Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

O-Peracetylated 1-(β-d-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated β-d-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-β-d-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(β-d-glucopyranosyl)-3,5-diphenyl- as well as 3-(β-d-glucopyranosyl)-1,5-diphenyl biurets besides 1-(β-d-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(β-d-glucopyranosyl)-5-(β-d-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated β-d-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of β-d-gluco, β-d-galacto and β-d-xylo configurations. O-Acyl protected 1-(β-d-glucopyranosyl)-3-(β-d-glycopyranosylcarbonyl)ureas were obtained from the reaction of β-d-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of β-d-gluco and β-d-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary α-amylase.     

N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods

N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated β-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3μM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the β-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.     

Transformation of thiols to disulfides by epolactaene and its derivatives

In this paper we report a disulfide formation of thiols induced by epolactaene and its derivatives. We previously reported the disulfide formation of N-acetylcysteine methyl ester by epolactaene in a 1:1 MeOH/0.5M NaHCO(3) aq solution. The present studies reveal that the disulfide formation proceeds under mild conditions such as in PBS at pH 7.3, suggesting that epolactaene may induce disulfide formation of cellular thiols. This compound induces the disulfide formation of several thiols in a 1:1 MeOH/0.5M NaHCO(3) aq solution at room temperature. Moreover, our results show that the acyl side-chain of epolactaene greatly influences the products of the reaction. We analyzed the reaction mechanism by using thiolysis products of epolactaene derivatives and propose a new reaction mechanism.     

N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases

A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide?>?carbamate?>?urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme inhibitory activity displayed pronounced effects with the activity improving substantially as one alters both the nature of the reacting carbonyl group (urea?>?carbamate?>?amide) and the pyrazole C4 substituent (CN?>?H?>?Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase. Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent K_i?=?100–200?pM), dual inhibitors of FAAH and monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC₅₀?=?10–20?nM) while simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).     

Specificity of acyl-CoA:phospholipid acyltransferases: solvent and temperature effects

Acyltransfer from CoA thiol esters to either the 1- or 2-position of monoacylglycerophosphoryl choline, which is catalyzed by a microsomal preparation from rat liver, had a temperature optimum of 30-35 degrees C. No significant alteration was observed in the ability of the acyltransferases to distinguish among the various thiol esters tested in the range of 15-40 degrees C. Acyl-CoA:1-acylglycerophosphoryl choline acyltransferase activity is inhibited by urea, N-alkyl ureas, and short-chain alcohols. The effect is not equal for all acyl derivatives, and ethylene glycol has much less inhibitory effect on the transfer of acids with an n - 6 (omega6) double bond. On the other hand, this inhibition of acyltransfer was relatively insensitive to the configuration of the Delta(9)-double bond of octadecadienoates. The specificity of the enzyme-catalyzed transfer of different acids to the 2-position can be correlated in part with the dissociation constants for the urea clathrate complexes. Added glycol does not appreciably alter the specificity of enzyme-catalyzed transfer to the 1-position, but it inhibits the transfer of all acids in a similar fashion.     

Liquid-phase parallel synthesis of ureas

An efficient and general liquid-phase method has been developed for the synthesis of a piperazine containing urea library. Reactions of the polymer bound carbamoyl chloride with primary or secondary amines afford ureas at ambient temperature. Desired compounds are liberated from the polymer support under mild conditions in high yields and high purity by simple precipitation and washings.     

Synthesis and activity of N-acyl azacyclic urea HIV-1 protease inhibitors with high potency against multiple drug resistant viral strains

As part of our efforts to identify potent HIV-1 protease inhibitors that are active against resistant viral strains, structural modification of the azacyclic urea (I) was undertaken by incorporating acyl groups as P₁′ ligands. The extensive SAR study has yielded a series of N-acyl azacyclic ureas (II), which are highly potent against both wild-type and multiple PI-resistant viral strains.     

Structure-activity studies of a novel series of 5,6-fused heteroaromatic ureas as TRPV1 antagonists

Novel 5,6-fused heteroaromatic ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that 4-aminoindoles and indazoles are the preferential cores for the attachment of ureas. Bulky electron-withdrawing groups in the para-position of the aromatic ring of the urea substituents imparted the best in vitro potency at TRPV1. The most potent derivatives were assessed in in vivo inflammatory and neuropathic pain models. Compound 46, containing the indazole core and a 3,4-dichlorophenyl group appended to it via a urea linker, demonstrated in vivo analgesic activity upon oral administration. This derivative also showed selectivity versus other receptors in the CEREP screen and exhibited acceptable cardiovascular safety at levels exceeding the therapeutic dose.     

Indirect chiral separation and analyses in human biological fluids of the stereoisomers of a thienothiopyran-2-sulfonamide (TRUSOPT), a novel carbonic anhydrase inhibitor with two chiral centers in the molecule.

