Carbohydrate Chain Terminators: Rational Design of Novel Carbohydrate‐Based Antifungal Agents (2007)

The terminators. A chain-termination approach was employed to rationally design monosaccharide derivatives as inhibitors of chitin biosynthesis by replacement of the 4-hydroxyl group of N-acetyl glucosamine monomers. Antifungal activity was observed for a variety of acylated monosaccharide and oxazoline derivatives by means of fungal-spore germination and adhesion assays. As bacterial, viral and fungal drug resistance to currently administered treatments increase, the need for the development of new therapeutic strategies against infective agents becomes more urgent. Crucial to the survival of many pathogens are carbohydrate structures, which are either themselves structurally unique or specific to nonmammalian organisms. Indeed, inhibition or interference with correct biosynthesis of oligosaccharide materials represents an attractive and potentially highly selective strategy for the development of new classes of therapeutic agents.1 Over recent years substantial effort has been expended in the search for inhibitors of particular glycosyl transferase and glycosidase enzymes as a means of disrupting specific biosynthetic pathways.2 The design of such inhibitors can be problematic, since in many cases little is known about the precise enzymes involved. However, an alternative strategy, and one that does not rely on precise structural information about specific enzymes, in particular biochemical pathways, is to exploit structural analysis of the key targeted oligosaccharide structure. This strategy is to invoke chain termination of oligosaccharide biosynthesis. Chain-termination of oligonucleotide synthesis was originally developed by Sanger et al.3 as a means of sequencing DNA. Subsequently, chain termination strategies have found routine and widespread use in interference with oligonucleotide synthesis, and, moreover, have become the molecular basis of antiviral therapies in clinical use, perhaps most preeminently in the case of AZT.4 Although chain-termination processes have been implicated in the biological affects of some monosaccharide derivatives on mammalian glycoconjugate5 and glycosoaminoglycan biosynthesis,6 it is curious that a chain termination approach has not been widely promulgated as a strategy for the development of new classes of inhibitors of pathogenic oligosaccharide biosynthesis. Although there could be potential pitfalls in this approach, it is our considered opinion that this research route merits further investigation. For example, in cases in which multiple-repeat units of carbohydrate structures are essential for pathogenic survival (e.g., in cell-wall formation), then the statistical chances of incorporations that lead to chain termination, would be more favourable. Moreover, there is now good evidence that glycosyl transferases do process activated donor substrates that are modified in a minimal way at a single hydroxyl group.7 Although certain of these compounds have been shown to act as enzyme inhibitors, many are good substrates that are readily processed.8 Indeed, as alluded to above, there is also literature precedent that chain-terminating modified carbohydrates have already been incorporated into mammalian glycoprotein oligosaccharides5 and glycosaminoglycans,6 with the net results of inhibition of their biosynthesis. As part of a program aimed at investigating the potential opportunities that chain termination of oligosaccharide biosynthesis offers to disease control, we became interested in the rational design of novel antifungal agents. Fungal infections represent a serious hazard to human and animal health,9 and—just as for bacterial infection—drug resistance to current therapies is increasing.10 The fungal cell wall11 consists of large sections of oligosaccharide materials including chitin, a polymer of β(1–4)N-acetylglucosamine (GlcNAc), and also β-glucan. The biosynthesis of both of these nonmammalian oligosaccharides could be potentially targeted12 by using a chain termination approach. The present study focuses on attempts to inhibit chitin biosynthesis. Chitin is assembled stepwise by the enzyme chitin synthase,13 which transfers single GlcNAc residues to a growing oligomeric chain; the donor substrate for the enzyme is UDP–GlcNAc. Potential chain terminators of this