Synthesis of Thiazolidin-4-Ones Derivatives, Evaluation of Conformation in Solution, Theoretical Isomerization Reaction Paths and Discovery of Potential Biological Targets.

Thiazolin-4-ones and their derivatives represent important heterocyclic scaffolds with various applications in medicinal chemistry. For that reason, the synthesis of two 5-substituted thiazolidin-4-one derivatives was performed. Their structure assignment was conducted by NMR experiments (2D-COSY, 2D-NOESY, 2D-HSQC and 2D-HMBC) and conformational analysis was conducted through Density Functional Theory calculations and 2D-NOESY. Conformational analysis showed that these two molecules adopt exo conformation. Their global minimum structures have two double bonds (C=N, C=C) in Z conformation and the third double (C=N) in E. Our DFT results are in agreement with the 2D-NMR measurements. Furthermore, the reaction isomerization paths were studied via DFT to check the stability of the conformers. Finally, some potential targets were found through the SwissADME platform and docking experiments were performed. Both compounds bind strongly to five macromolecules (triazoloquinazolines, mglur3, Jak3, Danio rerio HDAC6 CD2, acetylcholinesterase) and via SwissADME it was found that these two molecules obey Lipinski's Rule of Five.

Anticancer Potential of Novel Cinnamoyl Derivatives against U87MG and SHSY-5Y Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is probably the most malignant and aggressive brain tumor belonging to the class of astrocytomas. The considerable aggressiveness and high malignancy of GBM make it a tumor that is difficult to treat. Here, we report the synthesis and biological evaluation of eighteen novel cinnamoyl derivatives (3a-i and 4a-i) to obtain more effective antitumor agents against GBM. METHODS: The chemical structures of novel cinnamoyl derivatives (3a-i and 4a-i) were confirmed by NMR and MS analyses. The physicochemical properties and evaluation of the ADME profile of 3a-i and 4a-i were performed by the preADMETlab2.0 web program. Cinnamoyl derivatives 3a-i and 4a-i were tested in vitro for their cytotoxicity against the human healthy fibroblast (HDFa) cells using an MTT cell viability assay. Derivatives with no toxicity on HDFa cells were tested both on human glioblastoma (U87MG) and neuroblastoma (SHSY- 5Y) cells, chosen as an experimental model of brain tumors. Cell death mechanisms were analyzed by performing flow cytometry analyses. RESULTS: Cinnamoyl derivatives 3a-i and 4a-i showed good physicochemical and ADME properties suggesting that these compounds could be developed as oral drugs endowed with a high capability to cross the blood-brain barrier. Compounds (E)-1-methoxy-4-(2-(phenylsulfonyl)vinyl)benzene (2c) and (E)-N-benzyl-N-(2- (cyclohexylamino)-2-oxoethyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (3e) did not show cytotoxicity on healthy human fibroblast cells up to 100 µg/mL. The most anticarcinogenic molecule, compound 3e, emerged as the most potent anticancer candidate in this study. Flow cytometry results showed that compound 3e (25 µg/mL) application resulted in nearly 86% and 84% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. Compound 2c (25 µg/mL) resulted in 81% and 82% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. CONCLUSION: Cinnamoyl derivative 3e inhibits the proliferation of cultured U87MG and SHSY-5Y cells by inducing apoptosis. Further detailed research will be conducted to confirm these data in in vivo experimental animal models.

Novel styryl-thiazole hybrids as potential anti-Alzheimer's agents.

In this study, combining the thiazole and cinnamoyl groups into the styryl-thiazole scaffold, a series of novel styryl-thiazole hybrids (6a-p) was rationally designed, synthesized, and evaluated by the multi-target-directed ligands strategy as potential candidates for the treatment of Alzheimer's disease (AD). Hybrids 6e and 6i are the most promising among the synthesized hybrids since they are able to significantly increase cell viabilities in Aß1-42-exposed-human neuroblastoma cell line (6i at the concentration of 50 µg mL-1 and 6e at the concentration of 25 µg mL-1 resulted in ∼34% and ∼30% increase in cell viabilities, respectively). Compounds 6e and 6i exhibit highly AChE inhibitory properties in the experimental AD model at 375.6 ± 18.425 mU mL-1 and 397.6 ± 32.152 mU mL-1, respectively. Moreover, these data were also confirmed by docking studies and in vitro enzyme inhibition assays. Compared to hybrid 6e and according to the results, 6i also has the highest potential against Aß1-42 aggregation with over 80% preventive activity. The in silico prediction of the physicochemical properties confirms that 6i possesses a better profile compared to 6e. Therefore, compound 6i presents a promising multi-targeted active molecular profile for treating AD considering the multifactorial nature of AD, and it is reasonable to deepen its mechanisms of action in an in vivo experimental model of AD.

