Balancing potency and basicity by incorporating fluoropyridine moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitor that affords robust and sustained central Aß reduction.

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aß reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine bound to the A-A mismatch in the CAG/CAG base triad in dsRNA.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine (MCND) connected at the C2 position with a three-atom linker was examined for the binding to mismatches in double stranded RNA. Despite the fully complementary hydrogen bonding groups to guanine, MCND did not bind to guanine-guanine mismatch but did to adenine-adenine mismatch. The base pairs flanking the mismatch had weak effect on the binding, with showing the strongest binding to the A-A mismatch in the CAG/CAG sequence. The A-A mismatch in the GAC/GAC sequence was a poor substrate for the MCND binding. A monomeric derivative of MCND and another derivative lacking a methylcarbamate group showed negligilble binding to the A-A mismatch and the sequence selectivity. These results are important clues for the better molecular design of RNA binding small molecules.

Bioactivation of isothiazoles: minimizing the risk of potential toxicity in drug discovery.

Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.

Antiviral and anticancer optimization studies of the DNA-binding marine natural product aaptamine.

Aaptamine has potent cytotoxicity that may be explained by its ability to intercalate DNA. Aaptamine was evaluated for its ability to bind to DNA to validate DNA binding as the primary mechanism of cytotoxicity. Based on UV-vis absorbance titration data, the K(obs) for aaptamine was 4.0 (+/-0.2) x 10(3) which was essentially equivalent to the known DNA intercalator N-[2-(diethylamino)ethyl]-9-aminoacridine-4-carboxamide. Semi-synthetic core modifications were performed to improve the general structural diversity of known aaptamine analogs and vary its absorption characteristics. Overall, 26 aaptamine derivatives were synthesized which consisted of a simple homologous range of mono and di-N-alkylations as well as some 9-O-sulfonylation and bis-O-isoaaptamine dimer products. Each product was evaluated for activity in a variety of whole cell and viral assays including a unique solid tumor disk diffusion assay. Details of aaptamine's DNA-binding activity and its derivatives' whole cell and viral assay results are discussed.

Nonpeptide alpha V beta 3 antagonists. Part 10: In vitro and in vivo evaluation of a potent 7-methyl substituted tetrahydro-[1,8]naphthyridine derivative.

Subtle modifications were incorporated into the structure of clinical candidate 1. These changes were designed to maintain potency and selectivity while inducing changes in physical properties leading to improved pharmacokinetics in three species. This approach led to the identification of 4 as a potent, selective alphaVbeta3 receptor antagonist that was selected for clinical development based on an improved PK profile and efficacy demonstrated in an in vivo model of bone turnover.

Synthesis and biological evaluation of 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives as new ligands of cannabinoid receptors.

Cannabinoid receptors have been studied extensively in view of their potential functional role in several physiological and pathological processes. For this reason, the search for new potent, selective ligands for subtype CB receptors, CB(1) and CB(2), is still of great importance, in order to investigate their role in various physiological functions. The present study describes the synthesis and the biological properties of a series of 1,8-naphthyridine derivatives, characterised by the presence of some important structural requirements exhibited by other classes of cannabinoid ligands, such as an aliphatic or aromatic carboxamide group in position 3, and an alkyl or arylalkyl substituent in position 1. These compounds were assayed for binding both to the brain and to peripheral cannabinoid receptors (CB(1) and CB(2)). The results obtained indicate that the naphthyridine derivatives examined possess a greater affinity for the CB(2) receptor than for the CB(1) receptor. In particular, derivatives 6a and 7a possess an appreciable affinity for the CB(2) receptor, with K(i) values of 5.5 and 8.0 nM respectively; also compounds 4a, 5a and 8a exhibit a good CB(2) affinity, with K(i) values in the range of 10-44 nM. Furthermore, compounds 3g-i and 18 revealed a good CB(2) selectivity, with a CB(1)/CB(2) ratio >20.

Synthesis and biological evaluation of halogenated naphthyridone carboxamides as potential ligands for in vivo imaging studies of substance P receptors.

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone-6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC(50) = 100 nM +/- 20). The IC(50) value was not altered in the case of 2-chloro compound 1 (IC(50) = 100 nM +/- 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC(50) = 500 nM +/- 80). A drastic reduction in binding (IC(50) > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.

In vitro activity, pharmacokinetics, clinical safety and therapeutic efficacy of enoxacin in the treatment of patients with complicated urinary tract infections.

Minimal inhibitory concentrations (MIC) of enoxacin, nalidixic acid, pipemidic acid, norfloxacin, ciprofloxacin, ofloxacin and pefloxacin against isolates from 400 urological in-patients with complicated urinary tract infections (UTI) were determined by means of an agar dilution technique (10(4) cfu, multipointer). 28 patients (21 male, seven female) aged 36 to 84 years with complicated UTI due to sensitive bacteria were treated orally with 200 mg enoxacin b.i.d. for six to 14 days. Plasma and urine samples were collected from 19 patients, at intervals prior to and following a 400 mg dose of enoxacin, and enoxacin concentrations were determined by a high pressure liquid chromatography (HPLC) method. The MICs of enoxacin against all but one of the gram-negative isolates cultured from 265 urological patients were between 0.03 and 4 mg/l. The MICs against 134 gram-positive isolates were between 0.25 and 16 mg/l except for two strains of streptococci. At a concentration of 4 mg/l (8 mg/l), 90.3% (98%) of the total spectrum of isolates were inhibited by enoxacin. Of the quinolones tested, ciprofloxacin appeared to be the most active compound in vitro and cinoxacin the least active antimicrobial agent. The in vivo activity of enoxacin was comparable to that of norfloxacin, ofloxacin and pefloxacin. Oral administration of 400 mg of enoxacin to elderly patients resulted in peak serum concentrations between 0.7 and 6.3 mg/l (mean 3.6 mg/l) attained between 1.0 and 6.0 h following drug ingestion. The mean urinary recovery of parent drug within 24 h was 31.2% of the administered dose. 25 of 28 patients treated orally with enoxacin could be followed-up for five to 14 days after the end of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)