Chapter 22 Medicinal Chemistry of steroids

Chapter 22

Medicinal Chemistry of Steroids F. J. ZEELEN

427 428 431 434 436 438 442 443 444 445 445 447 448 448 456

Introduction Cholesterol The Bile Acids The Steroid Hormones of the Gonads

Biosynthesis The Menstrual Cycle Contraception Therapeutic Use of Progestagens Therapeutic Use of Estrogens Breast Cancer Therapeutic Use of Androgens Anti-Androgens The Adrenal Corticoids Hydrocortisone Aldosterone

459

Neurosteroids Summary

460

INTRODUCTION In the human organism, steroids fulfill a diverse number of functions. Cholesterol is a key constituent of cell membranes, while the bile acids are important for

Principles of Medical Biology, Volume 8B Molecular and Cellular Pharmacology, pages 427-463. Copyright 9 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-813-7

427

428

FJ. ZEELEN

the absorption of fats from the intestinal tract. The steroid hormones of the gonads induce sexual functions and the adrenal corticosteroids regulate the electrolyte balance of body fluids and decrease inflammatory and immune responses. Even in the nervous system steroids may have a function. What these compounds have in common is their biosynthesis. Although the organs, where these steroids are formed differ, the biosynthetic route and the enzymes involved are similar or even identical. For that reason the steroids are dicussed as one class of compounds.

CHOLESTEROL Cholesterol is the principal sterol of higher animals, a key constituent of cell membranes and lipoproteins, and a precursor of bile acids and steroid hormones. It is found in all body tissues, especially in the brain, spinal cord and animal fats. In fungi, ergosterol is the major steroid (Figure 1). Cholesterol can be synthesized by essentially all animal cells. In the human, the principal sites of synthesis are skin, liver and intestinal mucosa. Biosynthetic activity is also detectable in lung, kidney, gonad, muscle, brain and adipose tissue. The loss of cholesterol via conversion into bile acids and by direct secretion into bile is about 1.0-1.5 gram/day. From food, about 0.2-1.0 gram/day of cholesterol is taken up and the rest has to be synthesized by the body itself. The biosynthesis of cholesterol (see Figure 2) starts with the acetate pool. As is seen, in a series of steps, the 3-hydroxy-3-methylglutaryl coenzyme A complex is formed, which is reduced by the enzyme 3-hydroxy-3-methylglutaryl reductase (HMG-CoA reductase) to(3R)-mevalonic acid. This reductase is a major regulatory enzyme in cholesterol biosynthesis. An increase in concentration of oxygenated cholesterol metabolites leads to a decrease in synthesis of the enzyme. In a series of steps, six mevalonic acid molecules are coupled to squalene. In this process, six molecules of carbon dioxide are formed. The enzyme squalene epoxidase converts squalene into the (3S)-2,3-oxidosqualene, which is cyclized by

H

Figure 1. Cholesterol and ergosterol.

H

H I., CH3

429

Medicinal Chemistry of Steroids

OH

,, ~ acetate

~

~

H3C

OH

COOH

,, ~ ~.~ ~.~ H3C

~.T~o

%/o.

-~

mevalonic acid I

Seoer~m~ !

!

COOH

aq

squalene

H

2,3-oxidosqualeneH

--

~.~

H

lanosterol

H

2.4

H

H H

zymasteroi Figure 2.

cholesterol

Biosynthesisof cholesterol.

another enzyme to lanosterol. Next, the 14ct-methyl group is removed in three consecutive steps. All three oxidations are catalyzed by a single enzyme, a cytochrome P-450 (cytochrome P-45014DM) In a further series of steps the 14-15 double bond is reduced and the two methyl groups at position 4 are removed. In the human,

430

F.J. ZEELEN

H s

O

I I

H/,--O0~ ~ . / t CH3H

I I

H

0H CH3

C,~~~~,~

/

H

H

~.~ CH3

CH3

t

H

H

"~

.

CH3 Figure 3.

CH3

Lovastatin.

O

L CH3 CH3

C(CH~3 H

N

"

~ L ~ . ~1 N ~ O

Q

terbinafine

Figure 4.

ketoconazole

Antifungal Icompounds.

zymasterol is converted into cholesterol, whereas in fungi zymasterol is converted into ergosterol. Efficient regulation of cholesterol synthesis is important. In man, cholesterol is an essential component of cell membranes and a starting material for the biosynthesis of cholic acids and the steroid hormones. On the other hand, an elevated serum level of low-density lipoprotein bound cholesterol is a major risk factor in the development of generalized atherosclerosis and coronary artery disease. For that reason, inhibition of cholesterol synthesis plays an important role in the treatment of patients with hypercholesterolemia.

Medicinal Chemistry of Steroids

431

Since cholesterol synthesis is regulated by the enzyme HMG-CoA reductase, competitive inhibitors of that enzyme have been developed and are used clinically as an adjunct to diet. The lactone lovastatin is an example; in the body this lactone is in equilibrium with the open form (Figure 3). As already pointed out, the sterols have an important function in cell membranes. For this reason compounds that selectively inhibit ergosterol synthesis in fungi and do not disturb cholesterol synthesis in humans have found a place in therapy to treat fungal infections. These inhibitors may act on squalene epoxidase (for example, terbinafine) or the cytochrome P-450DM (for example, ketoconazole) (see Figure 4).

THE BILE A C I D S Human bile is produced in the liver, stored in the gall bladder and secreted into the intestine. It has emulsifying properties and promotes fat absorption. Bile acids are the chief constituents of the solid matter of the bile, which also contains inorganic salts, lecithin, cholesterol, bilirubin diglucuronide and other bile pigments. Cholic acid, chenodeoxycholic acid and deoxycholic acid are the major bile acids in human bile. The bile acids may be conjugated with taurine or glycine and, to a lesser extent. with glucuronic acid, glucose or N-acetylglucosamine. The bile acids are absorbed from the gastro-intestinal tract, conjugated in the liver and recirculated in the bile. Deoxycholic acid is a secondary bile acid; it is not synthesized by the human body, but is formed by intestinal bacteria via 7-dehydroxylation of cholic acid. Cholic acid and chenodeoxycholic acid are the primary bile acids (see Figure 5). The biosynthesis of cholic acid (Figure 6) starts with cholesterol, which is hydroxylated at the 7a-position by cholesterol 7a-hydroxylase, a cytochrome P-450 enzyme (P4507a). This hydroxylation is the rate-limiting step of cholic acid biosynthesis. The enzyme concentration increases with high cholesterol levels and decreases when bile acid levels are high. The next steps are the oxidation of the 3-hydroxyl group and isomerization of the 5,6 double bond. These two reactions are carried out by a single enzyme, 3~3-hydroxy-C27-steroid oxidoreductase. The 12o~-hydroxyl group is introduced by another cytochrome P-450 (P-45012a). The major pathway continues with reduction of the conjugated 4,5-double bond, whereby the 5-hydrogen comes at the 513-position, which is characteristic for the bile acids, followed by reduction of the 3-keto group. This reduction is carried out by a 3o~-hydroxysteroid dehydrogenase. In the human, there may be several isoenzymes with specific functions. The degradation of the side-chain starts with the formation of the (25R)-26-hydroxy derivative.[In the literature it is sometimes also described as 27-hydroxyderivative]. The enzyme involved is again a cytochrome P-450. A series of steps then leads to cholic acid.

