Nh2oh Arso2cl And Ketone
Marker degradation (Figure 5.119) I
Beckmann rearrangement: oxime ^ amide
Marker degradation (Figure 5.119) I
nh2oh oxime formation
nh2oh
ArSO2Cl / Py
Beckmann rearrangement
hydrolysis of amide function gives enamine; tautomerism to imine then allows further hydrolysis to ketone
dehydropregnenolone acetate
Oppenauer
hydrolysis of ester at C-3
dehydropregnenolone acetate
Oppenauer
liq NH3
ethisterone
liq NH3
ethisterone nucleophilic attack of acetylide anion on to carbonyl from less-hindered a face hydrolysis of ester at C-3
- androstenolone
- O
19-nor-14ß,17a-progesterone norethisterone norethisterone acetate
Figure 5.130
19-nor-14ß,17a-progesterone norethisterone norethisterone acetate
Figure 5.130
attack of acetylide anion on to a C-17 carbonyl (Figure 5.129), attack coming from the a-face, the methyl C-18 hindering approach from the p-face. The substrate androstenolone is readily obtained from the Marker degradation intermediate dehydropregnenolone acetate (Figure 5.119). The oxime (Figure 5.129) is treated with a sulphonyl chloride in pyridine and undergoes a Beckmann rearrangement in which C-17 migrates to the nitrogen giving the amide. This amide is also an enamine and can be hydrolysed to the 17-ketone. Acetylation or other esterification of the 17-hydroxyl in progestogens increases lipid solubility and extends the duration of action by inhibiting metabolic degradation. Examples include norethisterone acetate, medroxyprogesterone acetate, and hydroxyprogesterone caproate, discussed below.
Though considerably better than progesterone, the oral activity of ethisterone is still relatively low, and better agents were required. An important modification from ethisterone was the 19-nor analogue, norethisterone and its ester norethisterone acetate (Figure 5.130). Attention was directed to the 19-norsteroids by the observation that 19-nor-14p,17a-progesterone (Figure 5.130), obtained by degradation of the cardioactive glycoside strophanthidin (see page 250), displayed eight times higher progestational activity than progesterone, despite lacking the methyl C-19, and having the unnatural configurations at the two centres C-14 (C/D rings cis-fused) and C-17. Norethisterone can be synthesized from the oestrogen estrone (see page 279) which already lacks the C-9 methyl, or from androstenolone (Figure 5.129) by a sequence which allows oxidation of C-19 to a carboxyl, which is readily lost by decarboxylation when adjacent to the a,p-unsaturated ketone system.
Although ethisterone and norethisterone are structurally C21 pregnane derivatives, they may also be regarded as 17-ethynyl derivatives of testosterone (see page 282), the male sex hormone, and 19-nortestosterone respectively. Many of the commonly used progestogens fall into these two classes. Semi-synthetic analogues of progesterone, still containing the 17-acetyl side-chain, tend to be derivatives of 17a-hydroxyprogesterone, another biosynthetic intermediate on the way to hydrocortisone (Figure 5.114) that also has progesterone-like activity. Examples include hydroxyprogesterone caproate, and gestonorone (gestronol) caproate (Figure 5.131). Medroxyprogesterone acetate (Figure 5.131) contains an additional 6a-methyl, introduced to block potential deactivation by metabolic hydroxylation, and is 100-300 times as potent as ethisterone on oral administration. Megestrol acetate (Figure 5.131) contains a 6-methyl group and an additional A6 double bond. Norgestrel (Figure 5.131) is representative of progestogens with an ethyl group replacing the 13-methyl. Although these can be obtained by semi-synthesis from natural 13-methyl compounds, norgestrel is produced by total synthesis as the racemic compound.
hydroxyprogesterone caproate (hexanoate)
-O2CC5H11
hydroxyprogesterone caproate (hexanoate)
O2CC5H11
gestonorone (gestronol) caproate (hexanoate)
O2CC5H11
etynodiol (ethynodiol) diacetate gestonorone (gestronol) caproate (hexanoate)
O -OAc megestrol acetate
O -OAc medroxyprogesterone acetate megestrol acetate medroxyprogesterone acetate
desogestrel dydrogesterone etonogestrel
Me2N
desogestrel dydrogesterone etonogestrel
etynodiol (ethynodiol) diacetate
gestodene
norgestimate
Figure 5.131
Me2N
gestodene
norgestimate
- mifepristone
Figure 5.131
Since only the laevorotatory enantiomer which has the natural configuration is biologically active, this enantiomer, levonorgestrel, is now replacing the racemic form for drug use. In desogestrel (Figure 5.131), further features are the modification of an 11-oxo function to an 11-methylene, and removal of the 3-ketone. Structures of some other currently available progestogen drugs are shown in Figure 5.131.
