Drug metabolism (Phase-I)

Transcription

1 Drug metabolism (Phase-I) د. محمد نورالدين محمود 2 nd edition 2017 Several slides where adopted from Dr. Pran Kishore Deb lectures

2 Brief introduction The body treats drugs as foreign substances and has methods of getting rid of such chemical invaders. If the drug is polar, it will be quickly excreted by the kidneys. However, non-polar drugs are not easily excreted and the purpose of drug metabolism is to convert such compounds into more polar molecules that can be easily excreted. Change the structure of drug to more polar molecule usually affects the drug interaction with specific and non-specific receptors available in the tissues. The metabolism is carried out by two sets of reactions (i.e. phase-i and phase-ii). Both sets of reactions can also be regioselective and stereoselective. This means that metabolic enzymes can distinguish between identical functional groups or alkyl groups located at different parts of the molecule (regioselectivity), as well as between different stereoisomers of chiral molecules (stereoselectivity).

3 Brief introduction Drug metabolism or biotransformations are the chemical reactions that are responsible for the conversion of drugs into other products (metabolites) within the body before or after they have reached their sites of action (i.e. wherever drug is in free form). Drug metabolism or biotransformations Metabolites Since metabolism involve enzymesubstrate interaction, the properties of specificity is involved i.e. enzymes can distinguish between identical functional groups or alkyl groups located at different parts of the molecule (regioselectivity), as well as between different stereoisomers of chiral molecules (stereoselectivity). It is thought that biotransformation of molecule is intended 1. Directly to increase molecule polarity increase molecular excretion 2. Indirectly affects molecular interaction with specific and non-specific receptors molecular activity

4 The outcomes of metabolism Metabolism of a compound is the chemical modification of the molecular structure catalyzed by enzymes (biotransformation) Therefore, changing the molecular structure of a drug by metabolism will have effects on dynamical behavior of drug interaction with receptors and consequently- the kinetical behavior of the molecule. Therefore, changes in drug molecular structure may affect: 1. Drug interaction with target receptor (pharmacological effect) 2. Drug interaction with cell membranes and active transporters (elimination) 3. Drug interaction with specific and non-specific receptors in tissue (tissue distribution and half life)

5 Brief introduction Absorption (Oral, Topical, IV, IM, SC, IP, Inhalation) Distribution (Site of action - target cells, tissues, receptors) Highly hydrophilic Elimination (Urine, other excretory fluids) Metabolism (Chemical change of drug metabolites) Produce pharmacologically inactive metabolite Produce pharmacologically less-active metabolite Produce pharmacologically toxic metabolite Produce pharmacologically more-active metabolite Activate an inactive drug (PRODRUG)

6 Oral absorption Hydrophilic Lipophilic Bile excretion Renal elimination Hydrophilic

7 If the drug is polar enough, part of it will be directly excreted If the drug already containing polar functional group, it can be directly conjugated (phase-ii) If the drug after functionalization (Phase-I) becomes polar enough, it will be directly excreted. Otherwise, the drug need to be functionalized, conjugated then excreted DRUG PHASE-I PHASE-II EXCRETION

8 What does metabolism do to drug molecule? Less active Receptor Inactive (prodrug) Unwanted receptor Active

9 What does metabolism do to drug molecule? A) Biotransformation may produce a pharmacologically inactive metabolite which is readily excreted. Hydrolysis of procaine (local anesthetic drug) by esterases or procainamide (antiarrhythmic agent) by amidases

10 What does metabolism do to drug molecule? (Cont.) B) Biotransformation may produce a pharmacologically less active metabolite. Cytochrome P450 oxidative N-dealkylation of propranolol to nor-propranolol : anti-hypertensive drug morphine to nor-morphine : opiate analgesic drug

11 What does metabolism do to drug molecule? (Cont.) C) Biotransformation may produce a pharmacologically more active metabolite. Cytochrome P450 oxidative O-dealkylation of codeine to morphine Esterase hydrolysis of diamorphine to morphine

12 What does metabolism do to drug molecule? (Cont.) D) Biotransformation may activate an inactive drug (Prodrug). Hydrocortisone: a steroid hormone Hydroxycamptothecin: anticancer agent Both drugs has low aqueous solubility so are linked to hydrophilic group to make it water soluble suitable for injection

13 What does metabolism do to drug molecule? (Cont.) E) Biotransformation may activate an pharmacologically toxic metabolites. Thalidomide is an anti-nausea and sedative drug that was introduced in the late 1950s to be used as a sleeping pill, and was quickly discovered to help pregnant women with the effects of morning sickness. It was sold until 1962, when it was withdrawn after being found to be a teratogen, which caused many different forms of birth defects. More than 20,000 children in 46 countries were born with deformities such as phocomelia as a consequence of thalidomide use. Racemisation of R-thalidomide to S-thalidomide by isomerases

14 Significance of Drug Metabolism Knowing drug metabolism helps us to: A. Know the rate of drug metabolism: which controls the duration and intensity of the action of many drugs by controlling the amount of the drug reaching its target site. B. Know the products of drug metabolism: which controls the activity of the drugs whether being inactivated (detoxified) or activated (as in prodrugs). C. Know the competitive and uncompetitive interaction during metabolism: which controls drug-drug interactions D. Know the possible products of drug metabolism: which need to be documented for newly discovered drugs. Drug metabolism has great importance in medicinal chemistry because it influences the deactivation, activation, detoxification and toxification of the vast majority of drugs.

15 Sites of metabolism Liver Primary site! Highly perfused organ Rich in enzymes Acts on endogenous and exogenous compounds First pass effect! Extrahepatic metabolism sites Intestinal wall Sulfate conjugation Esterase and lipases - important in prodrug metabolism i.e. β-glucuronidase enzymes hydrolyze glucuronides for reabsorption (Enterohepatic recirculation) Bacterial flora Reduction of Aromatic nitro and azo compounds Lungs, kidney, placenta, brain, skin, adrenal glands Limited ability and largely unknown the biotransformations that they carry out are often more substrate selective and more limited to particular types of reaction (e.g., oxidation, glucuronidation).

16 Sites of Drug Metabolism Reactions The primary site for drug metabolism is liver Others are- Kidney, intestine, lungs, plasma Types of Drug Metabolism Reactions Drugs metabolism reactions can be divided into two distinct categories or phases: 1. Phase I Reactions 2. Phase II Reactions

17 1. Phase I or Functionalization Reactions 2. Phase II or Conjugation Reactions A. OXIDATION REACTIONS Oxidation of aromatic moieties. Oxidation of olefins. Oxidation at benzylic, allylic carbon atoms, and carbon atoms α to carbonyl or imines. Oxidation at aliphatic and alicyclic carbon atoms. Oxidation involving carbon-hetero atom systems. Carbon-nitrogen systems (N-dealkylation, oxidative deamination, N-oxide formation, N-hydroxylation). Carbon-oxygen systems (O-dealkylation). Carbon-sulfur systems (S-dealkylation, S-oxidation, desulfuration). Oxidation of alcohols and aldehydes. Other miscellaneous oxidative reactions. A. Glucuronic acid conjugation. B. Sulfate conjugation. C. Acetylation. D. Methylation. E. Conjugation with glycine, glutamine, and other a.a. F. Glutathione or mercapturic acid conjugation. B. REDUCTION REACTIONS Reduction of aldehydes and ketones. Reduction of nitro and azo compounds. Other miscellaneous reduction reactions. C. HYDROLYSIS REACTIONS Hydrolysis of esters and amides. Hydration of epoxides and arene oxides.

18 Phase I Reactions Reactions which introduce or unmask a polar functional group (e.g., OH, COOH, NH 2, SH) into the molecule to produce a more water soluble compound. The compound now either be polar enough to be excreted or may undergo phase-ii reactions In this step drugs undergoes functionalization reaction of oxidation, reduction or hydrolysis. Phase-I oxidation reactions are catalyzed by the superfamilies of cytochrome P450s (CYPs), flavin-containing monooxygenases (FMO), epoxide hydrolyses (EHs).

