Brent Blackburn

Synthesis and β-lactamase-mediated activation of a cephalosporin-taxol prodrug

Abstract Background: Enzyme-activatable prodrugs in conjunction with antibody-enzyme fusion proteins may enhance the anti-tumor efficacy of antibodies and reduce the toxic side effects of conventional chemotherapeutics. Cephalosporins have proven to be highly versatile triggers for the enzymatic activation of such prodrugs. Results: A cephern prodrug of taxol (PROTAX) was synthesized by substituting the C-3′ position of cephalothin with 2′-(γ-arninobutyryl) taxol. Hydrolysis of PROTAX by β-lactamase rapidly released 2′-(γ-anainobutyryl) taxol (kcatKM = (1.4 ± 0.1) × 105 s−1 M−1), which yielded taxol following intramolecular displacement. PROTAX is inactive in a microtubule assembly assay in vitro but has similar activity to taxol following prolonged activation with β-lactamase. PROTAX is ≈10-fold less toxic than taxol against SK-BR-3 breast tumor cells in vitro but has activity approaching that of taxol following prolonged activation with a fusion protein comprising β-lactamase fused to a tumor-targeting antibody fragment. Conclusions: Tubulin polymerization activity is abolished and cytotoxicity is reduced in the PROTAX prodrug compared to taxol. Activation of PROTAX by β-lactamase followed by self-immolation restores the activity of PROTAX to that of free taxol.

Mono-N-alkylation of anthranilamides via quinazolinones. An efficient synthesis of G5598, a benzodiazepine dione gpIIbIIIa antagonist

Abstract The mono-N-alkylation of an anthranilamide derivative via the reductive ring opening of a quinazolinone precursor, enables the synthesis of benzodiazepine dione derivative G5598, a potent inhibitor of the in vitro binding of GpIIbIIIa to fibrinogen.

Chapter 9. Glycoprotein IIb IIIa Antagonists

Publisher Summary This chapter describes the work leading to the discovery of antagonists of GPllbllla/fibrinogen interactions as anti-aggretory and anti-thrombotic agents for the treatment of human vascular diseases. Glycoprotein llbllla is a noncovalent heterodimeric protein complex whose tertiary structure is dependent on the association of divalent cations. Each subunit has a single transmembrane domain and a short cytoplasmic tail. For some time, GPllbllla and other integrins were thought to have a purely adhesive function and as such were considered incapable of signal transduction. However, recent work has shown integrins in general and GPllbllla, in particular, are involved in both inside-out and outside-in signaling. In addition to these signaling and adhesive functions, GPllbllla is involved in the trafficking of proteins from serum into the platelet alpha-granules. A number of reports have implicated GPllbllla in the transport of calcium ions into the platelet upon activation. The affinity of GPllbllla for fibrinogen can also be modulated by the lipid composition of the platelet membrane as well as by partial proteolysis of the extracellular domain. Glanzmanns thrombasthenia, a lifelong bleeding disorder, manifests itself in bleeding from gums and mucotaneous tissue and is not typically life threatening even when no functional GPllbllla is expressed. The chapter discusses proteins, peptides, peptide hybrids, and nonpeptides in connection with glycoprotein antagonists. Vascular occlusion by thrombus formation has been implicated as a primary cause of a variety of cerebral and cardiovascular diseases. Animal models of thrombosis have been devised and used to examine the biological response to GPllbllla inhibition.

Stability of Alkoxycarbonylamidine Prodrugs

AbstractPurpose. Alkoxycarbonylamidine prodrug modification was used to mask the positively-charged amidine moiety of an Arg-Gly-Asp pepti-domimetic and enhance oral bioavailability. The aqueous stability of ethoxycarbonylamidine (EGA), ethanethiocarbonylamidine (ETCA) and phenoxycarbonylamidine (PCA) prodrugs was examined. Methods. Degradation was followed by RP-HPLC and rate constants were determined from a degradation scheme defined by product analysis. Results. EGA gave a pH of maximum stability at pH ∼7 and was independent of pH below pH ∼4. A novel degradation pathway of EGA, conversion to ethoxycarbonyl- aminocarbonyl, was observed below pH 7. The relative rates below pH 7 were ECA~ETCA<PCA, in the same order of decreasing pKa of the conjugate acid of the substituted amidino group. Base-catalyzed cleavage of EGA to yield the amidine derivative gave the relative rates ECA<ETCA<PCA, in agreement with the decreasing pKa of the leaving groups. Conclusions. The observed rate constants at all pHs were small enough that only 5-30% (depending on the substituent) undesirable degradation is predicted during transit time of the gut. The spontaneous post-absorptive conversion to the amidine drugs at neutral pH is predicted to be 6x greater for the PCA than the EGA prodrugs.

Preparation and biological activity of novel tricyclic GPIIb/IIIa antagonists

Antagonists of the glycoprotein GPIIb/IIIa are a promising class of antithrombotic agents offering potential advantages over present antiplatelet agents (i.e., aspirin and ticlopidine). Novel tricyclic nonpeptidal GPIIb/IIIa antagonists have been prepared and evaluated in vitro as antagonists of fibrinogen binding to the purified GPIIb/IIIa receptor and as inhibitors of platelet aggregation. The work presented demonstrates the robustness of the benzodiazepinedione (BZDD) scaffold, which can be functionalized at the N1-C2 amide as well as at C7, to provide structural diversity and allow optimization of the physiochemical and pharmacological properties of the BZDD based GPIIb/IIIa antagonists. In addition, the resulting new class of tricyclic GPIIb/IIIa antagonists could be used to probe for additional binding interactions on the GPIIb/IIIa receptor and perhaps lead to BZDD based GPIIb/IIIa antagonists with increased potency. The tricyclic molecules reported herein demonstrate that a heterocyclic ring can be fused to the benzodiazepinedione scaffold with retention of anti-aggregatory potency and in the case of tetrazole 30i, increased potency relative to the bicyclic analogue 1c.