Two diketopiperazines, XR334 (1) and the novel compound XR330 (2), were isolated from the lyophilised biomass of an unidentified Streptomyces sp. Their structures were elucidated on the basis of spectroscopic studies and confirmed by chemical synthesis. Both compounds inhibited plasminogen activator inhibitor-1 activity in an amidolytic assay of tissue plasminogen activator mediated plasmin generation. Compound1 also enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat. These diketopiperazines represent the first low molecular weight inhibitors of plasminogen activator inhibitor-1, a physiological regulator of fibrinolysis protein including a 23 amino acid signal peptide with a predicted, glycosylated mass of 52 kD3). PAI-1 inhibition of tPA is mediated through a "bait" residue (Arg 346-Met 347) which mimics the normal substrate40. The physiological importance of PAI-1 has been demonstrated in transgenic mice which express high levels of human PAI-1 and suffer severe venous thrombosis5). An increase in the plasma concentration of PAI-1 has been proposed as a risk factor in thrombotic disease6). During a screening programme for inhibitors of PAI-1 activity we discovered a series of low molecular weight, nonpeptidyl inhibitors of PAI-1 from fermentation of an unidentified Streptomyces sp. Twoexamples of this series, XR334 (1) and the novel compound XR330 (2) were purified from the mycelium7) (Fig. 1). A series of closely related metabolites has been isolated from Streptomyces thioluteus including 18). Several of these compounds were reported to show weak antibacterial activity. Synthetic analogues of 1 have also been reported previously9~11}. Related diketopiperazines with putative cytotoxic activity have also been isolated from Micromonospora neiheunsis12). Wereport here an important new biologi- Structures of 1 and 2 isolated from the culture biomass of Streptomyces sp. and the structure confirmed by synthesis. Structures for two additional minor metabolites, 3 and 4, are proposed.

SummaryA critical component in the regulation of thrombus formation and clearance is the balance between tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAM). An increase in the plasma concentration of PAH has been proposed as a risk factor in thrombotic disease. Inhibition of PAI-1 activity may have utility in the treatment of thromboembolic disease. We report here the evaluation of three diketopiperazine-based low molecular weight inhibitors of PAI-1 activity (XR334, XR1853 and XR5082). In vitro these compounds reversed the inhibitory effects of PAI-1 against both tPA and urokinase (UK) (IC50:5 to 80 μM). In contrast, other serpin-serine protease interactions, including α1-antitrypsin-trypsin, α2-antiplasmin-plasmin and antithrombin-thrombin, were not affected, neither did these inhibitors affect global tests of haemostasis. In the light of this promising in vitro profile these compounds were evaluated in a standard radioisotopic assay of clot lysis in whole rat blood following intravenous administration. In this assay these compounds dose-dependently enhanced fibrinolysis ex vivo. After intravenous bolus administration XR334, XR1853 and XR5082 at 5 mg/kg increased clot lysis by 32.0 ± 5.1% SEM (n = 25, p <0.01), 36.7 ± 3.5% SEM (n = 36, p <0.01) and 60.0 ± 2.8% SEM (n = 17, p <0.01) respectively compared to vehicle. Intravenous infusion of these compounds (1 mg/kg/min for 20 min) significantly prolonged (approximately twofold) the time to blood vessel occlusion in the rat electrically-stimulated carotid artery thrombosis model. Thus, these low molecular weight inhibitors of PAI-1 activity enhanced fibrinolysis ex vivo and protected against thrombus formation in the rat.

Inhibition of Plasminogen Activator Inhibitor-1 Activity by Two Diketopiperazines, XR330 and XR334 Produced by Streptomyces sp.-The title compounds (I) represent the first low molecular weight inhibitors of plasminogen activator inhibitor-1. -(BRYANS, J.; CHARLTON, P.; CHICARELLI-ROBINSON, I.; COLLINS, M.; FAINT, R.; LATHAM, C.; SHAW, I.; TREW, S.;