432

F.J. ZEELEN

H OH " " 4" " " ~

H

COR

-.

~

COOH

I

140

140

OH

H

R=OH R=NHCH2CH2SO3H R=NHCH2COOH

H

cholic acid taurocholic acid glycocholic acid

chenodeoxycholic acid H ~ COOH OH 9 . I

H

deoxycholic acid

Figure 5.

The bile acids.

Where many of the enzymes involved are not fully specific, other minor pathways of synthesis exist, whereby, for example, the degradation of the sidechain starts in an earlier stage and the formation of the 3o~-hydroxy-513-H system is completed in a later stage. Two different routes have been found for the biosynthesis of chenodeoxycholic acid (Figure 7). The first one branches from the cholic acid synthesis before the 12o~-hydroxylation. The normal side-chain degradation then leads to deoxycholic acid. The other route starts with side-chain hydroxylation. The 7(x-hydroxyl group is then introduced by a mitochondrial hydroxylase which is differs from the microsomal cholesterol 7o~-hydroxylase. Inborn errors of cholic acid metabolism are rare. Cerebrotendinous xanthomatosis (CTX) is caused by a defect in the 26-hydroxylation step. The failure to synthesize normal levels of bile acids and the resulting disruption in cholesterol metabolism leads to a build-up of this sterol and its 5o~-reduced derivative, cholestanol, in the tissues of the affected individuals. Accumulation in the central nervous system causes progressive neurological disfunction and eventual death. The disease is readily treated by oral primary bile acid therapy if recognized in the

H

H

HO

OH H ""

H ""

OH

O

'

OH

H

OH

H

H

H

26 I ' H

,' 1 /

~|

HO H

H H

OH ~ I

HO

Figure 6.

""

OH H FO . H"'- - ' " ~ ~ . ~

O

H OH ~I ~ ""~ "' ~

""

1

H

OH

Biosynthesis of cholic acid. 433

.~COOH

/

1"i4

434

F.J. ZEELEN

H

H

HO cholesterol ~

H

'~

H

-

H chenodeoxycholic acid Figure 7.

Biosyntheticroutes to chenodeoxycholic acid.

first decades of life prior to significant accumulation of sterols in the central nervous system. The bile acids then act by feedback inhibition. Inborn defects in activity of 313-hydroxy-C27-steroid oxidoreductase or of the 513-reductase enzyme lead to accumulation of potentially cholestatic and hepatotoxic a typical bile acids. Primary bile acid therapy has proven also here to be beneficial in normalizing liver dysfunction. A low rate of conversion of cholesterol to bile acids may result in bile which is super-saturated with cholesterol. Crystallization of cholesterol monohydrate is then possible and may lead to formation of cholesterol gallstones. Conversely, administration of large amounts of cholic acid or chenodeoxycholic acid may result in dissolution of cholesterol gallstone. Cholic acid when administered by local infusion gives unacceptable side-effects while chenodeoxycholic acid is effective after oral administration. It is used in the treatment of patients with small cholesterol gallstones when surgery is contra-indicated.

THE STEROID HORMONES OF THE GONADS Progesterone, estradiol and estrone are the most important female sex hormones (Figure 8). The estrogens, estradiol and estrone, are responsible for the develop-

Medicinal Chemistry of Steroids

435

ment and maintenance of the sexual characteristics of the female. These hormones stimulate, in the female, growth and development of the vagina, uterus and Fallopian tubes. They contribute to enlargement of the breast through promotion of ductal growth, stromal development and accretion of fat. In concert with progesterone, the estrogens are responsible for the progress of the menstrual cycle and play a role in the maintenance of pregnancy. In the male, testosterone and stanolone are the major sex hormones (Figure 8). Together, these two hormones are responsible for the development and maintenance of the sexual characteristics, but their functions are different. During embryogenesis of the male, testosterone is important for the development of those portions of the urogenital tract derived from the Wolffian ducts (epididymis, vas deferens, seminal vesicle and ejaculatory duct), while stanolone is important for the virilization of the external genitalia and urogenital sinus. In man at puberty, testosterone induces the male sex drive and performance, increases muscle mass and vocal cord enlargement, penis and scrotum enlargement and spermatogenesis. Stanolone induces an increase in facial and body hair, acne and, at a later stage, scalp hair recession and prostate enlargement.

o

oN

I-IO

o

o

I-IO

progesterone

estradiol

estrone

oH

oN

o

I-I testosterone

Figure 8.

Steroid hormones of the gonads.