Exploration of Thiourea-Based Scaffolds for the Construction of Bacterial Ureases Inhibitors.

A series of 32 thiourea-based urease inhibitors were synthesized and evaluated against native bacterial enzyme and whole cells of Sporosarcina pasteurii and Proteus mirabilis strains. The proposed inhibitors represented structurally diverse thiosemicarbazones and thiocarbohydrazones, benzyl-substituted thiazolyl thioureas, 1H-pyrazole-1-carbothioamides, and dihydropirimidine-2(1H)-thiones. Kinetic characteristics with purified S. pasteurii enzyme determined low micromolar inhibitors within each structural group. (E)-2-(1-Phenylethylidene)hydrazine-1-carbothioamide 19 (Ki = 0.39 ± 0.01 µM), (E)-2-(4-methylbenzylidene)hydrazine-1-carbothioamide 16 (Ki = 0.99 ± 0.04 µM), and N'-((1E,2E)-1,3-diphenylallylidene)hydrazinecarbothiohydrazide 29 (Ki = 2.23 ± 0.19 µM) were used in modeling studies that revealed sulfur ion coordination of the active site nickel ion and hydrogen bonds between the amide group and the side chain of Asp363 and Ala366 carbonyl moiety. Whole-cell studies proved the activity of compounds in Gram-positive and Gram-negative microorganisms. Ureolysis control observed in P. mirabilis PCM 543 (e.g., IC50 = 304 ± 14 µM for 1-benzyl-3-(4-(4-hydroxyphenyl)thiazol-2-yl)thiourea 52) is a valuable achievement, as urease is recognized as a major virulence factor of this urinary tract pathogen.

Thiocarbohydrazone and Chalcone-Derived 3,4-Dihydropyrimidinethione as Lipid Peroxidation and Soybean Lipoxygenase Inhibitors.

The potential of the 4,6-diphenyl-3,4-dihydropyrimidine-2(1H)-thione (abbreviated as KKII5) and (E)-N'-benzylidenehydrazinecarbothiohydrazide (abbreviated as DKI5) compounds as possible drug leads is investigated. KKII5 and DKI5 are synthesized in high yield of up to 97%. Their structure, binding in the active site of the LOX-1 enzyme, and their toxicity are studied via joint experimental and computational methodologies. Specifically, the structure assignment and conformational analysis were achieved by applying homonuclear and heteronuclear 2D nuclear magnetic resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and density functional theory (DFT). The obtained DFT lowest energy conformers were in agreement with the NOE correlations observed in the 2D-NOESY spectra. Additionally, docking and molecular dynamics simulations were performed to discover their ability to bind and remain stabile in the active site of the LOX-1 enzyme. These in silico experiments and DFT calculations indicated favorable binding for the enzyme under study. The strongest binding energy, -9.60 kcal/mol, was observed for dihydropyrimidinethione KKII5 in the active site of LOX-1. ADMET calculations showed that the two molecules lack major toxicities and could serve as possible drug leads. The redox potential of the active center of LOX-1 with the binding molecules was calculated via DFT methodology. The results showed a significantly smaller energy attachment of 2.8 eV with KKII5 binding in comparison to DKI5. Thus, KKII5 enhanced the ability of the active center to receive electrons compared to DKI5. This is related to the stronger binding interaction of KKII5 relative to that of DK15 to LOX-1. The two very potent LOX-1 inhibitors exerted IC50 19 µΜ (KKII5) and 22.5 µΜ (DKI5). Furthermore, they both strongly inhibit lipid peroxidation, namely, 98% for KKII5 and 94% for DKI5.

