Hydroxy-Propil-ß-Cyclodextrin Inclusion Complexes of two Biphenylnicotinamide Derivatives: Formulation and Anti-Proliferative Activity Evaluation in Pancreatic Cancer Cell Models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with poor outcomes largely due to its unique microenvironment, which is responsible for the low response to drugs and drug-resistance phenomena. This clinical need led us to explore new therapeutic approaches for systemic PDAC treatment by the utilization of two newly synthesized biphenylnicotinamide derivatives, PTA73 and PTA34, with remarkable antitumor activity in an in vitro PDAC model. Given their poor water solubility, inclusion complexes of PTA34 and PTA73 in Hydroxy-Propil-ß-Cyclodextrin (HP-ß-CD) were prepared in solution and at the solid state. Complexation studies demonstrated that HP-ß-CD is able to form stable host-guest inclusion complexes with PTA34 and PTA73, characterized by a 1:1 apparent formation constant of 503.9 M-1 and 369.2 M-1, respectively (also demonstrated by the Job plot), and by an increase in aqueous solubility of about 150 times (from 1.95 µg/mL to 292.5 µg/mL) and 106 times (from 7.16 µg/mL to 762.5 µg/mL), in the presence of 45% w/v of HP-ß-CD, respectively. In vitro studies confirmed the high antitumor activity of the complexed PTA34 and PTA73 towards PDAC cells, the strong G2/M phase arrest followed by induction of apoptosis, and thus their eligibility for PDAC therapy.

Novel Antiproliferative Biphenyl Nicotinamide: NMR Metabolomic Study of its Effect on the MCF-7 Cell in Comparison with Cisplatin and Vinblastine.

A 1H-NMR-based metabolomic study was performed on MCF-7 cell lines treated with a novel nicotinamide derivative (DT-8) in comparison with two drugs characterized by a well-established mechanism of action, namely the DNA-metalating drug cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and the antimitotic drug vinblastine (vinblastine, VIN). The effects of the three compounds, each one at the concentration corresponding to the IC50 value, were investigated, with respect to the controls (K), by the 1H-NMR of cells lysates and multivariate analysis (MVA) of the spectroscopic data. Relevant differences were found in the metabolic profiles of the different treatments with respect to the controls. A large overlap of the metabolic profiles in DT-8 vs. K and VIN vs. K suggests a similar biological response and mechanism of action, significantly diverse with respect to CDDP. On the other hand, DT8 seems to act by disorganizing the mitotic spindle and ultimately blocking the cell division, through a mechanism implying methionine depletion and/or S-adenosylmethionine (SAM) limitation.

Pharmacophore Modeling and 3D-QSAR Study of Indole and Isatin Derivatives as Antiamyloidogenic Agents Targeting Alzheimer's Disease.

Thirty-six novel indole-containing compounds, mainly 3-(2-phenylhydrazono) isatins and structurally related 1H-indole-3-carbaldehyde derivatives, were synthesized and assayed as inhibitors of beta amyloid (Aß) aggregation, a hallmark of pathophysiology of Alzheimer's disease. The newly synthesized molecules spanned their IC50 values from sub- to two-digit micromolar range, bearing further information into structure-activity relationships. Some of the new compounds showed interesting multitarget activity, by inhibiting monoamine oxidases A and B. A cell-based assay in tau overexpressing bacterial cells disclosed a promising additional activity of some derivatives against tau aggregation. The accumulated data of either about ninety published and thirty-six newly synthesized molecules were used to generate a pharmacophore hypothesis of antiamyloidogenic activity exerted in a wide range of potencies, satisfactorily discriminating the 'active' compounds from the 'inactive' (poorly active) ones. An atom-based 3D-QSAR model was also derived for about 80% of 'active' compounds, i.e., those achieving finite IC50 values lower than 100 µM. The 3D-QSAR model (encompassing 4 PLS factors), featuring acceptable predictive statistics either in the training set (n = 45, q2 = 0.596) and in the external test set (n = 14, r2ext = 0.695), usefully complemented the pharmacophore model by identifying the physicochemical features mainly correlated with the Aß anti-aggregating potency of the indole and isatin derivatives studied herein.

