Steroids

The two pathways leading to terpenoids are described: the mevalonate pathway and the recently discovered mevalonate-independent pathway via deoxyxylulose phosphate. Terpenoids may be classified according to the number of isoprenoid units incorporated, and hemiterpenes, monoterpenes and the variants irregular monoterpenes and iridoids, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes, and higher terpenoids are described in turn, representing groups with increasing numbers of isoprene units. Structures are rationalized through extensive use of carbocation mechanisms and subsequent Wagner-Meerwein rearrangements. Steroids as examples of modified triterpenoids are discussed in detail, including stereochemistry and molecular shape. There follows specific consideration of cholesterol, steroidal saponins, cardioactive glycosides, phytosterols, vitamin D, bile acids, corticosteroids and their semi-synthesis, progestogens, oestrogens, and androgens. Monograph topics giving more detailed information on medicinal agents include volatile oils, pyrethrins, valerian, feverfew, chamomile and matricaria, Artemisia annua and artemisinin, gossypol, trichothecenes, Taxus brevifolia and taxol, Ginkgo biloba, forskolin, liquorice, quillaia, ginseng, vitamin A, cholesterol, dioscorea, fenugreek, sisal, sarsaparilla, yucca, Digitalis purpurea, Digitalis lanata, strophanthus, convallaria, squill, soya bean sterols, fusidic acid, vitamin D, bile acids, corticosteroid drugs, progestogen drugs, oestrogen drugs, aromatase inhibitors, oestrogen receptor antagonists, and androgen drugs.

The terpenoids form a large and structurally diverse family of natural products derived from C5 isoprene units (Figure 5.1) joined in a head-to-tail fashion. Typical structures contain carbon skeletons represented by (C5)„, and are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterter-penes (C25), triterpenes (C30) and tetraterpenes (C40) (Figure 5.2). Higher polymers are encountered in materials such as rubber. Isoprene itself (Figure 5.1) had been characterized as a decomposition product from various natural cyclic hydrocarbons, and was suggested as the fundamental building block for these compounds, also referred to as 'isoprenoids'. Isoprene is produced naturally but is not involved in the formation of these compounds, and the biochemically active isoprene units were identified as the diphosphate (pyrophosphate) esters dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) (Figure 5.2). Relatively few of the natural terpenoids conform exactly to the simple concept of a linear head-to-tail combination of isoprene units as seen with geraniol (Cio), farnesol (Ci5), and geranylgeraniol (C20) (Figure 5.3). Squa-lene (C30) and phytoene (C40), although formed entirely of isoprene units, display a tail-to-tail linkage at the centre of the molecules. Most terpenoids are modified further by cyclization reactions, but the head-to-tail arrangement of the units can usually still be recognized, e.g. menthol, bisabo-lene, and taxadiene. The linear arrangement of

C5 isoprene unit isoprene

Figure 5.1

isoprene units can be more difficult to appreciate in many other structures when rearrangement reactions have taken place, e.g. steroids, where in addition, several carbons have been lost. Nevertheless, such compounds are formed via regular terpenoid precursors.

Many other natural products contain terpenoid elements in their molecules, in combination with carbon skeletons derived from other sources, such as the acetate and shikimate pathways. Many alkaloids, phenolics, and vitamins discussed in other chapters are examples of this. A particularly common terpenoid fragment in such cases is a single C5 unit, usually a dimethylallyl substituent, and molecules containing these isolated isoprene units are sometimes referred to as 'meroter-penoids'. Some examples include furocoumarins (see page 145), rotenoids (see page 155), and ergot alkaloids (see page 368). One should also note that the term 'prenyl' is in general use to indicate the dimethylallyl substituent. Even macromolecules like proteins can be modified by attaching terpenoid chains. Cysteine residues are alkylated with farnesyl or geranylgeranyl groups, thereby increasing the lipophilicity of the protein and its ability to associate with membranes.

The biochemical isoprene units may be derived by two pathways, by way of intermediates meval-onic acid (MVA) (Figure 5.4) or 1-deoxy-D-xylulose 5-phosphate (deoxyxylulose phosphate; DXP) (Figure 5.6). Mevalonic acid, itself a product of acetate metabolism, had been established as a precursor of the animal sterol cholesterol, and

Mevalonic acid

dimethylallyl PP (DMAPP) (C5)

C20 IPP -V

Deoxyxylulose phosphate

isopentenyl PP (IPP) (C5)

Hemiterpenes (C5)

Monoterpenes (C10) Iridoids

Sesquiterpenes (C15)

Diterpenes (C20)

Sesterterpenes (C25)

Triterpenoids (C30)

Steroids (C18-C30)

Tetraterpenes (C40) Carotenoids

geraniol farnesol

geranylgeraniol

geranylgeraniol

Building Blocks Menthol
phytoene

menthol bisabolene

Figure 5.3

bisabolene

Figure 5.3

taxadiene the steps leading to and from mevalonic acid were gradually detailed in a series of painstakingly executed experiments. For many years, the early parts of the mevalonate pathway were believed to be common to the whole range of natural terpenoid derivatives, but it has since been discovered that an alternative pathway to IPP and DMAPP exists, via deoxyxylulose phosphate, and that this pathway is probably more widely utilized in nature than is the mevalonate pathway. This pathway is also referred to as the mevalonate-independent pathway or the methylerythritol phosphate pathway.

