The sulfamate functional group as a new anchor for solid-phase organic synthesis

[reaction: see text] Sulfamate derivatives were loaded on trityl chloride resin, and two variants of cleavage were developed for this sulfamate anchor: an acid treatment to easily restore the free sulfamate and a nucleophilic treatment to generate the corresponding phenol. In addition to loading/cleavage assays and stability experiments, a model sequence of reactions was performed with the new sulfamate anchor to show its applicability in further combinatorial solid-phase synthesis of libraries of biologically relevant sulfamate derivatives.     

O-(Aminosulfonylation) of phenols and an example of slow hydrolytic release

Sequential replacement of imidazole from sulfonyldiimidazole by phenols and then amines leads to O-arylsulfamate esters. Application of this coupling method to 19 phenols and 6 amines generates a library of 114 sulfamate esters, Ar-OSO₂-NR₂. A sulfamate based conjugate of ethinyl estradiol was prepared by using the steroid 3-hydroxyl as the phenol component, and an amino amide derived from linoleic acid as the amine. Hydrolysis of this conjugate was studied in aqueous buffer at pH values 2, 5, and 7.4, and (essentially identical) respective half-lives of 6.8, 6.6, and 6.7 days were observed.     

Solid-phase synthesis of hydroxysteroid derivatives using the diethylsilyloxy linker

Four different types of hydroxysteroids (primary alcohol, secondary alcohols, and phenol), bearing either an oxirane or an azide as a precursor of molecular diversity, were linked in good yields to solid support using the butyldiethylsilane polystyrene (PS-DES) resin. These molecules were then used as scaffolds to generate hydroxysteroid derivatives containing two levels of diversity. The proposed libraries were tested by running steroidal alcohols through a model sequence of reactions (solid-phase coupling, aminolysis of oxirane or reduction of azide, amidation, and final cleavage). As a result, two linked secondary alcohols (17beta-hydroxy-spiro-3(R)-oxirane-5alpha-androstane and 3beta-hydroxy-spiro- 17(S)-oxirane-5alpha-androstane) and a primary alcohol (spiro-17(S)-oxirane-3-(hydroxymethyl)-1,3,5(10)-estratriene) afforded good overall yields (>45%) and high HPLC purities (>90%) of hydroxysteroids derivatized as alkylamides without purification. One limitation was noted for the fourth library: the phenolic steroid linked by the diethylsilyloxy linker gave a poor overall yield of 8% of the desired model compound. Finally, the diethylsilyloxy linker was used successfully for a rapid solid-phase synthesis of a model library of twenty C19-steroid derivatives (3beta-amido-3alpha-hydroxy-5alpha-androstane-17-ones), with an average yield of 53% and average HPLC purity of 97% without purification steps.     

Proton-in-flight mechanism for the spontaneous hydrolysis of N-methyl O-phenyl sulfamate: implications for the design of steroid sulfatase inhibitors

The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate, and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 10(11). Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with catalytic rate accelerations of ~10(4), whereas the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~10(15). Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO(2)X(-), are shown to be correlated by a two-parameter equation based on pK(a)(ArOH) and pK(a)(ArOSO2XH).     

THAL, a sterically unhindered linker for the solid-phase synthesis of acid-sensitive protected peptide acids

The 5-(4-hydroxyphenyl)-3,4-ethylenedioxythienyl alcohol (THAL, Thiophene Acid Labile) is described as a new linker for the solid-phase synthesis of peptide carboxylic acids. It is based on the electron-rich 3,4-ethylenedioxythenyl (EDOTn) moiety and allows the obtention of free and tert-butyl-protected peptides by cleavage with 90% and 0.5% TFA, respectively. This very high acid lability makes it useful for the synthesis of sensitive peptides. Free and tert-butyl-protected Leu-enkephalins have been synthesized as models to demonstrate the utility of the linker.     

