and easy accessibility to the ester groups ensure that rapid hydrolysis will occur (Figure 7.44).
when it entered the body. However, simply adding a single OH or NH2 group to a large molecule is generally not a sufficient structural change to alter its log P enough so that the molecule becomes water soluble and can be excreted.
This is the job of phase II metabolism. In phase II, parent compounds and phase I metabolites are generally altered by attaching a highly polar group to appropriate functionality. The resulting modified compounds thereby gain suf- ficient polarity to render them water soluble so they can be excreted, usually in the urine. The process of attaching another molecule to a substrate is known as conjugation. Among the more common classes of phase II metabolism are:
• Glucuronide conjugation • Sulfate conjugation
• Glycine and glutamate conjugation • Glutathione (GSH) conjugation • Acetylation
• Methylation
In this section of the chapter we will explore conjugation reactions beginning with glucuronide formation. Glucuronides are among the most common of phase II metabolites. Functional groups that are susceptible to conjugation with glucuronic acid include alcohols, phenols, carboxylic acids, amines, and thiols.
O
OH OH HOHO
HO2C
β-Glucuronic acid
Glucuronic acid is made in the body from the highly abundant sugar d-glu- cose (Figure 7.45). Glucose is often converted in the body into a phosphate ester, α-d-glucose-1-phosphate. This is a substrate for the enzyme uridine tri- phosphate phosphorylase, which appends a uridine-5′-phosphate to the existing phosphate to form uridine-5′-diphosphate-α-d-glucose (UDPG). This in turn is a substrate for another enzyme, UDPG dehydrogenase, which oxidizes the primary alcohol to the carboxylic acid, uridine-5′-diphospho-α-d-glucuronic acid. Examination of this structure reveals a tetrahydropyran (cyclic six-membered ether) having a carbon that is attached to the ring oxygen also attached to another OR group, in this case an O-diphosphate-uridine. This cre- ates a functional group known as an acetal. Acetals (sp3 carbons bearing two OR groups) are derived from aldehydes and are subject to attack by nucleophiles in
HOH2C O HOHO
OH O
P
O O
OH
O HOH2C HOHO
OH O
P
O O
O
NH O
O N
O
OH OH
H H
H H
P O O
O α-D-glucose-
1-phosphate Uridine triphosphate phosphorylase
Uridine-5'- diphosphate- α-D-Glucose (UDPG)
UDPG dehydrogenase
O HO2C HOHO
OH O
P
O O
O
NH O
O N
O
OH OH
H H
H H
P O O
O
Uridine-5'-diphospho-α-D-glucuronic acid (UDPGA)
R XH
R-XH
UDP-glucuronyl transferase
HO2C O HOHO
OH X
R X = O, N, or S
O P
O O
O
NH O
O N
O
OH OH
H H
H H
P O O
O +
A β-glucuronide conjugate
Figure 7.45 The formation of conjugates of glucuronic acid begins with α-d-glucose. Glu- cose is often converted in the body into its 1-phosphate. When needed, the phosphate becomes phosphorylated by a uridine phosphate to form UDPG. The primary alcohol on the glucose ring is then oxidized by UDPG dehydrogenase to form a phosphorylated glucuronic acid. Nucleophilic functional groups such as alcohols, phenols, carboxylic acids, amines, and thiols react at the acetal carbon of the sugar, inverting the configuration at that site to give a β-glucuronic acid conjugate (glucuronide). This is catalyzed by UDP-glucuronyl transferase.
the presence of a catalyst, with displacement of one of the OR groups. In this case the diphosphate-uridine group renders the oxygen to which it is attached into a good leaving group. Thus in the presence of a nucleophile such as an alcohol, amine, carboxylic acid, or thiol, the enzyme uridine-5′-diphosphate- glucuronyl transferase catalyzes the displacement of the O-diphosphate-uridine group to form a β-glucuronide conjugate.
Morphine is an example of a parent compound that undergoes extensive glucuronide conjugation on the phenol (Figure 7.46). Note the change in the calculated log P values for morphine (ClogP = +0.57) and its 3-glucuronide conjugate (ClogP = −3.68). Thus attaching the highly polar sugar group with its three secondary alcohols and one carboxylic acid group makes the com- pound highly water soluble allowing for its excretion in the urine. Figure 7.47 shows the sequential phase I and, phase II metabolism of diazepam. The parent compound undergoes α-oxidation at the 3-position to form an alcohol. The OH group lowers the ClogP value from 2.96 for the parent compound to 2.34. In phase II, the alcohol is converted into a glucuronide conjugate and it is seen that the ClogP value decreases substantially to 0.76.
