Synthesis of Novel Saccharin Derivatives.

The synthesis of saccharin (1,2-benzisothiazol-3-one-1,1-dioxide) derivatives substituted on the benzene ring has seen limited development despite the longevity of this compound's use as an artificial sweetener. This type of saccharin derivative would however present attractive properties for the development of new bioactive, drug-like small molecule compounds. Here we report the derivatisation of the benzene ring of saccharin using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) to synthesise a diverse library of novel saccharin-1,2,3-triazole conjugates. All library compounds retain the capability for interactions with biomolecules via the unmodified sulfonamide and lactam groups of the parent saccharin core heterocycle. The compounds also encompass alternate orientations of the 1,2,3-triazole heterocycle, thus further adding diversity to the potential hydrogen bonding interactions of these compounds with biomolecules of therapeutic interest. Our findings demonstrate that specifically functionalized derivatives of saccharin may be prepared from either saccharin azide or saccharin alkyne building blocks in high yield using CuAAC.

Characterisation of Photoaffinity-Based Chemical Probes by Fluorescence Imaging and Native-State Mass Spectrometry.

Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer-associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug-resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein-probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein-probe binding, and covalent protein-probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure-activity relationships to support the development of optimum chemical probes.

P-glycoprotein-mediated chemoresistance is reversed by carbonic anhydrase XII inhibitors.

Carbonic anhydrase XII (CAXII) is a membrane enzyme that maintains pH homeostasis and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Here, we investigated a panel of eight CAXII inhibitors (compounds 1-8), for their potential to reverse Pgp mediated tumor cell chemoresistance. Inhibitors (5 nM) were screened in human and murine cancer cells (colon, lung, breast, bone) with different expression levels of CAXII and Pgp. We identified three CAXII inhibitors (compounds 1, 2 and 4) that significantly (≥ 2 fold) increased the intracellular retention of the Pgp-substrate and chemotherapeutic doxorubicin, and restored its cytotoxic activity. The inhibitors lowered intracellular pH to indirectly impair Pgp activity. Ca12-knockout assays confirmed that the chemosensitizing property of the compounds was dependent on active CAXII. Furthermore, in a preclinical model of drug-resistant breast tumors compound 1 (1900 ng/kg) restored the efficacy of doxorubicin to the same extent as the direct Pgp inhibitor tariquidar. The expression of carbonic anhydrase IX had no effect on the intracellular doxorubicin accumulation. Our work provides strong evidence that CAXII inhibitors are effective chemosensitizer agents in CAXII-positive and Pgp-positive cancer cells. The use of CAXII inhibitors may represent a turning point in combinatorial chemotherapeutic schemes to treat multidrug-resistant tumors.

Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases.

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates.

Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.

Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

A prodrug approach toward cancer-related carbonic anhydrase inhibition.

The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs for selectively targeting the extracellular cancer-associated CA enzymes.

The dimethoxyphenylbenzyl protecting group: an alternative to the p-methoxybenzyl group for protection of carbohydrates.

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH2Cl2 and 3,4-(methylenedioxy)toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and p-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, p-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)benzyl ether as an effective and robust alternative to p-methoxybenzyl as a protecting group for alcohols.

Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides.

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract.