Total synthesis and biological evaluation of structural analogues of compactin and dihydromevinolin.

The full experimental details for the total synthesis of (+)-compactin and 19 structural analogues are reported. We have evaluated three classes of analogues as inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase: (1) functional and stereoisomeric analogues that possess the full carbon skeleton of compactin or dihydromevinolin, (2) functional analogues in which one carbon of the skeleton has been replaced by oxygen, and (3) analogues in which all of the 3,5-dihydroxyvaleric acid moiety has been omitted. Our most potent inhibitors belong to the first class of analogues. Compounds 42 (5-ketocompactin) and 69 (5-ketodihydromevinolin) are as active as the natural products compactin and dihydromevinolin, respectively (I50 = 1-20 nM). The corresponding enones 37 and 68 are less active, having I50 values 20-30 times larger. Inverting the stereochemistry at C-3 or C-5 or about the hexahydronaphthalene ring of compactin results in the elevation of the I50 to values in the micromolar range, comparable to the KM of the natural substrate 3-hydroxy-3-methylglutaryl coenzyme A. Class 2 analogues are active in this concentration range also. The synthetic sequence developed for compactin and its analogues includes a new method that permits the selective preparation of either the R or the S epimer at C-3 of the 3,5-dihydroxyvaleric acid moiety. This entails the reaction of anhydride 9 with either (R)- or (S)-1-phenylethanol in the presence of 4-(N,N-dimethylamino)pyridine and triethylamine. The prochiral recognition is surprisingly high; under optimum conditions, the reaction of 9 with (R)-1-phenylethanol leads to a 15:1 ratio of diesters 17 and 18.

Repromicin derivatives with potent antibacterial activity against Pasteurella multocida.

Reductive amination of repromicin with polyfunctional amines has led to new macrolide antibacterial agents, some of which are highly potent against the Gram-negative pathogen Pasteurella multocida both in vitro and in a mouse infection model. A key element in this discovery was the recognition that among certain known macrolides increasing lipophilicity results in diminished in vivo activity. One repromicin derivative, 20-[N-[3-(dimethylamino)-propyl]-N-L-alanylamino]-20-deoxorepro micin (35), was selected for advanced evaluation. At 5 mg/kg, a single subcutaneous dose was found to control induced pasteurellosis in swine and induced respiratory disease in cattle.

Quantitative structure-activity relationships among macrolide antibacterial agents: in vitro and in vivo potency against Pasteurella multocida.

Quantitative structure-activity relationships have been found among macrolide antibacterial agents in their potencies against the bacterial pathogen Pasteurella multocida both in vitro and in mouse infections. To obtain these relationships we measured, among other things, the pK(a)'s and log P's of 15 known macrolides of diverse structures. Among these compounds, in vitro potency [log(1/MIC)] is a function of log P, log D, and CMR (R = 0.86). In vivo potency is a function of the higher pK(a), the HPLC chromatographic capacity factor log k', log(1/MIC) and pNF (R = 0.93). pNF is defined as the negative logarithm of the fraction of neutral drug molecules present in aqueous solution at pH 7.4. The same physical properties were determined for 14 macrolides not used in developing the original QSAR models. Using the in vivo model, we calculated the mouse protection potency ranges for these new compounds. Ten estimates agreed with those observed, three were lower by a half-order of magnitude, and one was calculated to be active in the range of 15-50 mg/kg, but in fact was not active at 50 mg/kg, the highest level tested. When these new compounds were combined with the original 15, and the QSAR's updated, the new equations for the in vitro and in vivo potencies were essentially the same as those originally found. Hence, the physical properties indicated above are major determinants of macrolide antibacterial potencies.

Discovery of RWJ-54428 (MC-02,479), a new cephalosporin active against resistant gram-positive bacteria.

The discovery of RWJ-54428 (MC-02,479), a new cephalosporin displaying promising activity against sensitive and resistant Gram-positive bacteria, is described. Progressive structural modification from the previously reported 3-phenylthiocephem MC-02,331 afforded an overall increase in potency against MRSA while retaining other key properties such as acceptable solubility and serum binding. Evaluation of the in vitro potency and in vivo efficacy of a series of closely related compounds resulted in selection of RWJ-54428 (MC-02,479) for further studies.

SAR studies of anti-MRSA non-zwitterionic 3-heteroarylthiocephems.

