Design, Synthesis, and Cytotoxic Evaluation of Novel Tubulysin Analogues as ADC Payloads.

The tubulysin class of natural products has attracted much attention from the medicinal chemistry community due to its potent cytotoxicity against a wide range of human cancer cell lines, including significant activity in multidrug-resistant carcinoma models. As a result of their potency, the tubulysins have become an important tool for use in targeted therapy, being widely pursued as payloads in the development of novel small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs). A structure-based and parallel medicinal chemistry approach was applied to the synthesis of novel tubulysin analogues. These efforts led to the discovery of a number of novel and potent cytotoxic tubulysin analogues, providing a framework for our simultaneous report, which highlights the discovery of tubulysin-based ADCs, including use of site-specific conjugation to address in vivo stability of the C-11 acetate functionality.

Optimization of Tubulysin Antibody-Drug Conjugates: A Case Study in Addressing ADC Metabolism.

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.

Cephalosporin-derived inhibitors of beta-lactamase. Part 4: The C3 substituent.

New C3-substituted beta-lactamase inhibitors were prepared and evaluated against representative class A and class C enzymes. It was possible to improve simultaneous inhibitory activity of both classes of serine hydrolase. Other inhibitors showed high selectivity for either the class C cephalosporinases or the class A penicillinases. This represents the first time that cephalosporin-derived inhibitors have demonstrated selectivity for the class A beta-lactamases.

NB2001, a novel antibacterial agent with broad-spectrum activity and enhanced potency against beta-lactamase-producing strains.

Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance resulting from enzyme overexpression. beta-Lactamase overexpression is a common mechanism of bacterial resistance to beta-lactam antibiotics. We present here the results for one of the beta-lactamase ECTA compounds, NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold. Unlike conventional beta-lactam antibiotics, where hydrolysis of the beta-lactam ring inactivates the antibiotic, hydrolysis of NB2001 by beta-lactamase releases triclosan. Evidence supporting the proposed mechanism is as follows. (i) NB2001 is a substrate for TEM-1 beta-lactamase, forming triclosan with a second-order rate constant (k(cat)/K(m)) of greater than 77,000 M-1 s-1. (ii) Triclosan is detected in NB2001-treated, beta-lactamase-producing Escherichia coli but not in E. coli that does not express beta-lactamase. (iii) NB2001 activity against beta-lactamase-producing E. coli is decreased in the presence of the beta-lactamase inhibitor clavulanic acid. NB2001 was similar to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum antibacterial agent and demonstrate the potential of ECTA in overcoming beta-lactamase-mediated resistance.