Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes.

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 µM to 174 ± 21 µM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.

Rational design, synthesis and biological evaluation of modular fluorogenic substrates with high affinity and selectivity for PTP1B.

Protein-tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type-2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self-immolative 4-hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme-initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B-catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.

Synthesis and structure-activity relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid amide hydrolase inhibitors.

The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3'-carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.

Biphenyl-3-yl alkylcarbamates as fatty acid amide hydrolase (FAAH) inhibitors: steric effects of N-alkyl chain on rat plasma and liver stability.

Secondary alkylcarbamic acid biphenyl-3-yl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which include the reference compounds URB597 and URB694. Given the intrinsic reactivity of the carbamate group, the in vivo potency of these molecules in rats is strongly affected by their hydrolysis in plasma or hepatic metabolism. In the present study, in vitro chemical and metabolic stability assays (rat plasma and rat liver S(9) fraction) were used to investigate the structure-property relationships (SPRs) for a focused series of title compounds, where lipophilicity and steric hindrance of the carbamate N-substituent had been modulated. The resulting degradation rates indicate that a secondary or tertiary alkyl group at the carbamate nitrogen atom increases hydrolytic stability towards rat plasma esterases. The calculated solvent accessible surface area (SASA) of the carbamate fragment was employed to describe the differences observed in rate constants of hydrolysis in rat plasma (log k(plasma)), suggesting that stability in plasma increases if the substituent exerts a shielding effect on the carbamate carbonyl. Stability in rat liver S(9) fraction is increased when a tertiary carbon is bound to the carbamate nitrogen atom, while other steric effects showed complex relationships with degradation rates. The SPRs here described may be applied at the pharmacokinetic optimization of other classes of carbamate FAAH inhibitors.

Design, synthesis, and biological evaluation of a biyouyanagin compound library.

Modern drug discovery efforts rely, to a large extent, on lead compounds from two classes of small organic molecules; namely, natural products (i.e., secondary metabolites) and designed compounds (i.e., synthetic molecules). In this article, we demonstrate how these two domains of lead compounds can be merged through total synthesis and molecular design of analogs patterned after the targeted natural products, whose promising biological properties provide the motivation. Specifically, the present study targeted the naturally occurring biyouyanagins A and B and their analogs through modular chemical synthesis and led to the discovery of small organic molecules possessing anti-HIV and anti-arenavirus properties.

Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism.

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

A second generation of carbamate-based fatty acid amide hydrolase inhibitors with improved activity in vivo.

The fatty acid ethanolamides are a class of signaling lipids that include agonists at cannabinoid and alpha type peroxisome proliferator-activated receptors (PPARalpha). In the brain, these compounds are primarily hydrolyzed by the intracellular serine enzyme fatty acid amide hydrolase (FAAH). O-aryl carbamate FAAH inhibitors such as URB597 are being evaluated clinically for the treatment of pain and anxiety, but interactions with carboxylesterases in liver might limit their usefulness. Here we explore two strategies aimed at overcoming this limitation. Lipophilic N-terminal substitutions, which enhance FAAH recognition, yield potent inhibitors but render such compounds susceptible to attack by broad-spectrum hydrolases and inactive in vivo. By contrast, polar electron-donating O-aryl substituents, which decrease carbamate reactivity, yield compounds, such as URB694, that are highly potent FAAH inhibitors in vivo and less reactive with off-target carboxylesterases. The results suggest that an approach balancing inhibitor reactivity with target recognition produces FAAH inhibitors that display significantly improved drug-likeness.

Structure-property relationships of a class of carbamate-based fatty acid amide hydrolase (FAAH) inhibitors: chemical and biological stability.

Cyclohexylcarbamic acid aryl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors, which includes the reference compound URB597. The reactivity of their carbamate fragment is involved in pharmacological activity and may affect their pharmacokinetic and toxicological properties. We conducted in vitro stability experiments in chemical and biological environments to investigate the structure-stability relationships in this class of compounds. The results show that electrophilicity of the carbamate influences chemical stability, as suggested by the relation between the rate constant of alkaline hydrolysis (log k(pH9)) and the energy of the lowest unoccupied molecular orbital (LUMO). Introduction of small electron-donor substituents at conjugated positions of the O-aryl moiety increased the overall hydrolytic stability of the carbamate group without affecting FAAH inhibitory potency, whereas peripheral non-conjugated hydrophilic groups, which favor FAAH recognition, helped decrease oxidative metabolism in the liver.

Synthesis and quantitative structure-activity relationship of fatty acid amide hydrolase inhibitors: modulation at the N-portion of biphenyl-3-yl alkylcarbamates.

Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (FAAH IC50 = 63 nM) with a selected set of substituents of different size, shape, flexibility, and lipophilicity. Docking experiments and linear interaction energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the beta-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3'-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors.