Claudia Mugnaini

Investigations on the 4-quinolone-3-carboxylic acid motif. 1. Synthesis and structure-activity relationship of a class of human immunodeficiency virus type 1 integrase inhibitors.

A set of 4-quinolone-3-carboxylic acids bearing different substituents on the condensed benzene ring was designed and synthesized as potential HIV-1 integrase inhibitors structurally related to elvitegravir. Some of the new compounds proved to be able to inhibit the strand transfer step of the virus integration process in the micromolar range. Docking studies and quantum mechanics calculations were used to rationalize these data.

Investigations on the 4-quinolone-3-carboxylic acid motif. 2. Synthesis and structure-activity relationship of potent and selective cannabinoid-2 receptor agonists endowed with analgesic activity in vivo.

Quinolone-3-carboxamides 11 bearing at position 5, 6, 7, or 8 diverse substituents such as halides, alkyl, aryl, alkoxy, and aryloxy groups differing in their steric/electronic properties, were prepared. The new compounds were tested in vitro for CB1 and CB2 receptor affinity in comparison with the reference compounds rimonabant and SR144528. The tested compounds exhibited CB2 affinity in the range from 55.9 to 0.8 nM and CB1 affinity in the range from >10,000 to 5.3 nM, with selectivity indeces [Ki(CB1)/Ki(CB2)] varying from >2666.6 to 1.23. On the basis of the structure-selectivity relationship developed, the presence of a substituent at C6/C8 or C7 well accounts for the high or low CB2 selectivity, respectively. Compound 11c, characterized by high CB2 affinity and selectivity, showed analgesic activity in the formalin test of acute peripheral and inflammatory pain in mice as a result of selective CB2 agonistic activity.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 4. Identification of New Potent and Selective Ligands for the Cannabinoid Type 2 Receptor with Diverse Substitution Patterns and Antihyperalgesic Effects in Mice

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quinolone-3-carboxamides, further structural modifications of the heterocyclic scaffold were explored, leading to the discovery of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response in mice.

Characterization of COR627 and COR628, Two Novel Positive Allosteric Modulators of the GABAB Receptor

The potential efficacy of GABAB receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABAB receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABAB receptor may constitute a novel approach in the pharmacological manipulation of the GABAB receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABAB PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5′-O-(3-[35S]thio)-triphosphate binding to native GABAB receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABAB receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([3H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABAB receptor.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 3. Synthesis, Structure—Affinity Relationships, and Pharmacological Characterization of 6-Substituted 4-Quinolone-3-carboxamides as Highly Selective Cannabinoid-2 Receptor Ligands

A set of quinolone-3-carboxamides 2 bearing diverse substituents at position 1, 3, and 6 of the bicyclic nucleus was prepared. Except for six compounds exhibiting Ki>100 nM, all the quinolone-3-carboxamides 2 proved to be high affinity CB2 ligands, with Ki values ranging from 73.2 to 0.7 nM and selectivity [SI=Ki(CB1)/Ki(CB2)] varying from >14285 to 1.9, with only 2ah exhibiting a reverse selectivity (SI<1). In the formalin test of peripheral acute and inflammatory pain in mice, 2ae showed analgesic activity that was antagonized by a selective CB2 antagonist. By contrast, 2e was inactive per se and antagonized the effect of a selective CB2 agonist. Finally, 2g and 2p exhibited CB2 inverse agonist-like behavior in this in vivo test. However, two different functional assays carried out in vitro on 2e and 2g indicated for both compounds an overall inverse agonist activity at CB2 receptors.

Development and Characterization of Immobilized Cannabinoid Receptor (CB1/CB2) Open Tubular Column for On-line Screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2-OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2-OT column can be used to determine the binding affinities (K(i) values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2-OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.

Three-dimensional quantitative structure-selectivity relationships analysis guided rational design of a highly selective ligand for the cannabinoid receptor 2.