During pregnancy, the corpus luteum continues to secrete progesterone for the first three months, after which the placenta becomes the supplier of both progesterone and oestrogen. Progesterone prevents further ovulation and relaxes the uterus to prevent the fertilized egg being dislodged. In the absence of pregnancy, a decline in progesterone levels results in shedding of the uterine endometrium and menstruation. Progestogens are useful in many menstrual disorders, and as oral contraceptives either alone at low dosage (progestogen-only contraceptives, e.g. norethisterone, levonorgestrel) or in combination with oestrogens (combined oral contraceptives, e.g. ethinylestradiol + norethisterone, ethinylestradiol + levonorgestrel). The combined oestrogen-progestogen preparation inhibits ovulation, but normal menstruation occurs when the drug is withdrawn for several days each month. The low dosage progestogen-only pill appears to interfere with the endometrial lining to inhibit fertilized egg implantation, and thickens cervical mucus making a barrier to sperm movement. The progestogen-only formulation is less likely to cause thrombosis, a serious side-effect sometimes experienced from the use of oral contraceptives. There appears to be a slightly higher risk of thrombosis in patients using the so-called 'third generation' oral
" (Continued)
contraceptive pills containing the newer progestogens desogestrel and gestodene. Current oral contraceptives have a much lower hormone content than the early formulations of the 1960s and 1970s, typically about 10% of the progestogen and 50% of the oestrogen content. Deep muscular injections of medroxyprogesterone or norethisterone esters, and implants of levonorgestrel can be administered to provide long-acting contraception. A high dose of levonorgestrel, alone or in combination with ethinylestradiol, is the drug of choice for emergency contraception after unprotected intercourse, i.e. the 'morning-after' pill. Hormone replacement therapy (HRT) in non-hysterectomized women also uses progestogen-oestrogen combinations (see page 279), whilst progestogens such as norethisterone, megestrol acetate, medroxyprogesterone acetate, and gestonorone caproate also find application in the treatment of breast cancers.
Mifepristone (Figure 5.131) is a progestogen antagonist used orally as an abortifacient to terminate pregnancy. This drug has a higher affinity for the progesterone receptor than does the natural hormone, and prevents normal responses. This leads to loss of integrity of the uterine endometrial lining, and detachment of the implanted fertilized egg.
at position 3; they are not restricted to females since small amounts are produced in the male testis. The principal and most potent example is estradiol (also oestradiol, but US spelling has been generally adopted), though only low levels are found in urine, and larger amounts of the less active metabolites estrone (oestrone) and the 16a-hydroxylated derivative estriol (oestriol) are present (Figure 5.132). Estrone has also been found in significant quantities in some plant seeds, e.g. pomegranate and date palm. These compounds have an aromatic A ring, a consequence of which is that C-19, the methyl on C-10, is absent. There is now no carbon side-chain at C-17, and the basic C18 skeleton is termed estrane.
