Elena Guaita

Fragment based design of new H4 receptor-ligands with anti-inflammatory properties in vivo

Using a previously reported flexible alignment model we have designed, synthesized, and evaluated a series of compounds at the human histamine H 4 receptor (H 4R) from which 2-(4-methyl-piperazin-1-yl)-quinoxaline ( 3) was identified as a new lead structure for H 4R ligands. Exploration of the structure-activity relationship (SAR) of this scaffold led to the identification of 6,7-dichloro 3-(4-methylpiperazin-1-yl)quinoxalin-2(1 H)-one (VUF 10214, 57) and 2-benzyl-3-(4-methyl-piperazin-1-yl)quinoxaline (VUF 10148, 20) as potent H 4R ligands with nanomolar affinities. In vivo studies in the rat reveal that compound 57 has significant anti-inflammatory properties in the carrageenan-induced paw-edema model.

Discovery of Quinazolines as Histamine H4 Receptor Inverse Agonists Using a Scaffold Hopping Approach

From a series of small fragments that was designed to probe the histamine H(4) receptor (H(4)R), we previously described quinoxaline-containing fragments that were grown into high affinity H(4)R ligands in a process that was guided by pharmacophore modeling. With a scaffold hopping exercise and using the same in silico models, we now report the identification and optimization of a series of quinazoline-containing H(4)R compounds. This approach led to the discovery of 6-chloro-N-(furan-3-ylmethyl)2-(4-methylpiperazin-1-yl)quinazolin-4-amine (VUF10499, 54) and 6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine (VUF10497, 55) as potent human H(4)R inverse agonists (pK(i) = 8.12 and 7.57, respectively). Interestingly, both compounds also possess considerable affinity for the human histamine H(1) receptor (H(1)R) and therefore represent a novel class of dual action H(1)R/H(4)R ligands, a profile that potentially leads to added therapeutic benefit. Compounds from this novel series of quinazolines are antagonists at the rat H(4)R and were found to possess anti-inflammatory properties in vivo in the rat.

Pharmacological characterization of the new histamine H4 receptor agonist VUF 8430

Background and purpose:  We compare the pharmacological profiles of a new histamine H4 receptor agonist 2‐(2‐guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H4 receptor agonist, 4‐methylhistamine.

Searching for new NO-donor Aspirin-like molecules: a new class of nitrooxy-acyl derivatives of salicylic acid

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

Effects of Cannabinoid Receptor Agonists on Rat Gastric Acid Secretion: Discrepancy Between In Vitro and In Vivo Data

The effects of the cannabinoid (CB)-receptor agonists WIN55,212-2 and HU-210 and the selective CB1-receptor antagonist SR141716A were tested on in vitro and in vivo acid secretion assays from the rat. In the isolated gastric fundus from immature rats, WIN55,212-2 (0.001–30 μ M), HU-210 (0.001–10 μ M), or SR141716A (0.1–10 μ M) did not change the basal acid output or acid responses to histamine, pentagastrin, or electrical field stimulation. HU-210 (0.3 μ mol/kg, intravenously) inhibited the acid response to pentagastrin in anesthetized adult, young, or immature rats with lumen-perfused stomachs; moreover, HU-210 reduced vagally induced acid secretion in adult animals, its antisecretory effect being reversed by SR141716A (0.65 μ mol/kg, intravenously). In vitro and in vivo data indicate that CB1 receptors are not located on parietal cells but, rather, on vagal pathways (possibly at preganglionic sites) supplying the gastric mucosa. The lack of effect of CB-receptor ligands in vitro cannot be ascribed to the use of immature rats, since HU-210 inhibited stimulated acid secretion in vivo, irrespective of the animal age.

Searching for new NO-donor aspirin-like molecules: Furoxanylacyl derivatives of salicylic acid and related furazans.

A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furazans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid (pH 1) and physiological solutions (pH 7.4), and in human serum. No compound exhibited COX-inhibitory activity against COX-1 and COX-2 isoforms, when tested up to 100μM, respectively, on isolated platelets and on monocytes. Phenylsulfonyl- and cyano-substituted furoxans inhibited platelet aggregation induced by collagen in human platelet-rich plasma, through a cGMP dependent mechanism. Furoxan derivatives displayed cGMP-dependent vasodilator activities, tested on rat aorta strips precontracted with phenylephrine. All products showed anti-inflammatory activity similar to that of ASA, tested on rats by the carrageenan-induced paw edema assay. Unlike ASA, all products showed markedly reduced gastrotoxicity in a rat lesion model.