Three molecules of acetyl-coenzyme A are used to form mevalonic acid. Two molecules combine initially in a Claisen condensation to give acetoacetyl-CoA, and a third is incorporated via a stereospecific aldol addition giving the branched-chain ester P-hydroxy-0-methylglutaryl-CoA (HMG-CoA) (Figure 5.4). This third acetyl-CoA molecule appears to be bound to the enzyme via a thiol group, and this linkage is subsequently hydrolysed to form the free acid group of HMG-CoA. In the acetate pathway, an acetoacetic acid thioester (bound to the acyl carrier protein) would have been formed using the more nucleophilic thioester of malonic acid. The mevalonate pathway does not use mal-onyl derivatives and it thus diverges from the acetate pathway at the very first step. In the second step, it should be noted that, on purely chemical grounds, acetoacetyl-CoA is the more acidic substrate, and might be expected to act as the nucleophile rather than the third acetyl-CoA molecule. The enzyme thus achieves what is a less favourable reaction. The conversion of HMG-CoA into (3^)-MVA involves a two-step reduction of the thioester group to a primary alcohol, and provides an essentially irreversible and rate-limiting transformation. Drug-mediated inhibition of this enzyme (HMG-CoA reductase) can be used to regulate the biosynthesis of mevalonate and ultimately of the steroid cholesterol (see statins, page 112).

The six-carbon compound MVA is transformed into the five-carbon phosphorylated isoprene units in a series of reactions, beginning with

SCoA

Claisen reaction

SCoA

acetyl-CoA

( acetoacetyl-CoA V©

H \^SEnz stereospecific aldol reaction; also involves hydrolysis of acetyl-enzyme linkage

HO2C

+ EnzSH

SCoA

HMG-CoA

O enzyme-bound acetyl group

HMG-CoA reductase

NADPH

reduction of aldehyde to alcohol ho2c

2 4 mevalonic acid (MVA)

sequential phosphorylation of the primary alcohol to a diphosphate

HO2C

OH OH H

mevaldic acid hemithioacetal

HO2C

mevaldic acid reduction of thio ester to aldehyde via hemithioacetal

SCoA

2 x ATP

HO-P-O-ADP

stereospecific allylic isomerization; equilibrium favours DMAPP

ATP -CO2

ATP facilitates the decarboxylation-elimination; the anticipated phosphorylation of the tertiary alcohol to make a better leaving group is apparently not involved h^JL

hr hS

hr hS

isopentenyl PP (IPP)

isomerase

isopentenyl PP (IPP)

dimethylallyl PP (DMAPP)

Figure 5.4

NADPH

phosphorylation of the primary alcohol group. Two different ATP-dependent enzymes are involved, resulting in mevalonic acid diphosphate, and decarboxylation/dehydration then follow to give IPP. Whilst a third molecule of ATP is required for this last transformation, there is no evidence for phosphorylation of the tertiary hydroxyl, though this would convert the hydroxyl into a better leaving group. Perhaps ATP assists the loss of the hydroxyl as shown in Figure 5.4. IPP is isomerized to the other isoprene unit, DMAPP, by an isomerase enzyme which stereospecifically removes the pro-R proton (H^) from C-2, and incorporates a proton from water on to C-4. Whilst the isomerization is reversible, the equilibrium lies heavily on the side of DMAPP. This conversion generates a reactive electrophile and therefore a good alkylating agent. DMAPP possesses a good leaving group, the diphosphate, and can yield via an SN1 process an allylic carbocation which is stabilized by charge delocalization (Figure 5.5). In contrast, IPP with its terminal double bond is more likely to act as a nucleophile, especially towards the electrophilic DMAPP. These differing reactivities are the basis of terpenoid biosynthesis, and carbocations feature strongly in mechanistic rationalizations of the pathways.

