Medicinal Chemistry II Introduction to antibiotics

Transcription

1 Medicinal Chemistry II Introduction to antibiotics Mohammed Nooraldeen Al-Qattan (PhD) 14/7/2016 History of microbials and anti-microbials Targets, spectrum, resistance, susceptibility, bacteriostatic vs bactericidal

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3 Anti + bio + tics The term antibiotic has its origin in the word antibiosis (i.e. against life). Antibiotics are chemical substances obtained from various species of microorganisms (bacteria, fungi, actinomycetes) that suppress the growth of other microorganisms and eventually may destroy them. The probable points of difference amongst the antibiotics may be: 1) Physical 2) chemical 3) pharmacological properties 4) antibacterial spectra 5) mechanism of action.

4 Infection Infection is the colonization of the host organism with a microorganism like bacteria, parasite, virus, or even a macro organism like fungi and macro parasites such as worms and nematodes. The microorganism then will use the host resources to reproduce and grow that results in a disease. Host system normally use the immune system to fight against the invading organism, first by the innate immune system, then by the adaptive immune system.

5 Oldest antibiotics Sdf 1. Molded curd of soybean was used in Chinese folk medicine to treat boils and carbuncles. 2. Molded cheese was sued by Chinese and Ukrainian farmers to treat infected wounds

6 History of microbials discovery Bacteria are single-cell microorganism first identified in 1670s by van Leeuwenhoek following his invention of the microscope. The relation between bacteria and infection was not recognized until , the French scientist Pasteur and Joubert noticed that bacteria are crucial for milk fermentation and might be responsible for diseases. They also noticed that anthrax bacilli were killed if grown in culture with other specific bacteria The Edinburgh surgeon Lister struggled to prove that surgeons are infecting their patients in operation theater. Thus he introduced the carbolic acid as operating ward antiseptic. Later in 1800+, Koch identified specific microorganisms that are responsible for specific diseases such as tuberculosis, cholera and typhoid.

7 History of anti-microbials discovery Most of the early compounds were antiprotozoals but little antibacterials were developed In 1934, Proflavine was used as wound disinfectant in World War II, however, it was unsuitable for systematic use due to its high toxicity. In 1935, Prontosil was discovered as effective antibacterial agent in vivo. Later it found to be a prodrug that release sulfanilamide as the active metabolite.

8 History of anti-microbials discovery In 1936, the first use of chemotherapy to combat infection was by Paul Ehrlich. Chemicals (magic bullets) were used at concentrations tolerated by the host to selectively inhibit microorganism proliferation (selective toxicity). Later in 1910, Paul developed synthetic toxic (antimicrobial) compound as arsenic complex called salvarsan which was effective against trypanosome. Salvarsan is a mixture of dimer (A), trimer (B) and Pentamer (C) of 4- arsenio 2-amino phenol

9 History of anti-microbials discovery The discovery of sulfonamides or sulfa drugs class of antibacterial agents was a breakthrough in the treatment of systematic bacterial infections. In 1940, penicillin (a bacterio-toxic fungal metabolite) was isolated from mold culture, even its effect was discovered earlier in 1929 by Alexander Fleming.. The discovery revolutionized the fight against bacterial infection by proven more effective than sulfonamides. In , several antibiotics were isolated from microorganisms such as streptomycin & neomycin (aminoglycosides), chloramphenicol, chlortetracycline (tetracyclines),, erythromycin (macroloides), valinomycin & bacitracin (cyclic peptides), cephalosporin C (βlactams)

10 Vancomycin (peptidoglycans) Bacitricin (cyclic polypeptide) Streptomycin (aminoglycosides)

11 History of anti-microbials discovery In 1952, synthetic antibacterial agents were developed such as isoniazid (antituberculosis). isoniazid In 1962, antibacterial quinolone of nalidixic acid (first generation) was developed followed by ciprofloxacin in 1987 (second generation) nalidixic acid ciprofloxacin

12 History of anti-microbials discovery Wright, Peter M., Ian B. Seiple, and Andrew G. Myers. "The evolving role of chemical synthesis in antibacterial drug discovery." Angewandte Chemie International Edition (2014):

13 History of anti-microbials discovery Therefore, a substance is classified as an antibiotic if the following conditions are met: 1. It is a product of metabolism (although it may be duplicated or even have been anticipated by chemical synthesis). 2. It is a synthetic product produced as a structural analog of a naturally occurring antibiotic. 3. It antagonizes the growth or survival of one or more species of microorganisms. 4. It is effective in low concentrations.