19 Phase II Reactions Reactions which attach/conjugate polar or hydrophilic endogenous compounds to the functional groups of phase-i metabolites or parent compounds that already have suitable functional groups to form water soluble conjugated products, thereby facilitating drug elimination. OR Reactions which conjugate polar or hydrophilic endogenous compounds to the drugs or its metabolites to form water soluble conjugated products, thereby facilitating drug elimination. Conjugated metabolites have increased molecular weight, improved water solubility and are generally devoid of any pharmacological activity and toxicity in human. Phase-II reactions are catalyzed by several superfamilies of UDP-glucuronosyltransferases (UGT), Sulfonyltransferases (SULT), N-acetyltransferases (NAT), and Methyltransferases (MT).

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21 Differences between phase-i and phase-ii reactions PHASE I REACTIONS PHASE II REACTIONS 1 Functionalization reactions Conjugation reactions 2 Metabolites may or may not have increased molecular weight 3 Metabolites may undergo Phase-II conjugation reaction 4 Metabolites may be pharmacologically less active or inactive Metabolites always have increased molecular weight Metabolites never undergo Phase-I Functionalization reaction Metabolites are generally pharmacologically inactive 5 May produce toxic metabolites Generally does not produce toxic metabolites 6 It may activate an inactive drug (prodrug) It does not activate an inactive drug

22 Recent works involve activation of pro-drugs by phase-ii enzymes. Mechanism of activation of cis-3-(9h-purin-6-ylthio) acrylic acid (Prodrug) by GST enzyme to 6- mercaptopurine (active) by two possible pathways 6-mercaptopurine (active) Prodrug (inactive) is converted to 6- mercaptopurine by the sequential action of renal γgt dipeptidase and cysteine S- conjugate β lyase. RUZZA, P. & CALDERAN, A Glutathione transferase (gst)-activated prodrugs. Pharmaceutics, 5, GSH-Drug adduct (inactive)

23 The reactions in Phase-I are carried by enzymes which can accept foreign compound (whether drug or other molecule) as substrate Therefore, if drug can bind to enzyme it will be metabolized otherwise it will not. The drug may have affinity to more than single enzyme, thus the drug will have more than single metabolite. Note the products may be different if the sequence of metabolism is different

24 The enzymes belong to Phase-I can: - Add polar functional groups to a wide variety of drugs. - Unmask polar functional groups which might already be present in a drug. E.g. demethylate a methyl ether to reveal a more polar hydroxyl group. Once the polar functional group has been added, the overall drug is more polar and water soluble, and is more likely to be excreted when it passes through the kidneys. The enzymes involved are mainly non-specific enzymes which are present in liver (E.g. cytochrome P450 enzymes) or gut wall, plasma and other tissues (e.g. estrases, amidases) The drug may have different affinities to different enzymes. If the affinity is higher toward enzyme-1 than enzyme-2, the drug metabolite-1 will usually be higher than metabolite-2 In addition, each type of enzyme is available in different copies with minor differences in structure that lead to minor differences in substrate specificity E.g. enzyme-1 is available in isoforms A, B and C. + K d1 + K d2

25 Affected drug functional groups during phase-i Some functional groups of drugs are most prone to oxidation, e.g. 1. N-methyl 2. Aromatic rings 3. Terminal alkyl chain 4. Least hindered alicyclic rings Some are prone to reduction e.g. 1. Nitro 2. Azo 3. Carbonyl Some are prone to hydrolysis 1. Ester 2. Amide

26 Different metabolites are obtained from different orientations of drug in enzyme binding site. This drug give 2 metabolites from the same metabolizing enzyme Drug Drug Drug Drug Bind with Orinetation-1 and K d1 Drug Bind with Orinetation-2 and K d2 Drug Drug Metabolite-1 Metabolite-2 Q) How a drug can have different metabolites, even if it is bind to a single enzyme? A) The drug binds to the enzyme through different orientations Drug Drug cannot bind with Orinetation-3 Thus gives no metabolite at this site

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28 The reactions catalysed by cytochrome P450 enzymes can involve the oxidation of carbon, nitrogen, phosphorus, sulphur, and other atoms. The oxidation reactions are easily taking place on nitrogen, phosphorus and sulfur atoms The oxidation reactions are easily taking place on carbon atoms if the carbon atom is either exposed (i.e. easily accessible to the enzyme) or activated. For example, methyl substituents on the carbon skeleton of a drug are often easily accessible and are oxidized to form alcohols, which may be oxidized further to carboxylic acids. In the case of longer chain substituents, the terminal carbon and the penultimate Carbon are the most exposed carbons in the chain, and are both susceptible to oxidation. If an aliphatic ring is present, the most exposed region is the part most likely to be oxidized.