The biosynthetic pathway to estradiol and estrone (Figure 5.133) proceeds from cholesterol via pregnenolone and bears a resemblance to the hydrocortisone pathway (Figure 5.114) in the early 17-hydroxylation step. Indeed, the same cytochrome P-450-dependent enzyme catalyses 17-hydroxylation of both pregnenolone and
- estradiol (oestradiol)
estrone (oestrone)
estrone (oestrone)
Characteristic features of oestrogens:
• Cjg estrane skeleton
• aromatic A ring (consequent loss of C-10 methyl)
• no side-chain estriol (oestriol)
- OH
estriol (oestriol)
O2 NADPH
pregnenolone
O 17a-hydroxylation
O2 NADPH
O 17a-hydroxylation
O2 NADPH
17a-hydroxypregnenolone
pregnenolone
17a-hydroxypregnenolone
sequential oxidation of C-10 methyl to aldehyde
O2 NADPH
- O2 NADPH aromatase
estrone (oestrone)
aromatase oxidative loss of C-10 formyl and aromatization of ring A (Figure 5.135)
NADH/ NADPH
aromatase
O2 NADPH
aromatase estradiol (oestradiol)
Figure 5.133
estrone (oestrone)
estradiol (oestradiol)
oxidative removal of side-chain (Figure 5.134)
O2 NADPH
O2 NADPH
O2 NADPH
- dehydroepiandrosterone
oxidation of 3-hydroxyl and tautomerism to conjugated system O
- NAD+
androstenedione
NADH/ NADPH
androstenedione
NADH/ NADPH
testosterone
testosterone
Figure 5.133
aromatase
aromatase
progesterone, as well as the next step in oestrogen biosynthesis, and it plays a significant role in controlling the direction of steroid synthesis. Whilst 17a-hydroxyprogesterone is transformed by 21-hydroxylation for hydrocortisone biosynthesis, in oestrogen biosynthesis the a-hydroxyketone function is oxidized from 17a-hydroxypregnenolone, cleaving off the two-carbon side-chain as acetic acid. The product is the 17-ketone dehydroepiandrosterone, which is the most abundant steroid in the blood of young adult humans, with levels peaking at about 20 years of age, then declining as the person ages. Apart from its role as a precursor of hormones, it presumably has other physiological functions, though these still remain to be clarified. A mechanism for the side-chain cleavage reaction, initiated by attack of an enzyme-linked peroxide, is shown in Figure 5.134, and is analogous to that proposed for loss of the 14-methyl group during cholesterol biosynthesis (see page 235). Oxidation and tautomerism in rings A/B then give androstenedione (Figure 5.133). Either androstenedione, or its reduction product testosterone, is a substrate for aromatization in ring A, with loss of C-19. This sequence is also catalysed by a single cytochrome P-450-dependent enzyme, called aromatase, and the reaction proceeds via sequential oxidation of the methyl, with its final elimination as formic acid (Figure 5.135). The mechanism suggested is analogous to that of the side-chain cleavage reaction. Formation of the aromatic ring is then a result of enolization. As with other steroid hormones, the exact order of some of the steps, including formation of the A4-3-keto function, 17-hydroxylation, reduction of the 17-keto, and aromatization in ring A, can vary according to organism, or site of synthesis in the body. Since breast tumours require oestrogens for growth, the design of aromatase inhibitors* has become an important target for anticancer drug research.
The aromatic ring makes the oestrogen molecule almost planar (see page 233) and is essential for activity. Changes which remove the aromaticity, e.g. partial reduction, or alter stereochemistry, give analogues with reduced or no activity.
17a-hydroxylation O
O2 NADPH
peroxy adduct formation via nucleophilic attack of peroxy-enzyme on to carbonyl O
O2 NADPH
Enz-Fe-OOH
homolytic cleavage of peroxy bond OH A OH .
O-Fe-Enz
side-chain lost as acetic acid
CH3CO2H
- Af iJ^O-H
Figure 5.134
sequential oxidation of C-10 methyl to aldehyde HO
O2 NADPH
O2 NADPH
O2 NADPH
peroxy adduct formation via nucleophilic attack of peroxy-enzyme on to carbonyl homolytic cleavage of peroxy bond
O2 NADPH
O2 NADPH
Enz-Fe-OOH
homolytic cleavage of peroxy bond
- Enz-Fe-O* HCO2H H H
side-chain lost as formic acid
Figure 5.135
- lumiestrone
diethylstilbestrol (stilboestrol)
O O coumestrol
diethylstilbestrol (stilboestrol)
O O coumestrol
Thus, exposure of estrone to UV light leads to inversion of configuration at C-13 adjacent to the carbonyl function, and consequently formation of a ds-fused C/D ring system. The product, lumiestrone (Figure 5.136) is no longer biologically active. Some planar non-steroidal structures can also demonstrate oestrogenic activity as a result of a similar shape and relative spacing of oxygen functions. Thus, the synthetic diethylstilbestrol (stilboestrol) (Figure 5.136) has been widely used as an oestrogenic drug, and coumestrol, daidzein, and genistein (Figure 5.136) are naturally occurring isoflavonoids with oestrogenic properties from lucerne, clovers, and soya beans, and are termed
Oestrogen Drugs
