Metabolic Changes of Drugs Books: 1. Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry 11th ed. Lippincott, Williams & Wilkins ed. 2. Foye’s Principles of Medicinal Chemistry Introductory Concepts ■ Biochemically speaking: Metabolism means Catabolism (breaking down of substances) + Anabolism (building up or synthesis of substances) ■ But when we speak about drug metabolism, it is only catabolism ■ That is drug metabolism is the break down of drug molecules ■ So what is building the drug molecules? We use the word “synthesis”, then ■ Drugs are synthesized in laboratory and thus is not an endogenous event ■ Lipid soluble drugs require more metabolisms to become polar, ionizable and easily excretable which involve both phase I and phase II mechanisms. What Roles are Played by Drug Metabolism? ■ One of four pharmacokinetic parameters, i.e., absorption, distribution, metabolism and excretion (ADME) ■ Elimination of Drugs: Metabolism and excretion together are elimination ■ Excretion physically removes drugs from the body The major excretory organ is the kidney. The kidney is very good at excreting polar and ionized drugs without any major metabolism. The kidney is unable to excrete drugs with high LWPC ■ In general, by metabolism drugs become more polar, ionizable and thus more water soluble to enhance elimination ■ It also effect deactivation and thus detoxication or detoxification ■ Many drugs are metabolically activated (Prodrugs) ■ Sometimes drugs become more toxic and carcinogenic Metabolite Examples and notes activity Routes that result in the formation of inactive metabolites are often referred to as detoxification. Inactive OH O O (detoxification) Phenol sulphokinase S O 3'-Phosphoadenosine-5'phosphosulfate (PAPS) Phenol Similar activity to the drug OH Phenyl hydrogen sulfate The metabolite may exhibit either a different potency or duration of action or both to the original drug. CH3 CH3 O O O N Hydroxylation N Cl H N N OH N-Demethylation Ph Diazepam (Sustained anxiolytic action) N Cl N Cl OH Ph Oxazepam (short duration) Ph Temazepam (Short duration) CH3 CONHNHCH CONHNH2 CH3 Different activity N-Dealkylation N Ipronazid (Antidepressant) N Isoniazid (Antituberculosis) HO Toxic metabolites NCOCH3 NHCOCH3 NH2 Other substances responsible for hepatotoxicity Substances responsible for methemoglobinamia OC2H5 N-Hydroxyphenacetin (Hepatotoxic) OC2H5 Phenacetin (Analgesic) OC2H5 Phenetidine Stereochemistry of Drug Metabolism OH OH CH2COCH3 H Ph Ph H O O O R-(+)-Warfarin O S-(-)-Warfarin OH Major route OH CH2COCH3 HO OH H2C Ph O S-6-Hydroxywarf arin H CH3 OH Minor route O O R,S-(+)-alcohol derivative CH3 H Ph O O R,R-(+)-alcohol derivative COOH Metabolism H COOH R-(-)-Ibuprofen (inactive) H CH3 OH H2 C H Ph H O CH2COCH3 H CH3 S-(+)-Ibuprofen (active) Sites of Drug Metabolism Liver: Major site, well organized with all enzyme systems The first-pass effect Following drugs are metabolized extensively by first-pass effect: Isoproterenol, Lidocaine Meperidine, Morphine, Pentazocine, Propoxyphene, Propranolol, Nitroglycerin, Salicylamide Intestinal Mucosa: The extra-hepatic metabolism, contains CYP3A4 isozyme Isoproterenol exhibit considerable sulphate conjugation in GI tract Levodopa, chlorpromazine and diethylstilbestrol are also reportedly metabolized in GI tract Esterases and lipases present in the intestine may be particularly important carrying out hydrolysis of many ester prodrugs Bacterial flora present in the intestine and colon reduce many azo and nitro drugs (e.g., sulfasalazine) Intestinal b-glucuronidase can hydrolyze glucuronide conjugates excreted in the bile, thereby liberating the free drug or its metabolite for possible reabsorption (enterohepatic circulation or recycling) Enzymes Involved in Drug Metabolism CYP450, Hepatic microsomal flavin containing monooxygenases (MFMO or FMO) Monoamine Oxidase (MAO) and Hydrolases Cytochrome P450 system: localized in the smooth endoplasmic reticulum. Cytochrome P450 is a Pigment that, with CO bound to the reduced form, absorbs maximally at 450nm Cytochromes are hemoproteins (heme-thiolate) that function to pass electrons by reversibly changing the oxidation state of the Fe in heme between the 2+ and 3+ state and serves as an electron acceptor–donor P450 is not a singular hemoprotein but rather a family of related hemoproteins. Over 1000 have been identified in nature with ~50 functionally active in humans with broad substrate specificity Simplified apoprotein portion HOOC CH3 L N N Fe+3 N N CH3 CH2 CH3 HOOC CH3 CH2 O H R Substrate binding site Heme portion with activated Oxygen Cytochrome P450: Naming ■ Before we had a thorough understanding of this enzyme system, the CYP450 enzymes were named based on their catalytic activity toward a specific substrate, e.g., aminopyrine N-demethylase now known as CYP2E1 ■ Currently, all P450’s are named by starting with “CYP” (CYtochrome P450, N1, L, N2 - the first number is the family (>40% homology), the letter is the subfamily (> 55% homology), and the second number is the isoform. The majority of drug metabolism is by ~10 isoforms of the CYP1, CYP2 and CYP3 families in humans ■ Major human forms of P450: Quantitatively, in the liver the percentages of total P450 protein are: CYP3A4 – 28%, CYP2Cx – 20%, CYP1A2 – 12%, CYP2E1 – 6%, CYP2A6 – 4%, CYP2D6 – 4% ■ By number of drugs metabolized the percentages are: CYP3A4 – 35%, CYP2D6 – 20%, CYP2C8 and CYP2C9 – 17%, CYP2C18 and CYP2C19 - 8% CYP 1A1 and CYP1A2 -10%, CYP2E1 – 4%, CYP2B6 – 3% Few Important CYP450 Isozymes CYP family Main functions CYP1 Xenobiotic metabolism CYP2 Xenobiotic metabolism, Arachidonic acid metabolism CYP3 Xenobiotic and steroid metabolism CYP7 Cholesterol 7α-hydroxylation CYP11 Cholesterol side-chain cleavage, Steroid 11β – hydroxylation, Aldosterone synthesis CYP17 Steroid 17α-hydroxylation CYP19 Androgen aromatization CYP21 Steroid 21-hydroxylation CYP24 Steroid 24-hydroxylation CYP27 Steroid 27-hydroxylation EC Recommended name Family/gene 126.96.36.199 * secologanin synthase CYP72A1 188.8.131.52 * trans-cinnamate 