible co-stimulator (ICOS) with its ligand (ICOS-L) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. This study represents the development and validation of a virtual screening strategy to identify small molecules that bind a novel druggable binding pocket in human ICOS. We used a lipophilic canyon in the apo-structure of ICOS and the ICOS/ICOS-L interface individually as templates for molecular dynamics simulation to generate 3D pharmacophores subsequently used for virtual screening campaigns. Our strategy was successful finding a first-in-class small molecule ICOS binder (5P, KD value=108.08±26.76 µM) and validating biophysical screening platforms for ICOS-targeted small molecules. We anticipate that future structural optimization of 5P will result in the discovery of high affinity chemical ligands for ICOS.

First-in-class small molecule inhibitors of ICOS/ICOSL interaction as a novel class of immunomodulators.

The interaction of the inducible co-stimulator (ICOS) with its ligand (ICOSL) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. The ICOS/ICOSL pathway is a validated target for T-cell lymphomas induced by the proliferation of T-follicular helper (Tfh) cells. Moreover, the inhibition of ICOS/ICOSL interaction can decrease the enhancement of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. However, targeting ICOS/ICOSL interaction is currently restricted to monoclonal antibodies (mAbs) and there are no small molecules in existence that can block ICOS/ICOSL. To fill this gap, we report herein the first time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit ICOS/ICOSL interaction. Implementation of the developed TR-FRET assay in high-throughput screening (HTS) of a focused chemical library resulted in the identification of AG-120 as a first-in-class inhibitor of ICOS/ICOSL interaction. We further employed docking studies and molecular dynamics (MD) simulations to identify the plausible mechanism of blocking ICOS/ICOSL complex formation by AG-120. Using the structure-activity relationship (SAR) by catalog approach, we identified AG-120-X with an IC50 value of 4.68 ± 0.47 µM in the ICOS/ICOSL TR-FRET assay. Remarkably, AG-120-X revealed a dose-dependent ability to block ICOS/ICOSL interaction in a bioluminescent cellular assay based on co-culturing Jurkat T cells expressing ICOS and CHO-K1 cells expressing ICOSL. This work will pave the way for future drug discovery efforts aiming at the development of small molecule inhibitors of ICOS/ICOSL interaction as potential therapeutics for cancer as well as other diseases.

Discovery of Small-Molecule TIM-3 Inhibitors for Acute Myeloid Leukemia Using Pharmacophore-Based Virtual Screening.

T-cell immunoglobulin and mucin domain 3 (TIM-3) is a negative immune checkpoint that represents a promising target for cancer immunotherapy. Although encouraging results have been observed for TIM-3 inhibition in the context of acute myeloid leukemia (AML), targeting TIM-3 is currently restricted to monoclonal antibodies (mAbs). To fill this gap, we implemented a pharmacophore-based screening approach to identify small-molecule TIM-3 inhibitors. Our approach resulted in the identification of hit compounds with TIM-3 binding affinity. Subsequently, we used the structure-activity relationship (SAR) by a catalog approach to identify compound A-41 with submicromolar TIM-3 binding affinity. Remarkably, A-41 demonstrated the ability to block TIM-3 interactions with key ligands and inhibited the immunosuppressive function of TIM-3 using an in vitro coculture assay. This work will pave the way for future drug discovery efforts aiming at the development of small-molecule inhibitors TIM-3 for AML.

Discovery of First-in-Class Small Molecule Inhibitors of Lymphocyte Activation Gene 3 (LAG-3).

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint that plays a key role in downregulating the immune response to cancer. Inhibition of LAG-3 interactions allows T cells to regain cytotoxic activity and reduce the immunosuppressive function of regulating T cells. We utilized a combination approach of focused screening and "SAR by catalog" to identify small molecules that function as dual inhibitors of the interactions of LAG-3 with major histocompatibility complex (MHC) class II and fibrinogen-like protein 1 (FGL1). Our top hit compound inhibited both LAG-3/MHCII and LAG-3/FGL1 interactions in biochemical binding assays with IC50 values of 4.21 ± 0.84 and 6.52 ± 0.47 µM, respectively. Moreover, we have demonstrated the ability of our top hit compound to block LAG-3 interactions in cell-based assays. This work will pave the way for future drug discovery efforts aiming at the development of LAG-3-based small molecules for cancer immunotherapy.