Oestrogens suppress ovulation and with progestogens form the basis of combined oral contraceptives and hormone replacement therapy (HRT). They are also used to supplement natural oestrogen levels where these are insufficient as in some menstrual disorders, and to suppress androgen formation and thus tumour growth of cancers dependent on androgens, e.g. prostate cancers. Oestrogens appear to offer a number of beneficial effects to women, including protection against osteoporosis, heart attacks, and possibly Alzheimer's disease. However, some cancers, e.g. breast and uterine cancers, are dependent on a supply of oestrogen for growth, especially during the early stages, so high oestrogen levels are detrimental. Steroidal oestrogens for drug use were originally obtained by processing pregnancy urines, but the dramatic increase in demand due to the introduction of oral contraceptives required development of semi-synthetic procedures. Androstenolone formed via the Marker degradation of diosgenin (Figure 5.129) may be transformed to the dione by catalytic reduction of the A5 double bond and oxidation of the 3-hydroxyl (Figure 5.137). This then allows production of androstadienedione by dibromination and base-catalysed elimination of HBr. Alternatively, it is now possible to achieve the synthesis of androstadienedione in a single step by a microbiological fermentation of either sitosterol obtained from soya beans (see page 256), or of cholesterol obtained in large quantities from the woolfat of sheep, or from the spinal cord of cattle (see page 236). These materials lack unsaturation in the side-chain and were not amenable to simple chemical oxidation processes, as for example with stigmasterol (see page 266). Their exploitation required the development of suitable biotransformations, and use of Mycobacterium phlei has now achieved this objective (Figure 5.137). The aromatization step to estrone can be carried out in low yields by vapour-phase free-radical-initiated thermolysis, or more recently with considerably better yields using a dissolving-metal reductive thermolysis. In both processes, the methyl at C-10 is lost. This sequence gives estrone, from which estradiol (oestradiol) may be obtained by reduction of the 17-carbonyl. However, by far the most commonly used medicinal estrogen is ethinylestradiol (ethinyloestradiol) (Figure 5.137), which is 12 times as effective as estradiol when administered orally. This analogue can be synthesized from estrone by treatment with potassium acetylide in liquid ammonia, which attacks from the less-hindered a-face (see page 273). The ethynyl substituent prevents oxidation at C-17, as in the metabolism of estradiol to the less active estrone. The phenol group allows synthesis of other derivatives, e.g. the 3-methyl ether mestranol (Figure 5.137), which acts as a pro-drug, being oxidized in the liver to ethinylestradiol. To retain oestrogenic activity, structural modifications appear effectively limited to the addition of the 17a-ethynyl group, and to substituents on the 3-hydroxyl. The ester estradiol valerate (oestradiol valerate) facilitates prolonged action through slower absorption and metabolism.
The lower activity metabolites estriol (oestriol) (about 2% activity of estradiol) and estrone (oestrone) (about 33% activity) (Figure 5.132) are sometimes used in hormone replacement therapy (HRT). Oestrogen and progesterone levels decline naturally at menopause when the menstrual cycle ceases. The sudden reduction in oestrogen levels can lead to a number of unpleasant symptoms, including tiredness, hot flushes, vaginal dryness, and mood changes. HRT reduces these symptoms, and delays other long-term consequences of reduced oestrogen levels, including osteoporosis and atherosclerosis. HRT currently provides the best therapy for preventing osteoporosis, a common disease in post-menopausal women.
diosgenin
Marker degradation and I removal of T
side-chain (Figure 5.129)
Marker degradation and I removal of T
side-chain (Figure 5.129)
androstenolone reduction of double bond; oxidation O of 3-hydroxyl (
i H2 / catalyst ii CrO3
i H2 / catalyst ii CrO3
androstenolone
cholesterol sitosterol
brominations a to ketone
Br2 / HOAc Br cholesterol sitosterol
Mycobacterium phlei
Mycobacterium phlei
base-catalysed eliminations androstadienedione base-catalysed eliminations androstadienedione vapour-phase radical-initiated thermolysis tetralin / A / 600° or
mestranol
ethinylestradiol (ethinyloestradiol)
mestranol
ethinylestradiol (ethinyloestradiol)
nucleophilic attack of acetylide anion on to carbonyl from less-hindered a face
estrone (oestrone)
dissolving metal reduction O
estrone (oestrone)
Figure 5.137
Osteoporosis is characterized by a generalized loss of bone mass leading to increased risk of fracture, and a sharp reduction in endogenous oestrogen levels is recognized as a critical factor. Oestrogen and progestogen combinations are used in HRT unless the woman has had a hysterectomy, in which case oestrogen alone is prescribed. Natural oestrogen structures are preferred to synthetic structures such as ethinylestradiol or mestranol. Before the availability of plant-derived semi-synthetic oestrogens, extraction of urine from pregnant women and pregnant horses allowed production of oestrogen mixtures for drug use. Conjugated equine oestrogens are still widely prescribed for HRT and are obtained by extraction from the urine of pregnant mares and subsequent purification, predominantly in Canada and the USA. Mares are about 120 days pregnant when urine collection begins, and collection continues for another 150-160 days. During this period, a mare will produce about 400-500 litres of urine.