14 A substance is classified as medicinal antibiotic if it has the previous conditions in addition to the following characters: 1. It must exhibit sufficient selective toxicity to be decisively effective against pathogenic microorganisms or neoplastic tissue, on the one hand, without causing significant toxic effects, on the other. 2. It must be chemically stable enough to be isolated, processed, and stored for a reasonable length of time without deterioration of potency and can be converted to other forms suitable for oral and parenteral uses 3. The rates of biotransformation and elimination of the antibiotic should be slow enough to allow a convenient dosing schedule

15 The bacterial cell o The important feature for antibacterial agents is their selective action against bacterial (prokaryotic) cells than animal (eukaryotic) cells. o Prokaryotic cells differ significantly from eukaryotic cells by having: > 1-10 µm length whereas eukaryotic length is µm. > No nucleus. > Circular DNA, no chromosomal structure. > Most of the organelles are simpler than in eukaryotics. > Different biochemistry (e.g. synthesize vitamins) > Characteristic cell wall which differ from bacteria to another, but generally, it is thick and fatty envelope which protect the bacterial cell from lysis and invading by external environment.

16 Gram-positive and gram-negative bacteria Gram Only two layers of peptidoglycan Gram + Consists of peptidoglycan layers Peptidoglycan Gram-positive cocci and Gram-negative bacilli Hans C. J. Gram, a Danish microbiologist, developed the Gram staining (methyl violet-iodine) method for staining bacteria so that they were more readily visible under the microscope.

17 Gram-positive and gram-negative bacteria The Gram stain is a staining procedure of great value in the identification of bacteria. The staining technique involves the addition of a purple dye followed by washing with acetone. Bacteria with a thick cell wall (20 40 nm) absorb the dye and are defined as Gram-positive because they are stained purple. Bacteria with a thin cell wall (2 7 nm) absorb only a small amount of dye, and the excess dye is washed out with acetone. These bacteria are then stained pink with a second dye (safranin) and are said to be Gram-negative. o Gram-positive bacteria these cells have a thick cell wall and are coloured purple. o Gram-negative bacteria these cells have a thin cell wall and are coloured pink.

18 Examples of important gram+ and gram- bacteria

19 Bacterial nomenculature

20 Potential targets for antibacterial agents Although the target and mechanism of action are unknown for many antibiotics, those known can be classified into the following: Bacterial cell wall synthesis Cell metabolism (e.g. folate synthesis) Cytoplasmic membrane Protein synthesis Reduced selectivity The targets and mechanism of action determine the antibiotic selectivity toward microorganisms than host cells Nucleic acid synthesis

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22 Potential targets for antibacterial agents Cell metabolism (e.g. folate synthesis) Disruption of bacterial cell metabolism is achieved by inhibiting enzymecatalyzed reactions. Such enzymes could be unique to bacteria or even present in host, however with differences in structure. Sulfonamides are example of antimetabolites acting on dihyropteroate synthase that forms precursors for folic acid which is critical for nucleic acid synthesis. While trimethoprim act on folate reductase. Sulfonamides mimic Paraaminobensoic acid (PABA) component of folic acid dihydropeteroate synthase Folic acid folate reductase DHFA and THFA Bacteria Human

23 Potential targets for antibacterial agents Bacterial cell wall synthesis Unlike eukaryotic cells, the bacterial cell has a cell wall which controls the size of bacterial cell against the osmotic pressure. During cellular division new cell wall is created. Any disruption of cell wall synthesis and repair leads to cell lysis. Several agents derived from fungal (Penicillins, cephalosporins and glycoproteins o vancomycin) and bacterial source (bacitracin) act this way