Sulfate conjugates are formed mainly from phenols, although they can also be formed from alcohols and aromatic amines. In the body sul- fate is less available than glucuronic acid and so these conjugates are generally formed to a lesser extent than glucuronides. Sulfate needs to
N H CH3
O OH
HO
N H CH3
O OH
O O HO2C HOHO
OH
ClogP: 0.57 ClogP: – 3.68
UDPGA
Morphine Morphine-3-glucuronide
Figure 7.46 Morphine is an example of a parent drug that undergoes phase II metabo- lism by forming a glucuronide conjugate from its phenol group. Note that in the process the calculated log P value (ClogP) decreases from +0.57 for morphine to −3.68 for the conjugate, making it substantially more water soluble. (ClogP values were determined using ChemBioDraw Ultra 13.0, Perkin Elmer Informatics, Waltham, MA).
be activated before it can react with nucleophiles. This is done by attach- ing a good leaving group to the sulfate moiety. This is an adenosine phosphate group and the resulting cofactor is called 3′-phosphoadenosine- 5′-phosphosulfate (Figure 7.48). The enzyme sulfotransferase catalyzes the attack of nucleophilic groups on the sulfur, releasing 3′,5′-adenosine diphosphate and producing the sulfate conjugate of the nucleophile.
Acetaminophen is an example of a drug that forms a sulfate conjugate, particularly in children in whom this is the major urinary metabolite.
This is shown in Figure 7.49. As with glucuronides, the sulfate conjugate has a substantially lower ClogP value (−0.84) as compared to the parent compound (ClogP = +0.49).
The amino acids glycine and glutamine form conjugates with carboxylic acids. The products are known as carboxyamides and are more water soluble than the original carboxylic acids. Chemically the reaction is attack of a
N N H3C O
Cl N
N H3C O
Cl
OH Phase I
α-oxidation
ClogP: 2.96 ClogP: 2.34
N N H3C O
Cl
O
ClogP: 0.76 O HO HO
COOH2H
Diazepam 3-Hydroxydiazepam
3-Hydroxydiazepam glucuronide Phase II
UDPGA
Figure 7.47 Diazepam is an example of a drug that first undergoes phase I α-oxidation to its 3-hydroxy metabolite and then phase II glucuronide conjugation of the alcohol.
Addition of the alcohol group in phase I lowers the ClogP value only slightly from +2.96 to +2.34. Conjugation with glucuronic acid, however, lowers the ClogP substantially to +0.76. (ClogP values were determined using ChemBioDraw Ultra 13.0, Perkin Elmer Informatics, Waltham, MA).
N
N N N
NH2
O
OH O
H H
H H
P O
O– O
O– P
O S
O O S
O
O O
X R-XH R
Sulfotransferase
X = O, N H
X R
O O O
3'-Phosphoadenosine-5'-phosphosulfate (PAPS)
Sulfate conjugate
Figure 7.48 Phenols and occasionally alcohols and aromatic amines can serve as nucleophiles in reactions with 3′-phosphoadenosine-5′-phosphosulfate (PAPS). They attack the sulfur displacing an adenosine diphosphate to form, after loss of a proton, a sulfate conjugate. The process is catalyzed by sulfotransferase.
HN CH3
O
OH
HN CH3
O
O S
O O
O
ClogP: – 0.84 ClogP: 0.49
PAPS Sulfotransferase
Acetaminophen
Acetaminophen-O-sulfate (major urinary metabolite formed in children)
Figure 7.49 Acetaminophen has a phenol group that is subject to phase II metabolic conversion into a sulfate conjugate. This changes the ClogP value from +0.49 in the drug to −0.84, increasing the water solubility. This is the major urinary metabolite found in children in whom acetaminophen was administered. (ClogP values were determined using ChemBioDraw Ultra 13.0, Perkin Elmer Informatics, Waltham, MA).
