PPAR modulators and PPAR pan agonists for metabolic diseases: the next generation of drugs targeting peroxisome proliferator-activated receptors?

The Peroxisome Proliferator-Activated Receptors-PPAR alpha, PPAR gamma, and PPAR delta--are members of the nuclear receptor gene family that have emerged as therapeutic targets for the development of drugs to treat human metabolic diseases. The discovery of high affinity, subtype-selective agonists for each of the three PPAR subtypes has allowed elucidation of the pharmacology of these receptors and development of first-generation therapeutic agents for the treatment of diabetes and dyslipidemia. However, despite proven therapeutic benefits of selective PPAR agonists, safety concerns and dose-limiting side effects have been observed, and a number of late-stage development failures have been reported. Scientists have continued to explore ligand-based activation of PPARs in hopes of developing safer and more effective drugs. This review highlights recent efforts on two newer approaches, the simultaneous activation of all three PPAR receptors with a single ligand (PPAR pan agonists) and the selective modulation of a single PPAR receptor in a cell or tissue specific manner (selective PPAR modulator or SPPARM) in order to induce a subset of target genes and affect a restricted number of metabolic pathways.

Synthesis of carbocyclic analogs of 2',3'-dideoxysangivamycin, 2',3'-dideoxytoyocamycin, and 2',3'-dideoxytriciribine.

Syntheses and antiviral activity of new carbocyclic analogs of 2', 3'-dideoxysangivamycin, 2',3'-dideoxytoyocamycin and 2',3'-dideoxytriciribine is described. The key intermediate, carbocyclic 4-chloro-5-iodopyrrolopyrimidine. was synthesized in good yield via a novel iodination method using I2 and CF3COOAg. This carbocyclic 4-chloro-5-iodopyrrolopyrimidine then allowed for a concise synthesis of the desired 4,5-disubstituted carbocyclic nucleosides.

Stereocontrolled synthesis of cis-dibenzoquinolizine chlorofumarates: curare-like agents of ultrashort duration.

The quaternizations of dibenzoquinolizines 9 and 14 with 3-halo-1-propanols are highly cis-selective (94-100% cis), results consistent with the N-methylation of O-methylcapaurine (7b), but in contrast to the proposed trans-stereochemistry of dibenzo[a,h]quinolizine methiodide 10 and the analogous quaternizations of 1-benzyl- and 1-phenylisoquinoline congeners 5b and 5c. In this report, we describe stereoselective preparation of the unique cis-dibenzoquinolizinium propanols 15 and 16and their transformation into bis- and mixed-onium chlorofumarates 19, 20ab, and 26. Dibenzo[a,g]quinolizinium propanol 15 was prepared enantioselectively in three steps from dihydroisoquinoline 11. Asymmetric transfer hydrogenation of 11 in the presence of triethylamine/formic acid and Noyori's chiral ruthenium catalyst 12 produced R-(-)-5',8-dimethoxynorlaudanosine (13) in 98% yield and 87% ee. Pictet-Spengler cyclization of 13 in formalin/formic acid afforded the dibenzo[a,g]quinolizine 14 in 65% yield. Quaternization of 14 with 3-chloro-1-propanol under Finkelstein conditions generated cis-dibenzoquinolizinium propanol 15 in 85% yield with >94% cis-selectivity. The cis-dibenzo[a,h]quinolizinium propanol 16 was obtained as a single stereoisomer by reaction of the known tetramethoxyquinolizine 9 with neat 3-iodo-1-propanol. Bis-onium chlorofumarates 18 and 19 and the mixed-onium derivative 20ab were prepared by a pool synthesis procedure from (1R)-trans-6a, 16, and chlorofumaryl chloride (17). Mixed-onium alpha-chlorofumarate 26 was synthesized from (1S)-trans-6d, 15 and (+/-)-trans-2,3-dichlorosuccinic anhydride (22), employing a recently disclosed chlorofumarate mixed-diester synthesis. The title compounds (19, 20ab, and 26) displayed curare-like effects of ultrashort duration in rhesus monkeys.

Synthesis of ultra-short-acting neuromuscular blocker GW 0430: a remarkably stereo- and regioselective synthesis of mixed tetrahydroisoquinolinium chlorofumarates.

[formula: see text] The stereo- and regioselective synthesis of ultra-short-acting nondepolarizing neuromuscular blocker GW 0430 (5a) is described. Key steps involved the enantioselective transfer hydrogenation of imine 8 employing Noyori's catalyst, the stereoselective crystallization and methanolysis of trans-bataines 11 and 12, and the stereo- and regioselective trans elimination of hydrogen chloride from 14. The latter transformation allowed complete control of the position of the chloro substituent and stereochemistry at the double bond of the linker in 15.

Potent dipeptide inhibitors of the pp60c-src SH2 domain.

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate.

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Design and synthesis of conformationally constrained analogues of 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) as potent inhibitors of cAMP-specific phosphodiesterase.

The synthesis and biological evaluation of cAMP-specific phosphodiesterase (PDE IV) inhibitors is described. The PDE IV inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, 2) was used as a template from which to design a set of rigid oxazolidinones, imidazolidinones, and pyrrolizidinones that mimic Ro 20-1724 but differ in the orientation of the carbonyl group. The endo isomer of each of these heterocycles was more potent than the exo isomer in an enzyme inhibition assay and a cellular assay, which measured TNF alpha secretion from activated human peripheral blood monocytes (HPBM). Imidazolidinone 4a inhibited human PDE IV with a Ki of 27 nM and TNF alpha secretion from HPBM with an IC50 of 290 nM. By comparison, Ro 20-1724 is significantly less active in these assays with activities of 1930 and 1800nM, respectively.

Phosphodiesterase type IV inhibition. Structure-activity relationships of 1,3-disubstituted pyrrolidines.

The synthesis of 1,3-disubstituted pyrrolidines 2 and their activities as type IV phosphodiesterase (PDE) inhibitors are described. Various groups were appended to the nitrogen of the pyrrolidine nucleus to enable structure-activity relationships to be assessed. Groups which render the pyrrolidine nitrogen of 2 nonbasic yielded potent PDE-IV inhibitors. Analogs of amides, carbamates, and ureas of 2 were synthesized to determine the effects that substitution on these functional groups had on PDE-IV inhibitor potency. The structural requirements for PDE-IV inhibitor potency differed among the three classes. A representative amide, carbamate, and urea (2c,d,h) were shown to be > 50-fold selective for inhibiting PDE-IV versus representative PDEs from families I-III and V. Furthermore, these same three inhibitors demonstrated potent functional activity (IC50 < 1 microM) by inhibiting tumor necrosis factor-alpha (TNF-alpha) release from lipopolysaccharide (LPS)-activated purified human peripheral blood monocytes and mouse peritoneal macrophages. These compounds were also tested orally in LPS-injected mice and demonstrated dose-dependent inhibition of serum TNF-alpha levels.

Electron transfer in the nitric-oxide synthases. Characterization of L-arginine analogs that block heme iron reduction.

Heme iron reduction in the nitric-oxide synthases (NOSs) requires calmodulin binding and is associated with increased NO synthesis and NADPH oxidation (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci., U. S. A. 90, 10769-10772). Here, we examined how L-arginine and the analogs N omega-methyl-L-arginine (NMA), N omega-nitro-L-arginine methyl ester (NAME), and d-(thioureido)-L-norvaline (thiocitrulline) affect electron flux through neuronal and macrophage NOS. L-Arginine and NMA increased or decreased NOS NADPH consumption depending on the isoform, while thiocitrulline and NAME decreased NADPH oxidation in both NOS by 73-86% relative to their ligand-free rates. Kinetic studies showed that thiocitrulline and NAME inhibited NOS NADPH consumption through binding within the substrate binding site. Thiocitrulline and NAME did not affect the NADPH-dependent reduction of NOS flavins nor NOS cytochrome c reduction, indicating that they blocked electron flux at a point beyond the flavins in the electron transfer sequence. Thiocitrulline and NAME inhibited both NADPH-dependent and dithionite-mediated heme iron reduction in the NOS isoforms relative to the substrate-free NOS, whereas L-arginine and NMA did not. Thus, L-arginine and NMA increase or decrease electron flux through the NOS by coupling NADPH oxidation to NO synthesis (L-arginine), or by occupying the substrate binding site with minimal catalytic coupling (NMA). In contrast, thiocitrulline and NAME decrease electron flux through both NOS isoforms by decreasing the reduction potential of the heme iron. Inhibition of heme iron reduction by substrate analogs is unusual and represents a new means to modulate electron flow through the NOS.

A pharmacodynamic model to investigate the structure-activity profile of a series of novel opioid analgesics.

A simple mathematical model of analgesia in the rat is developed and utilized to determine quantitative structure-activity relationships for a series of novel 4-anilidopiperidine opioids. The compounds tested (selected alkyl carboxyethyl esters attached at the one position of the piperidine ring) were designed for rapid inactivation by blood and tissue esterases. Model parameters included potency and rate constants for loss of pharmacodynamic effect by hydrolysis dependent and independent processes. A significant correlation is observed between duration of pharmacological effect in vivo and the rate constant for hydrolysis in human blood (r = 0.89). In vivo potency shows a moderate correlation with log P2 (r = -0.77). The validity of the model is shown by comparing model-based parameters which characterize potency and duration of effect in vivo with graphically derived parameters. Significant correlations are observed between model and graphically based estimates of potency (r = 0.75) and between model and graphically based estimates of duration of effect (r = 0.70). This model has potential application in studies of other classes of compounds in which hydrolytic cleavage limits duration of pharmacologic effect.

Peracid oxidation of an N-hydroxyguanidine compound: a chemical model for the oxidation of N omega-hydroxyl-L-arginine by nitric oxide synthase.

