Francesco Venturoni

On the Way to Selective PARP-2 Inhibitors. Design, Synthesis, and Preliminary Evaluation of a Series of Isoquinolinone Derivatives

PARP‐1 and PARP‐2 are members of the family of poly(ADP‐ribose)polymerases, which are involved in the maintenance of genomic integrity under conditions of genotoxic stimuli. The different roles of the two isoforms under pathophysiological conditions have not yet been fully clarified, and this is partially due to the lack of selective inhibitors. We report herein the synthesis and preliminary pharmacological evaluation of a large series of isoquinolinone derivatives as PARP‐1/PARP‐2 inhibitors. Among them, we identified the 5‐benzoyloxyisoquinolin‐1(2 H)‐one derivative as the most selective PARP‐2 inhibitor reported so far, with a PARP‐2/PARP‐1 selectivity index greater than 60.

Avicholic Acid: A Lead Compound from Birds on the Route to Potent TGR5 Modulators.

Grounding on our former 3D QSAR studies, a knowledge-based screen of natural bile acids from diverse animal species has led to the identification of avicholic acid as a selective but weak TGR5 agonist. Chemical modifications of this compound resulted in the disclosure of 6α-ethyl-16-epi-avicholic acid that shows enhanced potency at TGR5 and FXR receptors. The synthesis, biological appraisals, and structure-activity relationships of this series of compounds are herein described. Moreover, a thorough physicochemical characterization of 6α-ethyl-16-epi-avicholic acid as compared to naturally occurring bile acids is reported and discussed.

Sequence Variants in Kynurenine Aminotransferase II (KAT II) Orthologs Determine Different Potencies of the Inhibitor S-ESBA

The kynurenine pathway (KP, Figure 1) constitutes the major metabolic route of the essential amino acid l-tryptophan (lTrp) in mammals. KP metabolism is initiated by oxidative opening of the indole ring of l-Trp and leads to the production of NAD . The catabolic intermediates of KP have recently attracted considerable attention for their role in central nervous system (CNS) physiology and in the etiology and progression of neurodegenerative and immunological disorders. The major neuroactive metabolites originate from two competing branches of the pathway, where the central metabolite l-kynurenine (l-Kyn, 1) is transformed into 3-hydroxykynurenine (3-OH-Kyn, 2) and then further into quinolinic acid (QUIN, 3), or into kynurenic acid (KYNA, 4). KYNA is a competitive antagonist of the glycine site of the NMDA receptor complex, a noncompetitive antagonist of the a7 nicotinic acetylcholine receptor, 6] and is endowed with neuroprotective properties. QUIN (3), in contrast, is a neurotoxic agonist of the NMDA receptor complex, and 3-OH-Kyn (2) is a free radical generator that can contribute to neuronal damage in the CNS. In view of the neuroprotective or neurotoxic activities of KP metabolites, enzymes of the KP have long been considered interesting targets for rational therapeutic intervention. One of the most promising targets is kynurenine aminotransferase (KAT), a pyridoxal-5’-phosphate (PLP)-dependent enzyme that catalyzes the irreversible transamination of l-Kyn (1) into KYNA (4). Four isoforms of this enzyme have so far been identified, namely KAT I, KAT II, KAT III, and KAT IV (or mitAAT). Although all four isoforms are present in the mammalian brain, the enzyme activity of KAT III has not been confirmed so far, and only KAT I and KAT II have been thoroughly characterized with regard to their role in cerebral KYNA synthesis. These two isoforms differ in substrate specificity, with KAT I showing a lower specificity for l-Kyn (1) than KAT II. 13] The development of KAT inhibitors may prove useful in disorders associated with learning and memory deficits, where lowering the levels of brain KYNA may counterbalance glutamatergic and cholinergic hypofunction. Recently, we reported the synthesis and biological characterization of (S)-4-ethylsulfonylbenzoylalanine (S-ESBA, 5), the first potent and selective inhibitor of KAT II, which is the dominant KYNA-forming enzyme in the rat brain. Biological assays conducted in vitro showed that S-ESBA (5) inhibits KAT II obtained from partially purified rat liver with an IC50 value of 6.1 mm. In vivo studies support these results, demonstrating that the administration of S-ESBA (5) lowers the extracellular levels of KYNA in the hippocampus of unanesthetized rats. Herein we report a novel and more efficient synthesis of SESBA (5) and an analysis of its inhibitory activity using purified recombinant human KAT II. The data are discussed in light of the crystal structure of human KAT II in complex with its natural substrate l-Kyn (1), and on the basis of conserved and nonconserved residues that feature the sequences of speciesspecific orthologs of KAT II (human, rat, and mouse). Figure 1. The kynurenine pathway (KP) of tryptophan metabolism.