Development of a high-throughput TR-FRET screening assay for LAG-3/FGL1 interaction.

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. Fibrinogen-like protein 1 (FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.

Multicomponent Petasis reaction for the identification of pyrazine based multi-target directed anti-Alzheimer's agents: In-silico design, synthesis, and characterization.

Multi-target directed ligands (MTDLs) have recently attracted significant interest due to their exceptional effectiveness against multi-factorial Alzheimer's disease. The present work described the development of pyrazine-based MTDLs using multicomponent Petasis reaction for the dual inhibition of tau-aggregation and human acetylcholinesterase (hAChE). The molecular structure of synthesized ligands was validated by 1H & 13C NMR and mass spectrometry. The screened compounds were shown to have a strong inhibitory effect at 10 µM concentration against tau-oligomerization and hAChE, but only moderate inhibitory activity against Aß42. Among all the compounds, the half-maximal inhibitory concentration (IC50) for 21 and 24 against hAChE were 0.71 µM and 1.09 µM, respectively, while they displayed half-maximal effective concentrations (EC50) values of 2.21 µM and 2.71 µM for cellular tau-oligomerization, respectively. Additionally, an MTT experiment using tau-expressing SH-SY5Y neuroblastoma cells revealed that 21 was more neuroprotective than the FDA-approved medication donepezil. Furthermore, an MD simulation study was performed to investigate the dynamics and stability of AChE-21 and AChE-24 complexes in an aqueous environment. The MM-PBSA calculations were performed to evaluate the binding of 21 and 24 with AChE, and the relative binding energy was calculated as -870.578 and -875.697 kJ mol-1, respectively. As a result, the study offered insight into the design of new MTDLs and highlighted 21 as a potential roadblock to the development of anti-AD medications.

Discovery of new phenyl sulfonyl-pyrimidine carboxylate derivatives as the potential multi-target drugs with effective anti-Alzheimer's action: Design, synthesis, crystal structure and in-vitro biological evaluation.

Alzheimer's disease (AD) is multifactorial, progressive neurodegeneration with impaired behavioural and cognitive functions. The multitarget-directed ligand (MTDL) strategies are promising paradigm in drug development, potentially leading to new possible therapy options for complex AD. Herein, a series of novel MTDLs phenylsulfonyl-pyrimidine carboxylate (BS-1 to BS-24) derivatives were designed and synthesized for AD treatment. All the synthesized compounds were validated by 1HNMR, 13CNMR, HRMS, and BS-19 were structurally validated by X-Ray single diffraction analysis. To evaluate the plausible binding affinity of designed compounds, molecular docking study was performed, and the result revealed their significant interaction with active sites of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The synthesized compounds displayed moderate to excellent in vitro enzyme inhibitory activity against AChE and BuChE at nanomolar (nM) concentration. Among 24 compounds (BS-1 to BS-24), the optimal compounds (BS-10 and BS-22) displayed potential inhibition against AChE; IC50 = 47.33 ± 0.02 nM and 51.36 ± 0.04 nM and moderate inhibition against BuChE; IC50 = 159.43 ± 0.72 nM and 153.3 ± 0.74 nM respectively. In the enzyme kinetics study, the compound BS-10 displayed non-competitive inhibition of AChE with Ki = 8 nM. Respective compounds BS-10 and BS-22 inhibited AChE-induced Aß1-42 aggregation in thioflavin T-assay at 10 µM and 20 µM, but BS-10 at 10 µM and 20 µM concentrations are found more potent than BS-22. In addition, the aggregation properties were determined by the dynamic light scattering (DLS) and was found that BS-10 and BS-22 could significantly inhibit self-induced as well as AChE-induced Aß1-42 aggregation. The effect of compounds (BS-10 and BS-22) on the viability of MC65 neuroblastoma cells and their capability to cross the blood-brain barrier (BBB) in PAMPA-BBB were further studied. Further, in silico approach was applied to analyze physicochemical and pharmacokinetics properties of the designed compounds via the SwissADME and PreADMET server. Hence, the novel phenylsulfonyl-pyrimidine carboxylate derivatives can act as promising leads in the development of AChE inhibitors and Aß disaggregator for the treatment of AD.

