M. Chiavarini

New polycyclic pyrimidine derivatives with antiplatelet in vitro activity: synthesis and pharmacological screening.

The preparation and the pharmacological screening of novel anti-aggregatory/antiphlogistic polycyclic pyrimidine derivatives are described. The compounds were developed starting from bioactive 2-aminobenzopyranopyrimidine derivatives in order to assess the importance of the benzopyrano[4,3-d]pyrimidine structure and the role of an amino basic moiety in position 2. Antiplatelet activity was assessed in vitro against ADP and arachidonic acid-induced aggregation in guinea-pig plasma. Anti-inflammatory/analgesic/antipyretic activities were studied in rat paw oedema, mouse writhing test and E. coli-induced rat fever. Ulcerogenic and gastroprotective effects were also investigated in vivo on rat gastric mucosa. Among the tested compounds, the 5-substituted benzopyranopyrimidine derivatives 3d and 4d proved to be the most active antiplatelet agents as potent as acetylsalicylic acid against arachidonic acid-stimulated aggregation. Furthermore the 2-methylthio derivative 4d was endowed with greater efficacy against ADP aggregation suggesting that additional non-TXA2 dependent mechanisms are involved in its biological activity. Orally administered at 100 mg kg(-1) in rats this latter compound displayed antiphlogistic acitivity comparable to indomethacin (10 mg kg(-1)) coupled with an unusual gastroprotective effect on ethanol-induced ulcers. In conclusion, these findings indicate that the 5-pyrrolidino-2-methylthiobenzopyrano[4,3-d]pyrimidine 4d fulfils the chemical requirements to exhibit antiplatelet activity associated with gastroprotective effect.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Interaction of selective compounds with muscarinic receptors at dispersed intestinal smooth muscle cells

1 The characterization of muscarinic receptors on single cells of the guinea‐pig ileum longitudinal smooth muscle, devoid of neuronal elements, was functionally studied by estimating the affinities of muscarinic antagonists on acetylcholine‐induced contractions. 2 Atropine (5 × 10−11 to 5 × 10−6 m), 4‐diphenylacetoxy‐N‐methyl‐piperidine methiodide (4‐DAMP, 5 × 10−8 to 5 × 10−6 m), cyclohexyl(4‐fluoro‐phenyl) (3‐piperidinopropyl) silanol (pFHHSiD, 5 × 10−7 to 5 × 10−5 m) as well as pirenzepine (5 × 10−7 to 5 × 10−5 m) competitively antagonized the acetylcholine‐dependent contractions with different affinities (atropine > 4‐DAMP > pFHHSiD > pirenzepine). 3 Methoctramine (5 × 10−7 to 5 × 10−5 m), and AF‐DX 116 (5 × 10−6 and 5 × 10−5 m) also showed antagonist properties but these deviated from simple competition. These compounds, which discriminate between M2 and M3 receptors, showed a potency lower than that of pirenzepine, the rank order of potencies being pirenzepine > methoctramine > AF‐DX 116. When concentrations of AF‐DX 116, methoctramine and pirenzepine were increased an unspecific contractile effect occurred. 4 McN‐A‐343, a partial agonist on intact guinea‐pig longitudinal smooth muscle strips, on this preparation induced a weak contraction (about 7% in comparison to control) that was not reversed by antimuscarinic agents. 5 These data indicate that M3 rather than M2 receptor sites are present on this tissue.

R‐α‐methylhistamine‐induced inhibition of gastric acid secretion in pylorus‐ligated rats via central histamine H3 receptors

1 The effect of central H3 histamine receptor activation on gastric acid and pepsin production has been investigated in pylorus‐ligated rats. 2 Intracerebroventricular injections (i.c.v.) of the selective H3 agonist, R‐α‐methylhistamine (0.5–50 nmol per rat) caused a dose‐dependent inhibition of gastric acid secretion while intravenous administration (5–500 nmol per rat) was completely ineffective. 3 I.c.v. microinjections of mepyramine, tiotidine and thioperamide (51 nmol per rat), selective antagonists at H1, H2‐ and H3‐sites respectively, failed to modify the acid secretory response to pylorus ligation. 4 The antisecretory effect of R‐α‐methylhistamine (5 nmol per rat, i.c.v.) was selectively prevented by the H3‐blocker, thioperamide (51 nmol per rat, i.c.v.), mepyramine and tiotidine pretreatment being completely inactive. 5 Unlike acid secretion, pepsin production was not significantly affected by all the tested compounds. 6 These findings provide the first pharmacological evidence that the activation of central H3 histamine receptors exerts a negative control in the regulation of gastric acid secretion in conscious pylorus‐ligated rats.

