1. Organic Pharmaceutical Chemistry I Lec8: Phase I Reactions Oxidative Reactions By: S. L. Altahir Assistant Lecturer in Pharmaceutical Chemistry

2. THE OXIDATIVE REACTIONS Are classified as: 1.Oxidation of Aromatic Moieties 2.Oxidation of Olefins 3.Oxidation at Benzylic Carbon Atoms 4.Oxidation at Allylic Carbon Atoms 5.Oxidation at Carbon Atoms α to Carbonyls and Imines 6.Oxidation at Aliphatic and Alicyclic Carbon Atoms 7.Oxidation Involving Carbon–Heteroatom Systems a.OXIDATION INVOLVING CARBON–NITROGEN SYSTEMS: i.Tertiary Aliphatic and Alicyclic Amines. ii.Secondary and Primary Amines. iii.Aromatic Amines and Heterocyclic Nitrogen Compounds. iv.Amides. b.OXIDATION INVOLVING CARBON–OXYGEN SYSTEMS c.OXIDATION INVOLVING CARBON–SULFUR SYSTEMS 8.Oxidation of Alcohols and Aldehydes 9.Other Oxidative Biotransformation Pathways

3. 1- Oxidation of Aromatic Moieties Aromatic hydroxylation refers to the mixed-function oxidation of aromatic compounds (arenes) to their corresponding phenolic metabolites (arenols). Almost all aromatic hydroxylation reactions are believed to proceed initially through an epoxide intermediate called an “arene oxide” Arene oxide rearranges rapidly and spontaneously to the arenol product in most instances.

4. Most foreign compounds containing aromatic moieties are susceptible to aromatic oxidation. In humans, aromatic hydroxylation is a major route of metabolism for many drugs containing phenyl groups. Important therapeutic agents such as: propranolol, phenobarbital, phenytoin, phenylbutazone, atorvastatin, 17α-ethinylestradiol, and (S)(-)-warfarin, THE OXIDATIVE REACTIONS

5. 1- Oxidation of Aromatic Moieties What controls position for aromatic oxidation and some examples of aromatic oxidation? 1- For mono-substituted benzene rings, oxidation occurs majorly at the para position with minor oxidation at the ortho position. These hydroxylated metabolites then further undergo glucuronide or sulfate conjugation becoming hydrophilic and thus easily excreted out of the body. Examples of drugs undergoing this metabolic oxidation are Phenytoin(anticonvulsant), Warfarin (anticoagulant), Propranolol (antihypertensive), A torvastatin (lowers cholesterol), Phenobarbital (sedative hypnotic). the major urinary metabolite of phenytoin found in humans is the O-glucuronide conjugate of p- hydroxyphenytoin. (polar and water soluble) Most phenolic metabolites formed from aromatic oxidation undergo further conversion to polar and water soluble glucuronide or sulfate conjugates, which are readily excreted in the urine.

7. 1- Oxidation of Aromatic Moieties 2- The presence of substituents on the aromatic ring also influence the process of hydroxylation. Electron withdrawing groups such as Cl, COOH, SO 2 NHR, -N + R 3 slow down or resistant to the process whereas electron donating groups (OH, OCH 3, NH 2 ) accelerate the process of oxidation. Examples of aromatic compounds with electron withdrawing groups are Clonidine-antihypertensive agent (- Cl groups) and Probenecid-uricosuric agent (-SO 2 NHR and -COOH). Probenecid has been reported to not undergo hydroxylation at all.

8. 1- Oxidation of Aromatic Moieties 3- If there are two or more phenyl rings, hydroxylation proceeds in the electron rich ring. For example, Diazepam (anti-anxiety) has two phenyl rings in its structure, but hydroxylation occurs primarily in the electron rich ring as the other ring has an electron withdrawing group (-Cl) attached. Other examples of such oxidation are Chlorpromazine (antipsychotic agent), p-Chlorobiphenyl etc.

9. 4- There are certain compounds which are resistant to aromatic hydroxylation despite of the aromatic rings. This is because of the presence of multi electronegative chlorine atoms attached to the rings. Thus these compounds escape metabolism and are present in the body for a longer time due to their lipophilic property. Examples are Polychlorinated biphenyls (PCBs) and 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). These compounds are present as pollutants and are toxic thus increasing the risks of health hazards. 1- Oxidation of Aromatic Moieties

10. The metabolic oxidation of olefinic carbon–carbon double bonds leads to the corresponding epoxide. Epoxides derived from olefins generally tend to be somewhat more stable than the arene oxides formed from aromatic compounds. A few epoxides are stable enough to be directly measurable in biological fluids (e.g., plasma, urine). Like their arene oxide counterparts, epoxides are susceptible to enzymatic hydration by epoxide hydrase to form trans-1,2- dihydrodiols (also called 1,2-diols or 1,2-dihydroxy compounds). In addition, several epoxides undergo GSH conjugation. 2- Oxidation of Olefins

11. A well-known example of olefinic epoxidation is the metabolism, in humans, of the anticonvulsant drug carbamazepine (Tegretol ® ) to carbamazepine-10,11- epoxide. Subsequent hydration of the epoxide produces 10,11- dihydroxycarbamazepine, an important urinary metabolite (10%–30%) in humans. Oxidation of Olefins

12. Epoxidation of the olefinic 10,11-double bond in the H1- histamine antagonist cyproheptadine (Periactin) also occurs. Frequently, the epoxides formed from the biotransformation of an olefinic compound are minor products, because of their further conversion to the corresponding 1,2-diols. Indirect evidence for the formation of epoxides comes also from the isolation of GSH or mercapturic acid metabolites. Oxidation of Olefins

13. After administration of styrene to rats, two urinary metabolites were identified as the isomeric mercapturic acid derivatives resulting from nucleophilic attack of GSH on the intermediate epoxide. *

14. 2- Oxidation of Olefins The toxicity of some olefinic compounds may result from their metabolic conversion to chemically reactive epoxides. as a biotoxification pathway involves aflatoxin B1 which combine to RNA or DNA, protein and kill the cell. Other olefinic compounds, such as vinyl chloride, stilbene, and the carcinogenic estrogenic agent diethylstilbestrol (DES). An interesting group of olefin-containing compounds causes the destruction of CYP. Compounds belonging to this group include allyl isopropylacetamide, secobarbital, and the volatile anesthetic agent fluroxene. This olefinic moiety present in these compounds is activated metabolically by CYP to form a very reactive intermediate that covalently binds to the heme portion of CYP.

16. 2- Oxidation of Olefins

17. Carbon atoms attached to aromatic rings (benzylic position) are susceptible to oxidation, thereby forming the corresponding alcohol metabolite. Primary alcohol metabolites are often oxidized further to aldehydes and carboxylic acids (CH 2 OH → CHO → COOH), and secondary alcohols are converted to ketones. Alternatively, the alcohol may be conjugated directly with glucuronic acid. The selective cyclooxygenase 2 (COX-2) anti- inflammatory agent celecoxib undergoes benzylic oxidation at its C-5 methyl group to give hydroxy celecoxib as a major metabolite. 3- Oxidation at Benzylic Carbon Atoms

18. Significant benzylic hydroxylation occurs in the metabolism of the β-adrenergic blocker metoprolol (Lopressor ® ) to yield α-hydroxymetoprolol.

19. Additional examples of drugs and xenobiotics undergoing benzylic oxidation

20. Microsomal hydroxylation at allylic carbon atoms is commonly observed in drug metabolism. Example: the psychoactive component of marijuana, 1- tetrahydrocannabinol 1-THC. This molecule contains three allylic carbon centres (C-7, C- 6, and C-3). Allylic hydroxylation occurs extensively at C-7 to yield 7- hydroxy- 1-THC as the major plasma metabolite in humans. - Pharmacological studies show that allylic hydroxylation at C7 give 1° alcohol ( major active metabolite ) or even more active than, 1-THC and may contribute significantly to the overall CNS psychotomimetic effects of the parent compound. Hydroxylation also occurs to a minor extent at the allylic C-6 position to give both the epimeric 6-α and 6 -hydroxy metabolites (2° alcohol). Metabolism does not occur at C-3, presumably because of steric hindrance. 4- Oxidation at Allylic Carbon Atoms

