Structure-activity relationship within a series of pyrazolidinone antibacterial agents. 1. Effect of nuclear modification on in vitro activity.

The synthesis and biological evaluation of a series of pyrazolidinone-containing mono- and bicyclic compounds are described. The results of this investigation indicate that the [3.3.0] ring system bearing strongly electron-withdrawing groups in the 3-position provides the optimal arrangement for antibacterial activity. Two highly potent derivatives, LY193239 and LY255262, have been selected for further preclinical evaluation.

Orally absorbable cephalosporin antibiotics. 3. Preparation of biologically active R isomer of 7-(3-benzothienylglycylamido)deacetoxycephalosporanic acid.

The methyl and isopropyl esters of (RS)-3-benzothienylglycine were resolved with (+)- and (-)-tartaric acid in acetonitrile to give the corresponding R and S salts. The R-salt 4 was hydrolyzed to (R)-3-benzothienylglycine (5). The amino group in 5 was protected with the Boc function and the protected R amino acid 6 coupled with the p-NB ester of 7-ADCA to give the diprotected cephalosporin 7. After removal of the Boc and p-NB groups, the R isomer of 7-(3-benzothienylglycylamido)deacetoxycephalosporanic acid (1) was obtained. The p-NB ester of epimeric cephalosporin 7 was separated by preparative chromatography into R and S isomers. After removal of the protective groups, the S epimer was isolated. The comparison of antibacterial activity of the R and S epimers and the RS mixture of cephalosporin 1 is reported.

Oral absorption of cephalosporin antibiotics. 2. Expanded structure-activity relationships of 7-(arylacetamido)-3-substituted cephalosporins.

The structure-activity relationship for 7-arylacetamido cephalosporins has been extended. Modifications of the 7-aryl group led to improvements in microbiological activity against Gram-positive organisms. However, Gram-negative activity was generally much poorer than that of the lead compound 7-[(2-aminothiazol-4-yl)acetamido]-3-chloro-cephalosporanic acid (A). Modifications of the 3-position did not significantly change the microbiological activity or spectrum. Of the compounds selected for mouse protection studies (ED50's), 7-[(benzothien-3-yl)acetamido]-3-chloro cephalosporin and A showed the best per oral to subcutaneous ED50 ratios.

Structure-activity relationship within a series of pyrazolidinone antibacterial agents. 2. Effect of side-chain modification on in vitro activity and pharmacokinetic parameters.

The structure-activity relationship among a series of novel pyrazolidinone antibacterial agents is described. Specifically, the effect of modification of the side chain attached to the nitrogen at C-7 was explored in an attempt to improve the potency and spectrum of activity. This approach was successful in identifying several compounds having good in vitro profiles. These top candidates were then evaluated for their activity in vivo, and their pharmacokinetic behavior in various animal models was explored. This information proved critical for the identification of candidates for clinical evaluation.

Orally absorbable cephalosporin antibiotics. 2. Structure-activity studies of bicyclic glycine derivatives of 7-aminodeacetoxycephalosporanic acid.

Three positional analogues (4-, 5-, and 7-) of benzothienylglycine and (N-acetylindolinyl)-5-glycine were prepared and coupled to 7-aminodeacetoxycephalosporanic acid (7-ADCA) to give the cephalosporins 17a-c. In addition two isomeric (2,3-b and 3,2-b) thienothiopheneglycines were synthesized and coupled to 7-ADCA to yield cephalosporins 30d and 30e. In vitro testing of these new cephalosporins indicates good activity against Gram-positive bacteria. Against Streptococcus pneumoniae infections compound 25 displayed better mouse protection (both orally and subcutaneously) than cephalexin.

Discovery and structure-activity relationship of a series of 1-carba-1-dethiacephems exhibiting activity against methicillin-resistant Staphylococcus aureus.

The synthesis and antimicrobial activity of several new 1-carba-1-dethiacephalosporins is described. The discovery of unique activity of some of the analogues against methicillin-resistant Staphylococcus aureus led to the development of a structure-activity relationship designed to optimize this activity. The results of this investigation along with the pharmacokinetic characteristics of select compounds are described.

Oral absorption of cephalosporin antibiotics. 1. Synthesis, biological properties, and oral bioavailability of 7-(arylacetamido)-3-chloro cephalosporins in animals.