stanolone

FJ. ZEELEN

436

Biosynthesis Progesterone is produced from cholesterol (Figure 9) in the ovary and placenta, as well as in the adrenals. The side-chain of cholesterol is removed in a series of oxidative steps to yield pregnenolone. This process is carried out by a single enzyme, cytochrome P- 450sc C (side chain cleavage). Oxidation of the 3-hydroxyl group and isomerization of the double bond leads to progesterone. Both reactions are catalyzed by a single enzyme, 313-hydroxysteroid dehydrogenase. The enzyme in the ovary differs from that in the placenta, but is identical with that in the adrenal and testis. When this enzyme is deficient, the synthesis of progesterone is hindered. The result is accumulation of pregnenolone, which is then degraded to 17-keto-steroids (see biosynthesis of the androgens). These are converted by the peripheral 313-hydroxysteroid dehydrogenase into androgens. Deficiency of 313-hydroxysteroid dehydrogenase is a possible cause of congenital adrenal hyperplasia, which can also be due to deficiency of the enzyme steroid 21-hydroxylase (see biosynthesis of hydrocortisone). Congenital adrenal hyperplasia is the most frequent cause of ambiguous genitalia and salt wasting in newborn infants. When growing up, symptoms of nonclassical 313-hydroxysteroid dehydrogenase deficiency can vary from premature puberty and accelerated growth in children, to hirsutism, acne, temporal balding, irregular menses and infertility in adults. Progesterone production rates range from mean values of 0 75 mg/day for young males and ovariectomized females to about 300 mg/day in late pregnancy. The normal production rates in the menstrual cycle are 0.75-2.5 mg/day in the first (follicular) phase and 15-50 mg/day in the second (lutual) phase. In men, the androgens are synthesized from cholesterol via pregnenolone (see Figure 1I) by the Leydig cells in the testis and to a lesser extent by the adrenals. In the male, 3-10 mg of testosterone is synthesized daily. A single enzyme, cytochrome P-45017 a, catalyzes both the introduction of the 17-hydroxyl group, leading to hydroxypregnenolone, and the ensuing oxidative removal of the pregnane side-chain, yielding 313-hydroxyandrost-5-en-1 7-one. Oxidation of the 3-hydroxyl group and isomerization of the double bond gives androstenedione. Reduction of the 17-keto group by the enzyme 1713-hydroxysteroid dehydrogenase (also termed 17-ketoreductase) produces testosterone. Alternatively, the order of reactions may be reversed. Deficiency of 1713-hydroxysteroid dehydrogenase is the cause of a rare, inherited form of male pseudohermaphroditism. The affected males are usually reared as females during childhood. During puberty marked virilization occurs, leading in many cases to the spontaneous adoption of a male gender role. Stanolone (5 -dihydrotestosterone) is formed through the action of the enzyme 5tx-reductase. In the human, two 5tx-reductases, chronologically identified as type I and type II, are known. The type II enzyme is the major reductase in the prostate, whereas the type I isoenzyme is also present in the skin. Inherited defects in the type

437

Medicinal Chemistry of Steroids

H

D,"

cholesterol

pregnenolone

L

o

0

0

progesterone Figure 9. Biosynthesisof progesterone. II isoenzyme lead to male pseudohermaphrodism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. Where stanolone is the male hormone stimulating the prostate, inhibitors of 5cx-reductase are being developed for the treatment of prostate cancer and benign prostatic hyperplasia. Finasteride (see Figure 10), a specific inhibitor of the type II reductase, was introduced recently for the treatment of benign prostatic hyperplasia. In women, estrone is synthesized in the ovary from cholesterol via androstenedione (see Figure 12). The whole reaction sequence proceeds in three oxidative steps catalyzed by a single enzyme, aromatase, a cytochrome P-450 enzyme (P-450AROM) The enzyme estradiol 17[3-dehydrogenase converts estrone into the true hormone estradiol. This enzyme differs from the 17-hydroxysteroid dehydrogenase, which is important in the synthesis of testosterone. The enzyme aromatase can also convert testosterone into estradiol. This route is less important than the one already mentioned. The amounts produced vary during the menstrual cycle. Estradiol production may rise from 0.1 mg/day in the early follicular phase to 0.5-1.0 mg/day in the late follicular phase, decreasing to

438

F.J. ZEELEN

CONHC(CH3)3

o

Finasteride H

Figure 10.

H

Inhibitor of 5o~-reductase type II.

0.3 mg/day in the mid-luteal phase. Estrone production follows a similar pattern: 0.1 mg/day-early follicular phase; 0.3-0.6 mg/day-late follicular phase; and 0.3 mg/day-mid-luteal phase. Estrogen biosynthesis occurs not only in reproductive tissues of the female but also in adipose tissues. In post-menopausal women, this is the major site of production whereby estrone becomes the main estrogen. Its production is about 40 ~tg/day. Small amounts of estrogens are synthesized in the male testis, prostate, adipose and muscle tissue, and skin fibroblasts as well, as in the liver and the brain. Estriol is a weak estrogen, which is produced in the placenta during pregnancy (see Figure 13).

The Menstrual Cycle The series of events which regulate the menstrual cycle of the female highlights the interplay between the female hormones progesterone and estradiol. The first half of the normal 28-day cycle is dominated by the ripening follicle and is therefore called thefollicularphase. A critical role is played by the follicular hormone (FSH), produced and secreted by the hypophysis under the influence of the gonadotrophin-releasing hormone from the hypothalamus. This same releasing hormone stimulates the release of luteinizing hormone (LH), which explains why it is often named the luteinizing hormone-releasing hormone (LHRH). FSH stimulates the growth of the ripening follicle and induces the formation of receptors for LH in the theca and granulosa cells of this follicle. Circulating LH can then interact with these receptors and stimulate the synthesis of progesterone, whereas the synthesis of estradiol is stimulated by FSH (see Figure 14). In the uterus, the estrogens stimulate the regeneration and growth of the endometrium and induce the synthesis of receptors for progesterone.

--0

~---o

OH

1to hydroxypregnenolone

pregnenolone 0

. _ _ . ~ HO

1to 0

~

o

0

testosterone

androstenedione ~

0

0

H

H

stanolone

Figure 11. Biosynthesisof the androgens.

439

440

F.J.ZEELEN o

0

> o

No

androstenedione

estrone

~t

OH

OH

O

HO

estradiol Figure 12.

Biosynthesisof the estrogens.

OH -OH

HO

Figure 13.

Estriol.

The whole process is regulated via a feedback mechanism whereby increased concentrations of estradiol and also of progesterone lead to a decreased release of LHRH from the hypothalamus. At this stage of the cycle, the rising levels of estradiol stimulate the synthesis of LH in the hypophysis. The release is not

Medicinal Chemistry of Steroids

441

BRAIN

HYPOTHALAMUS

1

LUTEINIZING HORMONE RELEASING HORMONE (LHRH)

l

J ESTRADIOL

-% j-.

HYPOPHYSIS

l

LUTEINIZING HORMONE

PROGESTERONE

FOLLICLE STIMULATING HORMONE

OVARY

l

Figure 14. Regulatory system for the estrus cycle.

stimulated, however, until a sufficiently high level of estradiol has been reached. When that level is reached, preovulatory gonadotrophin release is triggered. Serum concentrations of LH and, to a lesser extent, FSH increase rapidly which, provided a follicle is in the appropriate stage of maturity, leads to rupture of the follicle and expulsion of the ovum (ovulation). The remaining granulosa cells increase in size and form the corpus luteum. This marks the start of the luteal phase. The luteinized cells synthesize progesterone, which leads to a rapid rise in circulating progesterone levels. In the uterus, this hormone induces a transformation of the proliferative endometrium to a differentiated, secretory endometrium, which makes it ready for implantation of a fertilized ovum.