24 Potential targets for antibacterial agents Cytoplasmic membrane o The bacterial cell membrane is permeable to various compounds and has in/out gates o Affecting membrane permeability has fatal consequences. o Agents from bacterial source Polymyxins, Colistin act in this way. o Agent are more toxic if used systematically than those inhibiting cell wall

25 Potential targets for antibacterial agents Protein synthesis Most of structural (e.g. peptidoglygans) and catalytic (e.g. enzymes) components of the cell are fully or partially made of proteins. Disruption of protein synthesis have disastrous effect on cell survival. Agents may inhibit protein synthesis at RNA transcription (Rifampicin), at ribosome (aminoglycosides, tetracyclines, macrolides, chloramphenicol). Structural difference between prokaryotic and eukaryotic ribosomes and polymerases improve selectivity, however the agents are toxic at high doses

26 Potential targets for antibacterial agents Nucleic acid synthesis Inhibition of nucleic acid transcription and replication prevents cell division and synthesis of essential proteins.. Agents act this way include nalidixic acid and proflavine.

27 Bacterial resistance to antibacterial agents Resistance is the failure of microorganisms to be killed or inhibited by antimicrobial treatment. Resistance could be present before exposure to drug i.e. intrinsic (e.g. due to impermeability or lack of susceptible target) or developed after exposure i.e. acquired (e.g. due to genetic mutations or transfection by DNA plasmid). If two antimicrobial agents share the same target, resistance to the first affects the activity of other Resistance mechanisms include: penetration, efflux, destruction, Δ target Persistence is bacterial survival within host cells, cysts or abscesses thus it is hard to be reached by antibiotics.

28 Bacterial resistance to antibacterial agents (Cont.) Plasmid may encodes: 1. Enzymes that destroy the antibiotic 2. Enzymes that modify the antibiotics 3. Pumps that efflux antibiotics 4. Another copy of enzyme being targeted by antibiotic

29 Spectrum of antibacterial agents Antimicrobials that inhibit wide range of bacterial genera belonging to both grampositive and gram-negative cultures are termed broad spectrum such as tetracyclines Antimicrobials that inhibit only few bacterial genera are termed narrow spectrum such as the glycopeptides of vancomycin which act on gram-positive & anaerobics The earlier terms; broad spectrum and narrow spectrum are less meaningful nowadays due to emergence of microbes resistant to single and multiple agents.

30 Identification of pathogen and antibiotic selection Empirical-based therapy: specific bacteria are associated with specific diseases UTI gm-ve E. Coli skin infection gm+ve Staph aureus Experimental-based therapy: culturing microorganism on growth media, identifying genus and species followed by in vitro assays to determine MIC & MBC Susceptibility test Inhibition zone = f(type, conc)

31 Antibiotic-sensitivity testing. Petri dishes were spread-inoculated with Staphylococcus albus (white growth) or Micrococcus luteus (yellow growth) before antibiotic assay "rings" were placed on the agar surface. The coloured disks at the end of each spoke of the rungs are impregnated with different antibiotics. Clockwise from the top (arrow) these are: Novobiocin, Penicillin G, Streptomycin (white disk), Tetracycline, Chloramphenicol, Erythromycin, Fusidic acid (green disk) and Methicillin. Clear zones of suppression of bacterial growth around the individual antibiotic disks are evidence of sensitivity to these antibiotics

32 Bactericidal vs. bacteriostatic o Depending on the concentration being used, the antibiotic can act on bacteria as bactericidal (i.e. kill) or bacteriostatic (i.e. halt growth and replication). o While for most antibiotics the bactericidal concentrations are achievable in vitro, only few antibiotics can be used at bactericidal concentrations in vivo due to toxicity issues. x2 or x4 o Gentamycin: bacteriostatic bactericidal (safe to be used as bactericidal) x40 o Tetracycline: bacteriostatic bactericidal (toxic if used as bactericidal) o Bacteriostatic antibiotics retard the logarithmic growth of bacteria in order to give time for immune system to deal with it.

