Synthesis of phenylpyrimidinones as guanylyl cyclase C inhibitors.

Diarrhea is one of the most important causes of mortality in the developing world, being responsible for 2.5 million deaths each year. Many of these deaths are caused by enterotoxigenic strains of bacteria, like Escherichia coli, that produce enterotoxins that cause acute watery diarrhea, commonly defined as secretory diarrhea. Studies on symptomatic patients indicate a high prevalence of enterotoxigenic E. coli strains producing the heat-stable toxin, STa. STa is a small, cysteine-rich peptide that binds to the extracellular receptor domain of guanylyl cyclase C (GCC), located at the luminal membrane of intestinal epithelial cells. GCC and its endogenous peptide ligands, guanylin and uroguanylin, play a key role in balancing water absorption and hydration of the intestinal lumen, as exemplified by the finding that loss of GCC function causes severe dehydration of the intestinal lumen, culminating in intestinal obstruction. From a mechanistic viewpoint, reduction of GCC activity offers an efficient approach to limit enterotoxigenic E. coli- provoked secretory diarrhea. Inhibition of GCC-mediated cGMP production would not only reduce anion secretion, but would also restore NHE3 activity, resulting in a comprehensive antidiarrheal action. In the present study, two novel phenylpyrimidinone derivatives were simultaneously synthesized and tested for their ability to block STa-induced CFTR activity in T84 cells.

Diffusion weighted MR imaging of primary and recurrent middle ear cholesteatoma: an assessment by readers with different expertise.

INTRODUCTION AND PURPOSE: Diffusion weighted imaging (DWI) has been proven to be valuable in the diagnosis of middle ear cholesteatoma. The aims of our study were to evaluate the advantage of multi-shot turbo spin echo (MSh TSE) DWI compared to single-shot echo-planar (SSh EPI) DWI for the diagnosis of cholesteatoma. MATERIAL AND METHODS: Thirty-two patients with clinical suspicion of unilateral cholesteatoma underwent preoperative MRI (1.5T) with SSh EPI and MSh TSE. Images were separately analyzed by 4 readers with different expertise to confirm the presence of cholesteatoma. Sensitivity, specificity, diagnostic accuracy, and positive (PPV) and negative predictive values (NPV) were assessed for each observer and interrater agreement was assessed using kappa statistics. Diagnosis was obtained at surgery. RESULTS: Overall MSh TSE showed higher diagnostic accuracy and lower negative predictive value (NPV) compared to conventional SSh EPI. Interreader agreement between the observers revealed the superiority of MSh TSE compared to SSh EPI. Interrater agreement among all the four observers was higher by using MSh TSE compared to SSh EPI. CONCLUSION: Our findings suggest that MSh TSE DWI has higher sensitivity for detection of cholesteatoma and lower probability of misdiagnosis. MSh TSE DWI is useful in guiding less experienced observers to the diagnosis.

Synthesis and in vitro pharmacological evaluation of a new series of 5-HT1A 5-HT2A and 5-HT2C receptor ligands containing a norbornene nucleus.

A series of 4-substituted piperazine derivatives bearing a norbornene nucleus have been prepared and their affinity for serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors has been evaluated. Compounds showing the highest affinity have been selected and evaluated on dopaminergic (D1 and D2) and adrenergic (alpha1 and alpha2) receptors. The combination of structural elements (heterocyclic nucleus, oxyalkyl chain and 4-substituted piperazine) known to be critical in order to have affinity on serotonin receptors and the proper selection of substituents led to compounds with higher receptor specificity and affinity. In binding studies, several molecules showed affinity in nanomolar range towards 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate to no affinity for other relevant receptors (D1, D2, alpha1 and alpha2). Compound 2q 4-[2-[4-(3,4-dichlorophenyl)piperazin-1-yl]ethoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (Ki = 1.13 nM), was the most active and selective derivative for the 5-HT2C receptor with respect to other serotonin, dopaminergic and adrenergic receptors. Moreover, compound 3p showed mixed 5-HT2A/5-HT2C activity with affinity values in nanomolar range.