29 Nomenclature of CYPs CPY Arabic Number Capital Letter - Arabic Number There are at least 33 different cytochrome P450 (CYP) enzymes can be classified into Families(1-4), subfamilies (many) and members (many). Most drugs in current use are metabolized by five primary CYP enzymes (CYP3A4, CYP2D6, CYP2C9, CYP1A2, and CYP2E1). The isozyme CYP3A4 is particularly important in drug metabolism and is responsible for the metabolism of most drugs. Family CYP3A4 Subfamily Member no.

30 Classifications of CYPs RELATIVE HEPATIC CONTENT OF CYP ENZYMES % DRUGS METABOLIZED BY CYP ENZYMES CYP2D6 2% CYP2E1 7% CYP 2C 17% CYP 1A2 12% CYP 3A4-5 26% OTHER 36% CYP 2C9 14% CYP 1A2 14% CYP 2C19 11% CYP 3A4-5 33% CYP2D6 23% CYP2E1 5%

31 Important CYPs CYP3A4 has a large binding pocket and can accept large, bulky molecules that are neutral. It catalyses N-dealkylations; aliphatic benzylic hydroxylations; aromatic hydroxylations; epoxidations, oxidation of atoms near the end of a molecule. CYP2D6 prefers less bulky, basic molecules and catalyses O-demethylations; N- dealkylations; oxidation of atoms near the middle or end of a molecule. CYP2C9 has a strong preference for substrates with acidic functional groups. It catalyses O-demethylations; aliphatic benzylic hydroxylations; aromatic hydroxylations; oxidation of atoms near the middle or end of a molecule.

32 cytochrome P450 in the oxidation of xenobiotics. The enzymes that constitute the cytochrome P450 family are the most important metabolic enzymes and are located in liver cells. They are haemoproteins (containing haem and iron) and they catalyse a reaction that splits molecular oxygen, such that one of the oxygen atoms is introduced into the drug and the other ends up in water. As a result, they belong to a general class of enzymes called the monooxygenases. cofactors Simplified depiction of the proposed activated oxygen cytochrome P450-substrate complex. Note the simplified apoprotein portion and the heme (protoporphyrin IX) portion or cytochrome P450 and the proximity of the substrate R-H undergoing oxidation. H

33 Proposed catalytic reaction cycle involving cytochrome P450 in the oxidation of xenobiotics. The other oxygen is still bound to Fe, thus can be use to oxidize the substrate OH H H H H H One of the activated oxygen forms water H H H The Fe is now getting oxidized and give e to O 2 to make it activated The Fe is now reduced thus can either bind CO or O 2

34 Drug requirements for oxidation reactions For any compound to undergo oxidative metabolism it needs 1. The atom should bear hydrogen atom: to be replaced with OH 2. The atom need to be exposed to catalytic binding site of the enzyme 3. The atom need to be electron rich to attract the activated oxygen. Presence of H atom Electron rich e feed C H - Nitrogen - Oxygen - Sulfur - Pi system Exposed

35 A. Oxidation of Aromatic Moieties Aromatic compounds (arenes) undergoes aromatic hydroxylation through an epoxide intermediate called an arene oxide to their corresponding phenolic metabolites (arenols). R R R O OH Arene Arene oxide Arenol In most of the drugs, hydroxylation occurs at para position HN O O N H C 2 H 5 O HN O O N H C 2 H 5 O OH Phenobarbital

36 A. Oxidation of Aromatic Moieties (Cont.) Effect of substituents on aromatic hydroxylation: Electron donating group activate (electron-rich) the aromatic ring towards hydroxylation Electron withdrawing group deactivate the aromatic ring towards hydroxylation Compounds with two aromatic rings, hydroxylation occurs preferentially in the more electron-rich ring. Cl Cl N H Cl H N N H Clonidine hydrochloride (Anti-hypertensive drug) Very less aromatic hydroxylation CCOOH No report on aromatic hydroxylation SO 2 N(CH 2 CH 2 CH 3 ) 2 Probeniside (Uricosuric agent)

