Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.
The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into -sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and -sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-Asheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving -strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet. Plasminogen activator inhibitor type 1 (PAI-1)1 is a member of the serine proteinase inhibitor or serpin superfamily (1, 2). Serpins play an important role in the control of proteinases involved in blood coagulation, complement activation, and inflammation and are distinguished functionally from other types of protein inhibitors by their ability to form SDS stable complexes with target proteinases. Crystal structures have shown that members of the family share a highly conserved tertiary fold consisting of three large -sheets surrounded by nine ␣-helices. This scaffold presents the reactive loop as a pseudosubstrate that binds to and inhibits the target proteinase (3). The target proteinases of PAI-1 are urokinase-type plasminogen activator and tissue-type plasminogen activator (tPA) (4) and as such it is an important modulator of events of extracellular proteolysis in fibrinolysis and in the turnover of extracellular matrix (5).One of the most striking features of serpins is their ability to undergo a dramatic conformational rearrangement with the N-terminal portion of the reactive loop inserting into -sheet A (6). This transition with cleavage of the loop is central to the formation of a stable inhibitory complex (7-10), but it can also occur spontaneously in vivo, without cleavage of the loop, to form an inactive latent conformation (11-14). Moreover, antithrombin and ␣ 1 -antitrypsin can be induced to adopt a latent conformation by heating in stabilizing concentrations of sodium citrate (15-17). Serpins are also able to link their reactive loop to a -sheet of another molecule to form loop-sheet polymers, one form of which (18) has recently been crystallized (19,20). These polymers are of considerable importance because they underlie the deficiency of ␣...
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.
In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR work on these novel templates gave a number of potent MRP1 modulators with great selectivity against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies were demonstrated.
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