Effects of cyclooxygenase-1 and -2 inhibition on gastric acid secretion and cardiovascular functions in rats.

The discovery of a second isoform of cyclooxygenase has led to a re-evaluation of the mechanisms underlying the adverse effects of nonsteroidal anti-inflammatory drugs, focusing in particular on the gastrointestinal system. We investigated the involvement of cyclooxygenase-1 and -2 in the regulation of gastric acid secretion and cardiovascular functions in anesthetized rats, after acute intravenous administration of the selective cyclooxygenase-1 inhibitor SC-560, the selective cyclooxygenase-2 inhibitor celecoxib and the nonselective inhibitor indomethacin. Indomethacin, celecoxib and SC-560 did not significantly modify basal acid secretion. Indomethacin and celecoxib were also ineffective on the acid secretion stimulated by pentagastrin; by contrast, SC-560 significantly enhanced the acid secretion stimulated by pentagastrin, electrical vagal stimulation or histamine. The stimulatory effects of SC-560 were prevented by cervical vagotomy, atropine and famotidine. Indomethacin caused either no change, increasing or decreasing effects on mean arterial pressure and heart rate. By contrast, SC-560 was unable to change cardiovascular parameters at 5 mg/kg, while inducing a marked bradycardia at 10 mg/kg. Celecoxib was ineffective. Our findings indicate that cyclooxygenase-1-derived prostaglandins are involved in the regulation of stimulated acid secretion and of basal heart rate; the role of prostaglandins in the acute control of systemic blood pressure under resting conditions seems to be negligible.

Morphological Features of Rat Gastric Mucosa after Acute and Chronic Treatment with Amtolmetin Guacyl: Comparison with Non-Selective and COX-2-Selective NSAIDs

Background/Aims: The compound amtolmetin guacyl (AMG) has been characterized in both animal and human studies as a novel non-selective non-steroidal anti-inflammatory drug (NSAID) endowed with lower ulcerogenicity in comparison with traditional NSAIDs due to a unique mechanism of action, namely the increase in endogenous production of gastric nitric oxide. Methods: Conscious rats were treated either acutely (4 h) or chronically (3 and 14 days) with intragastric AMG (50 and 150 mg/kg), the non-selective NSAID tolmetin (TOL, 30 and 100 mg/kg) or the COX-2-selective NSAID celecoxib (CXIB, 20 and 60 mg/kg). Macroscopically visible and histologic lesions were evaluated. The ultrastructure of mucosal microvasculature was assessed. Results: (1) TOL and CXIB caused quantitatively greater endothelial damage and inflammatory cell infiltration than that induced by AMG; (2) AMG and CXIB, unlike TOL, did not cause epithelial damage after acute or chronic treatment, and (3) gastric lesions induced by TOL underwent adaptation during chronic treatment. Conclusion: Endothelial cell damage in the gastric microvasculature is an early event following both non-selective and COX-2-selective inhibitors. The low gastric mucosal toxicity of AMG is confirmed after acute and chronic treatment.

Gastroprotection by (R)-α-methylhistamine: involvement of nitric oxide?

In the gastrointestinal tract, nitric oxide (NO) has been implicated as a critical mediator both in mucosal protection and in the pathogenesis of damage [1–3]. Based on evidence that NO modulates a series of processes responsible for the defence of the gastric mucosa, it is considered that NO exerts a predominantly protective activity. The present study is aimed at evaluating the hypothesis that NO could play a role in the gastroprotective activity exerted by the histamine H3 receptor selective agonist (R)-amethylhistamine [(R)a-MeHA] in the rat [4, 5]. The acute effects of the non-selective NO synthase inhibitor NG-nitroL-arginine methyl ester (L-NAME) and the selective inhibitor of the inducible isoform of NO synthase 1400W [6] on the protective action of (R)a-MeHA against gastric mucosal lesions induced by absolute ethanol have been evaluated.

Multitarget Drugs: Synthesis and Preliminary Pharmacological Characterization of Zileuton Analogues Endowed with Dual 5‐LO Inhibitor and NO‐Dependent Activities

The leukotrienes (LTs) are a family of lipid-derived autacoids that originate from arachidonic acid (AA). 5-Lipooxygenase (5LO) is the key enzyme in this process. It transforms AA through a two-step process, first into 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and then into unstable leukotriene A4 (LTA4). This intermediate can be transformed either by leukotriene B4 synthase into leukotriene B4 (LTB4), or by leukotriene C4 synthase, which is a specific glutathione transferase, into peptide– lipid leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). [1–3] LTs are involved in a variety of inflammatory and allergic disorders, particularly rheumatoid arthritis and inflammatory skin and bowel diseases. They also display potent bronchoconstrictor activity. Consequently, the treatment of allergic disorders and asthma are the classical indications for 5-LO inhibitors. 5] Novel and interesting potential indications are emerging for these products; for example, an increasing amount of experimental evidence shows an involvement of the 5-LO pathway in tumor cell proliferation. In particular, inhibition of 5-LO was found to induce apoptosis in various cancer cell types. The evidence that LTs are involved in atherogenesis and arterial wall remodeling sets the stage for new strategies in treating the development and progression of atherosclerosis. 7, 8] 5-LO inhibitors can be classified into four different classes according to their mechanism of action: redox-active compounds, competitive reversible inhibitors, inhibitors of 5-LO activating protein (FLAP), and iron-chelating inhibitors. Many substances that belong to these classes were developed as potent 5-LO inhibitors, including natural products. Among them, only ( )-[1-(1-benzo[b]thien-2-yl)ethyl]-1-hydroxyurea (1, zileuton), a hydroxyurea derivative of the iron-ligand-type inhibitor class, entered into the market in 1996 as an anti-asthmatic drug. 13] The commercially available product is a racemic mixture of R and S enantiomers, both of which display in vitro 5-LO inhibitor activity. A number of studies have been carried out in recent years to design 5-LO inhibitors with dual activity: 5-LO/cyclooxygenase (COX) inhibitors have received particular attention as antiinflammatory agents, and compounds either with dual 5-LO/ thromboxane A2 synthase inhibitory activity or with 5-LO inhibitor and platelet-activating factor (PAF) receptor antagonist mixed properties have also been developed. These are examples of multitarget drugs, which should be able to modulate more than one target simultaneously; their development represents an alternative approach to using drug cocktails especially in the treatment of complex diseases such as atherosclerosis and inflammation. The most common strategy to obtain these products is the combination of two appropriate drugs, or their crucial parts, into a single molecule. To our knowledge there has not yet been any documented examples of nitric oxide (NO) donor/5-LO inhibitor hybrids, despite the clear interest in such a combination. Indeed, NO is a physiological messenger that triggers a variety of actions in different systems. In particular, it plays very important roles in the cardiovascular system in maintaining a number of homeostatic responses: preservation of endothelial integrity, arterial blood vessel dilation including pulmonary arterial vasculature, inhibition of platelet adherence and aggregation, attenuation of leukocyte adherence, and activation and inhibition of vascular smooth muscle cell proliferation. NO also triggers relevant action in airways, inducing relaxation of airway smooth muscle, pro-inflammatory or anti-inflammatory effects, and regulation of mucociliary clearance. The use of NO donors in the treatment of cardiovascular disease (CD) is well known, while the therapeutic potential of these kinds of products in the field of respiratory disease is still under examination. Herein we propose new dual-action products, obtained by combining zileuton with NO donor nitrooxy or furoxan moieties. The synthesis of the final products required the preliminary preparation of a number of intermediates (Scheme 1). The substituted benzothiophene 3 was easily obtained by treating 6hydroxybenzothiophene (2) with n-butyllithium and anhydrous acetaldehyde in THF at 20 8C. The triflates 8–11 were prepared by the action of trifluoromethanesulfonyl anhydride in dichloromethane on the appropriate nitrooxy-substituted alcohols 4–7 in the presence of 2,6-lutidine at 40 8C and were immediately used. Treatment of 3 with sodium hydride in THF, followed by the appropriate triflates 8–11 in dichloromethane afforded the expected nitrates 14–17. The action of 37 % hydrochloric acid on these products dissolved in THF/water in the presence of hydroxyurea at 50 8C afforded the target com[a] Prof. D. Boschi, Dr. M. Giorgis, Prof. C. Cena, Dr. N. C. Talniya, Prof. A. Di Stilo, Prof. R. Fruttero, Prof. A. Gasco Department of Drug Science and Technology University of Turin, Via Pietro Giuria 9, 10125 Torino (Italy) Fax: (+ 39) 0116707286 E-mail : alberto.gasco@unito.it [b] Dr. G. Morini, Prof. G. Coruzzi, Dr. E. Guaita Department of Human Anatomy, Pharmacology and Forensic Medicine Section of Pharmacology, University of Parma Via Volturno 39, 43100 Parma (Italy)