NEPHAR 305 Pharmaceutical Chemistry I

Drug : Phase I

Assist.Prof.Dr. Banu Keşanlı

¾ Drug’s biochemical modification or degradation, usually through specialized enzymatic systems ¾ Xenobiotic: a chemical which is found in an organism but which is not normally produced or expected to be present in it ¾ Drug metabolism often converts lipophilic chemical compounds into more readily excreted polar products ¾ Duration and intensity of the pharmacological action of drugs is important ¾ Drug metabolism can result in toxication if the metabolite of a compound is more toxic than the parent drug or chemical ¾ or detoxication (process of preventing toxic entities from entering the body in the first place) by the activation or deactivation of the chemical

A prodrug is a pharmacological substance (drug) that is administered in an inactive (or significantly less active) form. Once administered, the prodrug is metabolised in vivo into an active metabolite. Importance of Drug Metabolism

Basic premise:

Lipophilic Drugs Hydrophilic Metabolites (Not excreted) (Excreted)

Water soluble increased renal excretion and Decreased tubular re-absorption of lipophilics Importance of Drug Metabolism

Metbolism Termination of Drug - Bioinactivation - Detoxification - Elimination

Metabolism Bioactivation - Active Metabolites - Prodrugs - Toxification

Phase I or Functionalization Reactions Oxidative Reactions • Oxidation of aromatic moieties • Oxidation of olefins • Oxidation at benzylic, allylic carbon, carbon atoms α to carbonyl and imines • Oxidation at aliphatic and alicyclic carbon • Oxidation involving carbon-heteroatom systems: Carbon-nitrogen systems (aliphatic and aromatic amines; N-dealkylation, oxidative deamination, N-oxide formation, N-hydroxylation) Carbon-oxygen systems (O-dealkylation) Carbon-sulfur systems (S-dealkylation, S-oxidation, and desulfuration) • Oxidation of alcohols and aldehydes • Other miscellaneous oxidative reactions

Reductive Reactions • Reduction of aldehydes and ketones • Reduction of nitro and azo compounds • Miscellaneous reductive reactions

Hydrolytic Reactions • Hydrolysis of esters and amides • Hydration of epoxides and arene oxides by epoxide hydrase Phase II or Conjugation Reactions

• conjugation • Sulfate conjugation • Conjugation with glycine, glutamine, and other amino acids • Glutathione or mercapturic acid conjugation • Acetylation • Methylation Transformation of Xenobiotics by Biological Systems ¾ Phase I and Phase II reactions are biotransformations of chemicals that occur during drug metabolism ¾ Oxidative biotransformations require both molecular oxygen and the reducing agent NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) ¾ One atom of molecular oxygen (O2) is introduced into the substrate R-H to form R-OH and the other oxygen atom is incorporated into H2O

OH

O2

NADPH Aromatic Hydroxylation ¾ Major route of metabolism for many drugs containing a phenyl group (and aromatic) ¾ Proceeds initially with an “arene oxide” intermediate ¾ Hydroxylation occurs at para position Activated rings – electron donating substituents Deactivated rings don’t get oxidized – toxic ¾ Dihydrol metabolite formation is possible ¾ Undergoes further conversions to polar soluble glucuronide or sulfate conjugates, which are readily excreted in the urine

(a) TethedralTetrahedral ara ürün H H intermediate O (b)

H

H O OH Oxidation Involving Carbon-Heteroatom Systems ¾ Hydroxylation of α-carbon atom attached to the heteroatom (N, O, S) results in unstable intermediate which decomposes via cleavage of carbon – heteroatom bond

¾ Hydroxylation or oxidation of heteroatom (N, O, S) could fall under these mechanisms: N-hydoxylation, N-oxide formation, sulfoxide, sulfone formation

¾ Structural factors determine the metabolic pathway – complicated

¾ Nitrogen functionalities such as amines, amides are found in natural products (, etc) and in numerous drugs (antihistamines, phenothiazine etc) Tertiary aliphatic and Alicyclic amines

Oxidative N-dealkylation: Oxidative removal of alkyl groups Small groups such as methyl, ethyl, isopropyl removed rapidly, t-butyl is impossible H H O O