A number of 7-(arylacetamido)-3-substituted cephalosporins were prepared and tested in animals for oral absorbability. Bioavailability in mice, rats, dogs, and monkeys was determined after oral or parenteral administration. Oral bioavailability of five compounds selected for more intensive study was generally higher than that of penicillin V in all species tested. The results of ED50 testing against experimental infections in mice generally supported the bioavailability studies. Antibiotic activities were evaluated against Gram-positive and Gram-negative organisms with some derivatives expressing in vitro activity similar to cefaclor. The plasma half-life in rats was relatively short and the plasma curves were strongly influenced by probenecid, indicating rapid renal secretion. Some 7-(arylacetamido)-3-chloro cephalosporins are orally absorbed in animals to a greater extent than penicillin V, and antibacterial agent of proven clinical utility.

Orally absorbable cephalosporin antibiotics. 1. Structure-activity relationships of benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid.

A structure-activity relationship study of a number of orally absorbed cephalosporins together with their syntheses is described. These new cephalosporins are benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid. Several different synthetic methods for the glycine side chains, their protection, and the final acylations are reported. Several of these analogues were more active than cephalexin both in vitro and in vivo against commonly encountered Gram-positive bacteria. (R)-7-(3-Benzothienylglycylamido)-3-methyl-3-cephem-4-carboxylic acid (1R) has emerged as a potent antibacterial agent and is currently undergoing preclinical evaluation.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 2. Indole-3-acetamides with additional functionality.

As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 3. Indole-3-glyoxamides.

The preceding papers of this series detail the development of functionalized indole-3-acetamides as inhibitors of hnps-PLA2. We describe here the extension of the structure-activity relationship to include a series of indole-3-glyoxamide derivatives. Functionalized indole-3-glyoxamides with an acidic substituent appended to the 4- or 5-position of the indole ring were prepared and tested as inhibitors of hnps-PLA2. It was found that the indole-3-glyoxamides with a 4-oxyacetic acid substituent had optimal inhibitory activity. These inhibitors exhibited an improvement in potency over the best of the indole-3-acetamides, and LY315920 (6m) was selected for evaluation clinically as an hnps-PLA2 inhibitor.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 1. Indole-3-acetamides.

Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.

Transgenic model for the discovery of novel human secretory non-pancreatic phospholipase A2 inhibitors.

Transgenic mice were created which overexpress human secretory non-pancreatic phospholipase A2 (sPLA2) pansomatically as a potential disease and drug-testing model. The mice were produced using a DNA construct in which the inducible mouse metallothionein gene promoter drives expression of a human sPLA2 minigene. High levels of sPLA2 were detected in several tissues by immunofluorescence localization. Expression in the testes caused hypospermia and male infertility. Circulating catalytically active sPLA2 could be induced to levels observed in patients undergoing a systemic inflammatory response but had no detectable effect on the mice. Therefore, these results suggest that sPLA2 hyperphospholipasemia alone may have only limited pathophysiological consequences. We further show that 3-[3-acetamide-1-benzyl-2-ethylindolyl-5-oxy]propane phosphonic acid LY311727), a potent new inhibitor of phospholipase A2 catalysis developed by our group, dramatically suppresses the circulating enzyme activity in these animals whereas 3-[3-acetamide-1-benzyl-2-propylindolyl-5-oxy]propane phosphonic acid (LY314024), a substantially less potent LY311727 analog, is without effect. These later results thus motivate the further development of this compound as a potential new therapeutic agent and valuable research tool.

Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2.

A lead compound obtained from a high volume human non-pancreatic secretory phospholipase A2 (hnps-PLA2) screen has been developed into a potent inhibitor using detailed structural knowledge of inhibitor binding to the enzyme active site. Four crystal structures of hnps-PLA2 complexed with a series of increasingly potent indole inhibitors were determined and used as the structural basis for both understanding this binding and providing valuable insights for further development. The application of structure-based drug design has made possible improvements in the binding of this screening lead to the enzyme by nearly three orders of magnitude. Furthermore, the optimized structure (LY311727) displayed 1,500-fold selectivity when assayed against porcine pancreatic s-PLA2.

Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: A new class of anti-inflammatory drugs, SPI.

LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states.