Designing Soluble PROTACs: Strategies and Preliminary Guidelines.

Solubility optimization is a crucial step to obtaining oral PROTACs. Here we measured the thermodynamic solubilities (log S) of 21 commercial PROTACs. Next, we measured BRlogD and log kwIAM (lipophilicity), EPSA, and Δ log kwIAM (polarity) and showed that lipophilicity plays a major role in governing log S, but a contribution of polarity cannot be neglected. Two-/three-dimensional descriptors calculated on conformers arising from conformational sampling and steered molecular dynamics failed in modeling solubility. Infographic tools were used to identify a privileged region of soluble PROTACs in a chemical space defined by BRlogD, log kwIAM and topological polar surface area, while machine learning provided a log S classification model. Finally, for three pairs of PROTACs we measured the solubility, lipophilicity, and polarity of the building blocks and identified the limits of estimating PROTAC solubility from the synthetic components. Overall, this paper provides promising guidelines for optimizing PROTAC solubility in early drug discovery programs.

Discovery and SAR Evolution of Pyrazole Azabicyclo[3.2.1]octane Sulfonamides as a Novel Class of Non-Covalent N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA) Inhibitors for Oral Administration.

Inhibition of intracellular N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity is a promising approach to manage the inflammatory response under disabling conditions. In fact, NAAA inhibition preserves endogenous palmitoylethanolamide (PEA) from degradation, thus increasing and prolonging its anti-inflammatory and analgesic efficacy at the inflamed site. In the present work, we report the identification of a potent, systemically available, novel class of NAAA inhibitors, featuring a pyrazole azabicyclo[3.2.1]octane structural core. After an initial screening campaign, a careful structure-activity relationship study led to the discovery of endo-ethoxymethyl-pyrazinyloxy-8-azabicyclo[3.2.1]octane-pyrazole sulfonamide 50 (ARN19689), which was found to inhibit human NAAA in the low nanomolar range (IC50 = 0.042 µM) with a non-covalent mechanism of action. In light of its favorable biochemical, in vitro and in vivo drug-like profile, sulfonamide 50 could be regarded as a promising pharmacological tool to be further investigated in the field of inflammatory conditions.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Synthesis and characterization of the first inhibitor of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD).

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is a membrane-associated zinc enzyme that catalyzes the hydrolysis of N-acylphosphatidylethanolamines (NAPEs) into fatty acid ethanolamides (FAEs). Here, we describe the identification of the first small-molecule NAPE-PLD inhibitor, the quinazoline sulfonamide derivative 2,4-dioxo-N-[4-(4-pyridyl)phenyl]-1H-quinazoline-6-sulfonamide, ARN19874.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Potent α-amino-ß-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies.

4-Cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in animal models. In the present work, we describe structure-activity relationship (SAR) studies on 3-aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to elucidate the principal structural and stereochemical features needed to achieve effective NAAA inhibition. Investigations on the influence of the substitution at the ß-position of the 2-oxo-3-azetidinyl ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA inhibitors as therapeutic agents for the treatment of pain and inflammation.

Acid Ceramidase in Melanoma: EXPRESSION, LOCALIZATION, AND EFFECTS OF PHARMACOLOGICAL INHIBITION.

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This down-regulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC50 = 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.

Benzoxazolone Carboxamides as Potent Acid Ceramidase Inhibitors: Synthesis and Structure-Activity Relationship (SAR) Studies.

Ceramides are lipid-derived intracellular messengers involved in the control of senescence, inflammation, and apoptosis. The cysteine amidase, acid ceramidase (AC), hydrolyzes these substances into sphingosine and fatty acid and, by doing so, regulates their signaling activity. AC inhibitors may be useful in the treatment of pathological conditions, such as cancer, in which ceramide levels are abnormally reduced. Here, we present a systematic SAR investigation of the benzoxazolone carboxamides, a recently described class of AC inhibitors that display high potency and systemic activity in mice. We examined a diverse series of substitutions on both benzoxazolone ring and carboxamide side chain. Several modifications enhanced potency and stability, and one key compound with a balanced activity-stability profile (14) was found to inhibit AC activity in mouse lungs and cerebral cortex after systemic administration. The results expand our arsenal of AC inhibitors, thereby facilitating the use of these compounds as pharmacological tools and their potential development as drug leads.