22. The antiarrhythmic agent quinidine is metabolized by allylic hydroxylation to 3- hydroxyquinidine, the principal plasma metabolite found in humans. This metabolite shows significant antiarrhythmic activity in animals and possibly in humans. Other examples of allylic oxidation include the sedative–hypnotic hexobarbital (Sombulex ® ) and the analgesic pentazocine (Talwin ® ). The 3-hydroxylated metabolite formed from hexobarbital is susceptible to glucuronide conjugation as well as further oxidation to the 3-oxo compound. Hexobarbital is a barbiturate derivative that exists in two enantiomeric forms. Studies in humans indicate that the pharmacologically less active (R)(-) enantiomer is metabolized more rapidly than its (S)(+)-isomer. Pentazocine undergoes allylic hydroxylation at the two terminal methyl groups of its N-butenyl side chain to yield either the cis or trans alcohol metabolites. 4- Oxidation at Allylic Carbon Atoms

23. For the hepatocarcinogenic agent safrole, allylic hydroxylation is involved in a bioactivation pathway leading to the formation of chemically reactive metabolites. This process involves initial hydroxylation at the C-1carbon of safrole, which is both allylic and benzylic. The hydroxylated metabolite then undergoes further conjugation to form a sulfate ester. This chemically reactive ester intermediate presumably undergoes nucleophilic displacement reactions with DNA or RNA in vitro to form covalently bound adducts. 4- Oxidation at Allylic Carbon Atoms

24. As shown in the scheme, nucleophilic attack by DNA, RNA, or other nucleophiles is facilitated by a good leaving group (e.g., SO4 2- ) at the C-1 position. The leaving group tendency of the alcohol OH group itself is not enough to facilitate displacement reactions. Importantly, allylic hydroxylation generally does not lead to the generation of reactive intermediates except in biotoxification of safrole agent. 4- Oxidation at Allylic Carbon Atoms

25. The mixed-function oxidase system also oxidizes carbon atoms adjacent (i.e., α) to carbonyl and imino (C=N) functionalities. An important class of drugs undergoing this type of oxidation is the benzodiazepines. For example, diazepam (Valium ® ), flurazepam (Dalmane ® ), and nimetazepam are oxidized to their corresponding 3- hydroxy metabolites. Oxidation at Carbon Atoms α to Carbonyls and Imines

26. Hydroxylation of the carbon atom to carbonyl functionalities generally occurs only to a limited extent in drug metabolism. An example involves the hydroxylation of the sedative– hypnotic glutethimide (Doriden) to 4-hydroxyglutethimide

27. Alkyl or aliphatic carbon centres are subject to mixed-function oxidation. Metabolic oxidation at the terminal methyl group often is referred to as ω-oxidation, and oxidation of the penultimate carbon atom (i.e., next-to-the-last carbon) is called ω–1 oxidation. The initial alcohol metabolites formed from these enzymatic ω and ω–1 oxidations are susceptible to further oxidation to yield aldehyde, ketones, or carboxylic acids. Alternatively, the alcohol metabolites may undergo glucuronide conjugation. 6- Oxidation at Aliphatic and Alicyclic Carbon Atoms 6- Oxidation at Aliphatic and Alicyclic Carbon Atoms

28. Aliphatic ω and ω–1 hydroxylations commonly take place in drug molecules with straight or branched alkyl chains. Thus, the antiepileptic agent valproic acid (Depakene ® ) undergoes both ω and ω–1 oxidation to the 5-hydroxy and 4-hydroxy metabolites, respectively. Further oxidation of the 5-hydroxy metabolite yields 2-n-propylglutaric acid. The sedative–hypnotic amobarbital undergoes extensive ω–1 oxidation to the corresponding 3′-hydroxylated metabolite. Omega and ω–1 oxidation of the isobutyl moiety present in the anti- inflammatory agent ibuprofen (Brufen ® ) yields the corresponding carboxylic acid and tertiary alcohol metabolites. Oxidation at Aliphatic and Alicyclic Carbon Atoms Oxidation at Aliphatic and Alicyclic Carbon Atoms

29. Additional examples of drugs reported to undergo aliphatic hydroxylation include meprobamate, glutethimide, ethosuximide, and phenylbutazone.