37 A. Oxidation of Aromatic Moieties (Cont.) Electron donating group (EDG) - Electronegative atoms: O > N > C sp2 ( O > NR2 > NH2 > OH > OR > OCOR > CH2=cH2 or benezne) Electron withdrawing group (EWG) is any group which have: 1. Pi system than can take electrons toward the pi resonance (-SO3H>-COOH>-COOR). 2. Positive charge that can take electrons ( N + R3>-N + H3) 3. Or both 1 and 2 (- N + O2 - ) 4. Halogens Q) Halogens are electronegative atoms, but why they are not electron donators (being poor e withdrawals)? A) Halogens attract electron and not share it, thus induce positive charge in the connected atom.

38 A. Oxidation of Aromatic Moieties (Cont.) Non-toxic Non-toxic Other possible fates for arene oxide, some of them lead to toxic effect toxic

39 A. Oxidation of Aromatic Moieties (Cont.) Rearrangement of arene oxide through intramolecular hydride (deuteride) migration called the NIH Shift. Because of an isotope effect on cleavage of the C-D bond, the proton is preferentially removed. Competing pathway to NIH shift is simple loss of a proton or deuterium from the cation intermediate The more stabilizing the R group is the more deprotonation that occurs when R is electron donating, 40-54% retention of D is found) when R is electron withdrawing only 0-30% of the product retains deuterium;

40 A. Oxidation of Aromatic Moieties (Cont.) Environmental polutants, such as polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), have attracted considerable public concern over their toxicity and health hazards. These compounds appear to be resistant to aromatic oxidation because of the numerous electronegative chlorine atoms in their aromatic rings (which causes inactivation). Polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants that are formed from auto emission, refuse burning, industrial processes, cigarette smoke, and other combustion processes. E. g. Benzo[α]pyrene, a potent carcinogenic agent.

41 A. Oxidation of Aromatic Moieties (Cont.) Aromatic hydroxylation of benzo[α]pyrene, can occur at a number of positions. The hydroxylated product can covalently binds to DNA. Therefore, certain arene oxides of benzo[α]pyrene (e.g. 4,5-oxide, 7,8-oxide, 9,10-oxide) appear to display some mutagenic and tumorigenic activity. Although the formed arene oxides and epoxides improves hydrophilicity and renal excretions, the molecules are so reactive that can covalently linked to nucleophilic groups in DNA and proteins thus damages them. unless been eliminated by conjugation (Phase II)

42 1. Oxidation of Aromatic Moieties (Examples) Some metabolites are active e.g. the parahydroxylated metabolite of phenylbutazone, oxyphenbutazone, is pharmacologically active and has been marketed itself as an anti-inflammatory agent (Tandearil) Examples of drugs and xenobiotics that undergo aromatic hydroxylation in humans. Arrow indicates site of aromatic hydroxylation

43 B. Oxidation of alkenes (Olefins The metabolic oxidation of olefinic carbon-carbon double bonds leads to the corresponding epoxide (or oxirane) in a manner similar to aromatic oxidation. Olefinic epoxides are more stable than arene oxides (Therefore, less toxic than arene oxide). Olefinic epoxides are susceptible to enzymatic hydration by epoxide hydrase to form trans-1,2- dihydrodiols, in a manner similar to arene oxides. Frequently, the Or may conjugate to macromolecules toxic effect epoxides formed from the biotransformation of an olefinic compound are minor products, because of their further conversion to the corresponding 1,2-diols. Why the oxidation on alefinic carbon of aclofenac is easier than on aromatic carbon?

44 B. Oxidation of Olefins (Cont.) The conjugation of styrene to macromolecules after being oxidized is the cause of their toxicity. Conjugate to nucleic acids and proteins which leads to toxic effects

45 B. Oxidation of Olefins (Cont.) The safest pathway to get rid (detoxify) epoxide and arene oxide is by glutathione adduct formation. Epoxide

46 C. Oxidation At Benzylic Carbon Atoms Carbon atom attached to aromatic rings (benzylic position) are susceptible to oxidation, forming the corresponding alcohol (or carbinol) metabolites. Benzylic carbon is close to a pi system, thus has good electron density to undergo oxidation

47 D. Oxidation At Allylic Carbon Atoms An allylic carbon is a carbon atom bonded to a carbon atom that in turn is doubly bonded to another carbon atom. Allylic carbon is equivalent to the benzylic carbon by being close to a pi system Microsomal hydroxylation at allylic carbon atoms is commonly observed in drug metabolism.