Structure assignment, conformational properties and discovery of potential targets of the Ugi cinnamic adduct NGI25.

The structure assignment and conformational analysis of cinnamic derivative N-benzyl-N-(2-(cyclohexylamino)-2-oxoethyl) cinnamamide (NGI25) was carried out through Nuclear Magnetic Resonance (NMR) spectroscopy, Molecular Dynamics (MD) and Quantum Mechanics (QM), i.e. semiempirical and Density Functional Theory (DFT) calculations. Moreover, Homonuclear (COSY, NOESY) and heteronuclear (HSQC, HMBC) experiments were applied to assign its protons and carbons. After structure identification, NGI25 was subjected to computational calculations to reveal its most favorable conformations. In particular, MD studies were performed in two different solvents, DMSO of intermediate polarity and hydrophobic CHCl3. The obtained results suggest that NGI25 adopts similar conformations in both environments. In particular, the two aromatic rings of the molecule reside in spatial vicinity, while they remain quite distant from the cyclohexane. 2D NOESY experiments confirmed the in silico MD and QM calculations. Finally, molecular docking calculations were performed in order to reveal possible enzyme-targets for NGI25. Swiss target module was used to guide the discovery of new targets based on the structure of NGI. Indeed, it was predicted that NGI25 inhibited butyrylcholinesterase (BCHE) and lipoxygenase (LOX). Molecular docking experiments, followed by Molecular Dynamics studies, confirmed the favorable binding of NGI25 to both enzymes.Communicated by Ramaswamy H. Sarma.

Conformational Properties and Putative Bioactive Targets for Novel Thiosemicarbazone Derivatives.

The structure assignment and conformational analysis of the thiosemicarbazones, DKI21 and DKI24, were performed through homonuclear and heteronuclear 2D Nuclear Magnetic Resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-ROESY, 2D-HSQC, and 2D-HMBC) and quantum mechanics (QM) calculations, using Functional Density Theory (DFT). In addition, utilizing a combination of 2D-NOESY and 2D-ROESY spectra an exo structure was established for both of the analogs. This experimental results were confirmed by theoretical mechanistic studies, as the lowest minima conformations derived through DFT calculations were compatible with the spatial correlations observed in the 2D-NOESY and 2D-ROESY spectra. Finally, molecular binding experiments were performed to detect the potential targets for DKI21 and DKI24, derived from SwissAdme. In silico molecular binding experiments showed favorable binding energy values for the most of the enzymes studied. The ADMET calculations, using the preADMET and pKCSm software, showed that the two molecules appear as possible drug leads.

Conformational Properties of New Thiosemicarbazone and Thiocarbohydrazone Derivatives and Their Possible Targets.

The structure assignment and conformational analysis of thiosemicarbazone KKI15 and thiocarbohydrazone KKI18 were performed through homonuclear and heteronuclear 2D Nuclear Magnetic Resonance (NMR) spectroscopy (2D-COSY, 2D-NOESY, 2D-HSQC, and 2D-HMBC) and quantum mechanics (QM) calculations using Functional Density Theory (DFT). After the structure identification of the compounds, various conformations of the two compounds were calculated using DFT. The two molecules showed the most energy-favorable values when their two double bonds adopted the E configuration. These configurations were compatible with the spatial correlations observed in the 2D-NOESY spectrum. In addition, due to the various isomers that occurred, the energy of the transition states from one isomer to another was calculated. Finally, molecular binding experiments were performed to detect potential targets for KKI15 and KKI18 derived from SwissAdme. In silico molecular binding experiments showed favorable binding energy values for all four enzymes studied. The strongest binding energy was observed in the enzyme butyrylcholinesterase. ADMET calculations using the preADMET and pKCSm software showed that the two molecules appear as possible drug leads.

Rational Design and Synthesis of AT1R Antagonists.