A Prospective Repurposing of Dantrolene as a Multitarget Agent for Alzheimer's Disease.

The orphan drug dantrolene (DAN) is the only therapeutic treatment for malignant hyperthermia (MH), a pharmacogenetic pathology affecting 0.2 over 10,000 people in the EU. It acts by inhibiting ryanodine receptors, which are responsible for calcium recruitment in striatal muscles and brain. Because of its involvement in calcium homeostasis, DAN has been successfully investigated for its potential as neuroprotecting small molecule in several animal models of Alzheimer's disease (AD). Nevertheless, its effects at a molecular level, namely on putative targets involved in neurodegeneration, are still scarcely known. Herein, we present a prospective study on repurposing of DAN involving, besides the well-known calcium antagonism, inhibition of monoamine oxidase B and acetylcholinesterase, cytoprotection from oxidative insult, and activation of carnitine/acylcarnitine carrier, as concurring biological activities responsible for neuroprotection.

Structure-property relationship study of the HPLC enantioselective retention of neuroprotective 7-[(1-alkylpiperidin-3-yl)methoxy]coumarin derivatives on an amylose-based chiral stationary phase.

The enantiomer separation of a number of racemic 7-[(1-alkylpiperidin-3-yl)methoxy]coumarin derivatives, some of which show outstanding in vitro multitarget neuroprotective activities, was successfully achieved on a polysaccharide-based chiral stationary phase, bearing amylose tris(3,5-dimethylphenylcarbamate) as a chiral selector, in normal polar mode (methanol and acetonitrile as the mobile phases). The majority of the screened selectands, especially those bearing 1-(3-X-benzyl)piperidin-3-yl moieties, showed baseline enantiomer separations, and compound 8 (X = NO2 ) was the best resolved (α = 2.01; RS  = 4.27). Linear free energy relationships, usefully complemented by molecular docking calculations, have the key role in enantioselective retention of aromatic interactions between π-donor moieties in the chiral selector and π-acceptor moieties in selectand, strengthened by hydrogen bond interaction between a hydrogen bond donor in the chiral selector and the hydrogen bond acceptor group(s) in the selectand. Statistically, reliable equations highlighted the importance of the substituent's size and substitution pattern (meta better than para) to affect the enantiorecognition of the title compounds. The chromatographic data support the scalability of the optimized experimental conditions for preparative purposes.

Insights into Structure-Activity Relationships of 3-Arylhydrazonoindolin-2-One Derivatives for Their Multitarget Activity on ß-Amyloid Aggregation and Neurotoxicity.

Despite the controversial outcomes of clinical trials executed so far, the prevention of ß-amyloid (Aß) deposition and neurotoxicity by small molecule inhibitors of Aß aggregation remains a target intensively pursued in the search of effective drugs for treating Alzheimer's disease (AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the reference compound 1, we disclosed equipotent derivatives bearing alkyl substituents at the indole nitrogen, and fairly tolerated bioisosteric replacements at the arylhydrazone moiety. For most of the investigated compounds, the inhibition of Aß40 aggregation (expressed as pIC50) was found to be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear relationship. The N¹-cyclopropyl derivative 28 was tested in cell-based assays of Aß42 oligomer toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective effects. This study confirmed the versatility of isatin in preparing multitarget small molecules affecting different biochemical pathways involved in AD.

Design, synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase.

Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic and palliative, for the treatment of Alzheimer's disease (AD). Donepezil is one of most used AChEIs in AD therapy, acting as a dual binding site, reversible inhibitor of AChE with high selectivity over butyrylcholinesterase (BChE). Through a combined target- and ligand-based approach, a series of coumarin alkylamines matching the structural determinants of donepezil were designed and prepared. 6,7-Dimethoxycoumarin derivatives carrying a protonatable benzylamino group, linked to position 3 by suitable linkers, exhibited fairly good AChE inhibitory activity and a high selectivity over BChE. The inhibitory potency was strongly influenced by the length and shape of the spacer and by the methoxy substituents on the coumarin scaffold. The inhibition mechanism, assessed for the most active compound 13 (IC(50) 7.6 nM) resulted in a mixed-type, thus confirming its binding at both the catalytic and peripheral binding sites of AChE.

Synthesis and Biological Evaluation of Dantrolene-Like Hydrazide and Hydrazone Analogues as Multitarget Agents for Neurodegenerative Diseases.

Dantrolene, a drug used for the management of malignant hyperthermia, had been recently evaluated for prospective repurposing as multitarget agent for neurodegenerative syndromes, including Alzheimer's disease (AD). Herein, twenty-one dantrolene-like hydrazide and hydrazone analogues were synthesized with the aim of exploring structure-activity relationships (SARs) for the inhibition of human monoamine oxidases (MAOs) and acetylcholinesterase (AChE), two well-established target enzymes for anti-AD drugs. With few exceptions, the newly synthesized compounds exhibited selectivity toward MAO B over either MAO A or AChE, with the secondary aldimine 9 and phenylhydrazone 20 attaining IC50 values of 0.68 and 0.81 µM, respectively. While no general SAR trend was observed with lipophilicity descriptors, a molecular simplification strategy allowed the main pharmacophore features to be identified, which are responsible for the inhibitory activity toward MAO B. Finally, further in vitro investigations revealed cell protection from oxidative insult and activation of carnitine/acylcarnitine carrier as concomitant biological activities responsible for neuroprotection by hits 9 and 20 and other promising compounds in the examined series.

Nose-to-brain delivery: A comparative study between carboxymethyl chitosan based conjugates of dopamine.

Herein, the synthesis of a novel polymeric conjugate N,O-CMCS-Dopamine (DA) based on an amide linkage is reported. The performances of this conjugate were compared with those of an analogous N,O-CMCS-DA ester conjugate previously studied (Cassano et al., 2020) to gain insight into their potential utility for Parkinson's disease treatment. The new amide conjugate was synthesized by standard carbodiimide coupling procedure and characterized by FT-IR, 1H NMR spectroscopies and thermal analysis (Differential Scanning Calorimetry). In vitro mucoadhesive studies in simulated nasal fluid (SNF) evidenced high adhesive effect of both ester and amide conjugates. Results demonstrated that the amide conjugate exerted an important role to prevent DA spontaneous autoxidation both under stressed conditions and physiological mimicking ones. MTT test indicated cytocompatibility of the amide conjugate with Olfactory Ensheating Cells (OECs), which were shown by cytofluorimetry to internalize efficiently the conjugate. Overall, among the two conjugates herein studied, the N,O-CMCS-DA amide conjugate seems a promising candidate for improving the delivery of DA by nose-to-brain administration.

Scouting around 1,2,3,4-Tetrahydrochromeno[3,2-c]pyridin-10-ones for Single- and Multitarget Ligands Directed towards Relevant Alzheimer's Targets.

A number of 1,2,3,4-tetrahydrochromeno[3,2-c]pyridin-10-one derivatives have been synthesized and screened against different targets involved in the onset and progression of Alzheimer's disease (AD), such as acetyl- and butyrylcholinesterase (AChE and BChE), monoamine oxidases A and B (MAO A and B), aggregation of ß-amyloid (Aß) and reactive oxygen species (ROS) production. Derivatives 1 c, 3 b, 4 and 5 a showed multifaceted profiles of promising anti-AD features and returned well-balanced multitargeting inhibitory activities. Moreover, compound 1 f, a potent and selective human MAO B inhibitor (IC50 =0.89 µM), proved to be a safe neuroprotectant in a human neuroblastoma cell line (SH-SY5Y) by improving viability impaired by Aß1-42 and pro-oxidant insult. Furthermore, structure-activity relationships (SARs) and docking models were derived in order to assist further hit-to-lead optimization stage.