Discovery and Optimization of Small-Molecule Ligands for V-Domain Ig Suppressor of T-Cell Activation (VISTA).

V-domain Ig suppressor of T-cell activation (VISTA) is an immune checkpoint that affects the ability of T-cells to attack tumors. A FRET-based high throughput screening identified NSC622608 as the first small-molecule ligand for VISTA. Investigation of the interaction of NSC622608 with VISTA using STD NMR and molecular modeling enabled the identification of a potential binding site in VISTA for NSC622608. Screening NSC622608 against a library of single-point VISTA mutants revealed the key residues in VISTA interacting with NSC622608. Further structural optimization resulted in a lead with submicromolar VISTA binding affinity. The lead compound blocked VISTA signaling in vitro, enhanced T-cell proliferation, and restored T-cell activation in the presence of VISTA-expressing cancer cell lines. This work would enable future development of small molecules targeting VISTA as immunomodulators and imaging probes.

MicroRNA-Based Multitarget Approach for Alzheimer's Disease: Discovery of the First-In-Class Dual Inhibitor of Acetylcholinesterase and MicroRNA-15b Biogenesis.

The multitarget approach in drug design is a powerful strategy in tackling the multifactorial nature of Alzheimer's disease (AD). Herein, we report a novel strategy in the design of multitargeted therapeutics for AD through dual inhibition of acetylcholinesterase (AChE) and microRNA-15b biogenesis. We performed high-throughput screening (HTS) of a chemical library to identify binders of mircoRNA-15b which is identified as a biomarker and potential therapeutic target of AD. The hits from HTS were further screened for their AChE inhibitory activity, the most widely investigated target for the development of AD therapeutics. MG-6267 was identified as the first dual inhibitor of AChE and microRNA-15b biogenesis. Cellular assays revealed the superiority of MG-6267 to single-targeted inhibitors of AChE and microRNA-15b in protecting SH-SY5Y neuroblastoma cells from amyloid-beta (Aß)-induced cytotoxicity. This work paves the way for future research efforts aiming at the development of microRNA-based multitargeted therapeutics for AD.

High-Throughput Platform for Real-Time Monitoring of ATP-Generating Enzymes in Living Cells Based on a Lanthanide Probe.

Remarkable variation between cell-free and cellular measurements of enzyme activity triggered the unmet need to develop tools for monitoring enzyme activity in living cells. Such tools will advance our understanding of the biological functions of enzymes and their potential impact on drug discovery. We report in this study a universal assay for monitoring ATP-generating enzymes in living cells using a self-assembled Tb3+ complex probe. Modulation of the rheological properties of cell culture media enabled shifting the lifetime of the Tb3+ complex in the presence of ATP from micro-to-millisecond range. Based on the response of the Tb3+ complex to ATP, cellular assays for 5 ATP-generating enzymes were developed. Remarkably, assessment of the activity of these enzymes in living cells is made possible for the first time. The pyruvate kinase M2 (PKM2) assay has been optimized for high-throughput screening (HTS) and further implemented in the identification of novel scaffolds as PKM2 inhibitors.

Dual Targeting of Monomeric Tau and α-Synuclein Aggregation: A New Multitarget Therapeutic Strategy for Neurodegeneration.

Development of efficient multitargeted therapeutic strategies is crucial in facing the multifaceted nature of neurodegenerative diseases. Parkinson's disease (PD) and Alzheimer's disease (AD), the two most common neurodegenerative disorders, share a common hallmark of accumulation of misfolded protein aggregates which are Lewy bodies (LBs) and neurofibrillary tangles (NFTs), respectively. Tau protein and α-synuclein (α-syn), the precursors of LBs and NFTs, have demonstrated synergistic aggregation and neurotoxicity in both in vitro and in vivo models. Herein, we validate for the first time dual targeting of monomeric tau and α-syn aggregation as an efficient platform for development of multitarget therapeutics for neurological disorders. Cellular fluorescence resonance energy transfer (FRET)-based high-throughput screening for tau-binding compounds, followed by additional screening of the hits for their ability to impede α-syn aggregation identified MG-2119 as a potential lead. The high binding affinity of MG-2119 to monomeric tau was verified using cellular FRET assay, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and microscale thermophoresis (MSH). Moreover, MG-2119 inhibited α-syn aggregation as revealed by thioflavin T (ThT) assay and dynamic light scattering (DLS) measurements. Interestingly, MG-2119 was capable of rescuing combined tau and α-syn-induced cytotoxicity in SH-SY5Y neuroblastoma cells in a dose-dependent manner. Less pronounced cell-rescuing effects were observed for single-targeted tau and α-syn aggregation inhibitors showcasing the superiority of the multitargeted approach described in this study. The satisfactory pharmacokinetic profile and low toxicity of MG-2119 hold promise for future optimization to develop potential therapeutics for neurological disorders.

Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission.

Switchable luminescent bioprobes whose emission can be turned on as a function of specific enzymatic activity are emerging as important tools in chemical biology. We report a promising platform for the development of label-free and continuous enzymatic assays in high-throughput mode based on the reversible solvent-induced self-assembly of a neutral dinuclear Pt(II) complex. To demonstrate the utility of this strategy, the switchable luminescence of a dinuclear Pt(II) complex was utilized in developing an experimentally simple, fast (10 min), low cost, and label-free turn-on luminescence assay for the endonuclease enzyme DNAse I. The complex displays a near-IR (NIR) aggregation-induced emission at 785 nm in aqueous solution that is completely quenched upon binding to G-quadruplex DNA from the human c-myc oncogene. Luminescence is restored upon DNA degradation elicited by exposure to DNAse I. Correlation between near-IR luminescence intensity and DNAse I concentration in human serum samples allows for fast and label-free detection of DNAse I down to 0.002 U/mL. The Pt(II) complex/DNA assembly is also effective for identification of DNAse I inhibitors, and assays can be performed in multiwell plates compatible with high-throughput screening. The combination of sensitivity, speed, convenience, and cost render this method superior to all other reported luminescence-based DNAse I assays. The versatile response of the Pt(II) complex to DNA structures promises broad potential applications in developing real-time and label-free assays for other nucleases as well as enzymes that regulate DNA topology.

Platinum(II) Complexes with Sterically Expansive Tetraarylethylene Ligands as Probes for Mismatched DNA.

Deficiencies in DNA mismatch repair (MMR) machinery result in greater incidence of DNA base pair mismatches in many types of cancer cells relative to normal cells. Consequently, luminescent probes capable of signaling the presence of mismatched DNA hold promise as potential cancer diagnostic and therapeutic tools. In this study, a series of cyclometalated platinum(II) complexes with sterically expansive tetraarylethylene ligands were synthesized and examined for selective detection of mismatched DNA. Increased steric bulk of the tetraarylethylene ligands in these complexes was observed to correlate with greater preferential luminescence enhancement in the presence of hairpin DNA oligonucleotides containing a mismatched site compared to well-matched oligonucleotides, with the most effective complex displaying ∼14-fold higher emission upon binding CC mismatched oligonucleotides compared to well-matched oligonucleotides. The results indicate binding to mismatched sites in DNA oligonucleotides occurs through metalloinsertion, and the luminescence response increases as a function of thermodynamic destabilization of the mismatch. Luminescence quenching experiments with Cu(phen)22+ and NaI further indicate mismatch binding from the minor groove, consistent with metalloinsertion. Binding to CC mismatched oligonucleotides was also investigated by isothermal titration calorimetry and UV-melting studies. These results demonstrate the efficacy of tetraarylethylene-based platinum(II) complexes for detection of mismatched DNA and establish a new molecular platform for development of organometallic DNA binding agents.

Structure-based design, synthesis, and evaluation of structurally rigid donepezil analogues as dual AChE and BACE-1 inhibitors.

A new series of structurally rigid donepezil analogues was designed, synthesized and evaluated as potential multi-target-directed ligands (MTDLs) against neurodegenerative diseases. The investigated compounds 10-13 displayed dual AChE and BACE-1 inhibitory activities in comparison to donepezil, the FDA-approved drug. The hybrid compound 13 bearing 2-aminoquinoline scaffold exhibited potent AChE inhibition (IC50 value of 14.7 nM) and BACE-1 inhibition (IC50 value of 13.1 nM). Molecular modeling studies were employed to reveal potential dual binding mode of 13 to AChE and BACE-1. The effect of the investigated compounds on the viability of SH-SY5Y neuroblastoma cells and their ability to cross the blood-brain barrier (BBB) in PAMPA-BBB assay were further studied.