3,4-Dihydro-1,3,5-triazin-2(1H)-ones as the First Dual BACE-1/GSK-3ß Fragment Hits against Alzheimer's Disease.

One of the main obstacles toward the discovery of effective anti-Alzheimer drugs is the multifactorial nature of its etiopathology. Therefore, the use of multitarget-directed ligands has emerged as particularly suitable. Such ligands, able to modulate different neurodegenerative pathways, for example, amyloid and tau cascades, as well as cognitive and neurogenic functions, are fostered to come. In this respect, we report herein on the first class of BACE-1/GSK-3ß dual inhibitors based on a 3,4-dihydro-1,3,5-triazin-2(1H)-one skeleton, whose hit compound 1 showed interesting properties in a preliminary investigation. Notably, compound 2, endowed with well-balanced potencies against the two isolated enzymes (IC50 of 16 and 7 µM against BACE-1 and GSK-3ß, respectively), displayed effective neuroprotective and neurogenic activities and no neurotoxicity in cell-based assays. It also showed good brain permeability in a pharmacokinetic assessment in mice. Overall, triazinone derivatives, thanks to the simultaneous modulation of multiple points of the diseased network, might emerge as suitable candidates to be tested in in vivo Alzheimer's disease models.

Benzoxazolone carboxamides: potent and systemically active inhibitors of intracellular acid ceramidase.

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

Multitarget drug discovery for Alzheimer's disease: triazinones as BACE-1 and GSK-3ß inhibitors.

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3ß. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03±0.01) µM and (14.67±0.78) µM for BACE-1 and GSK-3ß, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.

Discovery of a new class of highly potent inhibitors of acid ceramidase: synthesis and structure-activity relationship (SAR).

Acid ceramidase (AC) is an intracellular cysteine amidase that catalyzes the hydrolysis of the lipid messenger ceramide. By regulating ceramide levels in cells, AC may contribute to the regulation of cancer cell proliferation and senescence and to the response to cancer therapy. We recently identified the antitumoral agent carmofur (4a) as the first nanomolar inhibitor of intracellular AC activity (rat AC, IC50 = 0.029 µM). In the present work, we expanded our initial structure-activity relationship (SAR) studies around 4a by synthesizing and testing a series of 2,4-dioxopyrimidine-1-carboxamides. Our investigations provided a first elucidation of the structural features of uracil derivatives that are critical for AC inhibition and led us to identify the first single-digit nanomolar inhibitors of this enzyme. The present results confirm that substituted 2,4-dioxopyrimidine-1-carboxamides are a novel class of potent inhibitors of AC. Selected compounds of this class may represent useful probes to further characterize the functional roles of AC.

Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity.

The expression of acid ceramidase (AC) - a cysteine amidase that hydrolyses the proapoptotic lipid ceramide - is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents.

Combining galantamine and memantine in multitargeted, new chemical entities potentially useful in Alzheimer's disease.

Herein we report on a novel series of multitargeted compounds obtained by linking together galantamine and memantine. The compounds were designed by taking advantage of the crystal structures of acetylcholinesterase (AChE) in complex with galantamine derivatives. Sixteen novel derivatives were synthesized, using spacers of different lengths and chemical composition. The molecules were then tested as inhibitors of AChE and as binders of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Some of the new compounds were nanomolar inhibitors of AChE and showed micromolar affinities for NMDAR. All compounds were also tested for selectivity toward NMDAR containing the 2B subunit (NR2B). Some of the new derivatives showed a micromolar affinity for NR2B. Finally, selected compounds were tested using a cell-based assay to measure their neuroprotective activity. Three of them showed a remarkable neuroprotective profile, inhibiting the NMDA-induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC(50) = 0.28 nM).

Stereochemical and skeletal diversity arising from amino propargylic alcohols.

An efficient synthetic pathway to the possible stereoisomers of skeletally diverse heterocyclic small molecules is presented. The change in shape brought about by different intramolecular cyclizations of diastereoisomeric amino propargylic alcohols is quantified using principal moment-of-inertia (PMI) shape analysis.

Identification of biphenyl-based hybrid molecules able to decrease the intracellular level of Bcl-2 protein in Bcl-2 overexpressing leukemia cells.

