Stereoselective Synthesis of Highly Functionalized Arabinosyl Nucleosides through Application of an N-Nitro Protecting Group.

2'-Deoxy-2',5-disubstituted arabinosyl uridine derivatives bearing a halogen (Cl, Br or I) at C2' and an ethynyl group at C5 have been synthesized in 6 steps from 2',3',5'-tri- O-acetyl-5-iodo-uridine in overall yields of 61% (compound 3, Cl), 47% (compound 4, Br), and 19% (compound 5, I). Stabilization of a 2'- O-triflyl leaving group intermediate to overcome spontaneous intramolecular 2,2'-anhydro uridine formation was pivotal to the synthesis. Specifically, to favor SN2 reaction with a halogen nucleophile over intramolecular cyclization, the nucleophilicity of O-2 oxygen was reduced by incorporation of an adjacent electron withdrawing nitro substituent at N-3. The introduction of the 3- N-nitro group proceeded rapidly (nitronium trifluoroacetate, 1 min) and in quantitative yield. A one-pot method to remove the 3- N-nitro group by reductive nitration (zinc metal in acetic acid, 5 min) and the silyl protecting groups of the alkyne and 3',5' hydroxyls (fluoride reagent, 16 h) was established as the final synthetic step. This application of the 3- N-nitro protecting group addresses the significant shortfalls of the conventional approach to synthesis of 2' modified nucleosides, wherein condensation of a 2' modified sugar fragment with a pyrimidine base provides poor stereocontrol of N-glycosylation, low yields and incompatibility with 2' iodo sugars.

Synthesis of acylated glycoconjugates as templates to investigate in vitro biopharmaceutical properties.

A series of novel glycopyranosyl azides were synthesised wherein the carbohydrate moiety was peracylated with four acetyl, propionyl, butanoyl, pentanoyl (valeryl) or 3-methylbutanoyl (isovaleryl) ester linked groups. A panel of glycoconjugates was synthesised from these glycopyranosyl azides using copper-catalysed azide-alkyne cycloaddition. The in vitro metabolic stability, plasma stability and plasma protein binding was then measured to establish the impact of the different acyl group when presented on a common scaffold. The acetyl, propionyl and butanoyl esters exhibited metabolism consistent with esterase processing, and various mono-, di- and tri-acylated hydrolysis products as well as the fully hydrolysed compound were detected. In contrast, the pentanoyl and 3-methylbutanoyl esters were stable.

Design and synthesis of thiourea compounds that inhibit transmembrane anchored carbonic anhydrases.

A library of 32 novel glycoconjugate thiourea-bridged benzene sulfonamides have been synthesized from the reaction of glycosyl isothiocyanates with a panel of simple benzene sulfonamides comprising either a free amine or hydrazide. All compounds were investigated for their ability to inhibit the enzymatic activity of five human carbonic anhydrase (hCA) isozymes: hCA I, II and membrane-associated isozymes IX, XII and XIV. A physicochemical feature of the free sugar thioureido glycoconjugates was high water solubility (> 20 mg/mL), as well many of these compounds exhibited a desirable potency and CA isozyme selectivity profile. From this library several inhibitors displayed excellent potency-selectivity profiles for transmembrane anchored CAs over off-target CA I and II. These molecules provide potential dual-acting candidates for the development of inhibitors that target the extracellular CAs (IX, XII and XIV)-either directly as free sugars (membrane impermeable) or indirectly as acetylated prodrugs, becoming free sugars upon esterase hydrolysis.

Synthesis of sulfonamide-bridged glycomimetics.

A flexible and short synthesis of sulfonamide-bridged di-, tri-, tetra-, and octasaccharide glycomimetics was accomplished by reaction of glycosyl thioacetates with amino sugar substrates. The chemistry to incorporate the sulfonamide linker in place of a native O-glycosidic bond was broadly scoped, allowing access to head-to-head (1↔1) and head-to-tail (1→2), (1→3), (1→4), and (1→6) sulfonamide-bridged glycomimetics. The synthesis proceeds with retention of configuration at the anomeric center and is compatible with variable stereochemical arrangements and with acid- and base-labile protecting groups.

Synthesis of S-glycosyl primary sulfonamides.

The synthesis of S-glycosyl sulfonamides wherein the primary sulfonamide functional group (-SO(2)NH(2)) is directly attached to the anomeric position of a carbohydrate moiety is reported. Our general approach consists of first introducing a thioacetate group at the anomeric center of a per-O-acetylated sugar derivative. From this follows formation of a glycosyl sulfenamide (sugar-SNR(2)), oxidation of the sulfenamide to give a glycosyl N-protected sulfonamide (sugar-SO(2)NR(2)), and removal of the sulfonamide protecting (R) group to yield a primary sulfonamide at the anomeric center (sugar-SO(2)NH(2)). A variety of mono- and disaccharide derivatives were synthesized using this new methodology.