The indirect chiral separation of the four stereoisomers (1)-(4) of a novel carbonic anhydrase inhibitor with two chiral centers in the molecule is reported. The method is based on chemical derivatization of the secondary amino group of the inhibitor with chiral isocyanate, formation of diastereomeric urea derivatives, each with three chiral centers in the molecule, and their separation under nonchiral HPLC conditions. The attempts to separate racemic mixture (1) + (2) from its diastereomeric counterpart (3) + (4) under nonchiral conditions, and to separate enantiomers (1) and (2) directly on a chiral stationary phase (CSP) are also reported. The indirect method was utilized for the assessment of an in vivo inversion of configuration at either one or both chiral centers of the molecule of (1). Analyses of selected whole blood and urine samples from human subjects after multiple bilateral topical ocular dosing with (1) did not reveal the presence of any of the three possible stereoisomers (2)-(4) of (1) indicating that the inversion of configuration at neither one nor two chiral centers of (1) occurs in vivo.     

Synthesis, SAR, and antibacterial activity of novel oxazolidinone analogues possessing urea functionality

The syntheses of a number of novel oxazolidinone analogues possessing an urea functionality are reported. While the urea derivatives possessing aliphatic and aromatic groups were prepared by the more conventional isocyanate method, the derivatives possessing heterocyclic rings were synthesized by a relatively uncommon but otherwise efficient carbamate chemistry. Though the SAR resulted in novel compounds possessing in vitro activity equivalent to Linezolid, the compounds possess a range of substituents that are amenable for altering physicochemical properties of the resultant drug. The antibacterial activity was found to be not sensitive to the functional groups attached to the urea site regardless of the size and electronic characteristics. Based on in vivo results, one molecule has been identified as a candidate and additional work such as salt selection, scale-up, etc., are currently underway to take the molecule further through development.     

5′-Uridyl derivatives of N-glycosyl allophanic acid and biuret

(2′,3′-O-Isopropylidene-5′-uridyl) 4-(2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl)allophanates were obtained in the reactions of 2′,3′-O-isopropylidene-uridine and O-peracetylated β-d-gluco-, galacto- and xylopyranosylamines, and OCNCOCl. 2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl isocyanate and N-(2′,3′-O-isopropylidene-5′-uridyl)urea gave 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-5-(2′,3′-O-isopropylidene-5′-uridyl)biuret. Deprotection of the β-d-gluco configured allophanate and biuret was carried out by standard methods.     

Automated generation of a dihydropyrimidine compound library using microwave-assisted processing

Here we report the generation of a small focused library of 12 diversely functionalized dihydropyrimidine (DHPM) derivatives via one-pot three-component Biginelli cyclocondensation of beta-ketoesters, aldehydes and (thio)ureas. By applying controlled microwave heating under sealed vessel conditions using a fully automated microwave instrument including a gripper and liquid handler, the sequential synthesis of DHPMs can be performed in a shorter reaction time (10-20 min per one DHPM derivative) compared to conventional heating methods, which normally require several hours of reflux heating. The solid products either crystallize directly upon cooling or can be precipitated upon addition of water, requiring only filtration for isolation. In this way, the DHPM derivatives are obtained in high purity and no further purification by recrystallization or chromatography is necessary. This can be ascribed to the microwave heating technology where less side-product formation is often seen. The preparation of this 12-membered DHPM library can be carried out within approximately 9 h.     

Synthesis of N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids as potential inhibitors of glycogen phosphorylase

O-peracetylated N-(beta-D-glucopyranosyl)imino trimethylphosphorane obtained in situ from 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl azide and PMe3 was reacted with saturated and unsaturated aliphatic and aromatic dicarboxylic acids, or their anhydrides, or monoesters to give the corresponding N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids or derivatives. The acetyl protecting groups were removed according to the Zemplén protocol to give a series of compounds which showed moderate inhibitory effects against rabbit muscle glycogen phosphorylase b. The best inhibitor was 3-(N-beta-D-glucopyranosyl-carbamoyl)propanoic acid (7) with Ki = 20 microM.     

Design and synthesis of urea and thiourea derivatives and their inhibitory activities on lipopolysaccharide-induced NO production

Series of ureas and thioureas were designed and synthesized, and their inhibitory activities of NO production in lipopolysaccharide-activated macrophages were evaluated. We found several essential moieties in the structure of the prepared compounds for the activity. Thiourea derivatives revealed higher inhibitory activity than the corresponding urea derivatives. Among these compounds, 7e having carboxymethyl group at N3 position of thiourea was the most potent in the inhibition of NO production. They inhibited NO production through the suppression of iNOS protein and mRNA expression.     