nucleophile (the amino group from the amino acid) on the carbonyl of the carboxylic acid with displacement of OH. For this to be favorable the OH must be activated, since OH− is a relatively poor leaving group. This is done in two steps (Figure 7.50). First the acid is phosphorylated by adenosine tri- phosphate to form an acyl monophosphate. This then reacts with the
N
N N N
NH2
O
OH O
H H
H H
P O
O– O
O– P
O P O NH
O HO O OH HO
NH O
O
SH Coenzyme A
(HSCoA)
O
R OH
ATP
O
R OAMP
O
R SCoA
HSCoA
An acyl phosphate An acyl CoA-complex A carboxylic acid
R'
H2N CO2H O
R N
H R'
CO2H Carboxyamides R' = H (Glycine)
R' = CH2CH2CONH2 (Glutamine) H2N
R
Figure 7.50 Top: structure of coenzyme A, showing the terminal thiol group, which is its most nucleophilic site. Bottom: carboxylic acids are converted by adenosine triphosphate (ATP) into acyl monophosphates. Coenzyme A displaces the phosphate group to form an acyl-CoA complex which is essentially a thioester. Thioesters make good substrates for nucleophilic attack because the sulfur is a good leaving group. Certain amino acids, including glycine and glutamine, can attack the acyl-CoA complex with their amino groups to form carboxyamides, which are amino acid conjugates of the carboxylic acids.
excellent nucleophile, coenzyme A (CoA), which contains a terminal thiol group. The resulting product is an acyl-CoA complex, but chemically it is a thioester. Thioesters react readily with nucleophiles with the elimination of a thioalkoxide. These make good leaving groups because sulfur can stabilize a negative charge due to its high polarizability (Chapter 1). The amino groups of the amino acids are the nucleophiles in this reaction and the process is catalyzed by an N-acyltransferase. One compound that undergoes this phase II process is the NSAID salicylic acid (Figure 7.51). The carboxylic acid group
is conjugated with glycine in the presence of N-acyltransferase to form the conjugate. The resulting carboxyamide is more water soluble than salicylic acid as determined by ClogP (1.28 vs 2.19 for the parent compound).
GSH conjugates are usually formed as a means of detoxification of reactive electrophilic species. Most often these electrophiles are formed during phase I metabolism by oxidative processes. Metabolites such as epoxides and arene oxides, as discussed earlier in this chapter, are highly reactive but are usually rendered harmless by reaction with water cata- lyzed by epoxide hydrolase, to form trans-dihydrodiols. Occasionally, how- ever, such epoxides may have sufficient half-life so that other nucleophiles, such as functional groups associated with proteins, enzymes, or nucleic acids, are able to react before water resulting in covalently modified bio- molecules with altered functionality. GSH is produced in the body to counter such threats. This is a tripeptide formed from glutamic acid, gly- cine, and cysteine, with the thiol group of cysteine being the most nucleo- philic site.
OH ClogP: 2.19
OH
ClogP: 1.28 HN
O
CO2H OH
O
Salicyclic acid Salicyclylglycineamide 1) ATP
2) HSCoA 3) Glycine
Figure 7.51 Salicylic acid is converted into a glycine conjugate mediated by ATP and coenzyme A. The resulting conjugate has a substantially lower ClogP value than salicylic acid. (ClogP values were determined using ChemBioDraw Ultra 13.0, Perkin Elmer Infor- matics, Waltham, MA).
HS
HN H
O
NH2
HO2C H
O NH
CO2H Glutathione Cysteine
Glutamic acid
Glycine
When an electrophilic metabolite is formed GSH is produced in sufficient concentration so that reaction with it becomes more likely than reaction with an essential nucleophilic biomolecule (Figure 7.52). The reaction between GSH and the electrophile is catalyzed by the enzyme glutathione S-transferase (GST).
HS
HN H
O
NH2 HO2C H
O NH
CO2H
S
HN H
O
NH2 HO2C H
O NH
CO2H Electrophile (E)
E
Glutathione S-transferase
Glutathione conjugate E
Cleavage
S
NH2 H
O NH
CO2H E
Cleavage S
NH2
CO2H H E
Degradation
Degradation
S
HN
CO2H H E
O CH3
N-acetylation
–H+
Figure 7.52 The thiol group of GSH reacts with electrophilic metabolites in the pres- ence of GST to form a conjugate. These usually undergo degradation by cleavage of the glutamic acid and then the glycine residues. The resulting S-modified cysteine is usually acetylated on the amine to give the final, deactivated conjugate.