SAR studies in a series of 3-heteroarylthio substituted cephalosporins established that high activity against methicillin-resistant Staphylococcus aureus (MRSA) can be achieved with various heteroaryl substituents. Early results showed that highly lipophilic 3-heteroarylthio substituents, which were necessary for anti-MRSA activity, caused high affinity of such cephems toward serum proteins. Our earlier published efforts described discovery of zwitterionic cephems MC-02,331 and RWJ-54428 (MC-02,479), where serum binding was reduced by employing basic, positively charged functionalities attached to the 3-heteroarylthio substituent. In order to avoid low solubility problems associated with most such zwitterionic cephalosporins a wide variety of non-basic heteroaryl substituents was tested (non-zwitterionic cephems are more easily formulated as water soluble sodium salts for intravenous administration). Considerable reduction in serum binding was obtained in some analogs while maintaining high anti-MRSA potency.

In vitro microbiological characterization of novel macrolide CP-163,505 for animal health specific use.

A novel 16-membered-ring macrolide agent (CP-163,505, a reductive amination derivative of repromicin) was identified as an antibacterial against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae, important etiological agents of livestock respiratory disease. In vitro MIC50/90 analysis revealed that CP-163,505 was more potent (4x) than tilmicosin against P. multocida, and equivalent to tilmicosin against P. haemolytica and A. pleuropneumoniae. In time kill kinetic studies, CP-163,505 showed bactericidal activity against P. haemolytica, P. multocida and A. pleuropneumoniae and bacteriostatic activity against E. coli at 8 times its MIC. In vitro, CP-163,505 was more potent in alkaline pH (16 approximately 32 x ) and less potent in the presence of excess cations (Mg+2 and Ca+2, 4x). EDTA and PMBN increased CP-163,505 potency against E. coli (4x) but not against the other species. Similar results were obtained with erythromycin A and tilmicosin, which were used as controls. From our data, we hypothesize that Pasteurella and Actinobacillus have an outer membrane significantly different from that of the typical enteric Gram-negative bacterium E. coli.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.

Antibiotic resistance versus small molecules, the chemical evolution.

Two discovery approaches directed to addressing the problem of increasing bacterial resistance are described. The first is a program to build activity against methicillin-resistant Staphylococcus aureus (MRSA) into the cephalosporin class of antibacterials, by enhancing affinity for PBP2a, the penicillin-binding protein responsible for this resistance. Through stepwise improvement in potency, human serum binding, solubility, and betalactamase stability, a stable of new compounds with excellent potential as anti-MRSA agents was realized. From this set was chosen MC-02, 479 (RWJ-54428), which is now undergoing extensive preclinical evaluation. The second approach explores the uridyl peptide family of antibiotics, inhibitors of bacterial translocase (mraY), whose members include the pacidamycins, mureidomycins, and napsamycins. Access to a diverse set of analogs by total synthesis was catalyzed by the discovery that hydrogenation of the 4'-exoenamidofuranosyl moiety causes no loss in biological activity. Indepth exploration of SAR required (1) establishment of the absolute stereochemistry of the central diaminobutyric acid (DABA) moiety and (2) determination of the stereochemistry of the 4'-substituent on the deoxyfuranose unit. The former was accomplished by comparison of DABA derived from degradation of a natural product pacidamycin with a sample synthesized from L-threonine. The biological activity of one member of a synthesized library of possible stereoisomers of the natural product established the absolute stereochemistry of the remaining centers. A variety of analogs of the natural product were prepared utilizing the synthetic methods developed, and their biological activities provide important insights into the specificity and spectrum of the antibiotic class.

New anti-MRSA cephalosporins with a basic aminopyridine at the C-7 position.

Incorporation of a basic aminopyridine into the C-7 position of 3-(amine-substituted arylthio)-3-norcephalosporins, as in 3, afforded high potency against MRSA and acceptable solubility for intravenous administration.

Stereochemical elucidation and total synthesis of dihydropacidamycin D, a semisynthetic pacidamycin.

Hydrogenation of the C(4') exocyclic olefin of the pacidamycins has been shown to produce a series of semisynthetic compounds, the dihydropacidamycins, with antimicrobial activity similar to that of the natural products. Elucidation of stereochemistry in the pacidamycins has been completed through a campaign of natural product degradation experiments in combination with the total synthesis of the lowest-molecular weight dihydropacidamycin, dihydropacidamycin D. The stereochemical identities of the tryptophan and two alanine residues contained in pacidamycin D have been shown to be of the natural (S) configuration, and the unique 3-methylamino-2-aminobutyric acid contained in this series of antibiotics has been shown to be of the (2S,3S) configuration. Finally, the stereochemistry obtained by hydrogenation of the C(4')-C(5') exocyclic olefin has been shown to be (R) at the C(4') nucleoside site.