Regional differences in motor responsiveness to antimuscarinic drugs in rabbit isolated small and large intestine

The pirenzepine-related analogue, nuvenzepine, and the antagonists selective for the three muscarinic receptor subtypes 4-DAMP (M1 and M3 receptors), pirenzepine (M1 receptors), methoctramine (M2 receptors) have been tested on rabbit isolated small and large intestinal regions provided with spontaneous motor activity. The employed drugs differently affected intestinal motility patterns. The ileum pendular movements as well as the proximal colon and taenia coli tone, spike amplitude and frequency were concentration-dependently inhibited by the compounds here employed. Their pIC50 values followed the rank order of potency generally reported for the involvement of the M3 muscarinic receptors (4-DAMP > or = atropine > nuvenzepine > or = pirenzepine > methoctramine). Unlike nuvenzepine and the other antimuscarinics assayed, the M1 selective antagonist pirenzepine, at nanomolar concentrations, was able to enhance the proximal taenia coli motility patterns suggesting that a M1-inhibitory pathway might operate in the physiological modulation of taenia coli motility. At variance with longitudinal ileum and colon contractile activity, proximal circular colon motility was resistant to muscarinic as well as to alpha 1-, H1-, 5-HT-blockade indicating that NANC neuronal mechanisms could act at this level. In summary, these data provide evidence that, at intestinal level, nuvenzepine is almost completely devoid of reliable M1-linked effect thus possessing a different pharmacological selectivity at muscarinic receptor subtypes with respect to pirenzepine. Furthermore, it emerges that rabbit spontaneous small and large intestinal motility is probably driven by different physiological mechanisms regional-related. The peculiar circular colon refractoriness deserves further studies to be extended to the human tissue.

Muscarinic M1 and M3 receptor antagonist effects of a new pirenzepine analogue in isolated guinea-pig ileal longitudinal muscle-myenteric plexus

The new pirenzepine analogue DF 545 has been tested for its muscarinic M1 and M3 receptor antagonist properties in guinea-pig longitudinal muscle-myenteric plexus preparations. McN-A-343-induced inhibition of twitch contractions was taken as a parameter for muscarinic M1 receptor activation while electrical and acetylcholine-induced contractions were considered as a model for muscarinic M3 receptor stimulation. An unexpected contractile effect evoked by McN-A-343 was also investigated. In contrast to pirenzepine, DF 545 only weakly counteracted the M1-mediated McN-A-343 inhibitory effect but blocked M3-related twitch- or acetylcholine-stimulated responses with a 2-fold higher affinity than pirenzepine. Therefore, in this preparation, our findings suggest that DF 545 does not share the selectivity profile exhibited by pirenzepine at ileal muscarinic receptors. Studies on the McN-A-343 contractile effect provide evidence that this agonist may interact with ileal muscarinic effector sites in a different way from other cholinergic agents.

Synthesis and preliminary pharmacological study of new amidinobenzisothiazoles

Abstract Some new N -(1,2-benzisothiazol-3-yl)amidines were synthesized and tested for antiinflammatory, antipyretic, analgesic and spasmolytic activities in comparison with indomethacin and papaverine respectively. The synthesis of the new compounds has been described. All the amidinobenzisothiazoles showed remarkable analgesic action and an interesting antiphlogistic action, while only one drug displayed a certain antipyretic activity. As regards spasmolytic activity, 3 compounds out of 6 demonstrated a papaverine-like effect.

Are histamine receptors involved in the stimulant activities of thiazolylethylamines supposed as cyclic models of dimaprit

A representative group of 2-aminothiazolylethylamine derivatives, in which the gastric acid secretion stimulatingS-aminoalkylisothiourea moiety can be recognized, was tested. The quite different responses observed suggest thatin vivo but notin vitro some events mimicking an H2-receptor agonist-like activity rather than a direct interaction with H2-receptors could take place. On the basis of structure-activity relationships, it can be speculated that the active conformation of dimaprit is not that resembled by these compounds which have been considered as cyclic models of one of its possible conformations.

Functional comparison between nuvenzepine and pirenzepine on different guinea pig isolated smooth muscle preparations

The antimuscarinic agents nuvenzepine and pirenzepine were tested on four guinea pig isolated smooth muscle preparations in order to better investigate the existence of differences in the functional activities of such antagonists, as suggested by previous reports. The effects of both compounds were compared to those of atropine. Nuvenzepine showed a four-fold higher affinity than pirenzepine in competitively antagonizing acetylcholine-induced contractions on isolated ileal musculature (pA2 = 7.08 +/- 0.15) and on longitudinal ileum dispersed cells (pA2 = 7.11 +/- 0.19). By contrast, unlike pirenzepine which was ineffective, nuvenzepine inhibited histamine-induced ileal motor activity in a dualistic manner, behaving as an irreversible competitive H1 antagonist (pA2 = 5.02 +/- 0.11). Nuvenzepine was almost equipotent to pirenzepine in competitively preventing bethanechol-induced gall-bladder contractions (pA2 = 7.23 +/- 0.16) and it displayed a four-fold higher potency than pirenzepine in blocking vagal-stimulated tracheal constrictions (pIC50 = 6.77 +/- 0.06). Both compounds were definitely less potent than atropine. On the whole, these findings indicate that, on the selected preparations, nuvenzepine substantially shares the antimuscarinic properties of pirenzepine but it is also endowed with a (weak) H1-blocking action. Furthermore, based on some observations, the presence in gallbladder smooth muscle of muscarinic receptors distinguishable from those of ileum could be speculated.

Pharmacological activities of two new histamine analogs

The pharmacological properties of 2-aminohistamine and 2-amino-5-methylhistamine were studied and compared with those of histamine, 5-methylhistamine and dimaprit. The introduction of an amino group in position 2 of the histamine imidazole ring caused a reduction of histamine potency, mostly with respect to H1 receptors. Such disactivation was much more evident in its corresponding 5-methyl derivative. The pharmacological activity related to the chemical structure will be discussed in the paper.