Antimycobacterial activity of 3,4-dichlorophenyl-ureas, N,N-diphenyl-ureas and related derivatives

Substituted urea derivatives were prepared by reacting 3,4-dichlorophenyl isocyanate with amino acids, dipeptides, histamine or dicyandiamide among others, or from N,N-diphenyl-carbamoyl chloride and amino acids, dipeptides, or histamine. Other derivatives were obtained by reaction of PABA or PAS with arylsulfonyl halides. Some of the new compounds showed appreciable activity as antimycobacterial agents against Mycobacterium tuberculosis H37Rv, producing an inhibition of growth in the range of 80-89%, at a concentration of 6.25 microM. Some derivatives of this series might constitute interesting lead molecules for designing novel types of drugs effective against M. tuberculosis, a re-emerging pathogen both in the developed and under-developed countries.     

Discovery libraries targeting the major enzyme classes: The serine hydrolases

Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent K_i = 100–200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.     

Retardation of leaf senescence by urea cytokinins in Raphanus sativus

Approximately 500 urea derivatives and related compounds were tested for ability to retard leaf senescence as measured by chlorophyll retention in radish (Raphanus sativus) leaf discs. Of the 90 compounds found to be active, some had activity at 10^?6 M of the same order as kinetin. There was a high correlation between ability to promote chlorophyll retention and initiation of cell division. Highly active compounds had a planar ring and a HNCONH bridge; substitution with a HNCSNH bridge reduced activity and all other tested arrangements of the bridge gave inactive compounds. Substitution of both amino hydrogen atoms on one or both sides of the bridge reduced or removed activity. Some N′-substituted phenyl ureas were highly active. Introduction of a N-phenyl ring to a N-phenyl urea increased activity except where one ring was substituted in the para position with chloro, bromo or iodo. The activities of symmetrical disubstituted ureas were generally less than the corresponding N-monosubstituted derivative. The results suggest that the receptor site for cytokinin activity is the same for senescence retadation and cell division initiation.     

Enantioseparation using urea- and imide-bearing chitosan phenylcarbamate derivatives as chiral stationary phases for high-performance liquid chromatography

Completely deacetylated chitosan was prepared by the treatment of commercial chitosan with 50% aqueous NaOH, and then derivatized into several new chitosan phenylcarbamate derivatives having a urea and an imide moiety at the 2-position of the glucosamine ring by the reaction with isocyanate and phthalic anhydride/isocyanate, respectively. The chitosan derivatives were coated on macroporous silica gel and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The chiral recognition ability of the chitosan derivative was improved using the completely deacetylated chitosan. Among the novel chitosan derivatives, the 3,5-dimethyl-, 3,5-dichloro-, and 3,4-dichlorophenylcarbamate derivatives were found to possess relatively high chiral resolution abilities. The CSPs based on the chitosan phenylcarbamate-urea and -imide derivatives were stable in the presence of chloroform and ethyl acetate as a component of the eluents, and some racemates were better resolved by such eluents. The dichlorophenylcarbamate-imide derivatives showed a high chiral recognition for metal acetylacetonate complexes. The enantiomerization of Al(acac)3 was performed on the chitosan 3,5-dichlorophenylcarbamate-imide derivative CSP and the resulting chromatogram showed a 26% (+)-isomer enrichment.     

Identification of diacylated ureas as a novel family of fungus-specific leukocyte-activating pathogen-associated molecules

Polymorphonuclear leukocytes represent primary components of the host's innate immune defenses against fungal infection, suggesting involvement of fungal leukocyte attractants. We have found in various fungi, but not in bacteria or host cells, unstable lipid-like leukocyte chemoattractants, which also induced adherence and degranulation in human neutrophils. Purification from bakers' yeast and structural analyses by electrospray mass spectrometry, (1)H NMR spectroscopy, and chemical synthesis revealed these inflammatory mediators as diacylated ureas, a novel class of unstable lipoids. The N,N'-dipalmitoleyl urea appeared to be the most potent innate immune responses inducing compound eliciting half-maximum neutrophil chemotactic activity at 140 nm. The all-trans isomer, N,N'-dipalmitelaidyl urea, was found to be inactive with respect to stimulation of degranulation in neutrophils, which indicates a Delta(9) cis-double bond to be essential for bioactivity of these diacyl ureas. N,N'-Dipalmitoleyl urea elicited Ca(2+) mobilization in neutrophils, which was found to be pertussis toxin-sensitive and sensitive toward a carboxylmethyltransferase inhibitor, indicating that these diacyl ureas activate leukocytes via a putative Galpha(i)-protein-coupled receptor. Their isolation exclusively from fungi suggests that these lipoids are fungus-specific pathogen-associated molecules that may alert the human innate immunity system to the presence of a fungal infection.