NR C NR C R NH + C 1 α 1 α 1

R2 R2 R2 Secondary Carbonyl Tertiary amine Carbinolamine amine (aldehyde or ketone)

Antiestrogenic agent Tamoxifen (Nolvadex)

H3C H3C

NCH2CH2O O HNH2CH2CO H3C + C CH2CH3 CH2CH3 H H

Tamoxifen Secondary and Primary Amines • Oxidative N-dealkylation • Oxidative deamination • N-oxidation

9 Carbinolamine pathway to give corresponding primary amine metabolite through N-dealkylation

9 Which then is susceptible to oxidative deamination • initial α-C hydroxylation followed by C-N bond cleavage to give carbonyl metabolite and ammonia ¾ Metabolism of Propranolol both by direct deamination and deamination of its primary amine metabolite

OH OH OH H O O CH O CH O O CH C CH2 C CH C C H H 2 H2 2 HN HN H CH3 CH3 C C H H

CH CH3 Propranolol 3 Aldehyde metabolite NH Direct oxidative Carbinolamine 2 deamination

OH NH3 OH oxidative O CH deamination through primary amine C CH2 O CH H2 C CH2 H HN CH3 2 C NH2 O H

CH3 O Primary amine metabolite C (Desisopropyl Propranolol) Carbinolamine H3C CH3 N-Dealkylation

Oxidative Deamination ¾ Metabolic N-oxidation of secondary amine leads to N-oxygenated products, N-hydroxylamines which are susceptible to further oxidation giving nitrone metabolites - OH O + NH N N

CH 3 CH3 CH2

Secondary Hydroxylamine Nitrone amine

CF3 CH3 CF3 CH3 CF3 CH3 CH + CH HN CH3 N 3 - N 3 OH O Hydroxylation Nitrone ¾ Secondary amines undergo oxidative dealkylation and deamination more than N-oxidation ¾ Primary aliphatic amines are biotransformed by oxidative deamination or by N-oxidation ¾ Monoamine oxidase (MAO) are responsible for oxidative deamination ¾ Structural features, e.g. α-substituents determine whether C or N oxidation will occur • If no hydrogen atom on α-C then α-C hydroxylation is impossible Aromatic Amines and Heterocyclic Nitrogen Compounds

¾ Tertiary aromatic amines undergo oxidative N-dealkylation and N-oxide formation ¾ Secondary aromatic amines undergo N-dealkylation and N-oxide formation ¾ Primary amines undergo N-oxidation generating N-hydroxylamine metabolite ¾ Oxidation of hydroxylamine derivative to nitroso derivative is possible ¾ N-oxidation is minor compared to other biotransformations such as N-acetylation aromatic hydroxylation ¾ Conjugation pathways are observed (e.g. sulfate conjugation)

CH3 a Ar N Ar NHCH3

CH3 CH2OH Ar N

CH3 b CH3 + Ar N O-

CH3 Oxidation of Alcohols and Aldehydes

¾ Primary alcohols are oxidized to aldehydes which often undergo facile oxidation to generate polar carboxylic acid derivatives

¾ Secondary alcohols are oxidized to ketones – more likely to form conjugates or be reduced back to alcohol form

¾ Catalyzed by soluble alcohol dehydrogenase present in and other tissues

+ + RCH2OH + NAD RCHO + NADH + H

RCHO + NAD+ RCOOH + NADH

COOH

NH

H3C CH2OH

R=COH Mefenamic acid R=COOH Reductive Reactions

¾ Plays an important role in the metabolism of many compounds containing carbonyl, nitro and azo groups ¾ Bioreduction of carbonyl compounds generates alcohol derivatives ¾ Nitro and azo reductions lead to amino derivatives ¾ Hydroxyl and amino moieties of the metabolites are more susceptible to conjugation than the functional groups of the parent compounds ¾ Facilitate drug elimination

Reduction of Aldehydes and Ketones

¾ Carbonyl containing drugs and metabolites are common ¾ Aldehydes are more readily oxidized to carboxylic acids then reduced to alcohols ¾ Ketones are resistant to oxidation, mainly reduced to secondary alcohols ¾ Aldo-keto reductase enzymes are responsible for reduction ¾Bioreduction of ketones often leads to the creation of asymmetric center thus 2 possible stereoisomeric alcohols O CH3 CHCH2CH2 N CHCH2CH CH3 Cl CH2 Cl CH2

