Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.

It was by way of total synthesis that the issues concerning the stereostructure of leiodermatolide (1) have recently been solved; with the target now being unambiguously defined, the mission of synthesis changes as to secure a meaningful supply of this exceedingly scarce natural product derived from a deep-sea sponge. To this end, a scalable route of 19 steps (longest linear sequence) has been developed, which features a catalytic asymmetric propargylation of a highly enolizable β-keto-lactone, a ring closing alkyne metathesis and a modified Stille coupling as the key transformations. Deliberate digression from this robust blueprint brought a first set of analogues into reach, which allowed the lead qualities of 1 to be assessed. The acquired biodata show that 1 is a potent cytotoxin in human tumor cell proliferation assays, distinguished by GI50 values in the ≤3 nM range even for cell lines expressing the Pgp efflux transporter. Studies with human U2OS cells revealed that 1 causes mitotic arrest, micronucleus induction, centrosome amplification and tubulin disruption, even though no evidence for direct tubulin binding has been found in cell-free assays; moreover, the compound does not seem to act through kinase inhibition. Indirect evidence points at centrosome declustering as a possible mechanism of action, which provides a potentially rewarding outlook in that centrosome declustering agents hold promise of being inherently selective for malignant over healthy human tissue.

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The Oligonucleotide Safety Working Group subcommittee on genotoxicity testing considers therapeutic oligonucleotides (ONs) unlikely to be genotoxic based on their properties and on the negative results for ONs tested to date. Nonetheless, the subcommittee believes that genotoxicity testing of new ONs is warranted because modified monomers could be liberated from a metabolized ON and incorporated into DNA and could hypothetically cause chain termination, miscoding, and/or faulty replication or repair. The standard test battery as described in Option 1 of International Conference on Harmonisation S2(R1) is generally adequate to assess such potential. However, for the in vitro assay for gene mutations, mammalian cells are considered more relevant than bacteria for most ONs due to their known responsiveness to nucleosides and their greater potential for ON uptake; on the other hand, bacterial assays may be more appropriate for ONs containing non-ON components. Testing is not recommended for ONs with only naturally occurring chemistries or for ONs with chemistries for which there is documented lack of genotoxicity in systems with demonstrated cellular uptake. Testing is recommended for ONs that contain non-natural chemical modifications and use of the complete drug product (including linkers, conjugates, and liposomes) is suggested to provide the most clinically relevant assessment. Documentation of uptake into cells comparable to those used for genotoxicity testing is proposed because intracellular exposure cannot be assumed for these large molecules. ONs could also hypothetically cause mutations through triple helix formation with genomic DNA and no tests are available for detection of such sequence-specific mutations across the entire genome. However, because the potential for triplex formation by therapeutic ONs is extremely low, this potential can be assessed adequately by sequence analysis.

Cr(VI) is a human and animal carcinogen. Cr(VI) does not interact directly with DNA and thus its genotoxicity is attributed to its intracellular reduction to Cr(III) via reactive intermediates. The resulting types of DNA damage can be grouped into two categories: (1) oxidative DNA damage and (2) Cr(III)-DNA interactions. This study examines the molecular mechanism of Cr(VI) and Cr(III) genotoxicity in an intact cell. A system screening for DNA deletions (DEL assay) was used to compare induction of chromosomal rearrangements in the yeast Saccharomyces cerevisiae following Cr(VI) and Cr(III) exposure. Both forms of chromium induced DNA deletions albeit with different dose-response curves. N-acetylcysteine had a protective effect against Cr(VI) genotoxicity at high exposure doses but had no protective effect at lower doses or against Cr(III). An oxidative DNA damage repair mutant was hypersensitive to Cr(VI) only at high exposure and the mutant was not hypersensitive to Cr(III) exposure. These data imply that oxidative stress is involved in Cr(VI) genotoxicity at high exposure concentrations and not so in Cr(III). The Cr(III)-DNA interaction appears to be an important genotoxic lesion following Cr(VI) exposure at low-exposure concentrations. The CAN forward mutation assay revealed that within the concentration ranges used for this study, Cr(III) does not cause point mutations and Cr(VI) causes a mild but statistically significant increase in point mutation only at the highest concentration tested. This study reveals that DNA deletions occurring as a result of intrachromosomal homologous recombination are a useful endpoint for studying chromium genotoxicity.

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