4-monooxygenase CYP73 184.108.40.206 * benzoate 4-monooxygenase CYP53 220.127.116.11 * calcidiol 1-monooxygenase CYP27 18.104.22.168 * cholestanetriol 26-monooxygenase CYP27 22.214.171.124 * -monooxygenase CYP7 126.96.36.199 * flavonoid 3'-monooxygenase CYP75 188.8.131.52 * 3,9-dihydroxypterocarpan 6a-monooxygenase CYP93A1 184.108.40.206 * leukotriene-B4 20-monooxygenase CYP4F 220.127.116.11 * methyltetrahydroprotoberberine 14-monooxygenase CYP93A1 18.104.22.168 * tyrosine N-monooxygenase CYP79 Drug Interactions & Metabolism The drug interactions depend upon: a) the isoform(s) required by the drug in question, b) the isoforms altered by concomitant therapy, c) the type of enzyme alteration (induction or inhibition). General Metabolic Pathways Hydrolytic Reactions Esters and amides Epoxides and arene oxides by epoxide hydrase Phase II Conjugation Phase I Functionalization Drug Metabolism Glucuronic acid conjugation Sulfate Conjugation Glycine and other AA Glutathion or mercapturic acid Acetylation Methylation Oxidation Aromatic moieties Olefins Benzylic & allylic C atoms and a-C of C=O and C=N At aliphatic and alicyclic C C-Heteroatom system C-N (N-dealkylation, N-oxide formation, N-hydroxylation) C-O (O-dealkylation) C-S (S-dealkylation, S-oxidation, desulfuration) Oxidation of alcohols and aldehydes Miscellaneous Reduction Aldehydes and ketones Nitro and azo Miscellaneous Tetrahydrocannabinol (D1-THC) Metabolism 7 CH3 6 5 1 4 2 CH2OH OH OH 3 H3C O CH3 D1-THC COOH C5H11 OH H3C H3C O CH3 C5H11 O CH3 7-Hydroxy-D1-THC C5H11 D1-THC-7-oic Acid COOR OR COO- Where R = O H3C O CH3 C5H11 Glucuronide conjugate at either COOH or phenolic OH group OH OH HO H b-Glucuronyl moiety The metabolite is polar, ionisable and hydrophilic Oxidative Reactions Arenols OH Arene Oxides O Epoxides O C C C C Benzylic, allylic aliphatic C Hydroxylation C OH C H R N H "Activated Oxigen" [FeO]3+ Miscellaneous Oxidations S C S P O C O P Desulfuration R N OH R N CH2R R O CH3 R N R NH + O CHR R N O S CH3 R OH O O-Dealkylation SH, S CH3 S-Dealkylation and S-Oxidation N-Hydroxylation N-Dealkyaltion and Oxidative Deamination N-Oxide Formation ■ Hydroxylation is the primary reaction mediated by CYP450 ■ Hydroxylation can be followed by non-CYP450 reactions including conjugation or oxidation to ketones or aldehydes, with aldehydes getting further oxidized to acids ■ Hydroxylation of the carbon α to heteroatoms often lead to cleavage of the carbon – heteroatom bond; seen especially with N, O and S, results in N–, S– or O–dealkylation. ■ Must have an available hydrogen on atom that gets hydroxylated, this is important!!! Aromatic Hydroxylation R1 R1 R1 Spontaneous CYP450 ■ ■ O Mixed function oxidation of arenes to arenols via an epoxide intermediate arene oxide Occurs primarily at para position ■ Substituents attached to aromatic ring influence the hydroxylation R1 R1 Epoxide hydrolase Epoxide Hydrase Major route of metabolism for drugs with phenyl ring ■ ■ OH Aromatase OH OH R1 Glutathione OH S Activated rings (with electron-rich substituents) are more susceptible while deactivated (with electron withdrawing groups, e.g., Cl, N+R3, COOH, SO2NHR) are generally slow or resistant to hydroxylation OH OH Glutathione R1 Macromolecule OH Macromolecule H H H CYP2C19 N O O N H Phenytoin H CH3 O O H O N HO N N Amphetamine p-hydroxyphenytoin O OH OH O C CH H N CH3 ONa CH3 O HO Warfarin sodium 17-a-Ethinylestradiol O HO C Propranolol Ca+2 O OH O CH3 N F N CH3 N H 3C HN CH3 O C O Phenylbutazone 2 Atorvastatin O Cl H N H3C N O N S OH O Cl HN H3C Antihypertensive drug clonidine undergo little aromatic hydroxylation and the uricosuric agent probenecid has not been reported to undergo any aromatic hydroxylation Probenecid Clonidine CH3 O N Cl N N N S CH3 CH3 Preferentially the more electron rich ring is hydroxylated Cl Diazepam Chlorpromazine NIH Shift: Novel Intramolecular Hydride shift named after National Institute of Health where the process was discovered. This is most important detoxification reaction for arene oxides R R Spontaneous Rearrangement NIH Shift + O Arene Oxide R R - OH Arenol H H O H OH Oxidation of olefinic bonds (also called alkenes) O Epoxide hydrolase Epoxide Alkene OHOH trans dihydrodiol derivative ■ The second step may not occur if the epoxide is stable, usually it is more stable than arene oxide ■ May be spontaneous and result in alkylation of endogenous molecules ■ Susceptable to enzymatic hydration by epoxide hydrase to form trans1,2-dihydrodiols (also called 1,2-diols or 1,2-dihydroxy compounds) ■ Terminal alkenes may form alkylating agents following this pathway HO O Epoxide hydrolase CYP3A4 N O N NH 2 Carbamazepine (Active) OH O N NH 2 Carbamazepine 10,11 epoxide (Active & Toxic) O NH2 Carbamazepine trans 10,11 diol (Inactive) Q. Any similarities or dissimilarities with aromatic – NIH Shift, Conjugation with macromolecules? Benzylic Carbon Hydroxylation R2 R2 R1 C R1 H O C O S N H ■ Hydroxylate a carbon attached to a phenol group (aromatic ring) ■ R1 and R2 can produce steric hindrance as they get larger and more branched ■ So a methyl group is most likely to hydroxylate ■ Primary alcohol metabolites are often oxidized further to aldehyde and carboxylic acids and secondary alcohols are converted to ketones by soluble alcohol and aldehyde dehydrogenase OH O O N H CH3 O S CYP2C9 H H3C HO Tolbutamide Metabolism C H O ONa N CH3 O H3C Tolmetin sodium Dicarboxylic acid is the major metabolite N H O N H CH3 Oxidation at Allylic Carbon Atoms H R1 C C H H C C R2 H 7 5 1 4 2 R3 R1 R4 C OH C C R3 R4 CH3 CH3 HO HO OH OH C H R2 H 7CH2OH CH3 6 H OH OH 3 + H3C H3C O CH3 O CH3 C5H11 D1-THC + H3C C5H11 O CH3 H3C O CH3 C5H11 6b-Hydroxy-D1-THC 6a-Hydroxy-D1-THC 7-Hydroxy-D1-THC H2C H2C 3 2 HO OH H HO N N 1 H3CO H3CO N Quinine N 3-Hydroxyquinine C5H11 O-Glucuronide Cojugate O O O CH3 O 2' CH3 Hexabarbital 3' O O CH3 O OH CH3 3'-Hydroxyhexabarbital Pentazocine O CH3 O O CH3 3'-Oxohexabarbital Hydroxylation at C a to C=O and C=N CH 3 N R O H C C H R' R O H C C CH 3 O N OH N-demethylation 3 N R' Cl H N O N Cl O OH Cl N OH The benzodiazepines