Efficacious N-protection of O-aryl sulfamates with 2,4-dimethoxybenzyl groups

Sulfamates are important functional groups in certain areas of current medicinal chemistry and drug development. Alcohols and phenols are generally converted into the corresponding primary sulfamates (ROSO(2)NH(2) and ArOSO(2)NH(2), respectively) by reaction with sulfamoyl chloride (H(2)NSO(2)Cl). The lability of the O-sulfamate group, especially to basic conditions, usually restricts this method to a later stage of a synthesis. To enable a more flexible approach to the synthesis of phenolic O-sulfamates, a protecting group strategy for sulfamates has been developed. Both sulfamate NH protons were replaced with either 4-methoxybenzyl or 2,4-dimethoxybenzyl. These N-protected sulfamates were stable to oxidising and reducing agents, as well as bases and nucleophiles, thus rendering such masked sulfamates suitable for multi-step synthesis. The protected sulfamates were synthesised by microwave heating of 1,1'-sulfonylbis(2-methyl-1H-imidazole) with a substituted phenol to give an aryl 2-methyl-1H-imidazole-1-sulfonate. This imidazole-sulfonate was N-methylated by reaction with trimethyloxonium tetrafluoroborate, which enabled subsequent displacement of 1,2-dimethylimidazole by a dibenzylamine (e.g. bis-2,4-dimethoxybenzylamine). The resulting N-diprotected, ring-substituted phenol O-sulfamates were further manipulated through reactions at the aryl substituent and finally deprotected with trifluoroacetic acid to afford a phenol O-sulfamate. The use of 2,4-dimethoxybenzyl was particularly attractive because deprotection occurred quantitatively within 2 h at room temperature with 10% trifluoroacetic acid in dichloromethane. The four key steps in the protocol described [reaction of 1,1'-sulfonylbis(2-methyl-1H-imidazole) with a phenol, methylation, displacement with a dibenzylamine and deprotection] all proceeded in very high yields.     

Synthesis and evaluation of 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid as a linker in solid-phase synthesis monitored by gel-phase (19)F NMR spectroscopy

Gel-phase (19)F NMR spectroscopy is a useful monitoring technique for solid-phase organic chemistry due to the high information content it delivers and swift acquisition times, using standard NMR spectrometers. This paper describes the synthesis of the novel linker 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid in 29% yield over seven steps, using nucleophilic aromatic substitutions on 2,4,5-trifluorobenzonitrile as key steps. Following standard solid-phase synthesis a peptide could be cleaved from the linker using 20% TFA in CH(2)Cl(2) in 30 minutes, in contrast to a previously described monoalkoxy linker that requires 90% TFA in water at elevated temperature. A resin-bound peptide could be successfully glycosylated using only two equivalents of a thioglycoside donor, activated with N-iodosuccinimide and trifluoromethanesulfonic acid, and subsequent cleavage and deprotection gave the target glycopeptide. Direct glycosylation of the linker itself followed by mild acidic cleavage gave a fully protected hemiacetal for further chemical manipulation.     

Promiscuity of carbonic anhydrase II. Unexpected ester hydrolysis of carbohydrate-based sulfamate inhibitors

Carbonic anhydrases (CAs) are enzymes whose endogenous reaction is the reversible hydration of CO(2) to give HCO(3)(-) and a proton. CA are also known to exhibit weak and promiscuous esterase activity toward activated esters. Here, we report a series of findings obtained with a set of CA inhibitors that showed quite unexpectedly that the compounds were both inhibitors of CO(2) hydration and substrates for the esterase activity of CA. The compounds comprised a monosaccharide core with the C-6 primary hydroxyl group derivatized as a sulfamate (for CA recognition). The remaining four sugar hydroxyl groups were acylated. Using protein X-ray crystallography, the crystal structures of human CA II in complex with four of the sulfamate inhibitors were obtained. As expected, the four structures displayed the canonical CA protein-sulfamate interactions. Unexpectedly, a free hydroxyl group was observed at the anomeric center (C-1) rather than the parent C-1 acyl group. In addition, this hydroxyl group is observed axial to the carbohydrate ring while in the parent structure it is equatorial. A mechanism is proposed that accounts for this inversion of stereochemistry. For three of the inhibitors, the acyl groups at C-2 or at C-2 and C-3 were also absent with hydroxyl groups observed in their place and retention of stereochemistry. With the use of electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), we observed directly the sequential loss of all four acyl groups from one of the carbohydrate-based sulfamates. For this compound, the inhibitor and substrate binding mode were further analyzed using free energy calculations. These calculations suggested that the parent compound binds almost exclusively as a substrate. To conclude, we have demonstrated that acylated carbohydrate-based sulfamates are simultaneously inhibitor and substrate of human CA II. Our results suggest that, initially, the substrate binding mode dominates, but following hydrolysis, the ligand can also bind as a pure inhibitor thereby competing with the substrate binding mode.     