442

F.J. ZEELEN

If the ovum is fertilized, it is then further transported, while dividing, in about three days through the Fallopian tube to the uterus, where it takes another three days to prepare for nidation. In the absence of implantation, the corpus luteum degenerates after approximately 10 days. Both progesterone and estradiol production decrease rapidly, initiating shedding of the uterine endometrium (menstruation), which marks the start of the new cycle. During pregnancy, the production of progesterone by the corpus luteum and later by the placenta suppresses gonadotrophin secretion and inhibits ovulation.

Contraception Some 65 million women all over the world use steroid contraceptives to control their fertility. The combination estrogen-progestagen oral contraceptives are widely used. The administration of such a combination of estrogen and progestagen for 20-22 days results in a number of effects. First, it blocks ovulation; secondly, it interferes with phasic development of the uterine endometrium which decreases the chance for successful implantation, and thirdly, the cervical mucus may become so viscous that it hinders sperm penetration. The fall of estrogen and progestagen levels at the end of the treatment stimulates shedding of the endometrium leading to regular bleeding. This is of importance since it proves to the user that the contraceptive is effective. Since both estradiol and progesterone have little or no oral activity, synthetic analogues had to be developed. Ethinylestradiol is the estrogen of choice (see Figure 15). The daily dose is 0.02-0.05 mg. A larger selection of progestagens is now available (see Figure 16). Norethindrone was one of the first compounds used for contraception. Norethindrone acetate, norethynodrel and ethynodiol diacetate are pro drugs. In the body, these compounds are converted into norethindrone. These progestagens are used in a dose of 0.5-2 mg/day. Levonorgestrel was developed later. It is used in a dose of0.125-0.25 mg/day. Recently, desogestrel, dienogest, gestoden, and norgestimate have been introduced (Figure 17).

OH m

C

HO Figure 15.

Ethinylestradiol.

CH

Medicinal Chemistry of Steroids

443

c-c.

OH

OCOCH3

0

norethindrone acetate

norethindrone OH

OCOCH3 C--

O

C-- CH

CH3COO

ethynodiol diacetate

norethynodrel

Figure 16. Progestagensused in contraception. ,., ~,,2..n OCOs~CH3 C "" CH

C2H - C~CH

O

HO,A,N levonorgestrel

norgestimate

OH

_ OH

C2Hsi C2Hsi ~~~~L~C--CH ~',,~C~CH H H .

desogestrel

gestoden

9I i ~ ~ -

OH

CH2CN

dienogest

Figure 17. The newer progestagens. Therapeutic Use of Progestagens The progestagens are also used in the treatment of gynecological disorders such as dysmenorrhea, amenorrhea and dysfunctional uterine bleeding. Such therapy is essentially substitutive, replacing the missing endogenous ovarian hormone. The above

444

F.J.ZEELEN

L-o OC

~ OCOCH3

O CH3

CH3

medroxyprogesterone acetate

R=CH3 megestrol acetate

R=H nomegestrol acetate

c.3

O

O

CH3

O promegestone Figure 18.

dydrogesterone

Progesteronederivatives used as orally active progestagens.

mentioned progestagens may be used for this purpose, but a number of other progesterone derivatives has been developed specifically for this purpose (see Figure 18). Medroxyprogesterone acetate is used, at oral doses of 2.5-10 mg daily, for the treatment of functional uterine bleeding and secondary amenorrhea. Related compounds are megestrol acetate and its 19-nor derivative nomegestrol acetate. Promegestone which is a potent progestagen, is used therapeutically at oral doses of 0.125-0.25 mg daily. Dydrogesterone has an unnatural (retro) configuration. It is used therapeutically at oral doses of 10-40 mg daily.

Therapeutic Use of Estrogens Estrogens are used as estrogen replacement therapy; for example, to moderate the severe symptoms of menopause. This therapy can also reduce the rate of bone loss in postmenopausal women. A choice of oral and injectable preparations,

Medicinal Chemistry of Steroids

445

vaginal creams and a transdermal system are available for this application. These preparations mostly contain estradiol or estradiol esters. From the days when pregnant mares' urine was the only source of estrogens, preparations containing estrone sulfate or mixtures of estrone sulfate and other sulfates present in this urine have remained in use ("Conjugated estrogens" and "Esterified estrogens") (see Figure 19).

Breast Cancer The tumors in breast cancer are often estrogen dependent. When these tumors cannot be removed by surgery, it is important to remove the estrogen stimulus. The first approach is to block the action of circulating estrogens at the receptor level with an anti-estrogenic substance. The non-steroid tamoxifen is widely used for this purpose (see Figure 20). The second approach is to inhibit steroid aromatase so that estrogen production is blocked. For this action the non-steroid aminoglutethimide is used, but this compound is not fully specific. Hence, much research is going on in this area to find selective steroidal or non-steroidal aromatase inhibitors Formestane, a steroidal aromatase inhibitor, has been introduced recently.

Therapeutic Use of Androgens Androgens are used as replacement therapy in male hypogonadism and eunuchoidism. They are also used in view of their stimulating effect on the production of erythrocytes in the treatment of anemias caused by decreased erythropoiesis. The oral activity of testosterone and testosterone esters is low, so that these preparations have to be administered by injection. A preparation containing testosterone undecanoate dissolved in oleic acid is an exception in that it is reasonably active after oral administration. This is due to lymphatic absorbtion of this ester, so that the circulation can be reached without passage through the liver (see Figure 21 ).

o

o

OH !

NO~::} estrone sulfate

equilin sulfate

Figure 19. Major estrogen sulfates present in pregnant mares' urine.

446

F.J. ZEELEN

OCH2CH2N(CH3)2

o

/ NH2

O" -NH

"O

OZ OH formestane

aminoglutethimide

Figure 20.

tamoxifen

Compoundsused inthe treatment of estrogen dependent tumors.

OCOCloH21

OH

OH

OH

o

testosterone undecanoate

Figure 21.

methyltestosterone

fluoxymesterone

Someandrogens used in therapy.

An alternative approach is provided by substitution with a 17tx-alkyl group. A 17tx-methyl substituent hinders the inactivation of testosterone through oxidation of the 1713-hydroxyl group but does not interfere with the binding of the steroid to the receptor. Methyltestosterone and fluoxymesterone are effective oral androgens but these 17tx-methylated steroids show an increased risk for adverse effects on liver function (see Figure 21). In view of their anabolic activity, androgens are also used in conditions such as chronic infections, extensive surgery, or severe trauma which require reversal of catabolic processes or protein-sparing effects. These agents are adjuncts to, and not replacement for, conventional treatment of these disorders. A large number of steroids with a more selective action have been developed ("anabolic steroids"). These compounds are either derivatives of stanolone such as stanolone valerate and stanozolol, or derivatives of 19-nortestosterone (see Figure 22). Despite the risk of unwanted side-effects, androgens are used in

Medicinal Chemistry of Steroids

447

OCOC4H9

H

H

stanolone valerate

oH

0COC9H19

H

stanozolol

nandrolone decanoate

Figure 22. Anabolic steroids.

oH

Figure 23.