process are therefore GlcNAc residues in which the 4-hydroxyl has been modified.14 If such materials are processed by chitin synthase, then their transfer to the terminus of the growing chitin chain will result in a chain-termination step, since the required 4-hydroxyl at which subsequent units would be added will then be absent. Before embarking on the synthesis and testing of potential chain terminators two factors were considered. Firstly, the stability and polarity profile of potential drug compounds was borne in mind. Secondly, as it is a requirement that the potential chain terminator must be actively processed by the chitin synthase, thought was given as to what types of activated glycosyl donor should be synthesised and tested. The most obvious approach was that of synthesising and testing the modified UDP donors themselves, since these are the actual substrates processed by chitin synthase.15 Alternatively, a potential prodrug molecule could be synthesised, which might then be converted to the active UDP donor once inside the cell. Such a prodrug would have the added advantages of thermal stability and more appropriate polarity as compared to the UDP derivative. In line with previous literature precedent,5 it was reasoned that perhaps esters of GlcNAc derivatives could act as suitable prodrugs that might be able to penetrate into the cell without relying on carbohydrate-transport mechanisms. Therefore, peresters of GlcNAc derivatives in which the 4-hydroxyl had been modified were targeted. However, after deliberation further structural modification was thought to be worth investigation. From previous experience on the use of oxazolines as transition-state mimics that are readily processed by hexosaminidases,16 it was reasoned that perhaps oxazoline materials could themselves serve as activated substrates for chitin synthase. Alternatively, if not actually substrates for chitin synthase, then at the least oxazolines could represent another class of potential prodrug. Therefore, the synthesis of a small selection of GlcNAc-derived oxazolines was also undertaken, in which the key 4-hydroxyl was replaced with a small isoteric group. As a first foray into the field, a decision was taken to access glycosyl acetates and oxazolines in which the normal 4-hydroxyl of GlcNAc was replaced by H, OMe, F and N3. The anomeric centre of GlcNAc was first protected as the α-allyl ether. Regioselective benzoylation then led to alcohol 3, in which the 4-hydroxyl remained unprotected (Scheme 1). Reduction of the OH-4 was achieved by a two-step Barton–McCombie free-radical approach;17 conversion to the xanthate ester 4 was then followed by reduction with tri-n-butyl tin hydride to yield the deoxy compound 5. Methylation of the free hydroxyl of 3 was achieved by treatment with methyl iodide and sodium hydride in DMF to give the methyl ether 6, albeit in modest yield. Introduction of azide and fluoride at C-4 with the retention of configuration required was achieved by the use of a double-inversion procedure. Inversion of OH-4 was achieved by treatment of 3 with trifluoromethansulfonic anhydride followed by sodium nitrite to yield the galacto alcohol 7. Introduction of fluorine at C-4 was then accomplished with another inversion of configuration by treatment of alcohol 7 with diethylaminosulfur trifluoride (DAST) to yield gluco fluoride 8. Finally, introduction of azide, again with a second inversion at C-4, was then achieved by chloromesylation18 of 7 at 0 °C in pyridine, followed by treatment with sodium azide in DMF at 90 °C to yield gluco azide 9. Selective functionalization of N-acetylglucosamine at C-4. Reagents and conditions: a) 1: allyl alcohol, BF3⋅Et2O, 90 °C; 2: Dowex 50WX8-200, H2O, 70 °C (53 % over two steps); b) BzCl, pyridine, CH2Cl2, −20 °C (56 %); c) CS(Im.)2, toluene, reflux (94 %); d) Bu3SnH, AIBN, toluene, reflux (91 %); e) MeI, NaH, DMF, −20 °C (53 %); f) 1: Tf2O, pyridine, CH2Cl2, −20 °C; 2: NaNO2, DMF, RT (62 % over two steps); g) DAST, CH2Cl2, −30 °C to RT (64 %); h) 1: chloromesyl chloride, pyridine 0 °C; 2: NaN3, DMF, 90 °C (78 % over two steps). With a selection of modified GlcNAc derivatives in hand, attention was turned to accessing the corresponding esters and oxazoline derivatives. It was anticipated that similar reaction sequences could be applied in the four series and this generally was the case, though