1-Deoxy-D-xylulose 5-phosphate is formed from the glycolytic pathway intermediates pyruvic acid and glyceraldehyde 3-phosphate with the loss of the pyruvate carboxyl (Figure 5.6). Thiamine diphosphate-mediated decarboxylation of pyruvate

DMAPP

Sn1 reaction

Note: when using this representation of the allylic cation, do not forget the double bond

resonance-stabilized allylic cation

(compare page 21) produces an acetaldehyde equivalent bound in the form of an enamine, which reacts as a nucleophile in an addition reaction with the glyceraldehyde 3-phosphate. Subsequent release from the TPP carrier generates deoxy-xylulose phosphate, which is transformed into 2-C-methyl-D-erythritol 4-phosphate by a rearrangement reaction, conveniently rationalized as a pinacol-like rearrangement (Figure 5.6), coupled with a reduction. The expected aldehyde product from the rearrangement step is not detectable, and the single enzyme catalyses the rearrangement and reduction reactions without release of any intermediate. Analogous rearrangements are seen in the biosynthesis of the amino acids valine, leucine, and isoleucine. The methylerythritol phosphate contains the branched-chain system equivalent to the isoprene unit, but the complete sequence of steps

•s.^co2h o pyruvic acid thiamine PP

nucleophilic attack of enamine on to aldehyde o)

h3c.

R1 OH

N^ S D-glyceraldehyde

EO OH

R1 OH

N^ S D-glyceraldehyde see Figure 215 j=< 2 3-P

/ R2 TPP/pyruvate-derived enamine

Vop yV

O OH 1-deoxy-D-xylulose 5-P

Rf^S TPP anion regenerated pinacol-like rearrangement favoured by stability of resultant oxonium ion

HC.o

0 1-deoxy-H D-xylulose 5-P

HO OH

fosmidomycin

O OH

reduction of aldehyde to alcohol; the aldehyde intermediate remains enzyme-bound

NADPH

OH OH

2-C-methyl-D-erythritol 4-P

nucleophilic attack of phosphate hydroxyl on ^h O diphosphate; formation of '

O—p—OH phosphoanhydride

compare formation of UDPglucose, Figure 2.27

OH OH

OH OH

2-phospho-4-(CDP)-2-C-methyl-D-erythritol

HO OH

OH OH

OH OH

4-(CDP)-2-C-methyl- HO D-erythritol

O OH

OH O

OH O

2-C-methyl-D-erythritol-

2,4-cyclophosphate intramolecular elimination; then enol-keto tautomerism

NADPH

OH OH

steps to be determined

NADPH

OH OH

dimethyMlyl PP there is evidence that DMAPP isopentenyl PP (DMAPP) may be formed independently, and (IPP) not via isomerization of IPP

NADPH

NADPH

leading to the intermediate isopentenyl phosphate has yet to be elucidated. Reaction of methylery-thritol phosphate with cytidine triphosphate (CTP) produces a cytidine diphospho derivative (compare uridine diphosphoglucose in glucosylation, page 29), which is then phosphorylated via ATP. The resultant 2-phosphate is converted into a cyclic phosphoanhydride with loss of cytidine phosphate. This cyclophosphate, by steps not yet known (a possible sequence is proposed in Figure 5.6), leads to IPP, and links the deoxyxylulose pathway with the mevalonate pathway. DMAPP may then be derived by isomerism of IPP, or may be produced independently; this also remains to be clarified. Deoxyxylulose phosphate also plays an important role as a precursor of thiamine (vitamin Bi, page 30) and pyridoxol phosphate (vitamin B6, page 33).

Whether the mevalonate pathway or the deoxyxylulose phosphate pathway supplies iso-prene units for the biosynthesis of a particular terpenoid has to established experimentally. Animals appear to lack the deoxyxylulose phosphate pathway, so utilize the mevalonate pathway exclusively. Many other organisms, including plants, are equipped to employ both pathways, often concurrently. In plants, the two pathways appear to be compartmentalized, so that the mevalonate pathway enzymes are localized in the cytosol, whereas the deoxyxylulose phosphate pathway enzymes are found in chloroplasts. Accordingly, triterpenoids and steroids (cytosolic products) are formed by the mevalonate pathway, whilst most other ter-penoids are formed in the chloroplasts and are deoxyxylulose phosphate derived. Of course there are exceptions. There are also examples where the two pathways can supply different portions of a molecule, or where there is exchange of late-stage common intermediates between the two pathways resulting in a contribution of isoprene units from each pathway. In the following part of this chapter, these complications will not be considered further, and in most cases there is no need to consider the precise source of the isoprene units. The only area of special pharmacological interest where the early pathway is of particular concern is steroid biosynthesis, which appears to be from mevalonate in the vast majority of organisms. Thus, inhibitors of the mevalonate pathway enzyme HMG-CoA reductase will reduce steroid production, but will not affect the formation of terpenoids derived via deoxyxylulose phosphate. Equally, it is possible to inhibit terpenoid production without affecting steroid formation by the use of deoxyxyxlulose phosphate pathway inhibitors, such as the antibiotic fosmidomycin from Streptomyces lavendulae. This acts as an analogue of the rearrangement intermediate (Figure 5.6). Regulation of cholesterol production in humans is an important health concern (see page 236).

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