Hypertension is one of the most common diseases nowadays and is still the major cause of premature death despite of the continuous discovery of novel therapeutics. The discovery of the Renin Angiotensin System (RAS) unveiled a path to develop efficient drugs to fruitfully combat hypertension. Several compounds that prevent the Angiotensin II hormone from binding and activating the AT1R, named sartans, have been developed. Herein, we report a comprehensive review of the synthetic paths followed for the development of different sartans since the discovery of the first sartan, Losartan.

Covalent Inhibition of Bacterial Urease by Bifunctional Catechol-Based Phosphonates and Phosphinates.

In this study, a new class of bifunctional inhibitors of bacterial ureases, important molecular targets for antimicrobial therapies, was developed. The structures of the inhibitors consist of a combination of a phosphonate or (2-carboxyethyl)phosphinate functionality with a catechol-based fragment, which are designed for complexation of the catalytic nickel ions and covalent bonding with the thiol group of Cys322, respectively. Compounds with three types of frameworks, including ß-3,4-dihydroxyphenyl-, α-3,4-dihydroxybenzyl-, and α-3,4-dihydroxybenzylidene-substituted derivatives, exhibited complex and varying structure-dependent kinetics of inhibition. Among irreversible binders, methyl ß-(3,4-dihydroxyphenyl)-ß-(2-carboxyethyl)phosphorylpropionate was observed to be a remarkably reactive inhibitor of Sporosarcina pasteurii urease (kinact/KI = 10 420 s-1 M-1). The high potential of this group of compounds was also confirmed in Proteus mirabilis whole-cell-based inhibition assays. Some compounds followed slow-binding and reversible kinetics, e.g., methyl ß-(3,4-dihydroxyphenyl)-ß-phosphonopropionate, with Ki* = 0.13 µM, and an atypical low dissociation rate (residence time τ = 205 min).

Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical characterization, and biological evaluation.

To date, drugs that hit a single target are inadequate for the treatment of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases. The development of multitarget ligands, able to interact with the different pathways involved in the progession of these disorders, represents a great challenge for medicinal chemists. In this context, we report here the synthesis and biological evaluation of phenol-lipoyl hybrids (SV1-13), obtained via a linking strategy, to take advantage of the synergistic effect due to the antioxidant portions and anti-amyloid properties of the single constituents present in the hybrid molecule. Biological results showed that SV5 and SV10 possessed the best protective activity against Aß1-42 induced neurotoxicity in differentiated SH-SY5Y cells. SV9 and SV10 showed remarkable antioxidant properties due to their ability to counteract the damage caused by H2O2 in SHSY-5Y-treated cells. Hovewer, SV5, showing moderate antioxidant and good neuroprotective activities, resulted the best candidate for further experiments since it also resulted stable both simulated and plasma fluids.

Catechol-based inhibitors of bacterial urease.

Targeted covalent inhibitors of urease were developed on the basis of the catechol structure. Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC50 = 518 nM andkinact/Ki = 1379 M-1 s-1. Inhibitory activity of this compound was better and toxicity lower than those obtained for the starting compound - catechol. The molecular modelling studies revealed a mode of binding consistent with structure-activity relationships.

Structural exploration of cinnamate-based phosphonic acids as inhibitors of bacterial ureases.

The conjugated system of cinnamic acid, α-substituted with a phosphonoalkyl residue, was previously validated as a scaffold that provided one of the most potent organophosphorus inhibitors of bacterial urease. Following the idea of using Morita-Baylis-Hillman adducts to introduce the terminal phosphonic side chain functionality to the α,ß-unsaturated system, we currently report the synthesis and activity of an extended series of compounds. Cinnamates modified with 3-phosphonopropyl and 4-phosphonobutyl side chains were obtained in a convenient two-step procedure, which involved Pd-mediated transformations of the Morita-Baylis-Hillman bromides as the key substrates. The introduction of a terminal alkenyl fragment, which was achieved by Stille coupling with stannanes, was followed by a tandem C-P bond formation/oxidation process. A submicromolar ligand of Sporosarcina pasteurii urease (Ki = 0.509 µM) was identified among the active molecules. In addition, inhibitors of Proteus mirabilis urease affected bacterial growth at the micromolar level. Based on the structure-activity relationship and the mechanism of inhibition, we suggest a nontypical mixed mode of action for the slow binding compounds. We presume that the molecular distance between the phosphonic group and the backbone double bond allows a dual activity: complexation of the acidic group with nickel ions and Michael addition of a cysteine forming the active site lid.