Arginine is oxidized by a class of enzymes called the nitric oxide synthases (NOS) to generate citrulline and, presumably, nitric oxide (.NO). N-Hydroxylation of a guanidinium nitrogen of arginine to generate N-hydroxyarginine (NOHA) has been shown to be a step in the biosynthesis of .NO. In an effort to elucidate the mechanism by which further oxidation of NOHA occurs, the oxidation of a model N-hydroxyguanidine compound by several peracids was studied in depth. This oxidative chemistry is a possible model for the enzymatic process since the corresponding urea (or citrulline equivalent product) is obtained along with an oxidized nitrogen species. The oxidized nitrogen product was, however, not .NO but rather HNO. .NO generation in this chemical system and in the enzymatic process would require another one-electron oxidation. The mechanistic details of this are further discussed.

Irreversible inactivation of macrophage and brain nitric oxide synthase by L-NG-methylarginine requires NADPH-dependent hydroxylation.

L-NG-Methylarginine (NMA) is an established mechanism-based inactivator of murine macrophage nitric oxide synthase (mNOS). In this report, NMA is shown to irreversibly inhibit both mNOS (k(inact) = 0.08 min-1) and the recombinant constitutive brain NOS (bNOS). For both NOS isoforms, metabolism of NMA parallels that of the natural substrate L-arginine (ARG), in that it undergoes a regiospecific, NADPH-dependent hydroxylation to form L-NG-hydroxy-NG-methylarginine (NOHNMA). This intermediate then undergoes further NADPH-dependent oxidation to form L-citrulline (CIT). Authentic NOHNMA, synthesized from L-ornithine, irreversibly inhibited both mNOS (k(inact) = 0.10 min-1) and bNOS in an NADPH-dependent reaction. The conversion of either NMA or NOHNMA to CIT correlated with irreversible enzyme inactivation. Thus, the data suggest that enzyme inhibition occurs as a consequence of oxidative metabolism of the intermediate, NOHNMA. A unified mechanism is proposed that accounts for NO biosynthesis from ARG, for the inactivation of NOS by NMA and for the intermediacy of hydroxylated ARG or NMA derivatives in these processes.

Opioid receptor activity of GI 87084B, a novel ultra-short acting analgesic, in isolated tissues.

GI 87084B (3-[4-methoxycarbonyl-4-[(1-oxopropyl) phenylamino]1-piperidine]propanoic acid, methyl ester, hydrochloride) was found to be a potent opioid agonist in the guinea pig ileum (EC50 = 2.4 +/- 0.6 nM), the rat vas deferens (EC50 = 387 +/- 44 nM) and the mouse vas deferens (EC50 = 39.5 +/- 7.4 nM). In the guinea pig ileum, GI 87084B, was roughly equivalent in potency to fentanyl (EC50 = 1.8 +/- 0.4 nM). GI 87084B was more potent in this tissue than alfentanil (EC50 = 20.1 +/- 1.2 nM) and less potent than sufentanil (EC50 = 0.3 +/- 0.09 nM). Schild analyses of antagonism of GI 87084B by naloxone yielded pKB values of 8.2 and slopes indistinguishable from unity in the guinea pig ileum and the mouse vas deferens. Insurmountable antagonism of GI 87084B by naloxone was observed in the rat vas deferens. However, an empirical measure of antagonist potency could be made: apparent pA2 = 8.1. The agonist dissociation constant (KA) for GI 87084B (220 +/- 90 nM) was determined by receptor alkylation with beta-chlornaltrexamine in the guinea pig ileum. Calculation of receptor occupancy suggested poor receptor-effector coupling and limited receptor reserve in the rat vas deferens, which could explain the insurmountable antagonism seen with higher concentrations of naloxone. These data suggest that GI 87084B acted through the mu class of opioid receptors to inhibit contraction induced by field stimulation in these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis, and pharmacological evaluation of ultrashort- to long-acting opioid analgetics.

In an effort to discover a potent ultrashort-acting mu opioid analgetic that is capable of metabolizing to an inactive species independent of hepatic function, several classes of 4-anilidopiperidine analgetics were synthesized and evaluated. One series of compounds displayed potent mu opioid agonist activity with a high degree of analgesic efficacy and an ultrashort to long duration of action. These analgetics, 4-(methoxycarbonyl)-4-[(1-oxopropyl)phenylamino]-1-piperidinepropanoi c acid alkyl esters, were evaluated in vitro in the guinea pig ileum for mu opioid activity, in vivo in the rat tail withdrawal assay for analgesic efficacy and duration of action, and in vitro in human whole blood for their ability to be metabolized in blood. Compounds in this series were all shown to be potent mu agonists in vitro, but depending upon the alkyl ester substitution the potency and duration of action in vivo varied substantially. The discrepancies between the in vitro and in vivo activities and variations in duration of action are probably due to different rates of ester hydrolysis by blood esterase(s). The SAR with respect to analgesic activity and duration of action as a function of the various esters synthesized is discussed. It was also demonstrated that the duration of action for the ultrashort-acting analgetic, 8, does not change upon prolonged infusion or administration of multiple bolus injections.

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.