Design and synthesis of donepezil analogues as dual AChE and BACE-1 inhibitors.

Multi-target-directed ligands (MTDLs) centered on ß-secretase 1 (BACE-1) inhibition are emerging as innovative therapeutics in addressing the complexity of neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through introduction of backbone amide linkers to the designed compounds which are capable of hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies revealed the dual mode of action of compounds 4-6 against hAChE and BACE-1. Notably, compound 4 displayed potent hAChE inhibition (IC50 value of 4.11 nM) and BACE-1 inhibition (IC50 value of 18.3 nM) in comparison to donepezil (IC50 values of 6.21 and 194 nM against hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood-brain barrier (BBB) in PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small ligands for the treatment of Alzheimer's disease.

A fluorescent turn-on probe for cyanide anion detection based on an AIE active cobalt(ii) complex.

A new tetradentate polypyridine ligand that displays aggregation induced emission (AIE) characteristics has been synthesized. A coordination complex with CoCl2 has been prepared and characterized by X-ray crystallography. The Co(ii) complex retains the AIE activity of the ligand in aqueous solution while also exhibiting a selective turn-on fluorescence response in the presence of cyanide anion. A complex : CN- binding stoichiometry of 1 : 2 was indicated via Job plot analysis and the limit of detection for CN- was determined to be 0.59 µM. The fluorescence response is attributed to coordination of CN- by the AIE active Co(ii) complex, resulting in decreased solubility in aqueous medium and concomitant generation of larger nanoaggregates as revealed by dynamic light scattering (DLS) measurements. The sensitivity and selectivity displayed by this sensor for CN- over other anions renders it a candidate probe for CN- detection in aqueous environments.

Microwave-assisted synthesis and antitumor evaluation of a new series of thiazolylcoumarin derivatives.

A new series of thiazolylcoumarin derivatives was synthesized. The designed strategy embraced a molecular hybridization approach which involves the combination of the thiazole and coumarin pharmacophores together. The new hybrid compounds were tested for in vitro antitumor efficacy over cervical (Hela) and kidney fibroblast (COS-7) cancer cells. Compounds 5f, 5h, 5m and 5r displayed promising efficacy toward Hela cell line. In addition, 5h and 5r were found to be the most active candidates toward COS-7 cell line. The four active analogs, 5f, 5h, 5m and 5r were screened for in vivo antitumor activity over EAC cells in mice, as well as in vitro cytotoxicity toward W138 normal cells. Results illustrated that 5r has the highest in vivo activity, and that the four analogs are less cytotoxic than 5-FU toward W138 normal cells. In this study, 3D pharmacophore analysis was performed to investigate the matching pharmacophoric features of the synthesized compounds with trichostatin A. In silico studies showed that the investigated compounds meet the optimal needs for good oral absorption with no expected toxicity hazards.

Isatin-ß-thiocarbohydrazones: Microwave-assisted synthesis, antitumor activity and structure-activity relationship.

A new series of isatin-ß-thiocarbohydrazones was synthesized based on the pharmacophoric features of triapine required for metal chelation. Our strategy involved the modifications of triapine basic skeleton by replacing pyridinyl moiety with isatin which retains the tridentate feature of triapine needed for metal chelation. The new compounds were esteemed for their antitumor efficacy against cervical cancer (Hela) and kidney fibroblast cancer (COS-7) cell lines. Compounds 4c, 4d, 5c and 5e exhibited remarkable efficacy against Hela cell line. In addition, compounds 4c, 4k, 4e, 5c and 5e displayed an outstanding efficacy against COS-7 cell line. Compounds 4c, 4k, 4e, 5c and 5e were examined for their in vivo antitumor efficacy against Ehrlich ascites carcinoma (EAC) in mice. Pharmacophore mapping was performed to study the structural features of the synthesized compounds compared to triapine and to identify the essential moieties required for potent and selective antitumor activity.