Solid-phase synthesis of sulfamate peptidomimetics

A synthetic method for the preparation of sulfamate peptidomimetics is described. The methodology allows sulfamoylation in the solid phase using sulfamoyl chloride in DMA, followed by the acylation of the corresponding sulfamoylated product. Following this approach, several derivatives have been prepared starting from distinct alcohol sources, including alpha-, beta-, and gamma-hydroxyacids and phenols. The presence of protected amino functions on the building blocks opens the possibility of the addition of more diversity. This approach, which is compatible with Fmoc/Boc/Alloc protection, provides a useful and efficient tool for the preparation of new sulfamate peptidomimetics.     

Dual aromatase-sulfatase inhibitors based on the anastrozole template: synthesis, in vitro SAR, molecular modelling and in vivo activity

The synthesis and biological evaluation of a series of novel Dual Aromatase-Sulfatase Inhibitors (DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished via selective radical bromination and substitution reactions to furnish a series of inhibitory aromatase pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed S(N)2, Heck and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC(50) 3.5 nM), comparable with that of Anastrozole (IC(50) 1.5 nM) whereas, only moderate activity against steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition. Compound 11 was modelled into the active site of a homology model of human aromatase and the X-ray crystal structure of steroid sulfatase.     

A rigid linker-scaffold for solid-phase synthesis of dimeric pharmacophores

Bifunctional linker-scaffolds (compounds 1-3) were designed to meet several criteria for solid-phase syntheses of bivalent ligands. They have two amine-functionalized arms that can be differentially protected. Elaboration of these arms could give ligand-pharmacophore dimers wherein the two active components are held reasonably rigidly at around 10 A separation. Their bifunctional design also enables reactions of libraries with libraries to amplify diversity in a truly combinatorial fashion. Molecules 1-3 are also designed so that cleavage of the linker liberates the scaffold entity into solution under conditions that create only byproducts that should not interfere with biological assays. Thus they contain 2-nitrobenzene sulfonamide components that cleave in the presence of good nucleophiles. In the event, the linker-scaffolds 1-3 were prepared (Schemes 1 and 2). The N-benzyl system 2 was shown to have good stability to the types of conditions that might be used to functionalize the scaffold arms and to be sufficiently labile to the cleavage nucleophile (vide infra). The nucleophiles generally used to cleave nitrobenzene sulfonamides either generate undesirable byproducts (thiophenol or alkane thiols) or proved to be insufficiently reactive for the required solid-phase transformations (n-propylamine). However, sodium sulfide was investigated as a new alternative and shown to be a highly reactive cleavage agent that gives only volatile byproducts and sodium hydroxide. It is suggested that sodium sulfide is a highly desirable nucleophile for cleavage of 2-nitrobenzene sulfonamides, in general. The linker-scaffolds 1-3 were used to prepare a small library of bivalent ligands targeted to a protein receptor having charged cavities separated by approximately 10 A. These systems were made from guanidine, pyridinium, carboxylic acid, and sulfonic acid constituents (Tables 1 and 2).     

Dual aromatase-sulfatase inhibitors based on the anastrozole template: synthesis, in vitro SAR, molecular modelling and in vivo activity

The synthesis and biological evaluation of a series of novel Dual Aromatase-Sulfatase Inhibitors (DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished via selective radical bromination and substitution reactions to furnish a series of aromatase inhibitory pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed SN2, Heck and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC50 3.5 nM), comparable with that of Anastrozole (IC50 1.5 nM) whereas, only moderate activity against steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition.Compound 11 was modelled into the active site of a homology model of human aromatase and the X-ray crystal structure of steroid sulfatase.     

