DOI: 10.1002/cmdc.201200224 b-d-Glucosyl Conjugates of Highly Potent Inhibitors of Blood Coagulation Factor Xa Bearing 2-Chorothiophene as a P1 Motif Gianfranco Lopopolo, [a] Modesto de Candia, [a] Luigi Panza, [b] Maria Rosaria Romano, [c] Marcello Diego Lograno, [c] Francesco Campagna, [a] and Cosimo Altomare* [a] Introduction The serine protease factor Xa (fXa), located at the confluence of the intrinsic and extrinsic pathways of the blood coagula- tion cascade, catalyzes the cleavage of prothrombin to form thrombin, thus initiating the common pathway that leads to the formation of fibrin clots. Among the newly developed anti- coagulant drugs with better safety profiles than those currently used, thus allowing fixed oral dosing and not requiring time- and resource-intensive monitoring, fXa inhibitors have emerged as attractive options for venous thromboembolism (VTE) treatment and stroke prevention in patients with atrial fi- brillation (AF). [1] As fXa inhibitors should prevent the genera- tion of new thrombin without affecting the basal thrombin level that ensures primary hemostasis, [2] they should have a lower risk of bleeding than current antithrombotic therapy (heparins and warfarin), [3] and an even higher therapeutic ratio than direct thrombin inhibitors (DTIs) [4] such as dabigatran etexilate. [5] Intensive efforts are underway toward the pharmaceutical development of direct fXa inhibitors as new antithrombotic agents, as evidenced by the growing number of publications and patents filed in the last decade. [6] Large clinical trial pro- grams have established the effectiveness of fXa inhibitors, such as apixaban and rivaroxaban, in patients experiencing (or at risk of) VTE in orthopedic surgery, [1] and these drugs are pre- dicted to have a great impact on the anticoagulant market. [7] The rational design of orally bioavailable small-molecule fXa inhibitors, achieving good enzyme potency and selectivity, has been considerably supported by the X-ray co-crystal structures of protein–ligand complexes, sophisticated molecular model- ing techniques, and 3D QSAR studies. [8] FXa contains a serine protease domain in a trypsin-like closed b-barrel fold, encom- passing the catalytic triad Ser 195-His 57-Asp 102 and two prox- imal binding pockets, namely S1 and S4. Potent fXa inhibitors are usually L-shaped, with a preorganized scaffold orienting two nearly orthogonal molecular fragments toward the proxi- mal S1 and S4 pockets in the enzyme binding site. Due to the high shape retention of the active site and the well-defined subpockets, fXa is a good model for molecular recognition studies. [9, 10] We recently reported N-(2-hydroxyphenyl)acetamide-based inhibitors of fXa bearing 5-chlorothien-2-yl and 1-isopropylpi- peridin-4-yl moieties as P1 and P4 fragments, respectively. [11] The most potent compound 1 (Figure 1) inhibited fXa with a K i value of 0.3 nm and very high selectivity over thrombin and other serine proteases, achieving in vitro anticoagulant activity in the low micromolar range, as assessed by the prothrombin time clotting assay (PT 2 = 3.30 mm). Based on these data, com- We synthesized a novel O-glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5-chlorothien- 2-yl moiety and 1-isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A b-d-glucosyl unit was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of 1. The synthesized b-d-glucose- based compound 16 achieved picomolar inhibitory potency against human fXa (K i = 60 pm) and high selectivity over thrombin and other serine proteases. In addition to the chloro- thienyl S1 binder, a large gain in DG resulted from the addition of protonated 1-isopropylpiperidine (DDG = 29.7– 30.5 kJ mol À1 ), which should bind to the aromatic S4 pocket through efficient cation–p and C ÀH···p interactions. Instead, the C3-alkyl-linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves DG by an average of 2.9–3.8 kJ mol À1 . Compound 16 showed sub-micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT 2 and aPTT 2 equal to 0.135 and 0.389 mm, respectively). Although com- pound 16 was 1.4-fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6-fold) prolongation of PT relative to controls (P < 0.05) 60 min after oral dosing (75 mg kg À1 ). [a] Dr. G. Lopopolo, Dr. M. de Candia, Prof. F. Campagna, Prof. C. Altomare Dipartimento Farmaco Chimico University of Bari A. Moro, Via E. Orabona 4, 70125 Bari (Italy) E-mail : altomare@farmchim.uniba.it [b] Prof. L. Panza Dipartimento di Scienze del Farmaco University of Piemonte Orientale A. Avogadro Via Bovio 6, 28100 Novara (Italy) [c] Dr. M. R. Romano, Prof. M. D. Lograno Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche University of Bari A. Moro, Via E. Orabona 4, 70125 Bari (Italy) ChemMedChem 0000, 00, 1 – 10  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ MED