With the aim of enhancing the structural complexity and diversity of an existing collection of bi- and terphenyl compounds, we synthesized hybrid molecules comprising of spirocyclic ketones (a complexity-bearing core) and bi/terphenyls (privileged fragments). Compounds 1, 3, 4, and 6 showed well-defined activity on apoptosis and differentiation, making them potential leads for development as new anticancer agents and chemical probes to study signaling networks in neoplastic cells.

Synthetic small molecule Cdc25 phosphatases inhibitors.

Inhibition of Cdc25 phosphatases is a strategy for the discovery and development of novel anticancer agents targeting the cell cycle. A number of potent small molecule Cdc25 inhibitors have been identified. They are derived from different chemical classes; the most potent and selective derivatives are quinones. The electrophilic properties of quinones suggest the possibility of inducing a sulphydryl arylation of a cysteine in the enzyme active site. It is also possible that inhibition is due to redox cycling activity and production of ROS. Thus, oxidation of the thiolate form of cysteine occurs, leading to inactivation of enzymatic activity. Many of these inhibitors are active on all three Cdc25 phosphatases, cause cell cycle arrest and inhibit the growth of several human tumor cell lines. The possibility of toxicities induced by ROS, prompted the search for non-quinoid antagonists. It is not yet clear how these compounds bind within the enzyme's active site. Generally, electrophilic moieties able to trap the catalytic cysteine play an important role. Another strategy for identifying Cdc25 inhibitors is the development of compounds able to interact with the conserved loop region instead of phosphate.. In this review a summary of the most interesting Cdc25 inhibitors is given together with their biological activity. SAR studies concerning the importance of some structural features will be described.

Antiproliferative agents that interfere with the cell cycle at the G1-->S transition: further development and characterization of a small library of stilbene-derived compounds.

In this continuation of our research on derivatives containing the stilbene privileged structure or that are derived from it, we report the results of further studies carried out on the previously initiated collection of compounds. We used a parallel synthetic approach to rapidly obtain small sets of compounds and started the annotation of the library in progress by calculating some physicochemical properties to be eventually correlated with biological activities. A pharmacophore for the antiproliferative activity was also built to summarize the features of the library. We evaluated the antiproliferative and pro-apoptotic activities of all compounds as well as the cell-cycle effects of some representative compounds. After in-depth investigations, 3'-phenyl-[1,1';4',1'']terphenyl-4,3'',5''-triol showed the most interesting biological profile, as it interferes with cell-cycle progression at the G(1)-->S transition, acting on retinoblastoma phosphorylation and inducing cell differentiation.

Nitrogen-containing heterocyclic quinones: a class of potential selective antitumor agents.

The development of prodrugs that are enzymatically activated into anticancer agents is a promising perspective in cancer therapy. Many nitrogen-containing quinoid heterocycles have been reported to show antitumor effect. The principal interest in these compounds lies on their potential to produce tumor-selective toxicity. Selectivity occurs by difference in oxygen tension between normal and tumor tissue and by levels of the required activating enzymes. In this review a summary of the most interesting heterocyclic quinones is given together with their biological property. SAR studies concerning the importance of some structural features will be described.

Identification of a terphenyl derivative that blocks the cell cycle in the G0-G1 phase and induces differentiation in leukemia cells.

To further explore the SAR of resveratrol-related trans-stilbene derivatives, here we describe the synthesis of (a) a series of 3,5-dimethoxy analogues in which a variety of substituents were introduced at positions 2', 3', 4', and 5' of the stilbene scaffold and (b) a second group of derivatives (2-phenylnaphthalenes and terphenyls) that incorporate a phenyl ring as a bioisosteric replacement of the stilbene alkenyl bridge. We thoroughly characterized all of the new compounds with respect to their apoptosis-inducing activity and their effects on the cell cycle. One of the new derivatives, 13g, behaved differently from the others, as it was able to block the cell cycle in the G(0)-G(1) phase and also to induce differentiation in acute myelogenous leukemia HL60 cells. Compared to resveratrol, the synthetic terphenyl 13g showed a more potent apoptotic and differentiating activity. Moreover, it was active on both multidrug resistance and Bcr-Abl-expressing cells that were resistant to resveratrol.