Bridging repair of the abdominal wall in a rat experimental model. Comparison between uncoated and polyethylene oxide-coated equine pericardium meshes.

Biological meshes improve the outcome of incisional hernia repairs in infected fields but often lead to recurrence after bridging techniques. Sixty male Wistar rats undergoing the excision of an abdominal wall portion and bridging mesh repair were randomised in two groups: Group A (N = 30) using the uncoated equine pericardium mesh; Group B (N = 30) using the polyethylene oxide (PEO)-coated one. No deaths were observed during treatment. Shrinkage was significantly less common in A than in B (3% vs 53%, P < 0.001). Adhesions were the most common complication and resulted significantly higher after 90 days in B than in A (90% vs 30%, P < 0.01). Microscopic examination revealed significantly (P < 0.05) higher mesh integrity, fibrosis and calcification in B compared to A. The enzymatic degradation, as assessed with Raman spectroscopy and enzyme stability test, affected A more than B. The PEO-coated equine pericardium mesh showed higher resistance to biodegradation compared to the uncoated one. Understanding the changes of these prostheses in a surgical setting may help to optimize the PEO-coating in designing new biomaterials for the bridging repair of the abdominal wall.

Human mitochondrial carnitine acylcarnitine carrier: Molecular target of dietary bioactive polyphenols from sweet cherry (Prunus avium L.).

The effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity.

1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-glycoprotein inhibitors: Molecular determinants for affinity and selectivity over multidrug resistance associated protein 1.

A series of coniugates bearing a 1,2,3,4-tetrahydroisoquinoline motif linked to substituted 7-hydroxy-2H-chromen-2-ones was synthesized and assayed through calcein-AM test in Madin-Darby Canine Kidney (MDCK) cells overexpressing P-glycoprotein (P-gp) and closely related multidrug resistance associated protein 1 (MRP1) to probe the interference with efflux mechanisms mediated by P-gp and MRP1, respectively. A number of substituents at C3 and C4 of coumarin nucleus along with differently sized and shaped spacers was enrolled to investigate the effects of focused structural modifications over affinity and selectivity. Linker length and flexibility played a key role in enhancing P-gp affinity as proved by the most potent P-gp modulator (3h, IC50 = 70 nM). A phenyl ring within the spacer (3k, 3l, 3o) and bulkier groups (Br in 3r, Ph in 3u) at coumarin C3 led to derivatives showing nanomolar activity (160 nM < IC50 < 280 nM) along with outstanding selectivity over MRP1 (SI > 350). Molecular docking calculations carried out on a human MDR1 homology model structure contributed to gain insights into the ligands' binding modes. Some compounds (3d, 3h, 3l, 3r, 3t, 3u) reversed MDR thereby restoring doxorubicin cytotoxicity when co-administered with the drug into MDCK-MDR1 cells.

Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget acetylcholinesterase-monoamine oxidase B inhibitors.

Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes' inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule.

3-benzazecine-based cyclic allene derivatives as highly potent P-glycoprotein inhibitors overcoming doxorubicin multidrug resistance.

Aim: Enamino 3-benzazecine compounds, incorporating the C6-C8 allene system, were synthesized and evaluated in vitro as inhibitors of P-glycoprotein (P-gp) and/or multidrug resistance-associated protein 1 (MRP1), two efflux pumps mainly connected with multidrug resistance (MDR) in cancer cells. Results & methodology: Most of the synthesized compounds were selective P-gp inhibitors in Calcein-AM uptake assay. Structure-activity relationships (SARs) pointed out that CO2Me derivatives are more potent than acetyl derivatives, and 10,11-dimethoxy compounds are five to tenfold more potent inhibitors than the respective unsubstituted compounds, and that the P-gp inhibition potency is mainly related to volume parameters. Conclusion: Nanomolar P-gp inhibitors, such as 23 (IC50 = 4.2 nM), restored the antiproliferative activity of doxorubicin in multidrug-resistant cells. The observed activities showed that 3-benzazecine-based compounds may be promising MDR reversers.