are classic examples with both functionalities (3S) N-Methyloxazepam or 3-Hydroxydiazepam Diazepam (CH3 CH 2 )2 NCH2 CH 2 CH 3 O N N 3 N Cl Oxazepam O 3 N O2 N F Flurazepam The sedative hypnotic glutethimide possesses C a to carbonyl function 4 CH2 CH 3 C6 H5 3 Nimetazepam HO 4 CH2 CH 3 C6 H5 1 O N O H Glutethemide O N O H 4-Hydroxyglutethemide Aliphatic hydroxylation R1 R1 H H H C C C H H H H H H C C C H H H ■ OH Catalyzes hydroxylation of the ω and ω-1 carbons in aliphatic chains Generally need three or more unbranched carbons ■ H R1 H OH H C C C H H H H O H O N H N CYP450 Pentobarbital Metabolism O O N O N OH H CH3 OH CH3 CH3 O O CYP450 OH H3C H 3C H CH3 O Ibuprofen Metabolism O OH + CH3 HOOC OH CH3 Alicyclic (nonaromatic ring) Hydroxylation ■ Cyclohexyl group is commonly present in many drug molecules ■ The mixed function oxydase tend to hydroxylate at the 3 or 4 position of the ring ■ Due to steric factors if position 4 is substituted it is harder to hydroxylate the molecules OH O O S H3C N H O O N H O S CYP450 H3C O O Acetohexamide Metabolism N H O N H Oxidation Involving CarbonHeteroatom Systems ■ C-N, C-O and occasionally C-S ■ Two basic types of biotransformation processes: 1. Hydroxylation of a-C attached directly to the heteroatom (N,O,S). The resulting intermediate is often unstable and decomposes with the cleavage of the C-X bond: H O H R X Ca R X Ca O R XH + Usually Unstable Oxidative N-, O-, and S-dealkylation as well as oxidative deamination reaction fall under this category 2. ■ Hydroxylation or oxidation of heteroatom (N, S only, e.g., Nhydroxylation, N-oxide formation, sulfoxide and sulfone formation) Metabolism of some N containing compounds are complicated by the fact that C or N hydroxylated products may undergo secondary reactions to form other, more complex metabolic products (e.g., oxime, nitrone, nitroso, imino) C-N systems ■ ■ ■ ■ Aliphatic (1o, 2o, 3o,) and alicyclic (2o and 3o) amines; Aromatic and heterocyclic nitrogen compounds; Amides ■ Enzymes: 1. CYP mixed-function oxidases: a-C hydroxylation and N-oxidation 2. Amine oxidases or N-oxidases (non-CYP, NADPH dependent flavoprotein and require O): N-oxidation 3o Aliphatic and alicyclic amines are metabolized by oxidative N-dealkylation (CYP) Aliphatic 1o, 2o amines are susceptible to oxidative deamination, N-dealkylation and N-oxidation reactions Aromatic amines undergoes similar group of reactions as aliphatic amines, i.e., both Ndealkylation and N-oxidation H O H R1 N Ca R1 N Ca O R1 R2 R2 3o or 2o amine NH + R2 Carbinolamine 2o or 1o amine H o H O Ca Ca NH2 NH2 1 amine Carbinolamine O + Carbonyl NH3 Ammonia N-Dealkylation (Deamination) H R1 C N R3 OH CYP450 R1 R2 R4 C Spontaneous N R1 R3 C R2 R2 R4 O + HN R3 R4 ■ Deamination and N-dealkylation differ only in the point of reference; If the drug is R1 or R2 then it is a deamination reaction and If the drug is R3 or R4 then it is an N-dealkylation ■ In general, least sterically hindered carbon (a) will be hydroxylated first, then the next, etc. Thus the more substituent on this C, the slower it proceeds; branching on the adjacent carbon slows it down, i.e. R1, R2 = H is fastest. ■ Any group containing an a-H may be removed, e.g., allyl, benzyl. Quaternary carbon cannot be removed as contain no a-H ■ The more substituents placed on the nitrogen the slower it proceeds (steric hindrance) ■ The larger the substituents are the slower it proceeds (e.g. methyl vs. ethyl). In general, small alkyl groups like Me, Et and i–Pro are rapidly removed; branching on these substituents slows it down even more OH N N CH3 CYP2C19 CH3 Imipramine N-Dealkylation N N CH2 CH3 Spontaneous N N H CH3 Alicyclic Amines Often Generate Lactams N OH N N CH3 N CH3 N CH3 N Cotinine Carbinolamine Nicotine 1 C6 H 5 O 2 3 H 3C C6 H 5 N H Phenmetrazine COOCH 3 HN Methylphenidate O C6 H 5 H 3C Hydrolysis N OH H Carbinolamine intermediate O N N CH3 CH3 Cyproheptadine H 3C N O H 3-Oxophenmetrazine COOH COOH HN HN Ritalinic Acid O O 6-Oxoritalinic Acid O Lactum metabolite CH3 3oAmine drugs H3C CH3 N N CH3 CH3 H N N C CH3 O CH3 H3C CH3 N O O CH3 Lidocaine NH2 CH3 Tamoxifen Disopyramide CH3 O N CH3 CH3 N N S N CH3 N CH3 CH3 N CH3 CH3 Cl Br Diphenhydramine Alicyclic Amine drugs Chlorpromazine Benzphetamine Brompheniramine CH3 CH3 CH3 N N N H O O Meperidine CH3 HO O Morphine OH O CH3 Dextromethorphan 2o & 1o Amines O CH3 HN CH2 CH3 NH3 NH 2 CH3 O Phenylacetone Ampetamine Methampetamine Cl NHCH 3 O Ketamine CH3 Cl NH 2 O Norketamine Generally, dealkylation of secondary amines occurs before deamination. The rate of deamination is easily influenced by steric factors both on the a-C and on the N; so it is easier to deaminate a primary amine but much harder for a tertiary amine. Exceptions: Some 2o and 3o amines can undergo deamination directly without dealkylation. OH O HN OH OH O O O H Direct Oxidative CH 3 Deamination HN CH 3 H2 N CH 3 CH 3 Propranolol OH OH O Oxidative Deamination Through Primary Amine O O H3C HN O H CH 3 CH 3 Aldehyde Metabolite NH3 Carbinolamine CH3 NH 2 Primary Amine Metabolite (Desisopropyl Propranolol) O N-Oxidation H H H OH N N Hydroxylamine Nitroso N O Aromatic amines 1 aromatic amine H H 1° amines R C H N R H H C N CH3 R C H H 2 amine H C N H 3 amine CH3 CH3 R C H H C N N CH3 N O CH3 N-Oxide R C H Nitroso Nitro H R OH O H CH3 Hydroxylamine H R OH H H 3° amines R Hydroxylamine H R N H 1 amine 2° amines C H H C N H Nitrone CH2 O O N O ■ The attack is on the unbonded electrons so 3o amines can be oxidized ■ Generally, only occurs if nothing else can happen, so it is a rare reaction ■ Performed by both amine oxidases and hepatic MFO’s ■ Good examples would include amines attached to quaternary carbons since they cannot be deaminated H H3C N H Cl Chlorphentermine N-Hydroxylation H H3 C N NH2 CH3 Hydroxylamine Nitroso H CH3 Phentermine N CYP450 CH3 Cl H H3C Nitro Amantadine OH Amides C-N bond cleavage via a-C hydroxylation (formation of carbinolamide) and Nhydroxylation reactions Oxidation involving C-O System (O-Dealkylation) H R1 C OH CYP450 O R3 R1 R2 C Spontaneous O