ChlorpheniramineKlorfeniramin AldehitAldehyde yapısımetabolitendaki metabolit

oxidationOksidasyon reductionRedüksiyon

CHCH2COOH CHCH2CH2OH

Cl CH2 Cl CH2 O HO H H OH C C C CH CH CH3 3 3 +

AcetophenoneAf S-(-)-Methyl R-(+)-Methyl Phenyl Carbinol Phenyl Carbinol

O OH H H OH C C C CH CH CH OH CH2 3 OH CH2 3 OH CH2 3

C C6H5 C C6H5 C C6H5 + H H H O O O O O O R(+)-Warfarin R,S-(+)-Alcohol R,R-(+)-Alcohol Major diastereomer Minor diastereomer Hydrolytic Reactions

Hydrolysis of Esters and Amides ¾Metabolism of ester and amide linkages in many drugs is catalyzed by hydrolytic enzymes in tissues and plasma ¾ Metabolic products formed (carboxylic acids, alcohols, phenols, amides) are polar ¾ Functionally more susceptible to conjugation and excretion than the parent ester or amide ¾ Hydrolysis is a major biotransformation pathway for ester functionality

¾ Many parent drugs have been chemically modified or derivatized to generate prodrugs to overcome some undesirable property (e.g. bitter taste, poor absorption, poor solubility, irritation at site of injection) ¾ Ester derivatives are ideal prodrug candidates ¾ Amides are hydrolyzed slower compared to esters COOH O COOH OCCH3 OH + CH3COOH Acetic acid Aspirin Salicylic acid (Acetylsalicylic acid)

O O O C OCH3 C OH C OCH CH N H 3 3 H O CH3 N H CH3 N C O OH + H O C H O H

Cocaine Benzoylecgonine Methylecgonine

O Slow hydrolysis H2NCNHCH2CH2N(C2H5)2 Yavaş hidroliz Prokainamit O Procainamide H2NCOH O H2NCOCH2CH2N(C2H5)2 HFastızlı hidroliz hydrolysis Prokain Procaine NEPHAR 305 Pharmaceutical Chemistry I

Drug Metabolism Phase II

Prof. Dr. Hakkı Erdoğan Assist.Prof. Banu Keşanlı PHASE II REACTIONS

1. Glucuronidation 2. Sulfate Conjugation 3. Acetylation 4. Amino Acid Conjugation 5. Methylation 6. Glutathione Conjugation Glucuronic Acid Conjugation Uridine-5’-α-D-glucuronic Acid C-1

¾ The microsomal glucuronyl transferase conducts the donation of glucuronic acid from the endogenously synthesized UDPGA to various substrates to form glucuronide conjugates. ¾ Examples of such substrates are morphine and acetaminophen. Glucuronic Acid Conjugation

¾ Most common conjugative pathway ¾ Greatly enhances water solubility ¾ Numerous functional groups can combine with it ¾ Readily available in body ¾ Has polar carboxyl and hydroxyl groups ¾ Products are called glucuronides RN-G; RO-G; RCOO-G; RS-G; RC-G glucuronides could form at C1 atom of β-glucuronide

¾ Phenolic and alcoholic hydroxyls are most common functional groups metabolized ¾ Glucuronidation is not fully developed in infants and children

C-1 Table 5.10. Substrates forming Glucuronides

(R. B. Silverman “The Organic Chemistry of Drug Design and Drug Action” 1992, s.331) Type Compound Formula a) O-Glucuridation Hydroxyl (ether glucuronide) Ace tam ino phen CH3CONH OH phenol OH H O2N N CHCl2 Alcohol Chloramphenicol O OH C6H5O CH3 Carboxyl (ester glucuronide) Fenoprofen OH O b) N- Glucuridation

Amine Desipramine NHCH 3 Amide OCONH2

Meprobamate CH Carbamate H3C 3 O CNH2 O

OCH3 Sulfonamide Sulfadimethoxine N H2N SO 2NH N OCH 3 c) S- Glucuridation N Methimazole Sulfahydryl N SH CH 3 (C2H5)2NSH Karboditiyonik asit Disulfiram S

