FLUOROQUINOLONES: from structure to activity and toxicity F. Van Bambeke, Pharm. D. & P. M. Tulkens, MD, PhD Unité de Pharmacologie Cellulaire et Moléculaire Université Catholique de Louvain, Brussels, Belgium SBIMC / BVIKM www.sbimc.org - www.bvikm.org www.md.ucl.ac.be/facm www.isap.org soon... Mechanism of action of fluoroquinolones: the basics... PORIN DNA Topo DNA gyrase isomerase Gram (-) Gram (+) 2 key enzymes in DNA replication: DNA gyrase topoisomerase IV bacterial DNA is supercoiled Ternary complex DNA - enzyme - fluoroquinolone DNA GYRASE catalytic subunits COVALENTLY CLOSED CIRCULAR DNA FLUOROQUINOLONES: DNA GYRASE ATP binding subunits 4 stacked molecules (Shen, in Quinolone Antimicrobial Agents, 1993) Resistance to fluoroquinolones: the basics decreased efflux pump permeability DNA mutation of DNA gyrase Topo isomerase the enzymes Gram (-) Gram (+) Fluoroquinolones are the first entirely man-made antibiotics: do we understand our molecule ? R5 O R COOH 6 R7 X8 N R1 Don’t panic, we will travel together…. Chemistry and Activity This is where all begins... The pharmacophore common to all fluoroquinolones BINDING TO DNA R5 O O R C 6 - BINDING TO O BINDING TO THE ENZYME THE ENZYME R7 X8 N R1 AUTO-ASSEMBLING DOMAIN (for stacking) From chloroquine to nalidixic acid... nalidixic acid N CH3 O O HN CH 3 C - O chloroquine CH N N Cl N 3 C2H5 1939 O O C O- 1962 Cl N 1958 C2H5 7-chloroquinoline (synthesis intermediate found to display antibacterial activity) Nalidixic acid * a • typical chemical features of O O fluoroquinolones (a, b, c) BUT a naphthridone C - O- b (N at position 8: ) H C N N 3 • limited usefulness as drug C H 2 5 • narrow antibacterial spectrum c (Enterobacteriaceae only) • short half-life (1.5h) • high protein binding (90%) * Belg. pat. 612,258 to Sterling Drugs, 1962 From nalidixic acid to the 1st fluoroquinolone (1 of 4) nalidixic acid oxolinic acid * O O 1. modify naphthyridone O O into quinolone C - O C O O- O H3C N N N C2H5 C2H5 shows reduced protein binding... * Ger. pat. to Warner Lambert, 1967 * quinoleine From nalidixic acid to the 1st fluoroquinolone (1 of 4) nalidixic acid flumequine * 2. discovery of O O O O flumequine * C F C - O- O N H3C N N C2H5 CH3 shows weak but broad Gram(-) activity * Ger pat. to Rikker Labs, 1973 * benzo-quinolizine From nalidixic acid to the 1st fluoroquinolone (1 of 4) nalidixic acid pipemidic acid * O O O O 3. introduce a C - C - N O O piperazine * N N N H3C N N HN C2H5 C2H5 shows longer half-life... * Ger. Pat. to Roger Bellon, 1974 * pyrido-2-3-pyrimidine From nalidixic acid to the 1st fluoroquinolone (1 of 4) norfloxacin * combine all 3 O O nalidixic acid 2 features *... F C O- O O C - N O N HN 1 CH3 H3C N N C2H5 1978 3 broader Gram(-) activity less protein binding (50%) longer half-life (3-4h) * Belgian patent 863,429, 1978 to Kyorin * 6-fluoro-7-pyrimidino-quinoleine From norfloxacin to the other 1st generation fluoroquinolones: pefloxacin norfloxacin OO F C O- N N HN CH3 Add a methyl OO C to still increase F - O- half-life N N N CH3 CH 3 * Ger. pat. 2,840,910 to pefloxacin * Roger Bellon/Dainippon,1979 From norfloxacin to the other 1st generation fluoroquinolones: ofloxacin norfloxacin OO tricyclic compound F C (as in flumequine but - O morpholine ring) N N HN CH3 O O O O F C O-- F C O-- N N N N N O H3C N CH3 H3C CH3 pefloxacin ofloxacin* * Eur. pat. Appl. 47,005 to Daiichi, 1982 From norfloxacin to the other 1st generation fluoroquinolones: ciprofloxacin norfloxacin ciprofloxacin * O O F C cyclopropyl to O O O- increase potency F C O- N N HN N N CH3 HN O O O F C -- F O O C N N N N O-- N N O H C CH H3C 3 3 CH3 pefloxacin ofloxacin * Ger. pat. 3,142,854 to Bayer AG, 1983 "1st generation" fluoroquinolones norfloxacin ciprofloxacin O O O O F C - C O F - O N N N N HN HN CH3 cyclo piperazine propyl O O O O F C -- C O F - O- N N N N N N H3C CH3 O methyl H3C CH3 pefloxacin ofloxacin morpholine The "first generation" of fluoroquinolones 1960 1970 1980 t1/2 activity • Nalidixic acid • Norfloxacin 3-4 h ++ • Oxolinic acid • Pefloxacin 11 h + • Cinoxacin • Ofloxacin improved 6 h ++ • Pipemidic acid • Ciprofloxacin anti Gram (-) 3-4 h +++ activity • Fleroxacin • Rufloxacin From ofloxacin to levofloxacin... Ofloxacin is a racemic mixture O O C N F - O- H Levofloxacin is the pure (-) S isomer * N N O N O H3C CH3 CH3 The active form of ofloxacin is the (-) S isomer * Eur. pat. 206,283 to Daiichi, 1987 The present "first generation" of fluoroquinolones ... 