This paper describes a three-dimensional quantitative structure-selectivity relationships (3D-QSSR) study for selectivity of a series of ligands for cannabinoid CB1 and CB2 receptors. 3D-QSSR exploration was expected to provide design information for drugs with high selectivity toward the CB2 receptor. The proposed 3D computational model was performed by Phase and generated taking into account a number of structurally diverse compounds characterized by a wide range of selectivity index values. The model proved to be predictive, with r2 of 0.95 and Q2 of 0.63. In order to get prospective experimental validation, the selectivity of an external data set of 39 compounds reported in the literature was predicted. The correlation coefficient (r2=0.56) obtained on this unrelated test set provided evidence that the correlation shown by the model was not a chance result. Subsequently, we essayed the ability of our approach to help the design of new CB2-selective ligands. Accordingly, based on our interest in studying the cannabinergic properties of quinolones, the N-(adamantan-1-yl)-4-oxo-8-methyl-1-pentyl-1,4-dihydroquinoline-3-carboxamide (65) was considered as a potential synthetic target. The log(SI) value predicted by using our model was indicative of high CB2 selectivity for such a compound, thus spurring us to synthesize it and to evaluate its CB1 and CB2 receptor affinity. Compound 65 was found to be an extremely selective CB2 ligand as it displayed high CB2 affinity (Ki=4.9 nM), while being devoid of CB1 affinity (Ki>10,000 nM). The identification of a new selective CB2 receptor ligand lends support for the practicability of quantitative ligand-based selectivity models for cannabinoid receptors. These drug discovery tools might represent a valuable complementary approach to docking studies performed on homology models of the receptors.

Synthesis and pharmacological characterization of 2-(acylamino)thiophene derivatives as metabolically stable, orally effective, positive allosteric modulators of the GABAB receptor

Two recently reported hit compounds, COR627 and COR628, underpinned the development of a series of 2-(acylamino)thiophene derivatives. Some of these compounds displayed significant activity in vitro as positive allosteric modulators of the GABAB receptor by potentiating GTPγS stimulation induced by GABA at 2.5 and 25 μM while failing to exhibit intrinsic agonist activity. Compounds were also found to be effective in vivo, potentiating baclofen-induced sedation/hypnosis in DBA mice when administered either intraperitoneally or intragastrically. Although displaying a lower potency in vitro than the reference compound GS39783, the new compounds 6, 10, and 11 exhibited a higher efficacy in vivo: combination of these compounds with a per se nonsedative dose of baclofen resulted in shorter onset and longer duration of the loss of righting reflex in mice. Test compounds showed cytotoxic effects at concentrations comparable to or higher than those of GS39783 or BHF177.

A novel CB2 agonist, COR167, potently protects rat brain cortical slices against OGD and reperfusion injury

Cannabinoid CB2 receptor activation has been shown to have many pharmacological but not psychotropic effects. The aim of this study was to investigate the potential protection of brain tissues afforded by the novel substituted 4-quinolone-3-carboxylic acid derivative COR167, a selective CB2 agonist, toward ischemia and reperfusion-induced injury, as well as the mechanism of this potential effect. Rat brain cortical slices subjected to oxygen and glucose deprivation (OGD) followed by re-oxygenation were used. Cell damage was quantified by measuring at the end of the reperfusion phase the release into the artificial cerebrospinal fluid (ACSF) of lactate dehydrogenase (LDH), glutamate, IL-6 and TNF-α and by evaluating in tissue the lipid-peroxides (thiobarbituric acid-reactive substances, TBARS), the free, reduced glutathione content (GSH) and the water gain (TWG), taken as an index of cell swelling. COR167 (10nM or 100 nM), added to ACSF during the entire reperfusion phase, markedly reduced LDH and glutamate release, as well as TWG. Lower (0.1-1 nM) or higher concentrations (1,000 nM) were ineffective, suggesting thereby an hormetic behavior. COR167 at 10nM concentration markedly reverted in tissues TBARS increase and GSH decrease, while reducing IL-6 and TNF-α release into ACSF. COR167 effects on glutamate and LDH release were abrogated by the selective CB2 inverse-agonists COR170 (1 nM) and AM630 (1μM) but not by the CB1 antagonist AM251 (1 μM). COR170 as well as AM630 per se were able to revert TWG. The CB2 receptor agonist COR167 potently protected rat brain cortical slices against OGD and reperfusion injury, partly through CB2 receptors activation.

Design, Synthesis, and Pharmacological Characterization of Indol-3-ylacetamides, Indol-3-yloxoacetamides, and Indol-3-ylcarboxamides: Potent and Selective CB2 Cannabinoid Receptor Inverse Agonists

In our search for new cannabinoid receptor modulators, we describe herein the design and synthesis of three sets of indole-based ligands characterized by an acetamide, oxalylamide, or carboxamide chain, respectively. Most of the compounds showed affinity for CB2 receptors in the nanomolar range, with K(i) values spanning 3 orders of magnitude (377-0.37 nM), and moderate to good selectivity over CB1 receptors. Their in vitro functional activity as inverse agonists was confirmed in vivo in the formalin test of acute peripheral and inflammatory pain in mice, in which compounds 10a and 11e proved to be able to reverse the effect of the CB2 selective agonist COR167.