Recent advances in synthesis of PAR ligands as therapeutic strategy for inflammatory diseases.

Several studies have been published on discovering involvement of PARs receptors in a number of disease states, including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. This mini-review will focus on recent advances in the synthesis of PAR ligands highlighting their therapeutic potential in the treatment of various inflammatory diseases.

Microwave assisted synthesis: a new technology in drug discovery.

The interest in the microwave assisted organic synthesis has been growing during the recent years. It results from an increasing knowledge of fundamentals of the dielectric heating theory, availability of an equipment designed especially for the laboratory use as well as the discovery of the special techniques of the microwave syntheses. The efficiency of microwave flash-heating chemistry in dramatically reducing reaction times (reduced from days and hours to minutes and seconds) has recently been proven in several different fields of organic chemistry and this aspect is of great importance in high-speed combinatorial and medicinal chemistry. In this contribution, the current state of the art is summarized providing examples of the most recent applications in the field of microwave assisted synthesis of biologically active compounds both in heterocyclic and in peptide and peptidomimetic optimization.

Synthesis of 6beta-D-glucosyl and 6-nitroxy (-)-galanthamine derivatives as acetylcholinesterase inhibitors.

Galanthamine is an alkaloid approved for the treatment of Alzheimer's disease. In this paper the syntheses and the anticholinesterase activities of new glucosyl and nitroxy derivatives substituted on position 6 are reported. Compounds 2, 3 and 5 presented a percentage of inhibition of 35.22%, 47.48% and 67.89% respectively.

Enhanced activity of a hydrogen sulphide-releasing derivative of mesalamine (ATB-429) in a mouse model of colitis.

BACKGROUND AND PURPOSE: Mesalamine is the first-line therapy for colitis, but it lacks potency and is only effective for mild-to-moderate forms of this disease. Hydrogen sulphide has been shown to be a potent, endogenous anti-inflammatory substance, modulating leukocyte-endothelial adhesion and leukocyte migration. The purpose of this study was to determine if an H(2)S-releasing derivative of mesalamine (ATB-429) would exhibit increased potency and effectiveness in a mouse model of colitis. EXPERIMENTAL APPROACH: Colitis was induced in mice with trinitrobenzene sulphonic acid and the effects of ATB-429 and mesalamine were compared in several treatment regimens. The severity of colitis was determined using several indices, including a disease activity score (comprised of scores for diarrhea, weight loss and fecal blood), colonic myeloperoxidase activity and macroscopic/microscopic scoring of tissue injury. KEY RESULTS: Irrespective of the treatment regiment, ATB-429 was more effective than mesalamine in reducing the severity of colitis. ATB-429 was particularly effective in reducing granulocyte infiltration into the colonic tissue (by approximately 70%), as well as reducing the expression of mRNA for several key proinflammatory cytokines/chemokines (e.g., TNFalpha, IFNgamma). Treatment with ADT-OH, the H(2)S-releasing moiety of ATB-429, did not affect severity of colitis. CONCLUSIONS AND IMPLICATIONS: ATB-429 exhibits a marked increase in anti-inflammatory activity and potency in a murine model of colitis, as compared to mesalamine. These results are consistent with recently described anti-inflammatory effects of H(2)S. ATB-429 may represent an attractive alternative to mesalamine for the treatment of inflammatory bowel disease.

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates.

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.

Comparative bioavailability of two losartan formulations in healthy human volunteers after a single dose administration.