Design, synthesis, and biological evaluation of glycine-based molecular tongs as inhibitors of Abeta1-40 aggregation in vitro.

A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4'-oxydianiline spacers, were prepared and tested as inhibitors of beta-amyloid peptide Abeta(1-40) aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC(50) values in the micromolar range. Structure-activity relationships suggested that pi-pi stacking and hydrogen-bonding interactions play a key role in the inhibition of Abeta(1-40) self-assembly leading to amyloid fibrils.

1,3-Dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines as potent A2B adenosine receptor antagonists: design, synthesis, structure-affinity and structure-selectivity relationships.

A number of 1,3-dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines were prepared and evaluated as ligands of recombinant human adenosine receptors (hARs). Several 1,3-dipropyl derivatives endowed with nanomolar binding affinity at hA(2B) receptors, but poor selectivity over hA(2A), hA(1) and hA(3) AR subtypes were identified. A comparison with the corresponding 7-OH- and 7,9-unsubstituted-deazaxanthines revealed that 9-OH-9-deazaxanthines are more potent hA(2B) ligands with lower partition coefficients and higher water solubility compared to the other two congeneric classes of deazaxanthines. An optimization of the para-substituent of the 8-phenyl ring of 9-OH-9-deazaxanthines led to the discovery of compound 38, which exhibited outstanding hA(2B) affinity (Ki=1.0 nM), good selectivity over hA(2A), hA(1) and hA(3) (selectivity indices=100, 79 and 1290, respectively) and excellent antagonist potency in a functional assay on rat A(2B) (pA(2B)=9.33).

A New Class of 1-Aryl-5,6-dihydropyrrolo[2,1-a]isoquinoline Derivatives as Reversers of P-Glycoprotein-Mediated Multidrug Resistance in Tumor Cells.

A number of aza-heterocyclic compounds, which share the 5,6-dihydropyrrolo[2,1-a]isoquinoline (DHPIQ) scaffold with members of the lamellarin alkaloid family, were synthesized and evaluated for their ability to reverse in vitro multidrug resistance in cancer cells through inhibition of P-glycoprotein (P-gp) and/or multidrug-resistance-associated protein 1. Most of the investigated DHPIQ compounds proved to be selective P-gp modulators, and the most potent modulator, 8,9-diethoxy-1-(3,4-diethoxyphenyl)-3-(furan-2-yl)-5,6-dihydropyrrolo[2,1-a]isoquinoline-2-carbaldehyde, attained sub-micromolar inhibitory potency (IC50 : 0.19 µm). Schiff bases prepared by the condensation of some 1-aryl-DHPIQ aldehydes with p-aminophenol also proved to be of some interest, and one of them, 4-((1-(4-fluorophenyl)-5,6-dihydro-8,9-dimethoxypyrrolo[2,1-a]isoquinolin-2-yl)methyleneamino)phenol, had an IC50 value of 1.01 µm. In drug combination assays in multidrug-resistant cells, some DHPIQ compounds, at nontoxic concentrations, significantly increased the cytotoxicity of doxorubicin in a concentration-dependent manner. Studies of structure-activity relationships and investigation of the chemical stability of Schiff bases provided physicochemical information useful for molecular optimization of lamellarin-like cytotoxic drugs active toward chemoresistant tumors as well as nontoxic reversers of P-gp-mediated multidrug resistance in tumor cells.

Novel bisphosphonates with antiresorptive effect in bone mineralization and osteoclastogenesis.

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 µM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 µM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

Solid-phase synthesis and insights into structure-activity relationships of safinamide analogues as potent and selective inhibitors of type B monoamine oxidase.

Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide (IC50 = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of alpha-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S alpha-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites.