Synthesis and SAR of selective benzothiophene, benzofuran, and indole-based peroxisome proliferator-activated receptor delta agonists.

Recent literature has suggested the benefit of selective PPARdelta agonists for the treatment of atherosclerosis and other disease states associated with the metabolic syndrome. Herein we report the synthesis and structure-activity relationships of a series of novel and selective PPARdelta agonists. Our search began with identification of a novel benzothiophene template which was modified by the addition of various thiazolyl, isoxazolyl, and benzyloxy-benzyl moieties. Further elucidation of the SAR led to the identification of benzofuran and indole based templates. During the course of our research, we discovered three new chemical templates with varying degrees of affinity and potency for PPARdelta versus the PPARalpha and PPARgamma subtypes.

Association of anemia with outcomes in men with moderate and severe chronic kidney disease.

Anemia is a common complication of chronic kidney disease (CKD), but the outcomes associated with lower hemoglobin (Hgb) levels in patients with CKD not yet on dialysis are not well characterized. Analyses exploring outcomes associated with a single baseline Hgb value also do not account for the longitudinal variation of this measure. After collecting all Hgb measurements (N=17 194, median (range): 12 (1-168)) over a median follow-up period of 2.1 years in a historical prospective cohort of 853 male US veterans with CKD Stages 3-5 not yet on dialysis, we examined the association of time-averaged Hgb levels with predialysis all-cause mortality, end-stage renal disease (ESRD), and a composite end point of both. Kaplan-Meier survival analysis and Cox models adjusted for age, race, body mass index, smoking status, blood pressure, diabetes mellitus, cardiovascular disease, categories of estimated glomerular filtration rate, serum concentrations of albumin and cholesterol, and proteinuria were examined. Lower time-averaged Hgb was associated with significantly higher hazard of the composite end point (hazard ratio (95% confidence interval) in the adjusted model for time-averaged Hgb of <110, 111-120 and 121-130, compared to >130 g/l: 2.57 (1.85-3.58), 1.97 (1.45-2.66), 1.19 (0.86-1.63), P(trend)<0.001). Lower time-averaged Hgb was associated with both significantly higher pre-dialysis mortality and higher risk of ESRD, when analyzed separately. Anemia (especially time-averaged Hgb <120 g/l) is associated with both higher mortality and increased risk of ESRD in male patients with CKD not yet on dialysis.

Medicinal chemistry in the development of societies. Biodiversity and natural products.

This document has been elaborated by the IUPAC Medicinal Chemistry section and is backed by a large number of scientists, many of whom have had direct involvement and whose names appear at the end of the article. This work discusses the role that the discovery of new medicinal agents has in the development of societies as well as in the conservation of biodiversity in terms of work carried out on natural products. Also included are several recommendations for countries which are presently in search of their own scientific and technological development in medicinal agents. The IUPAC Medicinal Chemistry section would appreciate the collaboration of the scientific societies in every country to aid in the diffusion of this document.

15-Lipoxygenase and its inhibition: a novel therapeutic target for vascular disease.

The disease process known as atherosclerosis is the leading cause of morbidity and mortality in the Western world. Current therapies have focused on treating the major risk factors identified to date including plasma lipid derangements, hypertension, clotting disorders, and diabetes. However, a significant number of individuals will be diagnosed with this malady in the apparent absence of known risk factors. Recent attention has turned toward treating the disease at the level of the vessel wall. In this review, we assess the relevancy of the oxygenating enzyme 15-lipoxygenase (15-LO) as a therapeutic target. In vitro studies suggest that this enzyme may be involved in processes that modify native LDL in such a way as to be avidly taken up by tissue macrophages. In support of this contention are reports demonstrating the colocalization of 15-LO with macrophage-rich arterial lesions and epitopes of modified LDL. Investigations using transgenic animals also suggest that the site of 15-LO expression may be an important factor in the development of the disease. The alteration of important cellular fatty acids may also generate intracellular signals that promote a pro-atherogenic phenotype in the absence of measurable changes in bulk lipid peroxidation. A limited number of studies have examined 15-LO inhibitors and those structural determinants necessary for inhibition of the enzyme. These include natural products and synthetic analogs. Structure activity relationships have been defined for a number of compounds including caffeic acid derivatives, propargyl ethers, and catechols. A novel, potent, specific inhibitor of 15-LO that lacks significant antioxidant activity was tested for its ability to inhibit atherosclerotic lesion formation in vivo. This benzothiopyranoindole virtually eliminated lesion formation in two animal models in the absence of significant changes in plasma lipids. Further, it prevented the progression of pre-established lesions in another study. Collectively, these data provide a strong scientific rationale for exploring the inhibition of 15-LO as a therapeutic strategy.

Novel nonpeptide CCK-B antagonists: design and development of quinazolinone derivatives as potent, selective, and orally active CCK-B antagonists.

We have designed a novel series of CCK-B receptor antagonists by combining key pharmacophores, an arylurea moiety of 1 and a quinazolinone ring of 3, from two known series. Our earlier studies showed that compounds with methylene linkers in our "target" produced moderate binding affinity and selectivity for CCK-B receptors, whereas its higher and lower homologues resulted in loss of affinity. Introduction of -NH- as a linker dramatically enhanced binding affinity and selectivity for CCK-B receptors, thus providing several compounds with single-digit nanomolar binding affinity and excellent selectivity. Analogous to the earlier studies of the series of quinazolinone derivatives 3, we also found 3-isopropoxyphenyl as a preferred substitution on the N-3 quinazolinone. Electron-withdrawing substitutions on the urea terminal phenyl ring enhanced the CCK-B potency. Representative compounds of this series were tested in the functional assay and showed pure antagonist profiles. Compounds 51 and 61 were orally active in the elevated rat X-maze test. These compounds were also evaluated for their pharmacokinetic profile. The absolute oral bioavailability of compound 61 was 22% in rats.

Second generation "peptoid" CCK-B receptor antagonists: identification and development of N-(adamantyloxycarbonyl)-alpha-methyl-(R)-tryptophan derivative (CI-1015) with an improved pharmacokinetic profile.

We have previously described the design and development of CI-988, a peptoid analogue of CCK-4 with excellent binding affinity and selectivity for the CCK-B receptor. Due to its anxiolytic profile in animal models of anxiety, this compound was developed as a clinical candidate. However, during its development, it was determined that CI-988 had low bioavailability in both rodent and nonrodent species. In the clinic, it was further established that CI-988 had poor bioavailability. Thus, there was a need to identify an analogue with an improved pharmacokinetic (PK) profile. The poor bioavailability was attributed to poor absorption and efficient hepatic extraction. We envisaged that reducing the molecular weight of the parent compound (5, MW = 614) would lead to better absorption. Thus, we synthesized a series of analogues in which the key alpha-methyltryptophan and adamantyloxycarbonyl moieties, required for receptor binding, were kept intact and the C-terminus was extensively modified. This SAR study led to the identification of tricyclo[3.3.1.1(3,7)]dec-2-yl [1S-[1 alpha(S*)2 beta]-[2-[(2-hydroxycyclohexyl)amino]-1-(1H-indol-3- ylmethyl)-1-methyl-2-oxoethyl]carbamate (CI-1015, 31) with binding affinities of 3.0 and 2900 nM for the CCK-B and CCK-A receptors, respectively. The compound showed CCK-B antagonist profile in the rat ventromedial hypothalamus assay with a Ke of 34 nM. It also showed an anxiolytic like profile orally in a standard anxiety paradigm (X-maze) with a minimum effective dose (MED) of 0.1 microgram/kg. Although the compound is less water soluble than CI-988, oral bioavailability in rat was improved nearly 10 times relative to CI-988 when dosed in HP beta CD. The blood-brain permeability of CI-1015 (31) was also enhanced relative to CI-988 (5). On the basis of the overall improved pharmacokinetic profile as well as enhanced brain penetration, CI-1015 (31) was chosen as a development candidate.

Inhibitors of acyl-CoA:cholesterol acyltransferase: novel trisubstituted ureas as hypocholesterolemic agents.

Our continued interest in developing novel, potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, and our discovery of several active series of disubstituted urea ACAT inhibitors, have led us to investigate a series of trisubstituted ureas that are structural hybrids of our disubstituted series and of a trisubstituted urea ACAT inhibitor series disclosed by scientists at Lederle. This investigation has led to the discovery of novel trisubstituted ureas, several of which inhibit ACAT in the nanomolar range and effectively lower total plasma cholesterol when administered as a diet admixture in an acute model of hypercholesterolemia in rats. One analogue (35) also lowered total cholesterol as efficaciously as CL 277,082 in our chronic hypercholesterolemic rat model. The most notable finding of this study is that the SAR of the trisubstituted ureas diverges from that seen in our previously disclosed disubstituted urea series. This series showed optimal activity with 2,4-difluoro and 2,4,6-trifluoro substitution on the urea N-phenyl, whereas the disubstituted series showed optimal activity with bulky 2,6-disubstitution on the phenyl ring.

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

1. 15-Lipoxygenase (15-LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15-LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol-fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. 2. PD 146176 inhibited rabbit reticulocyte 15-LO through a mixed noncompetitive mode with a Ki of 197 nM. The drug had minimal effects on either copper or 2,2'-azobis(2-amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. 3. Control New Zealand rabbits were fed a high-fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg-1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nM). During the 12 week study, there were no significant differences in weight gain haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. 4. The drug plasma concentrations achieved in vivo did not inhibit low-density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176-treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. 5. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15 +/- 4 to 0% (P < 0.02); esterified cholesterol content was reduced from 2.1 +/- 0.7 to 0 micrograms mg-1 (P < 0.02) in this region. Immunostainable lipid-laden macrophages present in aortic intima of control animals were totally absent in the drug-treated group. 6. Results of these studies are consistent with a role for 15-LO in atherogenesis.

In vitro and in vivo disposition of 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976). Identification of a novel five-carbon cleavage metabolite in rats.

The metabolism of CI-976, a potent inhibitor of liver and intestinal acyl coenzyme A:cholesterol acyltransferase, was investigated in isolated rat hepatocytes and Wistar rats after oral administration. The major metabolite observed both in vitro and in vivo was identified as the 6-carbon, chain-shortened 5,5-dimethyl-6-oxo-[(2,4,6-trimethoxyphenyl)amino]hexanoic acid (M-4). M-4 was determined to be formed from the omega-carboxylic acid 11,11-dimethyl-12-oxo-12-[(2,4,6-trimethoxyphenyl)amino]dodecanoic acid (M-1) via the 2- and 4-carbon, chain-shortened intermediate metabolites [9,9-dimethyl-10-oxo-10-[(2,4,6-trimethoxyphenyl)amino]decanoic acid (M-2) and 7,7-dimethyl-8-oxo-8-[(2,4,6-trimethoxyphenyl)amino]octanoic acid (M-3)], respectively. M-1 was, in turn, determined to be derived from omega-hydroxy CI-976. A minor metabolite, identified in vitro and in vivo, was a novel 5-carbon, chain-shortened derivative, 6,6-dimethyl-7-oxo-7-[(2,4,6-trimethoxyphenyl)amino]heptanoic acid (M-5). M-5 was shown not to be formed from either M-1 or the omega-hydroxy derivative. Separate incubation of CI-976 (omega-oxidation and beta-oxidation pathways) and M-1 (beta-oxidation only) indicated a potential gender difference in the omega-oxidation of CI-976. Both the omega-oxidation and beta-oxidation pathways were enhanced by clofibrate and phenobarbital induction, and CI-976 metabolism was completely inhibited when coincubated with SKF525A pointing to cytochrome P450-mediated metabolism, presumably CYP4A. Etomoxir and L-carnitine had minor effects on the beta-oxidation of M-1, indicating beta-oxidation occurs predominately within peroxisomes.

Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcription factor NF-kappa B.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

Nephrolithiasis: how it happens and what to do about it.

Nephrolithiasis is caused by several biochemical mechanisms, identification of which allows specific therapy that may prevent recurrences. In patients with their first kidney stone, careful evaluation is needed to identify those at highest risk for recurrence. Extensive evaluation of this subgroup should lead to proper diagnosis and management of nephrolithiasis, resulting in lower morbidity for these patients.

Cholecystokinin B antagonists. Synthesis and quantitative structure-activity relationships of a series of C-terminal analogues of CI-988.