48 E. Oxidation At Carbon Atoms α to Carbonyls and Imines Microsomal hydroxylation at Carbon Atoms α to Carbonyls and Imines is also observed in drug metabolism. The C α is close to a pi-system, thus has good electron density to undergo oxidation Hydroxylation of the carbon atom α to carbonyl only is generally rare in drug metabolism.

49 F. Oxidation At Aliphatic and Alicyclic Carbon Atoms Alkyl or aliphatic carbon centers are subject to mixed function oxidation. 1. ω oxidation: oxidation at terminal methyl group. 2. ω 1 oxidation: oxidation at penultimate carbon atom (i.e. next to last carbon). 3. Activated carbon atom, that is next to sp, sp 2 carbons The initial alcohol metabolites formed from these ω and ω 1 oxidations are susceptible to further oxidation to yield aldehyde, ketones, or carboxylic acids. Alternatively, alcohol metabolites may undergo glucuronide conjugation.

50 trans-4-hdyroxyacetohexamide is the major metabolite due to good exposure of C4 and stereoselectivity of enzyme

51 Up to this point we only considered the oxidations at carboncarbon interface (C-C) The next slides will consider the oxidation at carbonheteroatom interface like C-O, C-N, C-S which may lead to molecular fragmentation UNDERSTANDING

52 G. Oxidation Involving CARBON-HETEROATOM Systems Metabolic oxidation of Carbon-Nitrogen Carbon-Oxygen Carbon-Sulfur Involves two basic types of biotransformation process occurs for Carbon-heteroatom oxidations: 1. Hydroxylation of the α-carbon atom attached directly to the heteroatom (N, O, S) and decomposition with cleavage of carbon-hetero atom bond (dealkylation). E.g. N-, O-, S- dealkylation, deamination reactions. 2. Hydroxylation or oxidation of the heteroatoms (N, S, only).

53 G. Oxidation Involving CARBON-HETEROATOM Systems (Cont.) Oxidation of Tertiary Aliphatic and Alicyclic Amines: 1. N-dealkylation: oxidative removal of alkyl groups 2. Deamination 3. N-oxidation 1 amine Aldehyde Aldehyde 1 amine Hydroxyl amine

54 G1) Oxidation Involving CARBON-NITROGEN Oxidation of Tertiary Aliphatic and Alicyclic Amines: 1. N-dealkylation: oxidative removal of alkyl groups Mechanistically, oxidative dealkylation proceeds via an initially formed carbinolamide, which is unstable and fragments to form the N-dealkylated product. Small alkyl groups (methyl, ethyl, isopropyl) removed rapidly and preferentially. The first alkyl group of tertiary amine is removed more rapidly than second alkyl group. Bisdealkylation of aliphatic tertiary amine to corresponding primary amine occurs very slowly. N-dealkylation of t-butyl group is not possible by the carbinolamine pathway because α-carbon hydroxylation can not occur.

55 G1) Oxidation Involving CARBON-NITROGEN (cont.) Examples of oxidative N-dealkylation, which followed by oxidative deamination which may be followed by oxidation Several consecutive oxidation of CH3 to carboxylic acid which is removed as CO2, since the main carbon contains NO hydrogen

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57 G1) Oxidation Involving CARBON-NITROGEN (cont.) Alicyclic tertiary amine often generate lactam metabolites by α-carbon hydroxylation reaction.

58 G1) Oxidation Involving CARBON-NITROGEN (cont.) Some secondary alicyclic amines like tertiary amines are metabolized to their corresponding lactam derivatives. Endogenous primary amines (e.g. dopamine, norepinephrine, tryptamine, and serotonin) and xenobiotics are metabolised via oxidative deamination by a specialized family of enzymes called monoamine oxidases (MAOs). MAO is a flavin (FAD)-dependent enzyme found in two forms, MAO-A & MAO-B. MAO-A & MAO-B have about 70% amino acid sequence homology. MAO enzymes are located on the outer membrane of mitochondria. MAOs are mostly found in LIVER and intestinal mucosa.