OH O
equilin dihydroequilin estropipate H tibolone
equilin dihydroequilin estropipate H tibolone
diethylstilbestrol (stilboestrol)
fosfestrol dienestrol (dienoestrol)
diethylstilbestrol (stilboestrol)
fosfestrol
NMe2
NMe2
estramustine
Figure 5.138
dienestrol (dienoestrol)
Me2N
estramustine
formestane
formestane
Me2N
- clomifene (clomiphene)
Figure 5.138
Horses are maintained in an almost continuous state of pregnancy, and need to be kept in confined stalls and fitted with a suitable urine collection device, though the use of catheters to collect urine has been discontinued. Animal welfare groups urge women to reject these drug preparations in favour of plant-derived alternatives. Conjugated equine oestrogens provide a profile of natural oestrogens based principally on estrone and equilin (Figure 5.138). It consists of a mixture of oestrogens in the form of sodium salts of their sulphate esters, comprising mainly estrone (50-60%) and equilin (20-30%), with smaller amounts of 17a-dihydroequilin, 17a-estradiol, and 17^-dihydroequilin. The semi-synthetic estropipate is also a conjugated oestrogen, the piperazine salt of estrone sulphate.
The structure of tibolone (Figure 5.138) probably resembles that of a progestogen more than it does an oestrogen. Although it does not contain an aromatic A-ring, the 5(10)-double bond ensures a degree of planarity. This agent combines both oestrogenic and progestogenic activity, and also has weak androgenic activity, and has been introduced for treatment of vasomotor symptoms of menopause.
Diethylstilbestrol (stilboestrol) and dienestrol (dienoestrol) (Figure 5.138) are the principal non-steroidal oestrogen drugs, used topically via the vagina. Fosfestrol (Figure 5.138) has value in the treatment of prostate cancer, being hydrolysed by the enzyme phosphatase to produce diethylstilbestrol as the active agent.
In estramustine (Figure 5.138), estradiol is combined with a cytotoxic alkylating agent of the nitrogen mustard class via a carbamate linkage. This drug has a dual function, a hormonal effect by suppressing androgen (testosterone) formation, and an antimitotic effect from the mustine residue. It is of value in treating prostate cancers.
" (Continued)
Phyto-oestrogens are predominantly isoflavonoid derivatives found in food plants and are used as dietary supplements to provide similar benefits to HRT, especially in countering some of the side-effects of the menopause in women. These compounds are discussed under isoflavonoids (see page 156). Dioscorea (wild yam) root or extract (see page 239) is also marketed to treat the symptoms of menopause as an alternative to HRT. Although there is a belief that this increases levels of progesterone, which is then used as a biosynthetic precursor of other hormones, there is no evidence that diosgenin is metabolized in the human body to progesterone.
Aromatase Inhibitors
Formestane (Figure 5.138), the 4-hydroxy derivative of androstenedione, represents the first steroid aromatase inhibitor to be used clinically. It reduces the synthesis of oestrogens and is of value in treating advanced breast cancer in post-menopausal patients.
Oestrogen Receptor Antagonists
Breast cancer is dependent on a supply of oestrogen, and a major success in treating this disease has been the introduction of tamoxifen (Figure 5.138). This drug contains the stilbene skeleton seen in diethylstilbestrol and related oestrogens, but acts as an oestrogen-receptor antagonist rather than as an agonist in breast tissue, and deprives the cells of oestrogen. However, it is an agonist in bone and uterine tissue. The chlorinated analogue toremifene is also available, but is used primarily in post-menopausal women. Oestrogen antagonists can also be used as fertility drugs, occupying oestrogen receptors and interfering with feedback mechanisms and leading to ova release. Clomifene (clomiphene) (Figure 5.138), and to a lesser extent tamoxifen, are used in this way, but can lead to multiple pregnancies.
phyto-oestrogens (see page 156). Dietary natural isoflavonoids are believed to give some protection against breast cancers, and are also recommended to alleviate the symptoms of menopause.
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