A study of structure-activity relationships of a series of 'dipeptoid' CCK-B receptor antagonists was performed in which variations of the phenyl ring were examined while the [(2-adamantyloxy)carbonyl]-alpha-methyl-R)-tryptophan moiety of the potent antagonist CI-988 was kept constant. Since the main focus of this study was phenyl substituent variation, series design techniques were employed to insure an adequate spread of physicochemical properties (lipophilic, steric, electronic), as well as positional substitution. A QSAR analysis on sets of 26 and 16 analogues revealed that CCK-B affinity was related to a combination of the overall size and, marginally, lipophilicity of the phenyl ring substituents (i.e., smaller groups were associated with increased potency with an optimum pi near zero, respectively). Further exploration revealed that the dimensions and electronics of the para-phenyl substituent could be related to CCK-B affinity. Increased affinity was seen with short, bulky (branched) electron withdrawing groups. Analogs with small para-substituents appeared to be about 1000-fold CCK-B selective, indicating that selectivity for CCK-B binding is sensitive to phenyl ring substitution. The 4-F-phenyl dipeptoid, derived from this study, has extraordinary high affinity at the CCK-B receptor (IC50 = 0.08 nM) and was also very selective (940-fold CCK-B selective). Consistent with previous reports, (S)-configuration at the substituted phenethylamide center, a carboxylic acid and the presence of a phenyl ring were found to be associated with increased affinity at both CCK-A and CCK-B receptors.

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of a series of substituted N-phenyl-N'-[(1-phenylcyclopentyl)methyl]ureas with enhanced hypocholesterolemic activity.

We recently described our initial structure-activity relationship (SAR) studies on a series of N-phenyl-N'-aralkyl- and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). From this series of analogs, compound 1 (PD 129337) was identified as a potent inhibitor of ACAT with an IC50 value of 17 nM. It was also shown to dose-dependently lower plasma cholesterol in cholesterol-fed rats. However, further investigation led to the suggestion that this compound was poorly absorbed, due to a lack of efficacy when administered by gavage in an aqueous vehicle. To overcome this deficiency, we continued our SAR study on this novel series of ACAT inhibitors using an acute in vivo screen in which the compounds are administered to rats in an aqueous, CMC/Tween suspension vehicle. Modification of the N'-phenyl moiety by incorporating functional groups which were amenable to forming salts and/or polar groups to reduce lipophilicity led to the identification of several inhibitors which displayed excellent efficacy employing this protocol. Overall, substitution on the phenyl ring in the ortho, meta, or para positions led to inhibitors with only a slight decrease in potency in vitro compared to the parent unsubstituted compound. Bulkier groups in the para position tended to lower the ACAT inhibitory activity in vitro. Polar groups, such as carboxyl (33,34), lowered in vitro activity significantly, suggesting that polar-ionic interactions are disfavored for the enzyme activity. From this series, compound 28 was evaluated further in secondary in vivo screens. In a chronic cholesterol-fed rat model of hypercholesterolemia, compound 28 dose-dependently reduced nonHDL cholesterol and significantly elevated HDL cholesterol. It showed significantly greater aqueous solubility than the parent compound 1. However, it was shown to cause adrenal toxicity in guinea pigs. This led us to design a series of homologs (44-51) with increased basicity and lower lipophilicity. Some of these compounds were more potent ACAT inhibitors in vitro and demonstrated excellent hypocholesterolemic activity in vivo. Interestingly, compound 45, unlike 28, did not produce adrenal toxicity in guinea pigs and demonstrated excellent lipid-modulating activity in the chronic model of preestablished dyslipidemia in rats.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 5. Identification and structure-activity relationships of novel beta-ketoamides as hypocholesterolemic agents.

A study of structure-activity relationships of substituted beta-ketoamide ACAT inhibitors I and II was performed. The results of this study suggest that whereas the beta-keto group was tolerated with no loss in activity, beta-hydroxy and oxime moieties led to significantly reduced activity in vitro and in vivo. The most potent inhibitor from the acyclic series (I) (11, IC50 = 0.006 microM) contained a C-13 alkyl chain. This compound reduced plasma total cholesterol by 38% and 66% at 3 and 30 mg/kg, respectively, in cholesterol-fed rats. Dimethylation alpha to the anilide core (5) and subsequent N-methylation of the amide NH (6) decreased in vitro potency significantly. It was also found that high potency was retained with inhibitors which incorporated the carbonyl into a lactam ring (II).