33 Combination therapy Combinations of antibiotics would - broaden the antimicrobial spectrum. - Decrease appearance of resisitance - Decrease doses of components in the combination - Increase therapeutic effectiveness Combination of bactericidal antibiotics such as β-lactams and aminoglycosides are used for urgent treatments before pathogen identification. Combination of bacteriostatic antibiotics such as macrolides and sulfonamides are commonly used for Upper RTI by Haemophilus inlfuenzae Combination of bacteriostatic (e.g. tetracycline) and bactericidal (β-lactams) is antagonistic.

34 Three possible outcomes are expected for the combination therapy 1. Synergism: the antibiotic effects of the combination are greater than sum of the effect of each antibiotic alone (A+B) > A+B 2. Additivity: the effect of the combination is equal to the sum of the effects of the two (A+B)= A+B 3. Antagonism: one of the components decreases the effectiveness of the other /

35 - The known example for antagonism is between tetracyclien and penicillin is due to the fact that penicilin is active only on growing bactreria. Tetracycline blocks growth and the bacteria remain in physiological phase insensitive to penicillin. - The example of synergism is that two chemotherapeutic agents with antimetabolite action, sufamethoxazole and trimethoprim double inhibit the bacterial metabolic pathway to synthesize folic acid - d

36 Useful combinations of antibiotics include 1. Cell wall inhibitors + aminoglycosides 2. Beta-lactams + beta-lactamase inhibitors 3. Beta-lactams act on different transpeptidases 4. Streptogramin combinations 5. Sulfonamides + trimethoprime

37 Others include - Doxycycline + aminoglycosides brucillosis - Amoxycillin ± tetracycline ± macroldies ± metronidazole f H. pylori - Vancomycin ± rifampin ± aminoglycosides MRSA - Penicillin + clindamycin group A streptococci - Fluroquinolones + macrolides L. pneumophila

38 Effect of serum protein binding on antibiotic activity The protein-bound antibiotic is not readily available for treatment of infection. Available amount = total amount protein bound amount Antibiotics tightly bound to plasma proteins can not be use to treat deep tissue infections Antibiotics not significantly bound to plasma proteins or easily released from the proteins have relatively shorter half-life.

39 Sulfonamides (on metabolic enzymes) Quinolones Rifampicin Penicillins Cephalosporins Aminoglycosides Tetracycline Chloramphenicol

40 Chemical classification of antibiotics Chemical classification of antibiotics is usually of limited value due to high chemical variability of antibiotics. Structurally similar antibiotics derived from different microorganisms may have similar mechanism of action. I. Sulfonamides VII. Fused ring systems II. III. IV. β-lactams: include penicillins and cephalosporins Aminoglycosides: amino sugars such as streptomycins, kanamycins, neomycins, gentamycins Polypeptides: such as tyrothiricin and polymyxin VIII. Lincomycins IX. Polyenes: Antifungal such as nystatin and amphotericins X. Unclassified antibiotics V. Macrolides: large lactone ring VI. Tetracyclines:

41 Sulfonamides Mohammed Nooraldeen Al-Qattan (PhD) 14/7/2016 Discovery, Mechanism of action, SAR, solubility problems and solutions, sulfonamides prodrugs

42 Sulfonamides The sulfonamides are bacteriostatic when administered to humans in achievable doses. They inhibit the enzyme dihydropteroate synthase, an important enzyme needed for the biosynthesis of folic acid derivatives and, ultimately, the thymidine required for DNA They do this by competing at the active site with p -aminobenzoic acid (PABA), a normal structural component of folic acid derivatives. Humans have no dihydropteroate synthase, which explains sulfonamides selectivity for bacterial cells. Indeed, the antimicrobial efficacy of sulfonamides can be reversed by adding significant quantitiesof PABA into the diet

43 Sulfonamides (Sulfa or sulpha drugs) Diseases like pneumonia, meningitis, dysentry etc., could not be treated effectively until the discovery of sulfa drugs which were suitable for internal use against gram+ bacteria. p-aminobenzenesulphonamide (sulfanilamide) was initially synthesized in 1908 as intermediate for azo dyes, later on, it was observed that it is effective against streptococci In 1935, a red dye (4-sulfonamide-2, 4 -diaminoazobenzene) was prepared and showed curative properties against infections and named Prontosil The inactivity of prontosil as antibacterial agents in vitro suggests that it should be converted to another metabolite to exert its antibacterial activity in vivo. Due to resistance development, only few sulfonamides are still in use such as (sul+trim)