59 G1) Oxidation Involving CARBON-NITROGEN (cont.) Oxidation of Secondary Primary Amines: 2. Oxidative deamination N-dealkylation of secondary amines proceeds via carbinolamine pathway (similar to tertiary amines) and gives rise to primary amine metabolite. Primary amine metabolites are susceptible to oxidative deamination following the process similar to N-dealkylation. In general, dealkylation of secondary amines is believed to occur before oxidative deamination. In some cases, direct deamination of the secondary amine is also possible.

60 N-dealkylation and Oxidative deamination α α If α-carbon hydroxylation can not occur, then oxidative deamination is not possible. α

61 G1) Oxidation Involving CARBON-NITROGEN (cont.) 3. N-oxidation: Mostly for primary and secondary amines as well as aromatic amines Primary amines will be converted to hydroxylamines, nitrone then nitrogen dioxide Secondary amines will be converted to hydroxylamines then nitrone. N-oxidation of secondary amines occurs much less than oxidative dealkylation and deamination Toxic metabolites

62 G1) Oxidation Involving CARBON-NITROGEN (cont.) Although N-oxidation is less common, it will be the major metabolite for compounds which have no hydrogen atom on Cα

63 G1) Oxidation Involving CARBON-NITROGEN (cont.)

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65 G2) Oxidation Involving CARBON-OXYGEN Several drugs containing ether group undergo oxidative O-dealkylation. The biotransformation involves an initial α-carbon hydroxylation to form a either hemiacetal or a hemiketal, which undergoes spontaneous carbon-oxygen bond cleavage to yield the dealkylated oxygen species (phenol or alcohol) and a carbon moiety (aldehyde or ketone).

66 G2) Oxidation Alcohols and Aldehydes (least important) Many oxidative processess (e.g. benzylic, allylic, alicyclic or aliphatic hydroxylation) generate alcohol or carbinol metabolites as intermediate products. Alcohols can also be oxidized further :- 1. Primary alcohols The carbon is well exposed here Oxidation of primary alcohol generates aldehydes. Oxidation of aldehydes generates carboxylic acid derivatives. 2. Secondary alcohols (not common) - The carbon is NOT well exposed here - Oxidation of secondary alcohol to ketones is NOT often important as it reduces back to secondary alcohol. - Secondary alcohol group being more polar and functionalized, is more likely to be conjugated than the ketone moiety.

67 H1) Oxidation Involving CARBON-SULFUR Several drugs containing CARBON-SULFUR functional group are susceptible to 1. S-dealkylation 2. Desulfuration 3. S-oxidation The first two process involve oxidative carbon-sulfur bond cleavage. 1. S-dealkylation: Similar to N- and O-dealkylation.

68 H2-H3) Oxidation Involving CARBON-SULFUR (cont.) 2. Desulfuration : Oxidative conversion of carbon-sulfur double bonds (C=S) (thiono) to the corresponding carbon-oxygen double bond (C=O) is called desulfuration. 3. S-oxidation : S-oxidation yields the corresponding sulfoxide derivatives. Sulfoxide drugs/metabolites may further oxidised to sulfones (-SO2-)

69 Case study for oxidation reactions 2-acetylaminoflurorene is wellknown hepatocarcinogenic, it undergoes an N-hydroxylation reaction catalyzed by CYP to form the corresponding N-hydroxy metabolite (also called a hydroxamic acid) Hydroxamic acid undergoes conjugation to form O-sulfate ester, which ionizes to generate the electrophilic nitrenium species.

70 Case study for oxidation reactions Chlorpormazine undergoes S-oxidation, oxidative N-dealkylation and oxidative deamination S-oxidation

71 Case study for oxidation reactions Applications for oxidation favorable sites: 1. The least substituted aromatic ring will be favorably oxidized, especially at the least hindered carbon atom Deactivated ring (Rare metabolite) e rich and exposed (Major metabolite) Some steric hindrance (minor metabolite)

72 Reduction

73 Reductive Reactions Reductive process play an important role in the metabolism of many compounds containing carbonyl, nitro and azo groups - Carbonyl Reduction alcohols Conjugated O-conjugates - Nitro and Azo Reduction amines Conjugated N-conjugates Phase-I Phase-II

74 1. Reduction of Aldehydes and Ketones Aldehydes reduces to primary alcohols. Ketones reduces to secondary alcohols. Reactions mediated by Aldo-Keto reductase enzymes Bioreduction of ketones often leads to the creation of an asymetric centre and thereby, two possible stereoisomeric alcohols. One of the stereoisomer may preferentially form predominantly over other stereoisomer and thus shows product stereo selectivity in drug metabolism.