1960 1970 1980 t1/2 activity • Nalidixic acid • Norfloxacin 3-4 h ++ • Oxolinic acid • Pefloxacin 11 h + • Flumequine • Ofloxacin improved 6 h ++ • Pipemidic acid • Ciprofloxacin anti Gram (-) 3-4 h +++ activity • Fleroxacin • Rufloxacin • Levofloxacin 6 h ++++ twice as active as ofloxacin per g How to improve the chemotherapeutic usefulness of the "first generation" fluoroquinolones 1. Maintain broad Gram(-) activity “2d generation” 2. Improve Gram(+) activity 3. Acquire activity against anaerobes “3d generation” The “second generation” fluoroquinolones 1960 1970 1980 1990 2000 a • Temafloxacin • Gram (-); • Sparfloxacin b • Grepafloxacin c • improved Gram (+) • Gatifloxacin d anti-anaerobe a: Toyama, 1988 (?) ; b: Dainippon, 1985-1987; c: Otskuda, 1989; d: Kyorin, 1988 The “third generation” fluoroquinolones 1960 1970 1980 1990 2000 • Clinafloxacin a • Trovafloxacin b anti-Gram (-) anti-Gram (+) • Moxifloxacin c anti-anaerobe • Gemifloxacin d a:Kyorin, 1987; b: Pfizer, 1993; c: Bayer, 1994; d: LG Chemical Ltd., S. Korea, 1994-98 1. maintenance of anti - Gram (-) activity Gram (-) NH2 R5 O F COOH piperazine N Gram (-) R7 C N N MIC R1 R bulky group Cl, F halogen MIC O-CH3 methoxy Gram (-) activity (E. coli) I II O O CH3 O O CH3 O O F C F C F C O- O- O- H3C H3C N N N N N N HN HN HN H 3 CO ciprofloxacin grepafloxacin gatifloxacin 0.125 - 0.5 0.06 - 2 0.06 2. improving Gram (+) activity (S. pneumoniae) Gram (+) CH3 R5 O pyrimidine F COOH N Gram (+) F H 2N R7 X8 N MIC F R1 bulky group N Gram (+) if X = C naphthyridone 8 Cl, F halogen MIC O-CH3 methoxy Activity against S. pneumoniae I II NH2 OO III O O F C F C O- O- H3C N N N N HN HN F H CO 3 H3C O O sparfloxacin moxifloxacin F C O- 0.125 - 0.5 0.01 - 0.5 CH3 O O O O N N HN F C F C O- O- H C 3 H N N N N N HN F F ciprofloxacin + H3N 0.5 - 2 H F F temafloxacin trovafloxacin 0.5 - 1 0.007 - 0.25 3. obtaining activity against anaerobes ... R5 O F COOH F R7 X8 N R1 F bulky group N if X8 = C naphthyridone O-CH3 methoxy Activity against B. fragilis OO CH3 O O I II III F C F C O- O- H3C N N N N HN HN H CO H 3 CO 3 O O F C gatifloxacin moxifloxacin O- 0.25 - 8 0.25 - 8 N N HN O O O O F C F C O- O- H ciprofloxacin N N N N N N N 2 - 128 F H3CO + H3N H CH HN2 2 gemifloxacin trovafloxacin F 0.5 - 64 0.125 - 8 Is there a SAR for emergence of resistance ? The "Mutant Prevention Concentration" * "When Mycobacterium bovis BCG and Staphylococcus aureus were plated on agar containing increasing concentrations of fluoroquinolone, colony numbers exhibited a sharp drop, followed by a plateau and a second sharp drop. The plateau region correlated,vith the presence of first-step resistant mutants. Mutants were not recovered at concentrations above those required for the second sharp drop, thereby defining a mutant prevention concentration (MPC). A C8-methoxy group lowered the MPC for an N-1-cyclopropyl fluoroquinolone" Is there a SAR for emergence of resistance ? Bactericidal activity of FQs against Mycobaterium bovis CH O O MIC (99) 3 F C - 1 O H5C2 N N 10-2 HN R 10-4 10-6 PD160793 PD161148 MPC (10) R = OCH3 R = H 10-8 Fraction of Fraction survivors 10-10 MIC 99 0.25 0.8 MPC 10 0.9 9 0.01 0.10 1.00 10.00 MPC/MIC 3.6 12 FQ concentration Dong et al; AAC 43:1756-1758 Fluoroquinolones with a C8-methoxy I II III O O CH3 O O O O F C F C F C O- O- O- H3C N N N N N N HN HN HN H CO H 3 CO 3 ciprofloxacingatifloxacin moxifloxacin Toxicity This is where all may fail... Frequent side effects of fluoroquinolones: is there a SAR ? COMPLEXATION WITH METALLIC IONS (Fe, Al, Mg, Ca) PHOTOTOXICITY DRUG INTERACTIONS: INHIBITION OF cyt P450 (1A2) ? CNS TOXICITY (BINDING TO GABA RECEPTOR) ? GASTRO-INTESTINAL DISCOMFORT ? CARTILAGE and MUSCULOSQUELETAL TOXICITY SAR of frequent side effects R R Ca++, Al+++, Fe++ N N complexation HN HN R Binding to R R5 O Penetration GABA receptor All FQs in CNS F COO- N R X N cipro, 7 8 grepa ... Inhibition of P450 R 1 sparflo, C flero, lomeflo F Inhibition of P450 Phototoxicity Fluoroquinolones with low or no drug interactions.. Trova O O Gati O O F C O- F C O- H H3C N N N N N F HN H + 3N H CO H 3 F O O Moxi Grepa CH3 O O F C O- F C O- N N HN H3C N N H CO 3 HN Fluoroquinolones with high phototoxicity ..