44 a colorless cleavage product formed by reductive liver metabolism of the administered Prontosil dye

45 Pteridine diphosphate

46 Sulfonamides inhibit DHPS by competing PABA. some bacteria can resist sulfonamide competition by making more PABA, decrease cell membrane permeability to sulfonamide or by getting mutated DHPS.

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48 Structure-activity relationship Any substitution in the ring abolish activity Should not be substituted (i.e. R 1 =H), and if substituted should be metabolized back to 1 amine (i.e act as prodrug) 4 Should be directly connected Only para substitution is allowed The benzene ring and sulfonamide are critical and should be directly connected Replacement of -SO 2 NH by -CONH or by SO 3 H reduces the activity The only possible variable site. Electron withdrawing group improve ionizability of NH and thus activity and solubility Should be 1 or 2 amine

49 Ionization of sulfonamides Sulfonamide group (SO2NH2) is unstable and get stabilized by losing a proton which results in negative charge being stabilized by resonance with sufone group. Therefore, the SO2NH2 group can be considered as HA acid similar to carboxyls (- COOH), phenols (benzene-oh) and thiols (-SH). The R group in SO2-NH-R affects the ionizability of NH. If R is electron withdrawing group, the antibacterial activity and solubility of the drug is improved. Pyrimidine is more electron withdrawing than benzene and thiazole rings. Thiazole substitution produces toxic sulfonamide derivatives. The lipid solubility influences the pharmacokinetic and antibacterial activity, and so increases the half-life and antibacterial activity in vivo.

50 Crystalluria and pka of sulfonamides Despite the good ability to treat infections, the sulfanilamide are associated with sever renal damage due to crystallization in the kidneys The pka of sulfonamido group (-SO2NH-) of sulfanilamide is 10.4, therefore at urine ph of 6 only 0.004% of sulfanilamide is ionized (water-soluble) to be excreted in urine. The precipitated sulfanilamide in urine lead to crystalluria. Sodium bicarbonate was administered before each dose of sulfanilamide to improve solubility and thus excretion in urine.

51 Case study: Sulphathiazole (metabolism problem) unchanged (63%) N4-acetyl (29%) N4-glucuronide (0.8%) N4-sulfate (0.5%) N1-glucuronide (3.8%) Kidney Acetylation of N4 of sulfonamide ionization of N1 (NH) solubility 4 1 pka=7.1 Highly ionizable pka > 7.1 Less ionizable Electron withdrawing group (pyrimidine) more soluble and less toxic Parke, D. V. The Biochemistry of Foreign Compounds. Oxford: Pergamon Press, 1968., p. 180

52 How to reduce crystalurea for sulfonamides So the methods used to reduce the crystalurea of sulfonamides is: 1. Increase urine flow to reduce the opportunity for crystals to seed in kidney 2. Increase the urine ph. The closer the urine ph to 10.4 (i.e. the pka of sulfanilamide), the more ionized, water soluble-salt will form. increase urine ph can be achieved by taking sodium bicarbonate. 3. Preparing derivatives of sulfanilamide that have low pka as close as possible to urine ph (ph 6) to ensure part of the dose is ionized in the kidney tubules. The pka can be lowered by using electron withdrawing group at N1.

53 Impaired Oral bioavailability of sulfonamide offers advantage to locally treat gastrointestinal infection. Oral bioavailability (i.e. absorption through GIT) requires balanced hydrophobic/hydrophilic characters. Too hydrophilic drug will not be absorbed as N4-succinyl derivatives Too hydrophobic drug will not be absorbed as N4-benzoyl derivatives

54 Sulfamethazine naming options 4-Amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide; N1-(4,6-dimethyl-2-pyrimidinyl)sulfanilamide; 2-sulfanilamido-4,6-dimethylpyrimidine. pka of 7.2

55 Classification of sulfonamides on the basis of Chemical structure N-substituted sulphonamide: Sulphadiazine, Sulphacetamide, Sulphadimidine. N-4 substituted sulphonamides (prodrugs): Prontosil. Both N-1 and N-4 substituted sulphonamides: Succinyl sulphathiazole, Phthalylsulphathiazole. Miscellaneous: Mafenide sodium.