(reduced metabolite) d) C- Glucuridation O C6H5 N Phenilbutazone N CH3 C6H5 O

Glucuronidation of Benzoic Acid

UGT= UDP-α-D-Glucuronsyltransferase Glucuronidation of Aniline Morphine Metabolism Morphine → Morphine -6-glucuronide (active metabolite) Morphine → Morphine -3-glucuronide (inactive metabolite)

1

2

4 5

Morphine -3-glucuronide is the major metabolite A small amount of morphine undergoes N-demethylation Sulfate Conjugation

3’-Phosphoadenosine-5’-phosphosulfate (PAPS)

¾The cytosolic enzyme sulfotransferase conducts the donation of sulfate from the endogenously synthesized PAPS to various substrates to form sulfate conjugates. ¾An example of such substrate is acetaminophen. Sulfate Conjugation

¾ Occurs primarily with phenols and occasionally with alcohols, aromatic amines and N-hydroxy compounds ¾ Sulfate amount in body is limited ¾ Leads to water soluble and inactive metabolites ¾ Glucuronidation of phenols is a competing reaction and may predominate Sulfate Conjugation

PAP: 3’-phosphoadenosine- 5’-phosphate

OH

HO R = H Albuterol R = SO3H Metabolite RO NHC(CH3)3 Acetylation N-Acetyltransferase

• A soluble enzyme • Isoniazid is a substrate • Genetic variation occurs – Some individuals are fast acetylators – Some individuals are slow acetylators • Acetyl coenzyme A is the endogenous donor molecule

¾ Important metabolic route for drugs containing primary amino groups

¾ Aromatic amines (ArNH2), sulfonamides (H2NC6H4SO2NHR), ¾ Hydrazine (-NHNH2), hydrazides (-CONHNH2) and primary aliphatic amines ¾ Gives inactive and nontoxic metabolites but does not enhance water solubility much Acetyl CoA

•Various acetylases, for examples, choline acetylase and N-acetyl transferase, all soluble enzymes, conduct the transfer of the acetyl group of acetyl CoA to various substrates. •For example, N-acetylation of isoniazid. Genetic polymorphism occurs with N- acetyltransferase. N-Acetyltransferase

N4

N1

(Antibacterial)

H O H H O N O N N O 7 7 7 N CH3 N O2N H2N N NH Cl Cl Cl

Klonazepam Clonazepam 7-Amino metaboliti 7-acetylamino7-Asetamino metaboliti metabolite 7-amino metabolite (anticonvulsant, muscle relaxant) Methylation S-Adenosylmethionine (SAM)

9 Cytosolic enzymes such as catechol-O-methyl transferase (COMT) and phenylethanolamine-N-methyl transferase (PNMT) conducts the donation of the methyl group from the endogenously synthesized SAM to various substrates to form methylated conjugates. 9 Norepinephrine is N-methylated by PNMT to form epinephrine. Norepinephrine, epinephrine, dopamine, and L-DOPA are O-methylated by COMT. Methyltransferases

• A family of soluble enzymes that conducts

– N-methylation; N-CH3

– O-methylation; O-CH3

– S-methylation; S-CH3 • S-adenosylmethionine (SAM)is the endogenous donor molecule. It is demethylated to S-adenosylhomocysteine Methylation 9 Methylation reactions play an important role in biosynthesis of many endogenous compounds and inactivation of numerous active biogenic amines 9 Minor pathway for conjugation of drugs and xenobiotics 9 Does not give polar, water soluble metabolites but pharmacologically inactive products 9 Catechols, phenols, amines and N-heterocyclic and thiol compounds 9 Substrates undergoing O-methylation by COMT must contain an aromatic1,2-dihydroxy group N-Methyltransferases PNMT- Phenylethanolamine-N-methyltransferase

Norepinephrine PNMT Epinephrine SAM

(Neurotransmitters) O-Methylation Of Catecholamines

COMT- catechol-O-methyltransferase O-Methylation of Norepinephrine

COMT- catechol-O-methyltransferase S-Methylation of 6-Mercaptopurine

(Immunosuppressive)

TPMT - thiopurinemethyltransferase; some individuals are deficient in this enzyme that is critically important for the metabolism of this agent AMINO ACID CONJUGATION