R3 R1 C R2 O + HO R3 R2 ■ Converts an ether to an alcohol plus a ketone or aldehyde ■ Steric hindrance discussion similar to N-dealkylation OH O H3C H 3C H3C O O NH2 N NH2 CY P4 50 O O N us eo tan on Sp H 3C O CH2 CH3 OH N NH2 N NH2 H 3C H 3C Trimethoprim O-Dealkylation O O N NH2 N NH2 CH3 N H3C O H N O O OH CH3 CH3 N O O O OH O CH3 Codeine H3C H3 C O CH3 Cl Phenacetin N NH2 OH O N N O Indomethacin N O O H3C Prazosin O H N CH3 Metoprolol ■ One exception that appears to be a form of O-dealkylation is the oxidation of ethanol by CYP2E1 ■ In this case R3 is hydrogen instead of carbon to form the terminal alcohol rather than an ether ■ The enzyme involved is CYP2E1 and has been historically referred to as the Microsomal Ethanol Oxidizing System (MEOS) H H3C C H OH CYP450 OH H3C C H Spontaneous OH H3C C H CH3 O Oxidation involving C-S System H ■ S-Dealkylation R1 C OH CYP450 S R3 R1 R2 C S R3 Spontaneous R1 C R2 R2 Steric hindrance discussion similar to N-dealkylation O S ■ Desulfuration R1 C R1 R2 C R2 O ■ R1 S-Oxidation S R1 R2 S O R2 R1 S N N N H 6-(Methylthio)-purine N S CH2 OH N N N N H R2 O Sulfone Sulfoxide CH3 S O CH2 SH N N N H 6-Mercaptopurine N O + HS R3 O H3C S COOH H N O N O H Methitural CF3 2-Benzylthio-4trifluoromethyl benzoic acid H3C H3C O S P O O Parathione O S N O H Pentobarbital N O H Thiopental NO2 H3C H3C O H N N S CH2C6H5 S O H O P O O Paraoxone NO2 N O N S S CH3 S Ring Sulfoxide N CH3 CH3 S Thioridazine N CH3 N S N CH3 S CH3 O Mesoridazine N O S O N CH3 CH3 S Ring Sulfone N N CH3 S S CH3 O O Sulforidazine Oxidative Dehalogenation H R C OH CYP450 Cl R Cl ■ C O Spontaneous O R C R C +H2O Cl OH + + Cl Cl Requires two halogens on carbon H ■ With three there is no hydrogen available to replace ■ With one, the reaction generally won’t proceed ■ The intermediate acyl halide is very reactive OH OH O2N OH NHCOCHCl2 O2N Chloramphenicol O2N OH O2N Q. What is Gray Baby Syndrome? Cl OH HCl OH NHCOCCl2 OH H Cl OH NHCOC OH O Oxamic Acid Derivative OH NHCOCCl O Oxamyl Chloride Derivative Tissue Nucleophiles Covalent Binding (Toxicity) Hepatic Microsomal Flavin Containing Monooxygenases (MFMO or FMO) ■ Oxidize S and N functional groups ■ Mechanism is different but end products are similar to those produced by S and N oxidation by CYP450 ■ FMO’s do not work on primary amines ■ FMO’s will not oxidize substrates with more than a single charge ■ FMO’s will not oxidize polyvalent substrates H3C S NH N H N H N N C MFMO H3C CH3 S NH N Cimetidine MFMO S-Oxidation Q. What is the difference with MFO? N O H N H N N C CH3 N Non-Microsomal Oxidation Reactions ■ Monoamine oxidase (outer membrane of mitochondria, flavin containing enzyme ) ■ Dehydrogenases (cytoplasm) ■ Purine oxidation (Xanthene oxidase) Monoamine oxidase H R1 C N R2 R3 H R1 C R2 O + H N H R3 ■ Two MAOs have been identified: MAO–A and MAO–B. Equal amounts are found in the liver, but the brain contains primarily MAO–B; MAO–A is found in the adrenergic nerve endings ■ MAO–A shows preference for serotonin, catecholamines, and other monoamines with phenolic aromatic rings and MAO–B prefers non–phenolic amines ■ Metabolizes 1° and 2° amines; N must be attached to α-carbon; both C & N must have at least one replaceable H atom. 2° amines are metabolized by MAO if the substituent is a methyl group ■ b–Phenylisopropylamines such as amphetamine and ephedrine are not metabolized by MAOs but are potent inhibitors of MAOs Alcohol dehydrogenase R2 R1 C Aldehyde dehydrogenase R2 OH R1 H C R1 C O O R1 C H O OH Metabolizes 1° and 2° alcohols and aldehydes containing at least one “H” attached to a-C; 1° alcohols typically go to the aldehyde then acid; 2° alcohols are converted to ketone, which cannot be further converted to the acid. The aldehyde is converted back to an alcohol by alcohol (keto) reductases (reversible), however, it goes forward as the aldehyde is converted to carboxylic acid; 3° alcohols and phenolic alcohols cannot be oxidized by this enzyme; No “H” attached to adjacent carbon H2 C Ethanol Metabolism H3C Alcohol Dehydrogenase OH H3C Aldehyde Dehydrogenase H C OH H3C O C O Purine oxidation O O N HN N N H Hypoxanthine Xanthine oxidase N HN O N H Xanthine Molybdenum Containing O O N H Xanthine oxidase N HN O H N HN O OH N H N H Uric acid (hydroxy tautomer) O N H N H Uric acid (keto tautomer) Reductive Reactions ■ Bioreduction of C=O (aldehyde and keton) generates alcohol (aldehyde → 1o alcohol; ketone → 2o alcohol) ■ Nitro and azo reductions lead to amino derivatives ■ Reduction of N-oxides to their corresponding 3o amines and reduction of sulfoxides to sulfides are less frequent ■ Reductive cleavage of disulfide (-S-S-) linkages and reduction of C=C are minor pathways in drug metabolism ■ Reductive dehalogenation is a minor reaction primarily differ from oxidative dehalogenation is that the adjacent carbon does not have to have a replaceable hydrogen and generally removes one halogen from a group of two or three Reduction of Aldehydes & Ketones H R C O H Aldehyde R C H OH H 1 alcohol R C O R2 Ketone R1 C OH R2 2 alcohol ■ C=O moiety, esp. the ketone, is frequently encountered in drugs and additionally, ketones and aldehydes arise from deamination Ketones tend to be converted to alcohols which can then be glucuronidated. Aldehydes can also be converted to alcohols, but have the additional pathway of oxidation to carboxylic acids ■ Reduction of ketones often leads to the creation of an asymmetric center and thus two stereoisomeric alcohols are possible ■ Reduction of a, b –unsaturated ketones found in steroidal drugs results not only in the reduction of the ketone but also of the C=C ■ Aldo–keto oxidoreductases carry out bioreductions of aldehydes and ketones. Alcohol dehydrogenase is a NAD+ dependent oxidoreductase that oxidizes alcohols but in the presence of NADH or NADPH, the same enzyme can reduce carbonyl compounds to alcohols O H O + C R1 O H H HO H2N R2 Chiral Alcohol O OH H2C HO OH H 2C CH3 H O O CH2 O O OH OH H2C CH3 H O R,R (+)-Warfarin O OH C6H5 CH3 N OH OH H3 C Naloxone H O N O Ox Nicotinamide moiety of NADP+ or NAD+ + R,S (+)-Warfarin R (+)-Warfarin O N+ C 6H5 H HO + H CH3 C6H 5 O R2 H2N R R Red Nicotinamide moiety of NADPH or NADH Ketone C R1 N H O O H3 C OH O OH O H2N OH Daunomycin HO O O Naltrexone CH3 CH3 OH C O OH H C CH CH H HO Norethindrone H2 C CH3 CH H H2 C C NH2 O Amphetamine Phenylacetone OH OH H H C C CH3 NHCH3 (-)-Ephedrine C 3b,5b-Tetrahydronorethindrone CH3 H2 C CH3 CH OH 1-Phenyl-2-propanol OH H C CH3 O 1-Hydroxy-1-phenylpropane-2-one C H CH CH3 OH 1-Phenyl-1,2-propandiol Reduction of Nitro & Azo Compounds H R C N R O H N C N R O H R2 R1 H H N R H N H N R N N Azido NH H NH2 + H 2N Two 1 amines R NH2 + N Amine H 1 amine Hydrazo Azo N H R1 R2 C OH Hydroxylamine Nitroso N C H Nitro R1 H H O N N2 R2 R1 and R2 are almost always aromatic Usually only seen when the NO2 functional group is attached directly to an aromatic ring and are rare Nitro reduction is carried out by NADPH-dependent microsomal and soluble nitroreductases (hepatic) NADPH dependent multicomponent hepatic microsomal reductase system reduces the azo Bacterial reductases in intestine can reduce both nitro and azo O H2N O S O N H2 O S N N NH2 H2N Prontosil N H2 H2N + NH2 NH2 Sulfanilamide 1,2,3-Triaminobenzene H N O O O2N S N Cl HO N O N N N O Clonazepam N H O O2N O NNa O OH Sulfasalazine N Dantrolene Reduction of Sulfur Containing Compounds O O Sulfoxide reduction (Cannot reduce a sulfone) R1 S R1 R2 S X R2 Sulfoxide R1 S R2 O Sulfone Disulfide reduction R1 S S R2 H3C SH + N S S N HS R2 H3C CH3 S H3C R1 S H3C CH3 N SH S N,N-Diethylthiocarbamic Acid Disulfiram O F OH CH3 H H3 C S O Sulindac Hydrolytic Reactions Hydrolyzes (adds water to) esters and amides and their isosteres; the OH from water ends up on the carboxylic acid (or its isostere) and the H in the hydroxy or amine ■ ■ ■ Enzymes: Non-microsomal hydrolases; however, amide hydrolysis appears to be mediated by liver microsomal amidases, esterases, and deacylases Electrophilicity of the carbonyl carbon, Nature of the heteroatom, substituents on the carbonyl carbon, and substituents on the heteroatom influnce the rate of hydrolysis In addition, Nucleophilicity of attacking species, Electronic charge, and Nature of nucleophile and its steric factors also influence the rate of hydrolysis Table: Naming carbonyl - heteroatom groups R1 R1 R2 Name Susceptibility to Hydrolysis C O Ester Highest O C S Thioester C R2 + O O Carbonate C N Amide O N Carbamate N N Ureide Lowest The Reactions O Ester hydrolysis R1 O C O R2 R1 C OH O Amide hydrolysis (slower) R1 C HO R2 O H N R2 R1 C OH H2N R2 Carbonate hydrolysis O O R1 O C O R2 R1 HO + OH Carbonate O C O HO R2 R2 + HO Carbonic acid derivative C H OH O C O + O C O + O H O H Carbonic acid Carbamate hydrolysis O O R1 O C R2 N R1 OH + HO C HN N R3 + HO R3 R3 Carbamic acid derivative Carbamate O R2 R2 C H OH Carbonic acid Urea hydrolysis R1 R2 O N C N R3 R4 Urea derivative O R1 R2 NH + HO C HN R1 C + R3 R4 Carbamic acid derivative HO C H O OH Carbonic acid O O Hydrazide hydrolysis N O R2 R3 H N N Hydrazide R2 R3 R1 C OH + H2N N R2 R3 Hydrazine C O + O H Drug Examples OH O O H3C OH O OH O + O OH H3C CH3 N H3C O O Salicylic Acid HO CH3 O CH3 N Cl N O Indomethacin N H3C O H3 C Slow Hydrolysis O HO O N N N O NH2 Prazosin H 2N CH3 H2N OH N CH3 O Rapid Hydrolysis H N N O CH3 O CH3 CH3 Procaine CH3 Methylecgonine N Procainamide O O N Benzoylecgonine CH3 CH3 O O Cocaine H N H3C + O O H2 N O O Aspirin O H3C OH Lidocaine CH3 O Stereoselectivity of Hydrolysis Etomidate (Amidate, hypnotic): R-(+)-isomer is more rapidly hydrolyzed, but S-(-)-isomer is more rapidly hydroxylated. The Concept of Prodrugs and Antedrugs Prodrug M D M D activation M D M D Antedrug D M D M inactivation ID M = Barrier & ID = inactive drug, D = active drug, M = modifier (I) Prodrug: Need metabolic activation (II) Antedrug: Active drug that is quickly inactivated thereby minimizing systemic effects Prodrugs and Related Terms ■ Albert in 1958 coined the term prodrug to refer a pharmacologically inactive compound that is metabolically activated in the mammalian system ■ Hard Drugs are not susceptible to metabolic or chemical transformation, have high lipid solubility and thus accumulation or high water solubility O O S F3C N NH2 O N CH3 Celecoxib: t1/2 10-12 h in humans ■ S F3C N NH2 O N Cl t1/2 ca. 680 h (Liver toxicity) Soft drugs are active compounds that after exerting its action undergo inactivation to give a nontoxic product. Indeed soft drugs are a group of modified compounds that are also designed to delivery the drugs in to the brain (the chemical delivery system). Bodor coined the term. Basic Concepts of Prodrugs ■ Carrier-linked prodrugs: a pro-moiety is attached, which is not necessary for activity but may impart some desired property to the drug, such as increased lipid or water solubility, or site-directed delivery ■ Advantages may include: ■ 1. increased absorption 2. alleviation of pain at the site of injection if the agent is given parenterally 3. elimination of an unpleasant taste associated with the drug 4. decreased toxicity 5. decreased metabolic inactivation 6. increased chemical stability 7. prolonged or shortened action Bioprecursor prodrugs contain no pro-moiety but rather rely on metabolism to introduce the functionality necessary to create an active species O OH Cl H N Cl O O2N O O O OH HO O O-Na+ O ONa O Prodrug: Chloramphenicol Hemisuccinate Na Salt O Prodrug: Prednisolon Hemisuccinate Sodium Salt ■ Inactive as it is and activated by hydrolysis by plasma esterases to chloramphenicol/ prednisolon ■ Increased water solubility for parenteral administration, which otherwise would precipitate and cause pain by damaging surrounding tissues CH 3 OH N Cl H N H3C Cl O CH3 Cl O OH O O2N H N CH3 O HO O O Prodrug: Chloramphenicol Palmitate O S CH3 (CH2)14CH3 Prodrug: Clindamycin Palmitate ■ Inactive as it is; activated by hydrolysis by intestinal esterases to chloramphenicol/ clindamycin ■ Minimize their bitter taste and improve their palatability in pediatric liquid suspensions O S N H O O CH3 N CH3 O COONa Prodrug: Carbenicillin Indanyl Ester ■ Inactive