a slightly modified procedure was required for the azido series. Removal of the allyl protection for methoxy-, deoxy- and fluoro- compounds 5–8 was readily achieved by heating the products with tetrakistriphenylphosphine palladium in acetic acid. However, this procedure was found to also reduce the azide functionality in compound 9. A variety of alternative deprotection conditions were investigated until a successful procedure, which involved treatment with PdCl2 in methanol, was arrived at. Subsequently, conversion to the desired glycosyl acetates 14–17 was achieved by treatment with acetic anhydride in pyridine (Scheme 2). The corresponding oxazoline derivatives were then accessed by treatment of the acetates with TMSBr and BF3⋅OEt2, to yield benzoylated oxazolines 18–21. Finally, removal of benzoates was achieved by treatment with sodium methoxide in methanol, to yield the oxazoline diols 22–25. Synthesis of potential chain terminating GlcNAc-derived esters and oxazolines. a) Pd(PPh3)4, AcOH, 80 °C (81 % from 5, 83 % from 6, 79 % from 8); b) PdCl2, MeOH, RT (72 % from 9); c) Ac2O, pyridine, RT (81 % from 10, 84 % from 11, 80 % from 12, 81 % from 13); d) TMSBr, BF3⋅OEt2, collidine, (ClCH2)2, RT (50 % from 14, 52 % from 15, 60 % from 16, 49 % from 17); e) NaOMe, MeOH, RT (90 % from 18, 81 % from 19, 88 % from 20, 85 % from 21). A series of control compounds was also accessed in order to confirm whether any observed biological activity was entirely due to functionalization of the 4-position. Known benzoate 2619 was therefore acetylated to give the control glycosyl acetate 27, which was also converted into the control benzoylated oxazoline 28. In addition as a final control, the known oxazoline triol 2920 was accessed by following literature procedures (Scheme 3). Synthesis of control compounds. a) Ac2O, pyridine, RT, (87 %); b) TMSBr, BF3⋅OEt2, collidine, (ClCH2)2, RT (53 %). The antifungal action of the putative chain-termination compounds was assessed by using two assays on microconidia and germlings of the dermatophyte,21 Trichophyton rubrum, a fungal pathogen that causes superficial skin diseases in humans and animals. The assays measured the effects of the compounds both on adhesion (Figure 1) and germination (Figures 2 and 3) of fungal spores/germlings. Assessment of adhesion of T. rubrum microconidia in the presence of varying concentrations of compounds 14–17, 18–21 and 27–28, in hydrolysed keratin broth (0.1 %). Error bars represent the standard deviations within eight technical replicates per treatment; two independent experiments were performed, of which one is shown. In the adhesion assay, a doubling dilution of the putative inhibitors was prepared across a 96-well plate, starting at 1 mM concentration, in keratin broth (0.1 %; 100 μL per well). Spore suspension (100 μL) was then added to each well (final volume 200 μL), with four replicate wells for each test. Plates were incubated at 30 °C for 24 h, and then emptied and washed with water (3×). Crystal violet solution (100 μL; 0.05 % w/v) was added to each well and incubated at RT for 15 min. Plates were then washed again in water (3×); this left the stain within the fungal material on the plate. To release the stain, of 98 % ethanol (100 μL) was added to each well and the plates were incubated at RT for 15 min with gentle shaking. Absorbance of the remaining dye was quantified by using a spectrophotometer at λ 570 nm, and absorbance values reflected the strength of adhesion of the T. rubrum spores to the plate. Effect of compounds 14–17, 18–21 and 27–28 on germination of T. rubrum microconidia. T. rubrum conidia were harvested and spore suspensions were adjusted to the appropriate density. Test compounds were dissolved and added to the spore suspension to obtain concentrations of up to 1 mM of each test compound. The resulting suspensions were pipetted with the appropriate number of replicates onto keratin agar (0.1 %) and incubated for 24 h at 30 °C. After incubation images were taken of the germinated spores to allow assessment of germ-tube formation. A sample of 30 sets of 10 spores was counted to quantify germ-tube initiation. The number of germinated spores was represented as a proportion of the 30×10 spores counted per treatment; two independent biological replicates were performed, of which one is shown. Error bars represent the standard