Danazol.

excessive dosages by athletes. Documented usage of these medications is a basis for disqualification in many athletic events. There is nevertheless a huge black market for these drugs. In men, the androgens suppress the activity of the anterior pituitary. This is part of the feed-back mechanism regulating the synthesis of the androgens. In women, androgens also suppress the activity of the anterior pituitary, resulting in the suppression of ovarian activity. For that reason danazol with its weak and impeded peripheral androgenic activity and strong pituitary suppressive action is used in the treatment of endometriosis (Figure 23).

Anti-Androgens Anti-androgens are of interest in the treatment of androgen-dependent diseases, such as androgen-dependent prostate cancer, acne, seborrhea and hirsutism. Cyproterone acetate is widely used as an anti-androgen but this compound has also progestational activity. Recently, flutamide, a non-steroidal selective anti-androgen, has entered the market (Figure 24).

F.J. ZEELEN

448

0

NO2 CFa

OCOCH3

NNCOCH(CH~ 0 a

Figure 24.

cyproterone acetate

flutamide

Anti-androgens.

THE

ADRENAL CORTICOIDS

Steroid hormones produced by the cells of the adrenal cortex in response to appropriate stimuli are the glucocorticoid hydrocortisone, the major steroid secreted in response to stress, and the mineralocorticoid aldosterone, which plays an important role in electrolyte and water balance and regulation of blood pressure (see Figure 25). These two hormones are synthesized in different cells. Aldosterone is synthesized and released by the cells of the outer layer of the zona glomerulosa of the adrenal cortex, while hydrocortisone is synthesized by the cells of the next inner layer, the zone fasciculata. The secretion of the two hormones is controlled by different systems.

Hydrocortisone Hydrocortisone promotes the conversion of protein to carbohydrate (gluconeogenesis) and the storage of carbohydrate as glycogen. Hydrocortisone decreases or prevents tissue responses to inflammatory processes, thereby reducing the development of local heat, redness, swelling and tenderness, the symptoms by which inflammation is recognized. However, the underlying cause of the disease persists. Hydrocortisone also has a complex, suppressant action on the immune system. For example, the number of T-lymphocytes and, to a lesser extent, B-lymphocytes in the blood are reduced and the ability of macrophages to produce interleukin-1 is impaired by hydrocortisone. The synthesis and release of hydrocortisone is stimulated by adrenocorticotrophin (ACTH), a peptide consisting of 39 amino acids, which is released by the

Medicinal Chemistry of Steroids

0

o

0

0

o

aldosterone Figure 25.

449

hydrocortisone

Adrenal corticosteroids.

anterior pituitary (adenohypophysis) in response to corticotrophin releasing hormone (CRH). CRH, a peptide of 41 amino acids, is secreted by the hypothalamus in response to signals from the limbic system. A feedback mechanism operates in such a way that high levels of hydrocortisone suppress the response of the hypothalamus and hypophysis (Figure 26). When hydrocortisone is overproduced, often due to a pituitary tumor causing high levels of circulating ACTH, the resulting disease is known as Cushing's disease. When hydrocortisone is underproduced, the disease is known as Addison's disease, most frequently the result of adrenal atrophy. It can, however, also be caused by inborn errors of metabolism. These will be discussed later. The biosynthesis of hydrocortisone (Figure 27) starts from cholesterol, which is converted in the usual manner into pregnenolone. The enzyme cytochrome P-45017tz introduces the 17cz-hydroxylgroup. This same enzyme can also oxidize the steroid further to the 17-ketone on the path to androgens. The ratio of 17,20 lyase to 17tx-hydraoxylase activity seems regulated by the availability of reducing equivalents flowing to the enzyme. The next step is the oxidation of the 313-hydroxyl group and isomerization of the 5,6-double bond. These two reactions are carried out by a single enzyme, 3~-hydroxy-steroid dehydrogenase. The enzyme of the adrenal is identical with that of the ovary and testis, but different from that present in the placenta and skin. When this enzyme is deficient, the concentration of hydrocortisone is low, resulting in increased output of ACTH by the pituitary, owing to reduced feedback. The high ACTH levels result in adrenal hyperplasia and the concentration of the precursor steroid increases markedly. It is then converted to the 17-ketosteroid, which is converted by the peripheral 3 ~l-hydroxysteroid dehydrogenase into androgens. The result is excessive virilization (Congenital adrenal hyperplasia). The next step of the biosynthesis of hydrocortisone is the 21-hydroxylation, carried out by the enzyme steroid 21 hydroxylase, a cytochrome P-450 enzyme

450

F.J. ZEELEN

(P-45021). As already mentioned, deficiency of this enzyme is another cause of congenital adrenal hyperplasia. The final step is the l ll3-hydroxylation, which is executed by another cytochrome P-450 (P-45011B). This enzyme is specific for the zone fasciculata. The normal production rate of liydrocortisone is about 20 mg/day.

HYPOTHALAMUS

CORTICOTROPHIN RELEASING HORMONE

C .Y,,o,..Ys.s

ADRENOCORTICOTROPHIN (ACTH) ,

,,

"'~ADRENA L C O R T E X

ZONA FASCICULATA

HYDROCORTISONE Figure 26.

Regulatorysystem for hydrocortisone release.

Medicinal Chemistry of Steroids

H

451

0

OH

HO

o.

O

O

-OH

-OH

hydroxyprogesterone Figure 27.

OH

O OH

hydrocortisone

Biosynthesisof hydrocortisone.

Therapeutic Use of Corticosteroids The corticosteroids are used in physiological doses for replacement therapy in adrenal insufficiency. In primary adrenal insufficiency, such as Addison's disease or after adrenalectomy, both mineralocorticoid and glucocorticoid replacement is needed. It is then usually preferred to use orally active synthetic analogues with a combined action, for example, fludrocortisone acetate (Figure 28). In secondary adrenal insufficiency associated with inadequate adrenocorticotrophin secretion, glucocorticoid replacement is usually adequate. Corticosteroids are also used in the treatment of congenital adrenal hyperplasia resulting from deficiency of the 1113-or 21-hydroxylation. The corticosteroids then also suppress ACTH and so remove the stimulus for increased androgen production. In patients with known or suspected adrenal insufficiency, rapidly acting corticoids are administered intravenously or intramuscularly prior to surgery, or if shock, severe trauma or other stress conditions occur. Hydrocortisone is not very soluble in water, but its phosphate ester is. In the body this ester is hydrolyzed to the active hydrocortisone. The phosphate ester (Figure 28) is therefore used in formulations intended for intravenous administration. The corticosteroids are used in pharmaceutical doses for their antiinflammatory (antiphlogistic) and, imunosuppressant glucocorticoid properties. For these pur-

452

F.J. ZEELEN

OCOCH3

OPO(ONa)2

O

O

OH

OH

O

fludrocortisone acetate Figure 28.

hydrocortisone phosphate

Corticosteroids.

poses, the synthetic analogues, with reduced mineralocorticoid action, are preferred to hydrocortisone. Some examples are given in Figure 29. These drugs are all derivatives of hydrocortisone. Cortisone with its 11-ketogroup has no intrinsic activity but in the body it is reduced to hydrocortisone, so its systemic activity is comparable to that of hydrocortisone. The same holds for prednisone, which is converted to prednisolone. Both pro drugs are also used therapeutically (see Figure 30). For most indications, glucocorticoid administration provides symptomatic relief but has no effect on the underlying disease processes. Use of these medications does not eliminate the need for other therapies that may be required. Glucocorticoids may be used, for example, to treat selected collagen and rheumatic disorders. Intra-articular injections of glucocorticoids may be used to treat rheumatoid arthritis and osteoarthritis. For this application, the potent synthetic analogues may be preferred because of the small volume of the effective dose. Glucocorticoids are also used to treat allergic disorders such as asthma, rhinitis and allergic skin reactions. For those asthmatic patients who require chronic maintenance therapy, inhalation aerosol therapy is often preferred. Even with this route of administration, a large proportion of the administered dose may enter the systemic circulation via absorption from the respiratory and gastrointestinal tracts. For this reason glucocorticoids have been developed, which are rapidly metabolized to less active or inactive products. Examples are beclomethasone dipropionate and flunisolide (Figure 31). Topical administration is also indicated for the relief of inflammation and pruritus associated with a range of skin disorders varying from diaper rash and sunburn to psoriasis and discoid lupus erythematosis. Treatment includes lotions, creams and ointments. These dermatological preparations differ very much in potency, so that the physician can select the most suitable class for the disorder to be treated.

Medicinal Chemistry of Steroids

~

453

OCOCH3

OH

OH

OH

OH

O OH

-OH

o

OH

O OH

o I

hydrocortisone acetate

prednisolone

oH

,

0

dexamethasone

Figure 29.

OH

0"

v

~,

CH3

methylprednisolone

_

0

OH

Z

paramethasone acetate

triamcinolone

Some glucocorticoids intended for systemic administration.

The classical corticoid such as hydrocortisone and the synthetic analogues dexamethasone and its phosphate or the acetate of methylprednisolone show weak topical activity. Analogues have been developed by chemical modifications such as esterification and substitution, which have the desired potency of local action and lack systemic activity. This may be illustrated by the following series of triamcinolone derivatives with potencies varying from low (desonide) to high (halcinonide). One may note the influence of fluoro substitution at the 6- or 9-position, esterification of the 21-hydroxyl group and etherification of the 16- and 17-hydroxyl groups (Figure 32). Note that the availability of the active corticosteroids from these topical formulations is influenced by the vehicle, so that activities may vary depending on the pharmaceutical formulation.

454

F.J. ZEELEN

OH

.,OH

-'-0 OH

o

o

prednisone

cortisone

Figure 30.

Prodrugs.

-/_

o.

- OCOC 2Hs -~

o

CH3

0

I

beclomethasone dipropionate

Figure 31.

F flunisolide

Glucocorticoids used in the treatment of asthma.

It is impossible to discuss here all the corticosteroids used today in topical preparations. They seem to come in endless series! For example, the 16[3-methyl series. The potencies vary again from low to very high (clobetasol propionate) (Figure 33). Local corticoid therapy is also used to treat inflammatory ocular disorders, but long-term administration may induce side-effects such as increased intraocular pressure. Medryson and fluorometholone have been specifically developed for ocular administration (Figure 34). These steroids have a decreased tendency for inducing increased intra-ocular pressure. Systemic glucocorticoid administration is used in severe ocular disorders and disorders involving deep ocular structures.

--OH OH

OH

OH

OO _O_o

1--~ O

OH

O

OH

ooO

0 I

..iv F triamcinolone acetonide

desonide

flurandrenolide

~o~o~.. I:o -- 0

OCOCHs

._o./__

~

_o

0

o

Oo~

0 I

F fluocinolone acetonide

Figure 32.

amcinonide

halcinonide

Topical corticosteroids.

~O~O+,H+ [ ..,,,. v ~ .J-,./'-.~.L v.~

\H, /-c,+

I

F betamethasone valerate

Figure 33.

diflorasone diacetate

O t h e r topical corticoids.

455

clobetasoi propionate

O~,H,

456

F.J. ZEELEN

OH

F---O

oN

I--O

0 I OH3

medryson

fluorometholone

Figure 34. Steroidsfor ocular administration. lnhibitors of Hydrocortisone Synthesis Inhibitors of hydrocortisone synthesis may be used to treat Cushing's syndrome, a disorder characterized by over-production of hydrocortisone. Metyrapone is a relatively non-toxic, fairly selective inhibitor of Cytochrome P-450 -, the enzyme 1115 responsible for the 1113-hydroxylation. It is, however, mainly used clinically to test the ability of the pituitary to secrete ACTH in response to the decreased concentration of plasma cortisol (see Figure 35). Trilostan (Figure 35) is an inhibitor of the 313-hydroxysteroid dehydrogenase. This compound is used to treat Cushing's syndrome. Also with this compound a compensatory stimulation of the secretion of ACTH is found. Aldosterone

This hormone regulates electrolyte balance in body fluids by promoting retention of sodium ions and excretion of potassium ions. Excessive levels of aldosterone lead to hypertension. Appreciable levels of this hormone are normally generated only in stress. Aldosterone exists in solution as an equilibrating mixture of structural isomers. The two principal forms are the 11, 18-hemiacetal and the acetal (Figure 36). As shown in Figure 37, the synthesis and release of aldosterone is stimulated by angiotensin II, an octapeptide, which is formed in the circulatory system. A fall in blood volume, a decrease in Na + concentration in the blood, a fall in blood pressure or release of cathecholamines at the juxtaglomerular apparatus of the kidney stimulates the release of renin, an enzyme with proteolytic activity from the juxtaglomerular cells. Renin acts on angiotensinogen, a plasma ~2 -globulin, and releases angiotensin I. This decapeptide, an intermediate with no hormonal activity, is then attacked by angiotensin converting enzyme to give the hormone, angiotensin II. This hormone,

Medicinal Chemistry of Steroids

45 7

OH 0

D

O r

~,

0 H

metyrapone Figure 35.

trilostan

Inhibitors of hydrocortisone synthesis.