Inhibition of carbonic anhydrase isozymes with benzene sulfonamides incorporating thio, sulfinyl and sulfonyl glycoside moieties.

A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthesized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four human carbonic anhydrases (hCA): isozymes I, II and tumour-associated isozymes IX and XII. The oxidation state of the sulfur in the carbohydrate tail moiety did not influence either enzyme inhibition potency or isozyme selectivity even though presenting opportunities for differing interactions with the target isozymes.

Synthesis of 5-Alkynyl Substituted 2'-Arabinosyl 2'-Halogenated Uridine Nucleosides.

This unit describes the detailed preparation of 5-alkynyl-2'-halogenated arabinosyl uridine nucleosides (2'-halo-ara-EdU) from uridine. These compounds were synthesized as prospective chemical probes for the detection of DNA synthesis in proliferating cells. Currently, this is the only synthetic methodology reported to access these compounds. The key to success of the synthetic approach was to employ a 3-N-nitro-protecting group to stabilize the required 2'-triflate nucleoside precursor toward nucleophilic substitution. Several synthetic challenges were overcome to accommodate the combination of a 5-alkyne and 3-N-nitro functional group, including facile introduction and removal of the N-nitro group, and removal of the sugar acetyl groups under acidic conditions. © 2019 by John Wiley & Sons, Inc.

Carbonic anhydrase inhibitors: inhibition of isozymes I, II, and IX with triazole-linked O-glycosides of benzene sulfonamides.

We report the synthesis of a series of benzene sulfonamides containing triazole-O-glycoside tails for evaluation as carbonic anhydrase (CA) inhibitors. These glycoconjugates were synthesized by the 1,3-dipolar cycloaddition reaction of 4-azidobenzenesulfonamide with O-propynyl glycosides. Compounds were assessed for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA I and II and the tumor-associated isozyme hCA IX (h = human). Against hCA I these compounds were either micromolar or low-nanomolar inhibitors, while against hCA II and IX inhibition in the range of 6.8-53 and 9.7-107 nM, respectively, was observed. The most potent inhibitor against hCA IX was the galactose derivative 8 (Ki = 9.7 nM); this is so far the most potent glycoconjugate inhibitor reported for the tumor-associated hCA IX. These carbohydrate-tethered sulfonamides may prove interesting lead candidates to target tumor-associated CA isozymes, wherein the CA domain is located extracellularly.

N-Cinnamoyl-l-phenyl-alanine methyl ester.

As part of an ongoing investigation into the development of N-substituted amino acids as building blocks for dynamic combinatorial chemistry, we report the structure of the title compound, C(19)H(19)NO(3). This compound crystallizes as discrete mol-ecules. The cinnamoyl group is non-planar, with the phenyl ring and the amide twisted out of the ethyl-ene plane. The benzyl and ester groups lie above and below the amide plane. The mol-ecules stack along the crystallographic c axis, connecting through C(4) chains of N-H⋯O hydrogen bonds, with the extended structure stabilized by C-H⋯O inter-actions and π-π inter-actions [centroid-to-centroid distances 3.547 (8) and 3.536 (8) Å].

A novel class of carbonic anhydrase inhibitors: glycoconjugate benzene sulfonamides prepared by "click-tailing".

Aryl and heteroaryl sulfonamides (ArSO(2)NH(2)) are therapeutically used to inhibit the catalytic activity of carbonic anhydrases (CAs). Using a "click-tail" approach a novel class of glycoconjugate benzene sulfonamides have been synthesized that contain diverse carbohydrate-triazole tails. These compounds were assessed for their ability to inhibit three human CA isozymes in vitro: cytosolic hCA I and hCA II and transmembrane, tumor-associated hCA IX. This isozyme has a minimal expression in normal tissue but is overexpressed in hypoxic tumors and its inhibition is a current approach toward new cancer therapies. The qualitative structure-activity for all derivatives demonstrated that the stereochemical diversity present within the carbohydrate tails effectively interrogated the CA active site topology, to generate several inhibitors that were potent and selective toward hCA IX, an important outcome in the quest for potential cancer therapy applications based on CA inhibition.

Carbonic anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic halves of the carbonic anhydrase active site.

We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

Synthesis of sulfonamide-conjugated glycosyl-amino acid building blocks.

The efficient synthesis of novel glycoconjugate amino acid building blocks wherein the amino acid and carbohydrate moieties are linked via a sulfonamide functional group is reported. The general reaction sequence consists of coupling a glycosyl thioacetate to an amino acid methyl ester followed by oxidation and deprotection of the carbohydrate moiety. We demonstrate the synthesis of derivatives from a range of amino acids, with reaction at either the α-amino group of amino acid precursors or the sidechain ε-amino group of lysine precursors.