as it is and activated by hydrolysis by plasma esterases to carbenicillin ■ Lipophilic indanyl ester furnish improved oral bioavailability F O F O CH3 CH3 CH3 H H H H3C S OH OH OH H3C O F H3C S O S O O Sulfide (Active) Sulindac (Inactive) Sulfone (Inactive) Prodrugs of Functional Groups Carboxylic acids and alcohols: Most common Amines and azo linkages: Not been used much Carbonyl compounds: Not found to be used widely Carboxylic Acids and Alcohols Converted to ester prodrugs which are often hydrolyzed to active drug by different types of esterase enzymes: Ester hydrolase Lipase Cholesterol esterase Acetylcholinesterase O Drug O Promoiety Drug OH + O Drug HO Promoiety Esterase or Carboxypeptidase Cholinesterase O O O Promoiety Drug OH + HO Promoiety Microflora in the gut Manipulation of steric and electronic properties of promoiety allows control of rate and extent of hydrolysis Advantage of Prodrug Formation I: Increased absorption of hydrophilic drugs by making less hydrophilic or more lipophilic H3C CH3 CH3 OH O O H3C O H NH+ CH3 OH NH+ CH3 HO Esterase O CH3 CH3 Dipivefrin HO H Epineprine H3C O CH3 CH3 OH Pivalic Acid Prodrug of Epinephrine: Dipivefrin More lipophilic, thus achieve higher intraocular concentration Hydrolysis occur in cornea, conjunctiva, and aqueous humor after ophthalmic application Advantage of Prodrug Formation II: Masking unpleasant taste Chloramphenicol palmitate and Clindamycin palmitate has already been shown. Other drugs include CH3 O O S N O O CH3 H3C HO N CH3 O H2N H3C HO CH3 O CH3 H C O 3 H N CH3 O O OH O CH3 O CH3 H3C N-Acetyl sulfisoxazole CH3 O O O O CH3 OH CH3 O CH3 CH3 Erythromycin estolate O O H3C O H3C O N CH3 O O H3 C H3C H3C H 3C CH3 O O CH3 O O O O CH3 O Troleandomycin CH3 CH3 CH3 O CH3 O CH3 Not all carboxylic esters hydrolyzed in vivo where double ester approach is used H N R1 O S N O CH3 Esterase CH3 No Reaction COOR2 (R2 = Ethyl, Propyl, Butyl, Phenyl) Penicillin Esters H N R1 S Esterase O N O R3 COOR2 (R2 = Ethyl, Propyl, Butyl, Phenyl) Cephalosporin Esters No Reaction N H 2N S OCH3 N H N O S N OCH3 CH3 O O O O CH3 O Cefpodoxime Proxetil (Prodrug) N H2 N S O S N OCH3 CH3 O O H CH3 O O N H 2N S OCH3 N H N O CH3 Esterase OCH3 N H N O CH3 + CO2 + HO S N OCH3 O H3C O + O OH H3C CH3 CH3 Advantage of prodrug formation III: Increase hydrophilicity and thus water solubility to apply parenterally or also orally when compounds are too lipophilic to formulate in liquid dosage form O Drug O O C H2 O O-Na+ C H2 HO Drug OH + O C H2 C H2 Succinates O Drug O O P O-Na+ OH Drug OH + HO P O-Na+ OH Phosphates O - O O Drug Drug OH + O O O Rapid and thus the prodrug is unstable O O-Na+ H H3C CH3 H N N O OH CH3 CH3 N H2O H3PO4 Cl Phosphatase O HO O S CH3 P O OH OClindamycin Phosphate H N H3C O OH CH3 Cl O HO Clindamycin OH S CH3 Chemical Delivery System The site specific delivery of drugs is an important way of increasing drug’s therapeutic index. The knowledge of prodrug and drug metabolism is used to concentrate drugs at its target site thus minimizing the systemic toxicity. HO CH2CH COOH HO NH2 BBB; Active transport to CNS by L-Amino acid delivery system HO HO HO CH2CH COOH HO CH2CH2NH2 NH2 L-Dopa Dopamine (Active) Antedrugs (Soft Drugs) I stopped taking medicine as I prefer original disease to side effects !! Because, Vioxx’ll treat pain but who’ll treat vioxx ?? Safety-Based Drug Withdrawals from U.S. Market (2006-2007) Drugs Vioxx (Rofecoxib) Ximelagatran (Exanta) Therapeutic activity Antiinflammatory (COX-2 inhibitor) Date approved Date withdrawn 05/99 09/04 Myocardial Infarction etc. 2006 Hepatotoxicity Anticoagulant Primary health risk Tegaserod (Zelnorm) IBS, constipation 2002 2007 Cardiovascular ischemic events Aprotinin (Trasylol) Induce bleeding during sergery) 1960s 2007 Ischemic colitis and Severe constipation Why the Adverse Drug Reactions Occur? Because of unintended systemic actions in most therapeutic classes of drugs To bring a drug from concept to market It takes about 10-15 years $897 millions to $1.7 billions Overall attrition rate 10,000:1 What is Antedrug? An active synthetic drug which is inactivated by a metabolic process upon entry into the systemic circulation. Therefore, a true antedrug acts only locally. True Antedrug Partial Antedrug Inactive Metabolite Less active metabolite Lee HJ and Soliman MRI (1982). Science, 215, 989. OCOR OH OH O O OH OH IA A Prodrug O OH Antedrug A O OH OH OH O O OH OH CO 2R Hydrocortisone (IA = Inactive Compound, A = Active Compound) IA CO 2- Chemical Approaches 1) The Carboxylic Esters and Amides 2) 20-Thioester Derivatives 3) g-Butyrolactone Derivatives O CH3 HO CH3 H H CO2R O OH HO CH3 hydrolysis in plasma H O CH3 H H H O Stable locally and active Inactive Inactivation of Steroid 21-ate Esters in Bood Plasma. COOH OH O CH3 HO CH3 H F H O OCOCH3 OH CO2Me t1/2 6.3 min plasma O O CH3 HO CH3 F O O HO CH3 H F H O OH OH CO2Me t1/2 90 min plasma CH3 HO CH3 F O O SCH2F OCOEt Me H CH3 CH3 HO CH3 Liver H O Inactivation of Fluticasone Propianate OCOEt Me H F F OH F Inactive H H H OH OH COOH Advantages of Antedrugs Localization of the drug effects Elimination of toxic metabolites, increasing the therapeutic index Avoidance of pharmacologically active metabolites that can lead to long-term effects Elimination of drug interactions resulting from metabolite inhibition of enzymes Simplification of PK problems caused by multiple active species M.O.F.Khan*, K.K.Park, H.J.Lee. Antedrugs: An Approach to Safer Drugs. Curr. Med. Chem., 12(19), 22272239, 2005. Phase II: Drug Conjugation Attachment of small polar endogenous molecules such as glucuronic acid, sulfate and amino acids to Phase I metabolites or parent drugs Products are more water-soluble and easily excretable Attenuate pharmacological activity and thus toxicity Trapping highly electrophilic molecules with endogenous nucleophiles such as glutathione prevent damage to important macromolecules (DNA, RNA, proteins) Regarded as true detoxifying pathway (with few exceptions) In general, appropriate transferase enzymes activate the transferring group (glucuronate, sulphate, methyl, acetyl) in a coenzyme form Glucuronic Acid Conjugation Glucuronidation is the most common conjugation pathway The coenzyme, UDP glucuronic acid is synthesized from the corresponding phosphate UDP-glucuronic acid contains D-glucuronic acid in the a-configuration at the anomeric center, but glucuronate conjugates are b-glycoside, meaning inversion of stereochemistry is involved in the glucuronidation Glucuronides are highly hydrophilic and water soluble UDP glucuronosyltransferase is closely associated with Cyp450 so that Phase I products of drugs are efficiently conjugated Four general classes of glucuronides: O-, N-, S-, and C- Neonates have undeveloped liver UDP-glucuronosyltransferase activity, and may exhibit metabolic problem. For example, chloramphenicol (Chloroptic) leads neonates to “gray baby syndrome” Neonatal jaundice may be attributable to their inability to conjugate bilirubin with glucuronic acid Formation of Glucuronide Conjugate UTP HO HO HO PPi O HO Phosphorylase OPO 32a-D-Glucose-1phosphate HO HO HO O O HO O O NH O P O P O O- O- O HO UDPG N O 2NAD U D OH PG + 2N de hy dr og en A DH as e HOOC HO O HO O O HO O P O P O OUDP-Glucuronyl-transferase (microsomal) HO HO HO O RXH O XR HO b-D-Glucuronide UDP O- O NH O HO N O OH Uridine-5'-diphosphoa-D-Glucose (UDPG) Types of Compounds Forming Glucuronides TYPE EXAMPLES CH3 O H N N CH3 O-Glucuronide OH Phenols Acetaminophen HO O morphine OH OH Cl H N CH3 CH3 O OH O2N Alcohols H N O Cl OH Chloramphenicol Propranolol OH O Enols O Hydroxycoumarine H2N N-hydroxyamines/amides S O2 NHOH CH3 N OH N-hydroxydapsone N-Hydroxy-2-acetylaminoflourene COOH OH Aryl acids Salicylic acid CH3 O OH O Fenoprofen Arylalkyl acids NH2 N-Glucuronides Arylamines O H N S N 7-Amino-5- Sulfonamides nitroindazole O2N N Alkylamines O N H2N O N CH3 N H CH3 Sulfisoxazole H CH3 3o Amines N CH3 Desipramine O NH2 H3C Amides O O H3C Meprobamate NH2 O Cyproheptadine N S-Glucuronides HS N CH3 Sulfhydryl Methimazole H3C S Carbodithioic acid H3C N SH Disulfirum (reduced form) O C-Glucuronides N CH3 N O Phenylbutazone Sulfate Conjugation Occurs less frequently than does glucuronidation presumably due to fewer number of inorganic sulfates in mammals and fewer number of functional groups (phenols, alcohols, arylamines and N-hydroxy compounds) Three enzyme-catalyzed reactions are involved in sulfate conjugation O - ATP O - - O S O O Sulfate PPi Mg+2 ATP sulfurylase O O O S O P O O - O O Ad ATP ADP - O O S O P O O - O +2 Mg APS phosphokinase HO OH Adenosine-5'phosphosulfate (APS) O Ad RXH PAP O Sulfotransferase (soluble) -2 O3 PO OH 3'-phosphoadenosine-5'phosphosulfate (PAPS) - O S XR O Sulfate conjugate Sulfation of Drugs Phenolic sulfation predominates Phenolic O-glucuonidation competes favorably with sulfation due to limited sulfate availability Sulfate conjugates can be hydrolyzed back to the parent compound by various sulfatases Sulfoconjugation plays an important role in the hepatotoxicity and carcinogenecity of N-hydroxyarylamides In infants and young children where glucuronyltransferase activity is not well developed, have predominating O-sulfate conjugation Examples include: a-methyldopa, albuterol, terbutaline, acetaminophen, phenacetin OH H HO N H3C HO H COOH N HO HO a-Methyldopa Albuterol OH H CH3 CH3 CH3 HO H N OH CH3 CH3 CH3 Terbutaline Possible Mechanism of Phenacetin Toxicity Electrophilic nitreneum Amino Acid Conjugation The first mammalian drug metabolite isolated, hippuric acid, was the product of glycine conjugation of benzoic acid R O COH Benzoic Acid, R = H Salicylic Acid, R = OH R O O CONHCH2COH Hippuric Acid, R = H Salicyluric Acid, R = OH Amino acid conjugation of a variety of caroxylic acids, such as aromatic, arylacetic, and heterocyclic carboxylic acids leads to amide bond formation Glycine conjugates are the most common Taurine, arginine, asparagine, histidine, lysine, glutamate, aspartate, alanine, and serine conjugates have also been found Mechanism of Amino Acid conjugation Drug-COOH An Acyl-CoA Intermediate Glycine Conjugate R = H Glutamine Conjugate R = CH2CH2CONH2 Brompheniramine Metabolism N CH3 N CH3 CH3 NH N P450 NH2 N P450 Br Brompheniramine Br N CHO N P450 Br H N N O Br Brompheniramine N-oxide COOH Aldehyde dehydrogenase Br CH3 N CH3 N Br Glycine conjugate Br Carboxylic Acid metabolite Glycine N-acyltransferase COOH Glutathione Conjugation NH 2 H N HO O HO HS H N O O N H O OH O O O O O N H S S H N NH 2 HO NH 2 Glutathione reduced form (GSH) N H OH O O OH O Glutathione oxidized form (GSSG) Glutathione is a tripeptide (Glu-Cys-Gly) – found virtually in all mammalian tissues Its thiol functions as scavenger of harmful electrophilic parent drugs or their metabolites Examples include SN2 reaction, SNAr reaction, and Michael addition SN2 Examples GSH R X SG + Y GlutathioneS-Transferase A. SN2 R X Y - 1. CH3O2SO Busulfan OSO2CH3 ONO2 2. H ONO2 SG X = C, O, S; Y = leaving group or epoxide CH3O2SO ONO2 - SG O NO2 H SG S+ G ONO2 ONO2 - SG H O SG ONO2 + OH Nitroglycerine CH 3 O O CH3 O Naproxcinod O O N O GSSG SNAr Examples X SG GSH B. SNRr Z Z N 1. N O + N O S H3C N - N - SG N N N H Azathioprine O + N OSG S N H3C N N N N N H N SH NO2 + N N SG H3C N N 1-Methyl-4-nitro-5H (S-glutathionyl) 6-Mercaptopurine imidazole Michael Addition H+ C. - Z SG Z SG Michael Addition CH3 N CH3 N CH3 N SG - SG HO O OH HO O O CH3 SG N O O O HO GS OH HO OH CH3 N O OH Mercapturic Acid Conjugates Drug O HO S H N O O O Amino Acid g-Glutamyl-AA (AA) OH N H NH 2 g -Glutamyl transpeptidase Drug O HO S H N NH 2 O Glutathione Conjugate Drug Glycine Cysteinyl Glycinase Acetyl CoA S HO NH 2 O S-substituted Cysteine Derivative CoASH Drug S H 2N O N H O Mercapturic acid conjugate CH 3 Acetyl Conjugation Metabolism for drugs containing a primary amino group, (aliphatic and aromatic amines), amino acids, sulfonamides, hydrazines, and hydrazides The function of acetylation is to deactivate the drug, although Nacetylprocainamide is as potent as the parent antiarrhythmic drug procainamide (Procanbid) or more toxic than the parent drug, e.g., N-acetylisoniazid