OBJECTIVE: To compare the bioavailability of two potassic losartan immediate release tablet (50 mg) formulations (Losartan from Laboratórios Cristália Ltd., Brazil, as a test formulation and Cozaar from Merck Sharp & Dohme Farmacêutica Ltd., Brazil, as a reference formulation) in 25 volunteers of both sexes. MATERIAL AND METHODS: The study was conducted in an open, randomized, 2-period crossover design and a 1-week washout period. Plasma samples were obtained over a 24-hour interval. The concentrations of losartan and its active metabolite losartan acid were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with negative ion electrospray ionization using a selected ion monitoring method. From the losartan and losartan acid plasma concentrations vs. time curves the following pharmacokinetic parameters were obtained: AUClast, AUC0-inf and Cmax. RESULTS: The geometric mean and respective 90% confidence interval (CI) of Losartan/Cozaar losartan percent ratios were 92.9% (82.2-105.0%) for Cmax, 99.0% (92.5-105.9%) for AUClast, and 99.1% (92.7-105.8%) for AUC0-inf. Furthermore, the geometric mean and respective 90% CI of Losartan/Cozaar losartan acid percent ratios were 98.5% (91.5-106.0%) for Cmax, 97.9% (93.3 102.7%) for AUClast, and 98.1% (93.6-102.9%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC0-inf were within the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the potassic losartan immediate release 50 mg tablet was bioequivalent to the Cozaar immediate release 50 mg tablet, according to both the rate and extent of absorption. While there were no significant differences in the bioequivalence assessed by either losartan or losartan acid, future bioequivalence studies on losartan may be performed by quantifying losartan alone as the parent compounds are more discriminative.

In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives.

In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.

Nesiritide utilization evaluation in a university teaching hospital.

BACKGROUND AND OBJECTIVES: Nesiritide is a new vasodilator approved for decompensated heart failure (DHF). Compared with nitroglycerin, nesiritide improves haemodynamics and symptoms in the first 3 h of therapy. However, nesiritide is more expensive than nitroglycerin (US$380-1500 daily vs. US$2-5 daily). Since its approval in the US in late 2001, nesiritide use has increased dramatically in our institution. Nesiritide has become a focus of our multidisciplinary drug utilization initiative, aimed at performing a nesiritide utilization evaluation (NUE) and developing a nesiritide usage guideline. METHODS: Medical records of patients who received nesiritide from 1 October 2003 to 31 March 2004 were reviewed. Nesiritide utilization pattern was presented to the initiative group for guideline development. RESULTS: A total of 162 records were reviewed. A 22.6% of inappropriate usage was reported. The most significant inappropriate usage was in patients who received the agent for precardiac valvular surgery optimization, followed by those for diuresis in non-cardiac-related fluid overload states. The median duration of nesiritide therapy was 6 days (range 1-94). The median length of stay (LOS) in our institution was 14 days (National statistics DHF LOS: 5.3 days). Eliminating inappropriate nesiritide usage can lead to a potential of US$141 886 savings per year. CONCLUSION: Based on the results, a 48-h nesiritide restriction policy was implemented. Usage beyond 48 h requires Heart Failure Service approval. Future NUE will evaluate the effectiveness of this policy. The overall management of DHF also needs to be evaluated to improve efficiency of care.

Derivatives as 5HT1A receptor ligands--past and present.

Serotonin is a neuromediator, well-know for its implication in mood regulation, anxiety, depression and, insomnia as well as in normal human function such as sleep, sexual activity and appetite. In this way, serotonin (5-hydroxytryptamine, 5-HT) is one of the most attractive targets for medicinal chemists and pharmaceutical companies. Among 5-HTRs, the 5-HT1A subtype is the best studied, and it is generally accepted that it is involved in psychiatric disorders such as anxiety and depression. Several structurally different compounds are known to bind 5-HT1A receptor sites such as aminotetralins, ergolines, arylpiperazines, indolylalkylamines, aporphines and aryloxyalkyl-amines. In this review, we report an overview of the 5-HT1A receptor ligands, belonging to different chemical classes.

The application of microwave irradiation as new convenient synthetic procedure in drug discovery.