O OH

OH

i

O

r--OH 9

OH

O

O

Figure 36.

OH

O

Different forms of aldosterone.

in addition to its effects on the zone glomerulosa cells of the adrenal cortex, sensitizes vascular smooth muscles to the contractile effects of norepinephrine. There is also evidence that atrial natriuretic factor, a recently discovered peptide hormone, inhibits the steroidogenic capacity of the adrenal zon glomerulosa. The normal production of aldosterone varies from 100-500 Ftg/day. The biosynthesis of aldosterone starts from cholesterol, which is converted in the usual manner into progesterone. The enzyme steroid 21 hydroxylase, a cytochrome P-450 enzyme, converts progesterone into deoxycortone. This cytochrome, P-45021, also plays an important role in the synthesis of hydrocortisone. The next step in the biosynthesis is the formation of corticosterone, which is followed by two further hydroxylations at the 18-position to give aldosterone (Figure 38). A single enzyme, cytochrome P-45018 catalyzes all three hydroxylations. Patients deficient in cytochrome P-45018 are subject to potentially fatal electrolyte abnormalities as neonates and a variable degree of hyponatremia and hyperkalemia combined with poor growth in childhood, but they may have no symptoms as adults.

458

FJ. ZEELEN

KIDNEY

JUXTAGLOMERULAR CELLS

RENIN

ANGIOTENSINOGEN

ANGIOTENSIN I

ANGIOTENSIN CONVERTING ENZYME

ANGIOTENSIN II

S

,o,E,,L

L zoNAGLOMEROLOSA) Figure 37.

Regulation of aldosterone synthesis and secretion.

It is of interest to note that patients have been found with mutations in this enzyme resulting in specific deficiencies; for example, in the last 18 hydroxylation or in the two 18 hydroxylations. So these three hydroxylations seem to occur in slightly different active sites on this enzyme.

Medicinal Chemistry of Steroids

459 ---OH

--0

OH

---0

OH

0

0

progesterone

deoxycortone H

corticosterone

OH 0

O'

OH 0

0

0

aldosterone

Figure 38.

Biosynthesisof aldosterone.

Therapeutic Use of Mineralocorticoids and Antagonists Aldosterone is one of the more potent of the steroid drugs but it has found little use in clinical practise. Steroidal aldosterone antagonists such as spironolactone and canrenone (see Figure 39) are important in the treatment of edematous states of different origins and other diseases with enhanced aldosterone production.

NEUROSTEROIDS Steroids have a role in the nervous system, although their exact functions remain to be determined. For example, 3ct-hydroxy-5ct-pregnan-20-one(see Figure 40) is present in the human brain and peripheral (sciatic) nerves. Its synthesis is independent of adrenal or gonadal function. The steroid proved to be a potent allosteric modulator of the y-aminobutyric acid (GABA)-A receptor complex in the frontal cortex, but a weak modulator of the GABA-A receptor complex in the spinal cord. In animal experiments, 3t~-hydroxy-5tx-pregnan20-one showed an anxiolytic effect. It is of interest to note old experiments which demonstrated that in man, infusion of a pharmacological dose of progesterone (500 mg !) induced 1 to 2 hour hypnosis.

460

FJ. ZEELEN

0

0

0

SCOCH3

0

spironolactone Figure 39.

canrenone

Aldosteroneantagonists.

I OI4

Figure 40.

O

H

3a-Hydroxy-5a-pregnan-20-one.

This effect is probably due to metabolites of progesterone, such as 3c~-hydroxy5(x-pregnan-20-one (Figure 40). Elucidation of the role of these neurosteroids forms an interesting area of current research.

SUMMARY In the human organism, steroids fulfill a diverse number of functions. The organs, where these steroids are formed, differ but the biosynthetic routes and the enzymes involved are similar or even identical so that inborn defects in these enzymes often affect the biosynthesis of a number of steroids. For this reason, the discussion of the biosynthesis of the steroid is discussed at length.

Medicinal Chemistry of Steroids

461

In recent years molecular biology has succeeded in elucidating the structures of the enzymes involved, thus providing a much better understanding of congenital diseases affecting steroid metabolism. These results are a part of the discussion. The biological functions and properties of the major natural steroids are also discussed and synthetic analogues used as therapeutics are mentioned. Examples of steroid drugs acting as antagonists or inhibitors of enzymes of the biosynthesis are also given.

R E C O M M E N D E D READINGS Books Bohl, M., & Duax, W.L. (1992). Molecular Structure and Biological activity of Steroids, CRC Press, Boca Raton. Norman, A.W., & Litwack, G. (1987). Hormones, Academic Press, Orlando. Zeelen, F.J. (1990). Medicinal Chemistry of Steroids, Elsevier, Amsterdam.

REVIEWS DESCRIBING RECENT DEVELOPMENTS Bile Acids Axelson, M. Shoda, J., Sj6vall. J., Toll, A., & Wikvall, K. (1992). Cholesterol is converted to 7o~-hydroxy-3-oxo-4-cholestenoic acid in liver mitochondria. J. Biol. Chem. 267, 1701-1704. Pandak, W.M., Chun Li, Y., Chiang, J.Y.L., Studer, E.J., Gudey, E.C., Heuman, D.M., Vlahcevic, Z.R., & Hylemon, P.B. (1991). Regulation of cholesterol 7a-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in chronic biliary diverted rat, J. Biol. Chem. 266, 3416-3421. Russell, D.W., & Setchell, K.D.R. (1992). Bile acid biosynthesis, Biochemistry 31, 4737-4749.