deviation within 30 technical replicates. Light micrographs of germination assays. Micrographs were taken at 400× magnification, and show T. rubrum germlings 24 h after treatment with the test compounds (17, 20 and 21) and controls (DMSO, 27 and 28) indicated, at a concentration of 1 mM; scale bar=25 μm. The rationale for performing adhesion assays was that any cell-wall damage caused as a result of inhibition of correct chitin synthesis would affect the transit of secreted proteins through the wall, which, in turn, would affect the composition and adhesion properties of the fungal extracellular matrix. Adhesion of the germlings was most compromised by methylation of the 4-hydroxyl both for the acetate series (15) and also for the oxazolines (19), as compared with their respective controls (27 and 28). This data implies that secretion is indeed affected by these potential chain terminators, and hints at the importance of a robust cell wall. A more direct assay of cell-wall propinquity is to assess germination and evaluate the formative effects of the various substitutions at the 4-postion (Figure 2). Germination assays were performed on germlings of T. rubrum (Figures 2 and 3). None of the completely deprotected putative chain terminators 22–25 or 29 displayed significant biological activity in the 1–1000 μM range that was examined.22 Moreover, the control compounds, namely acetate 27 and oxazoline 28, were not found to inhibit germination at concentrations of up to 1 mM. However, putative chain-terminating acetates 14–17 and oxazolines 19–21 all significantly inhibited T. rubrum germination at concentrations of 1 mM. In line with the adhesion assays 1 mM concentrations of methylated compounds 15 and 19 reduced germination by circa 50 %, as compared with their respective controls. However, the most potent inhibition was observed with fluorooxazoline 20 and azidooxazoline 21, which inhibited approximately 80 and 70 % of germination, respectively, at a concentration of 1 mM. Figure 3 shows a selection of light micrographs of germination assays performed in the presence of dimethyl sulfoxide (DMSO; control), fluoroacetate 17 and corresponding control acetate 27, and azido- and fluoroxazolines 20 and 21 and their corresponding control oxazoline 28. The putative chain-terminating acetate 17 and oxazolines 20 and 21 clearly inhibited germination at concentrations of around 1 mM, whereas the controls had no effect. The fact that the deprotected putative chain terminators 22–25 displayed little or no significant activity, whereas the protected variants all did, deserves comment. One rationalisation, in line with previous hypotheses,5d is that the better compound polarity profile23 of the benzoate esters, which are probably more able to penetrate into cells, and therefore, probably act as prodrugs. Once inside the cell they could be deacylated enzymatically, which would lead to production of the active drug molecule in situ. Direct proof of the precise mode of action of the most active compounds 20 and 21 will require substantial further investigation, but the fact that the control oxazolines 28 and 29 do not display any appreciable biological effects, taken together with extensive previous work on potential chain terminators,5, 6 does lead one to the probable conclusion that it is indeed replacement of the 4-hydroxyl which results in the observed antifungal activity. The question as to whether the active compounds themselves produce their biological effect by acting as chain terminators or as enzyme inhibitors,24 remains to be answered. However, whatever the precise molecular mode of action, this work has demonstrated that novel antifungal carbohydrates can be rationally designed by using a chain-termination approach. Further investigations into the potential uses of carbohydrate chain terminators as the basis for novel strategies against a variety of other infective agents are in progress, and the results will be reported in due course. The authors gratefully acknowledge the European Union (Marie Curie Intra-EU Fellowship to R.D.), the BBSRC and Stiefel Laboratories Inc. (CASE award to L.B.), and the EP Abraham Cephalosporin Fund for financial support. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2007/z700234_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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