Cyclic secondary sulfonamides: unusually good inhibitors of cancer-related carbonic anhydrase enzymes.

Carbonic anhydrase IX (CA IX) is a target for hypoxic cancer therapies, and the discovery of CA IX selective ligands is imperative for the development of these agents. Primary sulfonamides are broad specificity inhibitors of CA enzymes, while secondary sulfonamides are generally poor CA inhibitors. However, saccharin, a cyclic secondary sulfonamide, has unusually good inhibition of CA IX (Ki = 103 nM). In this study, we demonstrate that the affinity and selectivity of saccharin for CA IX can be further modulated when linked to hydrophobic or hydrophilic substituents. The hydrophilic glycoconjugate derivative (12) showed improved inhibition of CA IX (Ki = 49.5 nM) and extremely poor inhibition of the predominant off-target CAs (Ki > 50000 nM) compared to saccharin. This >1000-fold selectivity for CA IX over off-target CAs is unprecedented for classical primary sulfonamide CA inhibitors. Our study highlights the potential of cyclic secondary sulfonamides to be exploited for the discovery of potent, cancer-selective CA inhibitors.

Structural insights into carbonic anhydrase IX isoform specificity of carbohydrate-based sulfamates.

Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate "tail" moiety. Seven compounds inhibited CA IX with low nM Ki values of 1-2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.

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.

Design, synthesis, and biological evaluation of novel carbohydrate-based sulfamates as carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs) IX and XII are enzymes with newly validated potential for the development of personalized, first-in-class cancer chemotherapies. Here we present the design and synthesis of novel carbohydrate-based CA inhibitors, several of which were very efficient inhibitors (K(i)<10 nM) with good selectivity for cancer-associated CA isozymes over off-target CA isozymes. All inhibitors comprised a carbohydrate core with one hydroxyl group derivatized as a sulfamate. Five different carbohydrates were chosen to present a selection of molecular shapes with subtle stereochemical differences to the CA enzymes active site. Variable modifications of the remaining sugar hydroxyl groups were incorporated to provide an incremental coverage of chemical property parameters that are associated with biopharmaceutical performance. All sulfamate inhibitors displayed ligand efficiencies that are consistent with those reported for good drug lead candidates.

Targeting hypoxic tumor cell viability with carbohydrate-based carbonic anhydrase IX and XII inhibitors.

Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of which inhibit CA IX and XII within a narrow range of low nanomolar K(i) values (5.3-11.2 nM). We evaluate for the first time carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have potential for development as in vivo cancer cell selective inhibitors.

Sulfonamide linked neoglycoconjugates--a new class of inhibitors for cancer-associated carbonic anhydrases.

The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to optimize both SAR (structure-activity relationship) and SPR (structure-property relationship) characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors (K(i)s in the low nanomolar range) that target cancer-associated CAs.

Anti-mycobacterial activity of a bis-sulfonamide.

A bis-arylsulfonamide, 7, has been identified that exhibits growth inhibition of Mycobacterium smegmatis at less than 25 microg/mL, but has no such activity against Escherichia coli or Staphylococcus aureus. A closely related bis-arylsulfonamide (8) was much less active, but was the only other compound among 54 arylsulfonamides tested with detectable growth inhibition of M. smegmatis.

Inhibition of membrane-associated carbonic anhydrase isozymes IX, XII and XIV with a library of glycoconjugate benzenesulfonamides.

A library of glycoconjugate benzenesulfonamides that contain diverse carbohydrate-triazole tails were investigated for their ability to inhibit the enzymatic activity of the three human transmembrane carbonic anhydrase (CA) isozymes hCA IX, hCA XII and hCA XIV. These isozymes have their CA domains located extracellularly, unlike the physiologically dominant hCA II, and are of immense current interest as druggable targets. Elevated expression of isozymes IX and XII is a marker for a broad spectrum of hypoxic tumors-this physiology may facilitate a novel approach to discriminate between healthy cells and cancerous cells. Many of these glycoconjugates were potent inhibitors (low nM), but importantly exhibited different isozyme selectivity profiles. The most potent hCA IX inhibitor was the glucuronic acid derivative 20 (K(i)=23nM). This compound was uniquely hCA IX selective cf. all other isozymes (16.4-, 16.8- and 4.6-fold selective against hCA II, XII, and XIV, respectively). At hCA XII there were many inhibitors with K(i)s<10nM that also demonstrated excellent selectivity (up to 344-fold) against other isozymes. Potent hCA XIV inhibitors were also identified, several with K(i)s approximately 10nM, however no hCA XIV-selective derivatives were evidenced from this library. The sugar tails of this study have shown promise as a valuable approach to both solubilize the aromatic sulfonamide CA recognition pharmacophore and to deliver potent inhibition and isozyme differentiation of the transmembrane CAs.