The Steroid Hormones of the Gonads-Biosynthesis Andersson, S., & Moghrabi, N. (1997). Physiology and molecular genetics of 17[~ hydroxysteroid dehydrogenases, Steroids 62, 143-147. Hanukoglu, 1. (1992). Steroidogenic enzymes" structure, function, and role in regulation of steroid hormone biosynthesis, J. Steroid Biochem. Molec. Biol. 43, 779-804. Harris, G., Azzolina, B., Baginsky, W., Cimis, G., Rasmussen, G.H., Tolman, R.L, Raetz, C.R.H., & Elisworth, K. (1992). Identification and selective inhibition of an isoenzyme of steroid 5 -reductase in human scalp, Proc. Natl. Acad. Sci. USA 89, 10787-10791. Labrie, F., Simard, J., Luu-The, V., B61anger, A., & Pelletier, G. (1992). Structure, function and tissue-specific gene expression of 3~-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues, J. Steroid Biochem. Molec. Biol. 43, 805-826. Labrie, F., Van Luu-The, Lin, S.-X., Labrie, C., Simard, J., Breton, R., & B61anger, A. (1997). The key role of 1713-hydroxysteroid dehydrogenases in sex steroid biology, Steroids 62,148-158. Mendon~a, B.B., Russell, A.J., Vasconcelos-Leite, M., Arnhold, 1.J.P., Bloise, W., Wajchenberg, B.L., Nicolau, W., Sutcliffe, R.G., & Wallace, A.M. (1994) Mutation in 3fl-hydroxysteroid

462

F.J. ZEELEN

dehydrogenase type 11 associated with pseudohermaphroditism and premature pubarche or cryptic expression in females, J. Molec. Endocrinol. 12, 119-122. Peters, D.H. & Sorkin, E.M. (1993). Finasteride. A review of its potential in the treatment of benign prostatic hyperplasia, Drugs 46, 177-208. R6sler, A. (1992). Steroid 17l~-hydroxysteroid dehydrogenase deficiency in man: an inherited form of male pseudohermaphroditism, J. Steroid Biochem. Molec. Biol. 43, 989-1002. Thigpen, A.E., Davis, D.L., Milatovich, A., Mendonca, B.B., Imperato-McGinley, J., Griffin, J.E., Francke, U., Wilson, J.D., & Russell, D.W.(1992). Molecular genetics of steroid 5 -reductase 2 deficiency, J. Clin. Invest. 90, 799-809.

Therapeutic Use of Androgens Lukas, S.E. (1993). Current perspectives on anabolic-androgenic steroid abuse, TIPS 14, 61-68.

Hydrocortisone Karl, M., Lamberts, S.W.J., Detera-Watleigh, S.D., Encio, l.J., Stratakis, C.A., Hurley, D.M., Accili, D., & Chrousos, G.P. (1993). Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene, J. Clin. Endocrinol. Metab. 76, 683-689. Miller, W.L., Auchus, R.J., & Geller, D.H. (1997). The regulation of 17,20 lyase activity, Steroids 62, 133-142. White, P.C., Cumow, K.M., & Pascoe, L. (1994). Disorders of steroid 1 l~-hydroxylase isoenzymes, Endocrine Rev. 15, 421-438.

Aldosterone Jamieson, A., Connell, J.M.C., & Fraser, R. (1993). Glucocorticoid-suppressible hyperaldosteronism: from confusion to conclusion? J. Molec. Endocrinol., 10, 3-5. Kawamoto, T., Mitsuuchi, Y., Toda, K., Yokayama, Y., Miyahara, K., Miura, S., Ohnishi, T., Ichikawa, Y., Nakao, K., lmura, H., Ulick, S., & Shizuta, Y. (1992). Role of steroid 1ll]-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans, Proc. Natl. Acad. Sci, USA, 89, 1458-1462. Lombes, M., Kenouch, S., Souque, A., Farman, N., & Rafestin-Oblin, M.-E. (1994). The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 1113-hydroxysteroid dehydrogenase, Endocrinology, 135, 834-840. Miyahara, K., Kawamoto, T., Mitsuuchi, Y., Toda, K., lmura, H., Gordon, R.D., & Shizuta, Y. (1992). The chimeric gene linked to glucocorticoid-suppressible hyperaldosteronism encodes a fused P-450 protein possessing aldosterone synthase activity, Biochem. Biophys. Res. Commun. 189, 885-891. Mitsuuchi, Y. Kawamoto, T., Miyahara, K., Ulick, S., Morton, D.H., Niaki, Y., Kuribayashi, 1., Toda, K., Hara, T., Orii, T., Yasuda, K., Miura, K., Yamamoto, Y., lmura, H., & Shizuta, Y. (1993). Congenitally defective aldosterone biosynthesis in humans: inactivation of the P-450cl 8 gene (CYP11B2) due to nucleotide deletion in CMO I deficient patients, Biochem. Biophys. Res. Commun. 190, 864-869. Mitsuuchi, Y. Kawamoto, T., Rosier, A., Naiki, Y., Miyahara, K., Toda, K., Kuribayashi, 1., Orii, T., Yasuda, K., Miura, K., Nakao, K., Imura, H., Ulick, S., & Shizuta, S. (1992). Congenitally defective aldosterone biosynthesis in humans: the involvement of point mutations of the P-450Cl 8 gene (CYP11B2) in CMO II deficient patients, Biochem. Biophys. Res. Commun. 182, 974-979. Mulay, S., D'Sylva, S., & Varma, D.R. (1993). Inhibition of the aldosterone-suppressant activity of atrial natriuretic factor by progesterone and pregnancy in rats, Life Sci. 52, 1121-1128.

Medicinal Chemistry of Steroids

463

White, P.C., Pascoe, L., Curnow, K.M., Tannin, G., & R6ssler, A. (1992). Molecular biology of 11 [3-hydroxylase and 11 [3-hydroxysteroid dehydrogenase enzymes, J. Steroid Biochem. Molec. Biol. 43, 827-835.

Neurosteroids Bitran, D., Purdy, R.H., & Kellogg, C. (1993). Anxiolytic effect of progesterone is associated with increases in cortical allopregnanolone and GABA A receptor function, Pharmacol. Biochem. Behavior, 45, 423-428. Mathur, C., Prasad, V.V.K., Raju, V.S., Welch, M., & Lieberman, S. (1993). Steroids and their conjugates in the mammalian brain, Proc. Natl. Acad. Sci. USA, 90, 85-88. Mellon, S.H. (1994). Neurosteroids: biochemistry, modes of action, and clinical relevance. J. Clin. Endocrinol. Metab. 78, 1003-1008. Mok, W.M., Bukusoglu, C., & Krieger, N.R. (1993). 3o~-hydroxy-5tz-pregnan-20-one is the only active anesthetic steroid in anesthesized mouse brain, Steroids 58, 112-114. Giusti, L., Belfiore, M.S., Martini, C., & Lucacchini, A. (1993). 3o~-Hydroxy-5o~-pregnan-20-one modulation of solubilized GABA/benzodiazepine receptor complex, J. Steroid Biochem. Molec. Biol. 45, 309-314.