Inhibitors of acyl-Coa:cholesterol acyltransferase. 4. A novel series of urea ACAT inhibitors as potential hypocholesterolemic agents.

We have synthesized a series of N-phenyl-N'-aralkyl and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). This intracellular enzyme is thought to be responsible for the esterification of dietary cholesterol; hence inhibition of this enzyme could reduce diet-induced hypercholesterolemia. For this series of compounds, the in vitro ACAT inhibitory activity was improved by increasing the bulk of the 2,6-substituents on the phenyl ring. Additionally, we found that spacing of the aromatic rings was critical for ACAT inhibitory activity. A phenyl ring five atoms away from the requisite 2,6-diisopropylphenyl moiety was optimal for in vitro activity. Substitution alpha to the N'-phenyl moiety enhanced in vitro potency. In the case of phenylcycloalkyl ureas, ACAT inhibitory activity was independent of the size of the cycloalkyl ring. From this series of analogs, compound 25, which had excellent in vitro potency for inhibiting ACAT, was found to lower plasma cholesterol by 73% in vivo when administered in the diet at 50 mg/kg in an animal model of hypercholesterolemia. In this model, compound 25 lowered plasma cholesterol dose dependently and was as efficacious as the Lederle ACAT inhibitor CL 277082.

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 2. Modification of fatty acid anilide ACAT inhibitors: bioisosteric replacement of the amide bond.

In order to further define the structural features necessary for potent inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in vitro and cholesterol lowering in vivo, systematic study of bioisosteric replacements for the amide bond in our previously identified series of fatty acid anilide ACAT inhibitors was undertaken. Only replacement of amide bonds with isosterases having both hydrogen bond donor and acceptor functionalities yielded compounds retaining ACAT inhibitory activity. Replacement of the amide bond with the urea bioisostere yielded compounds that were potent ACAT inhibitors in vitro and efficacious hypocholesterolemic agents in vivo. Examination of the structure activity relationships in the phenyl ring and alkyl portion of the N-phenyl-N'-alkylureas revealed that 2,6-diisopropyl substitution was optimal in the phenyl ring. When the 2,6-diisopropyl moiety was kept constant, potency in vitro and in vivo was maintained with straight and branched alkyl groups from 6 to 18 carbons in length.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 1. Identification and structure-activity relationships of a novel series of fatty acid anilide hypocholesterolemic agents.

A series of fatty acid anilides was prepared, and compounds were tested for their ability to inhibit the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) in vitro and to lower plasma total cholesterol and elevate high-density lipoprotein cholesterol in cholesterol-fed rats in vivo. The compounds reported were found to fall into two subclasses with different anilide SAR. For nonbranched acyl analogues, inhibitory potency was found to be optimal with bulky 2,6-dialkyl substitution. For alpha-substituted acyl analogues, there was little dependence of in vitro potency on anilide substitution and 2,4,6-trimethoxy was uniquely preferred. Most of the potent inhibitors (IC50 less than 50 nM) were found to produce significant reductions in plasma total cholesterol in cholesterol-fed rats. Additionally, in vivo activity could be improved significantly by the introduction of alpha,alpha-disubstitution into the fatty acid portion of the molecule. A narrow group of alpha,alpha-disubstituted trimethoxyanilides, exemplified by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (39), was found to not only lower plasma total cholesterol (-60%) in cholesterol-fed rats but also elevate levels of high-density lipoprotein cholesterol (+94%) in this model at the screening dose of 0.05% in the diet (ca. 50 mg/kg).

Application of polymerase chain reaction techniques to study of rabbit renin gene expression.

Studies were performed to develop techniques to assess renin mRNA in a single microdissected juxtaglomerular apparatus (JGA) of rabbit using the polymerase chain reaction (PCR) method of amplification of cDNA sequences. In preliminary studies synthetic oligonucleotide primers corresponding to regions in the rat renin cDNA sequence, which are highly conserved between mouse, rat, and man, were found to yield good amplification efficiency with a rat renin cDNA template, but little product was observed with rabbit cDNA template. We therefore employed a nested primer PCR cloning technique to clone an 839 base pair portion of the rabbit renin cDNA to obtain species-specific sequence information for primer design. Here we report the nucleotide sequence of a partial rabbit renin cDNA clone and the use of species-specific primers that permit semiquantitative assessment of rabbit mRNA levels in the single JGA.