75 2. Reduction of Nitro and Azo Compounds Bioreduction of aromatic nitro and azo compounds leads to aromatic primary amine metabolites. Aromatic nitro compounds are reduced initially to the nitroso and hydroxylamine intermediates that subsequently further reduced to amine + Ar N O Ar N O Ar NHOH Ar NH 2 O Nitro Nitroso Hydroxylamine Amine Azo reduction proceed via hydrazo intermediate (-NH-NH-) that subsequently cleaved reductively to yield the corresponding amines. Ar N N Ar' Ar NH NH Ar' H 2 N Ar + H 2 N Ar' Azo Hydrazo Amine

76 Case study for reduction reactions Bacterial reductases play a role in enterohepatic recirculation of nitro or azo containing drugs. Sufasalazine is an azo containing prodrug which is activated to sulfanilamine by intestinal bacteria

77 Case study oxidation-reduction

78 Case study oxidation-reduction

79 Case study for reduction of secondary alcohol

80 Hydrolysis

81 Hydrolytic Reactions Hydrolysis of Esters and Amides is catalyzed by widely distributed hydrolytic enzymes. Esters alcohols, phenols and carboxylic acids - Non-specific esterases (liver, plasma, kidney, and intestine) - Plasma pseudocholinesterases also participate - Hydrolysis of esters is major metabolic pathway for ester drugs Amides amines and carboxylic acids - Liver microsomal amidases, esterases and deacylases

82 Hydrolytic Reactions (Cont.) Aspirin is hydrolyzed to salicylic acid by plasma estrases to releas salicylic acid which have antiinflammatory effect. COOH O O CH 3 COOH OH OH O CH 3 Asprin (Acetylsalicyclic acid) Salicyclic Acid Acetic Acid Hydrolysis of procaine (local anesthetic drug) by esterases is faster (t 0.5 =1 min) than Hydrolysis of procainamide (antiarrhythmic agent) by amidases (t 0.5 =4 hours)

83 Hydrolytic Reactions esters bond is weaker than amide bond - The reactivity of ester and amide bond depend on how much the carbonyl carbon is electropositive - Nitrogen atom is less electronegative than oxygen, so it will be weaker electron withdrawing atom - Therefore, the carbonyl carbon attached to oxygen atom will be more electropositive, and more reactive toward nucleophilic attack of water molecule during hydrolysis.

84 Case study for hydrolytic reactions Dipivefrine is a prodrug of adrenaline, which is used to treat glaucoma. Dipivefrine: is a di-tertbutylcarboxy ester of adrenaline, thus it is more lipophilic and can better penetration through the corneal membrane then hydrolyzed to give the active form (adrenaline)

85 General notes for phase-i reactions Hydrolysis normally catalyzed by carboxylesterases: Cholinesterase. Hydrolyzes cholinelike esters (such as succinylcholine), procaine and acetylsalicylic acid. Arylcarboxyesterase. Liver carboxyesterase Zwitterion can be easily excreted

86 General notes for phase-i reactions (Cont.) Esters that are sterically hindered are hydrolyzed more slowly and may be appeared unchanged in urine Steric hindrance for estrases Amides are more stable to hydrolysis than esters.large fraction of amide containing drugs are normally excreted unchanged.

87 Bioactivation of omeprazole (case study) Proton-pump are present in parietal cells to exchange of K+ with H+ Proton-pump inhibitors are bioactivated next to the parietal cells (i.e. At highly acidic environment) Omeprazole (a proton-pump inhibitor) is activated by Protonation at benzimidazole ring followed by attachment of pyridine nitrogen to form another ring The new ring is opened and loses a water molecule to generate sulfonamide The sulfonamide is highly susceptible to nucleophilic attach by SH of cysteine residue of the proton pump which leads to pump damage.