A novel and efficient synthesis of 3,4-dihydroxyphenylacetic ester and amide derivatives/conjugates and assessment of their antioxidant activity.

Phenolic acids, a sub-class of polyphenols, are widely studied. By contrary, 3,4-dihydroxyphenylacetic acid is scarcely studied. For this purpose, a series of 3,4-dihydroxyphenylacetic acid ester and amide derivatives/conjugates were synthesised for the first time. A systematic study has been performed to quantitatively identify the functional groups present in these compounds using different techniques such as 1H NMR, 13C NMR and ESI MS. The synthesised compounds were evaluated for their in vitro antioxidant activity by a DPPH radical-scavenging assay. Their physico-chemical profile is also studied using Molinspiration tool. Among all tested compounds, amidoester 36 showed the best scavenging activity possessing an EC50 17 µΜ and improved physico-chemical properties compared to the parent compound.

Novel organophosphorus scaffolds of urease inhibitors obtained by substitution of Morita-Baylis-Hillman adducts with phosphorus nucleophiles.

The reactivity of Morita-Baylis-Hillman allyl acetates was employed to introduce phosphorus-containing functionalities to the side chain of the cinnamic acid conjugated system by nucleophilic displacement. The proximity of two acidic groups, the carboxylate and phosphonate/phosphinate groups, was necessary to form interactions in the active site of urease by recently described inhibitor frameworks. Several organophosphorus scaffolds were obtained and screened for inhibition of the bacterial urease, an enzyme that is essential for survival of urinary and gastrointestinal tract pathogens. α-Substituted phosphonomethyl- and 2-phosphonoethyl-cinnamate appeared to be the most potent and were further optimized. As a result, one of the most potent organophosphorus inhibitors of urease, α-phosphonomethyl-p-methylcinnamic acid, was identified, with Ki = 0.6 µM for Sporosarcina pasteurii urease. High complementarity to the enzyme active site was achieved with this structure, as any further modifications significantly decreased its affinity. Finally, this work describes the challenges faced in developing ligands for urease.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Structure-guided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases.

Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π-π stacking interaction between a pyridine ring and Tyr372.

Mapping the pathway toward thiophosphinic pseudopeptides. Synthesis of suitably protected PG-Phe-Ψ[P(S)(OX)CH2]-Gly-OY analogues as thiophosphinyl dipeptide isosters (TDI), a comparative study for selective deprotection and further elongation.

In the present study, we describe in detail the first synthesis of a new class of phosphorus compounds, thiophosphinyl pseudopeptides. We prepared several fully protected thiophosphinate pseudodipeptides of the general formula PG-Phe-Ψ[P(S)(OX)CH2]-Gly-OY starting from the corresponding phosphinate pseudodipeptide using Lawesson's reagent. Selective deprotection, further elongation, and stability of these compounds were studied, and the results are disclosed. These compounds can be used as transition-state-mimicking inhibitors for several zinc metalloproteases.

Computer-aided optimization of phosphinic inhibitors of bacterial ureases.

Urease inhibitors can be considered as a tool to control the damaging effect of ureolytic bacteria infections in humans which occur commonly in the developed countries. Computer-aided optimization of the aminomethylphosphinate structures by modifying both their N- and P-termini led to the invention of a novel group of inhibitors of bacterial ureases. Introduction of P-hydroxymethyl group into the molecule resulted in considerable increase of the inhibitory activity against enzymes purified from Bacillus pasteurii and Proteus vulgaris as compared with their P-methyl counterparts described previously. The designed compounds represent a competitive reversible class of urease inhibitors. The most potent, N-methyl-aminomethyl-P-hydroxymethylphosphinic acid, displayed K(i) = 360 nM against P. vulgaris enzyme.

Design, synthesis, and evaluation of novel organophosphorus inhibitors of bacterial ureases.

A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in vitro against urease from Bacillus pasteurii and Proteus vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with Ki = 170 nM (against B. pasteurii enzyme) for the most active N-( N'-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid.