Pharmacological Research, Vol. 46, No. 5, 2002 doi:10.1016/S1043-6618(02)00208-6, available online at http://www.idealibrary.com on NEW INSIGHTS INTO THE PHARMACOLOGICAL PROPERTIES OF POTENT ANTIPLATELET 2-AMINO-BENZO[d]ISOTHIAZOL-3-ONE DERIVATIVES VIGILIO BALLABENI a , MASSIMILIANO TOGNOLINI a , FRANCESCO CALCINA a , MARIANNINA IMPICCIATORE a , PAOLA VICINI b and ELISABETTA BAROCELLI a,∗ a Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy, b Dipartimento Farmaceutico Università di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy Accepted 2 August 2002 A series of 2-amino-benzo[d]isothiazol-3-one derivatives (1–8), previously described as in vitro potent antiaggregatory agents endowed with spasmolytic properties, was evaluated for in vitro an- tiplatelet activity in guinea-pig platelet rich plasma and for in vivo effects on experimental thrombosis and bleeding time in mice. All the 2-amino-benzo[d]isothiazol-3-one derivatives 1–8 were more po- tent antiplatelets against collagen than acetylsalicylic acid and, unlike this drug, strongly inhibited thromboxane agonist U46619-induced aggregation. Subacutely administered (5 mg kg -1 day i.p. for 5 days), compounds 6 and 7 protected mice from collagen/epinephrine induced pulmonary throm- boembolism at about 20-fold lower doses than acetylsalicylic acid and they prolonged bleeding time like the most part of the other derivatives. The potent antithrombotic activity was coupled with the absence of any lethal and ulcerogenic effect up to 200 mg kg -1 os. © 2002 Elsevier Science Ltd. All rights reserved. Key words: antiplatelet drugs, experimental thrombosis, bleeding, subacute treatment, benzoisothiazolone. INTRODUCTION Although evidence has accumulated about the clinical ef- fectiveness of aspirin in the treatment of thromboembolic diseases [1–3], there is currently a request for the develop- ment of antiplatelet agents free from the gastrointestinal side effects associated with a specific cyclooxygenase in- hibition [4,5]. Within a research program directed toward the design and synthesis of new inhibitors of platelet aggregation, a potent antiplatelet activity against differ- ent aggregating agents was previously evidenced in vitro and ex vivo for 2-amino-benzo[d]isothiazol-3-one [6]. Starting from this investigation we developed derivatives bearing substituents on the 2-amino group, proved to be crucial for biological activity [7]. Such compounds (1–8) possessed in vitro marked antiplatelet activity against ADP and arachidonic acid coupled with spasmolytic properties [8]. The present report is aimed at the further characterisation of the pharmacological profile of com- pounds 1–8 (Fig. 1) through additional investigations addressed at the evaluation of their in vitro antiplatelet ∗ Corresponding author. Dipartimento di Scienze Farmacologiche, Bio- logiche e Chimiche Applicate, Universit` a di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy. E-mail: barocell@unipr.it activity on aggregation induced in guinea-pig plasma by collagen or by thromboxane A 2 agonist U46619 and their in vivo antithrombotic effectiveness and tolerability. In detail, these latter effects were studied after subacute intraperitoneal administration on acute pulmonary throm- boembolism and tail bleeding time in mice. Furthermore, data on acute ulcerogenicity and toxicity (expressed as LD 50 ) were obtained in the same species. Moreover, ex- periments were performed to detect the possible interac- tion of 2-amino-benzo[d]isothiazol-3-one derivatives 1–8 with histamine and acetylcholine receptors on guinea-pig ileal smooth muscle. MATERIALS AND METHODS Materials For in vivo tests, calf collagen (Type III: Sigma Chem- ical Co., St. Louis, MO) was suspended (2.5 mg ml -1 ) according to Momo et al. [9] and lysine acetylsalicy- late (Flectadol ® , Carlo Erba, Milan, Italy) was used as reference drug. Epinephrine bitartrate, histamine dihy- drochloride, acetylcholine chloride, acetylsalicylic acid, were purchased from Sigma Chemical Co., St. Louis, MO. 9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin 1043-6618/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.