30. Oxidation at Aliphatic and Alicyclic Carbon Atoms Oxidation at Aliphatic and Alicyclic Carbon Atoms 6.Alicyclic (nonaromatic ring) Hydroxylation The cyclohexyl group is commonly found in many medicinal agents, and is also susceptible to mixed-function oxidation (alicyclic hydroxylation). mixed function oxydase tend to hydroxylate at the 3 or 4 position of the ring to give cis and trans stereo isomers. trans- 4-hydroxyacetohexamide

31. Nitrogen and oxygen functionalities are commonly found in most drugs and foreign compounds; sulfur functionalities occur only occasionally. Metabolic oxidation of carbon–nitrogen, carbon–oxygen, and carbon–sulfur systems principally involves two basic types of biotransformation processes: 1.Hydroxylation of the α-carbon atom attached directly to the heteroatom (N, O, S). The resulting intermediate is often unstable and decomposes with the cleavage of the carbon–heteroatom bond: 2. Hydroxylation or oxidation of the heteroatom (N, S only, e.g., N-hydroxylation, N- oxide formation, sulfoxide, and sulfone formation). Oxidation Involving Carbon–Heteroatom Systems:

32. Metabolism of nitrogen functionalities (e.g., amines, amides) is important because such functional groups are found in many natural products (e.g., morphine, cocaine, nicotine) and in numerous important drugs (e.g., phenothiazines, antihistamines, tricyclic antidepressants, β-adrenergic agents, sympathomimetic phenylethylamines, benzodiazepines). Oxidation Involving Carbon–Nitrogen Systems: Oxidation Involving Carbon–Nitrogen Systems:

33. This system can be divided into three basic classes: 1.Aliphatic (primary, secondary, and tertiary) and alicyclic (secondary and tertiary) amines. 2.Aromatic and heterocyclic nitrogen compounds 3.Amides Oxidation Involving Carbon–Nitrogen Systems: Oxidation Involving Carbon–Nitrogen Systems:

34. 1- Tertiary Aliphatic and Alicyclic Amines The oxidative removal of alkyl groups (particularly methyl groups) from tertiary aliphatic and alicyclic amines is carried out by hepatic CYP mixed-function oxidase enzymes. This reaction is commonly referred to as oxidative N-dealkylation. The N-hydroxylation or N-oxidation reactions, however, appear to be catalyzed not only by CYP but also by a second class of hepatic mixed-function oxidases called amine oxidases (sometimes called N- oxidases. These enzymes are NADPH-dependent flavoproteins and do not contain CYP. They require NADPH and molecular oxygen to carry out N-oxidation

35. STEPS 1- The initial step involves α-carbon hydroxylation to form a carbinolamine intermediate, which is unstable. 2- carbinolamine undergoes spontaneous heterolytic cleavage of the C–N bond to give a secondary amine and a carbonyl moiety (aldehyde or ketone). 1- Tertiary Aliphatic and Alicyclic Amines 1- Tertiary Aliphatic and Alicyclic Amines

36. Secondary and Primary Amines Secondary amines (either parent compounds or metabolites) are susceptible to oxidative N-dealkylation, oxidative deamination, and N-oxidation reactions. As in tertiary amines, N-dealkylation of secondary amines proceeds by the carbinolamine pathway. Dealkylation of secondary amines gives rise to the corresponding primary amine metabolite. Primary amines (whether parent drugs or metabolites) are biotransformed by oxidative deamination (through the carbinolamine pathway) or by N-oxidation. Endogenous primary amines (e.g., dopamine, norepinephrine, tryptamine, and serotonin) and xenobiotics based on the structures of these endogenous neurotransmitters are metabolized, however, via oxidative deamination by a specialized family of enzymes called monoamine oxidases (MAOs)

37. Secondary and Primary Amines

38. Secondary and Primary Amines Secondary and Primary Amines N-oxidation appears to be the major biotransformation pathway for primary aliphatic amines, such as phentermine, chlorphentermine (p-chlorphentermine), and amantadine, because α-carbon hydroxylation cannot occur. In humans, chlorphentermine is N-hydroxylated extensively. Metabolic N-oxidation of secondary aliphatic and alicyclic amines leads to several N-oxygenated products. N-hydroxylation of 2˚ amines generates the N- hydroxylamine metabolites. Often, these hydroxylamine products undergose to further oxidation (either spontaneous or enzymatic) to the corresponding nitrone derivatives.