Continuous flow synthesis and scale-up of glycine- and taurine-conjugated bile salts

A multi-gram scale protocol for the N-acyl amidation of bile acids with glycine and taurine has been successfully developed under continuous flow processing conditions. Selecting ursodeoxycholic acid (UDCA) as the model compound and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as the condensing agent, a modular mesoreactor assisted flow set-up was employed to significantly speed up the optimization of the reaction conditions and the flow scale-up synthesis. The results in terms of yield, in line purification, analysis, and implemented flow set-up for the reaction optimization and large scale production are reported and discussed.

Discovery and characterization of novel potent PARP-1 inhibitors endowed with neuroprotective properties: From TIQ-A to HYDAMTIQ

Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in controlling cell survival or death. As a consequence, therapeutic interventions with PARP-1 inhibitors are sought in different pathological conditions such as cancer, cardiovascular and inflammatory diseases, as well as brain ischemia. In the first part of this work, as a continuation of our efforts in the field, we report the design, synthesis and biological appraisal of novel potent PARP-1 inhibitors. A crystallization experiment is carried out to ascertain the mode of binding to PARP-1 of the most potent compound, namely 2-((dimethylamino)methyl)-9-hydroxythieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), whilst molecular modeling studies are performed to infer the role of water molecules in ligand binding. In the second part of the work, we discuss the results of HYDAMTIQ in models of brain ischemia as well as its preliminary physicochemical and pharmacokinetic characterization. Collectively, the data obtained qualify HYDAMTIQ as a novel lead candidate for advancement to clinical settings of brain ischemia.

Exploring the synthetic versatility of the Lewis acid induced decomposition reaction of α-diazo-β-hydroxy esters. The case of ethyl diazo(3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)acetate.

Ethyl diazo(3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)acetate was prepared by aldol-type condensation of ethyl diazoacetate with isatin. A systematic and mechanistic study on the Lewis acid induced decomposition reaction of this valuable diazo precursor was carried out with the aim to gain new insights into the mechanistic aspects of the reaction as well as to further understand the factors and experimental conditions which affect the relative product distribution. The reaction, which may proceed via cationic and noncationic mechanisms, was found to be significantly influenced by the reaction environment determined by the characteristics of the Lewis acid employed, by the ability of the Lewis acid to form a complex with the alcohol functionality of the α-diazo-β-hydroxy ester, and by the polarity and nucleophilicity of the solvent used.

BF3·Et2O-Induced Decomposition of Ethyl 2-Diazo-3-hydroxy-3,3-diarylpropanoates in Acetonitrile: A Novel Approach to 2,3-Diaryl β-Enamino Ester Derivatives

The BF(3).Et(2)O-induced decomposition of ethyl 2-diazo-3-hydroxy-3,3-diarylpropanoates, prepared by the addition of a series of benzophenones to ethyl diazo(lithio)acetate, is reported and studied. By using acetonitrile as a solvent, the corresponding N-acyl beta-enamino ester derivatives are obtained in good yields and with a diverse regioselectivity as the result of 1,2-aryl migration in the vinyl cation intermediates. The factors that govern the migratory aptitude as well as the mechanistic aspects of the reaction are discussed.

Conformational properties of cholic acid, a lead compound at the crossroads of bile acid inspired drug discovery

Cholic acid is an endogenous primary bile acid endowed with endocrine signaling functions and digestive properties. In this work its conformational profile and the impact of the steroid nucleus are investigated using DFT studies and NMR spectroscopy. As a result, three major minima conformations are found with ‘extended’, ‘semi-extended’ and ‘folded’ side chains, respectively. While the ‘extended’ and ‘semi-extended’ conformations are often found as bioactive conformations in the binding site of co-crystallized biological targets, the ‘folded’ conformation is only experimentally observed in NMR studies. Overall, the results of this study provide additional basis for the interpretation of biological functions of bile acids, and disclose new pharmacophoric templates for the design of constrained bile acid analogues.

HPLC/ELSD analysis of amidated bile acids: An effective and rapid way to assist continuous flow chemistry processes

The employment of the flow N-acyl amidation of natural bile acids (BAs) required the in-line connection with suitable analytical tools enabling the determination of reaction yields as well as of the purity grade of the synthesized glyco- and tauro-conjugated derivatives. In this framework, a unique HPLC method was successfully established and validated for ursodeoxycholic (UDCA), chenodeoxycholic (CDCA), deoxycholic (DCA) and cholic (CA) acids, as well as the corresponding glyco- and tauro-conjugated forms. Because of the shared absence of relevant chromophoric moieties in the sample structure, an evaporative light scattering detector (ELSD) was profitably utilized for the analysis of such steroidal species. For each of the investigated compounds, all the runs were contemporarily carried out on the acidic free and the two relative conjugated variants. The different ELSD response of the free and the corresponding conjugated BAs, imposed to build-up separate calibration curves. In all the cases, very good precision (RSD% values ranging from 1.04 to 6.40% in the long-period) and accuracy (Recovery% values ranging from 96.03 to 111.14% in the long-period) values along with appreciably low LOD and LOQ values (the former being within the range 1-27 ng mL(-1) and the latter within the range 2-44 ng mL(-1)) turned out.