9 Amino acids, glycine and glutamine are used to conjugate carboxylic acids, particularly aromatic acids and arylalkyl acids 9 Carboxylic acid substrate is activated with ATP and coenzyme A (CoA) to form an acyl-CoA complex 9 Limited amount of amino acids in body is available so few conjugation reactions occur 9 Competes with conjugation with glucuronic acid 9 Polar and water soluble metabolites 9 Glycine conjugation occurs with aromatic acids and arylalkyl acids 9 Glutamine conjugation occurs with arylacetic acids

Glycine Glutamine AMINO ACID CONJUGATION

(mitochondria) Salicyluric Acid is the Glycine Conjugate of Aspirin

9 Multiple Metabolic Pathways Exist for Aspirin’s Metabolism

• Hydrolysis produces salicylic acid metabolite

Amide linkage

Salicylic acid

¾ Salicyluric acid, the glycine conjugate of salicylic acid, is the main metabolite of aspirin ¾ Approximately 76% of aspirin is metabolized through amino acid conjugation. Glutamine Conjugation

GlutamineGlutamin N-acyltransferaseN-açiltransferaz COOH O O OCH2CONHCHCH2CH2CONH2 COOH

N(CH3)2

DiphenhydramineDifenhidramin GlutamineGlutamin konjügatconjugateı Glutathione (GSH) Conjugation

¾ Important pathway for detoxifying chemically reactive electrophilic compounds ¾ Covalent interaction of metabolically generated electrophilic intermediates with cellular nucleophiles leads to drug ¾ GSH protects cellular constituents by bonding to metabolites via –SH group

Glutathione

¾ Xenobiotics conjugated with GSH usually are not excreted as such but undergo further biotransformation to give S-substituted N-acetylcysteine products called mercapturic acid ¾ Nucleophilic GSH reacts with electrophilic substrates * nucleophilic displacement at an electron deficient carbon or heteroatom * nucleophilic addition to an electron deficient double bond - ¾ Aliphatic and arylalkyl halides (Cl, Br, I), sulfates (OSO3 ),

sulfonates (OSO 2R),

¾ Nitro compounds (NO2), and organophosphates (OP[OR]2) have electron deficient carbon atoms to conjugate with GSH ¾ If not sufficiently electron deficient (not enough electron withdrawing groups) GSH conjugation does not take place

δ+ δ− + HX GSH + CH2 X GS-CH2

R R

R= Alkyl, aryl, benzyl, allylic - X = Br, Cl, I, OSO3 , OSO2R, OPO(OR)2 ¾ is metabolised primarily in the liver, into non-toxic products

acetaminophen Bioactivation of Acetaminophen Stereochemical Aspects of Drug Metabolism

¾ Many drugs often are administered as racemic mixtures in humans e.g. warfarin, propranolol, hexobarbital, ibuprofen, glutethimide ¾ May differ in pharmacological activity ¾ Individual enantiomers of a racemic drug often are metabolized at different rates and could be metabolized by different pathways ¾ Substrate stereoselectivity: a preference for one stereoisomer as a substrate for a ¾ Metabolizing enzyme or metabolic process ¾ Biotransformations could lead to new asymmetric center e.g. bioreduction of ketone xenobiotics Metabolism of Warfarin Enantiomers: 9 More active (S)(-) isomer is 7-hydroxylated (aromatic hydroxylation) 9 (R)(+) isomer undergoes keto reduction to yield (R,S) warfarin alcohol as the major plasma metbolite HO H C OH H2C CH3

C C6H5

H O 5 O O C R(+) enantiomer OH H C CH 2 3 R, S(+)-Alcohol C H

C6H5 O O O S(-) C Warfarin OH H2C CH3

(Anticoagulant) C H

C6H5

HO O O

7-Hydroxywarfarin *local anesthetic

• hydrolysis • Conjugation *non-steroidal anti-inflammatory drug

• Sulfoxide to sulfide reduction *antidepressant • N-demethylation Meperidine (ethyl 1-methyl-4-phenylpiperidine-4-carboxylate) is a narcotic analgesic drug which undergoes phase 1 and phase 2 reactions. Show its metabolites forming from possible metbolism pathways.

O

C2H5O

N

CH3