Acetylation is two-step, covalent catalytic process involving N-acetyl transferase O H3 C X- O CoASH SCoA H3 C O X H2N R H3 C NHR X- N-Acetylation of amines Genetic polymorphism in N-acetyltransferase activity Multiple NAT2 alleles (NAT2*5, *6, *7, and *14) have substantially decreased acetylation activity and are common in Caucasians and populations of African descent. In these groups, most individuals carry at least one copy of a slow acetylator allele, and less than 10% are homozygous for the wild type (fast acetylator) trait. The ratio of NAT2 activity is 7 in Caucasians to 18 in the Chinese population. Example of Acetylated Drugs O O HO S NH O OH NH2 CH3 CH3 Cilastatin HO H3C N S H N O COOH Imipenem NH Fatty Acid and Cholesterol Conjugation Hydroxyl-containing drugs can undergo conjugation with a wide range of endogenous fatty acids such as saturated acids from C10 to C16 and unsaturated acids such as oleic and linoleic acids Cholesterol ester metabolites have been detected for drugs containing either an ester or a carboxylic acid O O HO O Cl N Cl OH Cholesterol Fatty Acid Cl O Prednimustine O (CH2) 10 COOH Methyl Conjugation Minor conjugation pathway, important in biosynthesis of epinephrine and melatonin; in the catabolism of norepinephrine, dopamine, serotonin, and histamine; and in modulating the activities of macromolecules (proteins and nucleic acids) Except for the formation of quarternary ammonium salts, methylation of an amine reduces the polarity and hydrophilicity of the substrates A variety of methyl transferase, such as COMT (catechol O-methyl transferase), phenol-O-methyltransferase, N-methyl transferase, Smethyltransferase etc are responsible for catalyzing the transfer of methyl group from SAM to RXH H2 N COOH H2N ATP H2 N COOH PPi + Pi Methyltransferase H3CS Methionine adenosyltransferase + S O Mthetionine HO Ad CH3 HX-R OH S-Adenosylmethionine Mechanism of methyl conjugation CH3 -X-R COOH + S O HO Ad OH Case Study Case 2. Imagine yourself as a drug information specialist at a poison control center. A technician from the coroner’s office is investigating a case and requires assistance in identifying the possible sources of benzodiazepines (BZDs) in the toxicology profile of a particular corpse. The technician has identified four distinct BZDs in this blood sample. She believes that the major component is diazepam (1) (72% of the identified BZDs) and that the remaining three components are metabolites (NOTE: the assay identifies only active compounds). O H N N Cl Q. What are the three structures of potential ACTIVE metabolites for diazepam? CH3 O O H N N 2 OH N Cl N Cl CH3 O N 1 4 OH N Cl 3 Assignment: Due by this Friday http://www-home.cr.duq.edu/~harrold/basic_concepts_index.html Study Guide 1. What Roles are Played by Drug Metabolism? Know with structural examples 2. Role of stereochemistry in metabolism of drugs with example of warfarin, ibuprofen and itomidate 3. What is first pass effect; enterohepatic circulation? Why and how they occur? Drug examples 4. Metabolisms in the intestinal mucosa 5. CYP450, Hepatic microsomal flavin containing monooxygenases (MFMO or FMO) Monoamine Oxidase (MAO) and Hydrolases. Drugs metabolised by these enzymes and the active sites of these enzymes. Types of metabolic reaction catalyzed by these enzymes 6. Specific CYP enzymes with the number of drugs they metabolize 7. Few CYP family with their main functions 8. Drug interaction basics related to metabolic enzymes Study Guide Cont. 9. Mechanism and routes of aromatic hydroxylation. The effects of electron donating and withdrawing groups in aromatic hydroxylation. Drug examples. What is NIH shift? 10. Oxidation of olefins. Role of epoxide hydrolase. Can olefenic epoxide be converted to alcohol as in aromatic epoxide by NIH shift? 11. What type of C in a drug molecule can not be hydroxylated? 12. What is allylic and benzylic hydroxylation? Show drug examples. 13. Show the drug examples where hydroxylation occur on Cα to C=O and C=N bonds 14. Show the drug examples where hydroxylation occur at aliphatic and alicyclic carbon atoms. Which carbons are more easily hydroxylated? 15. What is N-oxidatin and N-dealkylation. What enzymes are involved? How do you differentiate between N-dealkylation and deamination. Drug examples. What types of drugs generates lactams instead of causing dealkylation? 16. What is the difference between mixed function oxidases and amine oxidases? Study Guide Cont. 17. What is the difference between ethanol oxidation and O-dealkylation? 18. What is S-dealkylation, desulfuration and S-oxidation? Drug examples. 19. How does steric factors influence S- O- and N-dealkylations? 20. Oxidative dehalogenation with special example of chloramphenicol. Why chloramphenicol cause toxicity to the babies? 21. What is MFMO and its active site? What types of functional groups are metabolized by this enzyme? Drug examples. 22. MAO, dehydrogenases, xanthene oxidases and their functions with drug examples. Difference between MAO-A and MAO-B. 23. Alcohol and aldehyde dehydrogenases, the coenzymes and the types of drugs they work on. 24. Azo and nitro reductases, their coenzymes and the drugs they act on. Study Guide Cont. 25. Different types of hydrolytic enzymes. Compare rate of hydrolysis of esters, amides, carbonates and carbamates. 26. What are prodrugs and antedrugs? What are the advantages? Examples. 27. What are different types of conjugation reactions? 28. The enzymes and substrates involved in glucuronidation, and sulfate conjugation. 29. Why acetaminophen is toxic to neonates? Mechanism of phenacetin and acetaminophen toxicity. 30. What types of drugs or metabolites may form glycin conjugates? 31. What are different mechanisms involved in glutathione conjugation? What is mercapturic acid conjugate? Mercapturic acid conjugate of acetaminophen is a sign of its toxicity – why? 32. Mechanism of acetylation. What is slow and fast acetylator? 33. What is COMT? What coenzymes is involved in its action? What types of drugs and/or neurotransmitters are metabolized by COMT?