40. The biotransformation of aromatic amines parallels the carbon and nitrogen oxidation reactions seen for aliphatic amines. For tertiary aromatic amines, such as N,N-dimethylaniline, oxidative N-dealkylation as well as N-oxide formation take place. Secondary aromatic amines may undergo N-dealkylation or N-hydroxylation to give the corresponding N- hydroxylamines. Further oxidation of the N-hydroxylamine leads to nitrone products, which in turn may be hydrolyzed to primary hydroxylamines. Aromatic Amines and Heterocyclic Nitrogen Compounds:

42. Amides Amides  Amide functionalities are susceptible to oxidative carbon–nitrogen bond cleavage (via α-carbon hydroxylation) and N-hydroxylation reactions.  Oxidative dealkylation of many N-substituted amide drugs and xenobiotics has been reported. As oral hypoglycemic chlorpropamide.  Mechanistically, oxidative dealkylation proceeds via an initially formed carbinolamide, which is unstable and fragments to form the N-dealkylated product.  For example, diazepam undergoes extensive N-demethylation to the pharmacologically active metabolite desmethyldiazepam.

43. Oxidation Involving Carbon–Oxygen Systems Oxidative O-dealkylation of carbon–oxygen systems (principally ethers) is catalysed by microsomal mixed function oxidases. Steps : 1- the biotransformation involves an initial α-carbon hydroxylation to form either a hemiacetal or a hemiketal, 2- which undergoes spontaneous carbon–oxygen bond cleavage to yield the dealkylated oxygen species (alcohol) and a carbon moiety (aldehyde or ketone).

44. Oxidation Involving Carbon–Oxygen Systems

45. Oxidation Involving Carbon–Sulfur Systems Carbon–sulfur functional groups are susceptible to metabolic S- dealkylation, desulfuration, and S-oxidation reactions. The first two processes involve oxidative carbon–sulfur bond cleavage. S-dealkylation is analogous to O- and N-dealkylation mechanistically (i.e., it involves α-carbon hydroxylation) and has been observed for various sulfur xenobiotics. Desulfuration is Oxidative conversion of carbon–sulfur double bonds (C=S) (thiono) to the corresponding carbon–oxygen double bond (C=O).

46. Oxidation Involving Carbon–Sulfur Systems A well-known drug example of this metabolic process is the biotransformation of thiopental to its corresponding oxygen analogue pentobarbital. Organosulfur xenobiotics commonly undergo S-oxidation to yield sulfoxide derivatives. For example, Several phenothiazine derivatives as in thioridazine (Mellaril) and also The H2-histamine antagonists cimetidine (Tagamet) and metiamide. - Sulfoxide metabolites, such as those of thioridazine, reportedly undergo further oxidation to their sulfone -SO2- derivatives.

47. . Oxidation Involving Carbon–Sulfur Systems

48. Many oxidative processes (e.g., benzylic, allylic, alicyclic, or aliphatic hydroxylation) generate alcohol or carbinol metabolites as intermediate products. If not conjugated, these alcohol products are further oxidized to aldehydes (if primary alcohols) or to ketones (if secondary alcohols). The bioconversion of alcohols to aldehydes and ketones is catalysed by soluble alcohol dehydrogenases present in the liver and other tissues. NAD+ is required as a coenzyme, although NADP+ also may serve as a coenzyme. Oxidation of Alcohols and Aldehydes:

49. In addition to the many oxidative biotransformations discussed previously, oxidative aromatization or dehydrogenation and oxidative dehalogenation reactions also occur. Metabolic aromatization has been reported for norgestrel. Aromatization or dehydrogenation of the A ring present in this steroid leads to the corresponding phenolic product 17α-ethinyl-18-homoestradiol as a minor metabolite in women.

50. Many halogen-containing drugs and xenobiotics are metabolized by oxidative dehalogenation. For example, the volatile anesthetic agent halothane is metabolized principally to trifluoroacetic acid in humans. It has been postulated that this metabolite arises from CYP-mediated hydroxylation of halothane to form an initial carbinol intermediate that spontaneously eliminates hydrogen bromide (dehalogenation) to yield trifluoroacetyl chloride. The latter acyl chloride is chemically reactive and reacts rapidly with water to form trifluoroacetic acid.

51. Another example of oxidative dehalogenation concerns the antibiotic chloramphenicol. In vitro studies have shown that the dichloroacetamide portion of the molecule undergoes oxidative dechlorination to yield a chemically reactive oxamyl chloride intermediate that can react with water to form the corresponding oxamic acid metabolite or can acylate microsomal proteins. Thus, it appears that in several instances, oxidative dehalogenation can lead to the formation of toxic and reactive acyl halide intermediates.

53. What would be the primary metabolite of a phase I reaction for the following drug molecules?