TRAM linker: a safety-catch linker for the traceless release of acrylamides

[reaction: see text] A novel safety-catch linker for the solid-phase synthesis of small-molecule libraries containing electrophilic reactive groups has been developed. Upon cleavage from solid support, the linker generates a Michael acceptor (an acrylamide) on each library member. Utilization of a two-resin system in the final cleavage step provides crude products in high purity, allowing direct use in biological assays following filtration and evaporation.     

Solid-phase synthesis of substituted guanidines using a novel acid labile linker

A novel acid labile linker for solid-phase synthesis of substituted guanidines has been developed. Its synthetic utility is exemplified by high-yielding pyrazole displacement with structurally and electronically diverse sets of aliphatic and aromatic amines. The final cleavage is achieved by treatment with 95:5 trifluoroacetic acid/water for 1 h. The corresponding guanidines were obtained in high purity (80-95%) and good isolated yields (50-95%). The scope and limitations of this linker were further demonstrated by the solid-phase synthesis of an 880-member library of individual trisubstituted arylguanidines employing pyrazole displacement with a set of 11 anilines and two subsequent Mitsunobu N-alkylations with sets of 10 and 8 alcohols, respectively.     

Imidazolinium sulfonate and sulfamate zwitterions as chiral solvating agents for enantiomeric excess calculations

The synthesis of enantiopure unsymmetrical N-heterocyclic based zwitterions incorporating imidazolinium and alkylsulfonate or sulfamate groups is described. The desired compounds were prepared in good yields from 1,3-propanesultone or cyclic sulfamidates and imidazolines. The imidazolinium based zwitterions proved to be versatile chiral solvating agents for Mosher’s acid, alcohols, cyanohydrins, amino alcohols, nitro alcohols, thiols, and carboxylic acids with very high shifts in the ¹H and ¹⁹F NMR.     

A traceless perfluoroalkylsulfonyl (PFS) linker for the deoxygenation of phenols

[reaction: see text]. The synthesis of a novel perfluoroalkylsulfonyl (PFS) fluoride is described for use as a traceless linker in solid-phase organic synthesis. Attachment to the resin and subsequent coupling of a phenol affords a stable arylsulfonate that behaves as a support-bound aryl triflate. Palladium-mediated reductive cleavage of a wide variety of phenols generated the parent arenes. The resin-bound aryl triflate was shown to be stable to reductive amination conditions, and the traceless synthesis of Meclizine is reported.     

New stable backbone linker resins for solid-phase peptide synthesis

[reaction: see text] Two new 4-methoxybenzaldehyde backbone linker resins were developed for the solid-phase synthesis of peptides. The linkers are very stable during the cleavage of common protecting groups for amines (Fmoc, Boc) and carboxylic acids (Me, All, tBu) in peptide synthesis. Cleavage from the resin with refluxing TFA is sufficiently mild for peptides containing polar and nonpolar amino acids.     

Preparation of fluorinated linkers: use of 19F NMR spectroscopy to establish conditions for solid-phase synthesis of pilicide libraries

Three fluorinated linkers which are analogues of linkers commonly used in solid-phase peptide synthesis have been prepared. One of the linkers was used in combination with gel-phase 19F NMR spectroscopy to develop conditions for solid-phase synthesis of two libraries of pilicides, i.e. compounds designed to inhibit assembly of adhesive pili in uropathogenic Escherichia coli. Attachment to and cleavage from the linker could be monitored based on the chemical shift of the fluorine atom of the linker. In addition, use of the linker as internal standard allowed quantification and optimization of reactions occurring further away from the linker when fluorinated building blocks were employed. Importantly, high-quality 19F NMR spectra were obtained for compounds linked to a TentaGel resin in a standard NMR tube using an ordinary NMR instrument.     

New selenyl linker for solid-phase synthesis of dehydropeptides

A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide.     

A nonacid degradable linker for solid-phase synthesis

Synthesis and applications of two new nonacid degradable linkers as an alternative to the Wang linker for solid-phase synthesis are described. Resin from linker 2 looks superior to linker 1 in terms of yields for both anchoring of the first building block and cleavage and in terms of higher purity of the final product. Use of linker 2 avoids side reactions associated with the use of Wang resin due to an undesired cleavage during final acid treatment.