56 Classification of sulfonamides on the basis of Chemical structure: A) N-substituted sulfonamides R 1 R 2

57 Classification of sulfonamides on the basis of Chemical structure: B) N-4 substituted sulphonamides (prodrugs) Prontosil drug is inactive in vitro, but it is active in vivo since it is converted to sulphanilamide by azo reductase enzymes. Azo reductase 4-sulfonamide-2, 4 -diaminoazobenzene Note: Pro-drugs of amines are occasionally prepared by incorporating them in to an azo linkage. By the action of azo reductase the amino compounds are released in vivo.

58 Classification of sulfonamides on the basis of Chemical structure: B) N-4 substituted sulphonamides (prodrugs) Sulphasalazine by the action of azo reductase releases the 5-amino salicylic acid (5-ASA) and sulphapyridine. The generation of anti-inflammatory salicylic acid prior to absorption prevents the systemic absorption of the agents and enhances the concentration of it in active site (intestine). Therefore, sulphaslazine is mainly used to treat inflammatory bowel syndrome due to the released 5-ASA. pka=2.3 pka=6.5 Low absorption from GIT Due to low lipophilicity pka=8.4 Low absorption from GIT Mostly ionized at intestinal ph (6-7) pka=2.9 LogP =2.3 LogP =0.35 LogP =2.2

59 Classification of sulfonamides on the basis of Chemical structure: C) Both N-1 and N-4 substituted R 1 R 2

60 Classification of sulfonamides on the basis of Chemical structure: D) Miscellaneous It is NOT a true sulfanilamide-type compound, as it is not inhibited by PABA. Its antibacterial action involves a mechanism that differs from that of true sulfanilamide-type compounds.

61 Some kinetic information about sulfonamides Well oral absorption and tissue distribution Bound to plasma protein (sulfisoxazole and sulfamethoxaole 30%-70%). Therefore, sulfonamides may displace other protein-bound drugs or metabolites such as bilirubin Partly deactivated by acetylation or glucuronidation at t N4

62 Correlation between pka and plasma protein binding (other factors are excluded) Compound pka Plasma Pr. binding Acetic acid PABA Sulfisoxazole sulfamethoxine Sulfamethoxazole Sulfadiazine Sulfamerazine Sulfamethazine % 95% 60% 38%

63 Metabolism of sulfonamides Sulfonamids can be metabolized in human body by almost three pathways 1. Glucuronidation: occurs mainly at aromatic amine (N4) or other nucleophilic amine to form inactive water soluble metabolites 2. Acetylation occurs at both N1 and N4 to form inactive, usually with low water solubility at urine ph 3. Oxidation: occurs at aromatic amine to form cytotoxic hydroxylamine Oxidation Reduction Cytotoxic hydroxylamine

64 Mechanisms of Microbial Resistance to Sulfonamides The indiscriminate use of sulfonamides has led to the emergence of resistance strains of bacteria. Resistance is acquired likely- through: 1. Compensatory increase in biosynthesis of PABA. 2. Mutations at dihydropteroate synthase 3. Decrease cell membrane permeability to sulfonamides. 4. Active efflux of sulfonamides outside the cell 5. Acquisition of another copy of DHPS through plasmid transfection.

65 Synthesis of sulfonamides Synthesis of Sulphanilamide (from benzene)

66 Synthesis of sulfacetamide from sulfanilamide

67 1. -LACTAM ANTIBIOTICS S O O N O N O N 1. Penam 2. Carbapenam 3. Oxapenam S O N N O N O 4. Penem 5. Carbapenem 6. Monobactam S O O N O N O N 7. Cephem 8. Carbacephem 9. Oxacephem Antibiotics 67