Heterocyclic compounds hold a special place among pharmaceutically important natural and synthetic materials. The remarkable ability of heterocyclic nuclei to serve both as biomimetics and reactive pharmacophores has largely contributed to their unique value as traditional key elements of numerous drugs. In both lead identification and lead optimization processes there is an acute need for new organic small molecules. Traditional methods of organic synthesis are orders of magnitude too slow to satisfy the demand for these compounds. The fields of combinatorial and automated medicinal chemistry have been developed to meet the increasing requirement of new compounds for drug discovery, within these fields, speed is of the essence. The efficiency of microwave flash-heating chemistry in dramatically reducing reaction times (reduced from days and hours to minutes and seconds) has recently been proven in several different fields of organic chemistry. We believe that the time saved by using focused microwaves is potentially important in traditional organic synthesis but could be of even greater importance in high-speed combinatorial and medicinal chemistry. In this review, it is impossible to cover all significant developments in the area of microwave-assisted organic synthesis (MAOS). Rather, outlines the basic principles behind the technology and summarizes the areas in which microwave technology has made an impact, to date. Specific attention is given to application of microwave irradiation in liquid systems, and in the solid state as well of several representative biologically interesting nuclei. In addition we report some of the most recently disclosed applications in combinatorial chemistry.

Benign idiopathic partial seizures in the velocardiofacial syndrome: report of two cases.

We describe two children with the velocardiofacial syndrome and benign partial-onset seizures. Both presented with slight dysmorphic traits, mild to moderate mental delay, and high-arched palate. A cardiac defect was present in only one of them. In each patient, sporadic rolandic or occipital partial-onset seizures with the clinical and electroencephalographic features of benign idiopathic childhood epilepsy manifested at age 3 and 5 years, respectively. Treatment was started only in one patient, with complete seizure control. These two cases show that benign partial epilepsy can be a component manifestation of the central nervous system-related symptoms of the velocardiofacial syndrome.

Probing the shape of a hydrophobic pocket in the active site of delta-opioid antagonists.

The change of selectivity and the induction of antagonism by the insertion of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the second position of several opioid peptides have led to the interpretation of Tyr-Tic as a specific message domain for delta-opioid antagonists and to the discovery of dipeptides with substantial opioid activity. Selectivity and activity increase enormously when Tyr is substituted by 2',6'-dimethyl tyrosine (Dmt), hinting that the side chain of Dmt fits a hydrophobic cavity of the receptor very tightly and precisely. We have investigated the specificity of this fit by systematic changes of the substituents on the aromatic ring of ryr. Mono- and disubstitutions different from 2',6'- invariably lead to catastrophic decreases of activity. The only substitution compatible with retention of substantial antagonism is 2-methyl. An analysis of the conformational properties of all analogues reveals that substitutions do not affect the global shape of the molecule significantly. Accordingly, it is possible to use the shape of the different side chains to map the hydrophobic cavity of the receptor. The resulting complementary image is funnel shaped.

Synthesis of substituted benzamides as anti-inflammatory agents that inhibit preferentially cyclooxygenase 1 but do not cause gastric damage.

Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) benzamide) (5a), is a non-steroidal anti-inflammatory drug (NSAID), commercialised in Italy until 1985 with the brand name of Synovial(R), that has been widely used to treat arthritic patient. In addition, it was shown to spare gastric mucosa. Here we have synthesised a series of novel substituted benzamides, related to Parsalmide, and have evaluated their activity in vitro on COX-1 and COX-2 as well as in vivo in the carrageenin-induced rat paw edema, a classical in vivo anti-inflammatory assay. Compounds 5b, 11a and 11b, which showed a favourable profile in vitro and in vivo, were screened in comparison with Parsalmide for gastrointestinal (GI) tolerability in vivo in the rat. Results obtained showed that Parsalmide and compound 11b inhibited both COX-1 and COX-2 in vitro as well as they were active in vivo. Both compounds were devoid of gastric effect at the efficacious dose. In addition, both prevented indomethacin-induced gastric damage. Thus, these compounds may guide the definition of a new leading structure with anti-inflammatory activity that may allow designing new safer NSAIDs.

Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.