Pharmac. Therap. B. 1975, Vol. I, No. 2. pp. 233-275.

Pergamon Press.

Printed in Great Britain

Specialist Subject Editor:

Z. LARON

STRUCTURE

AND

EFFECTS

OF

ANABOLIC

STEROIDS

B. CAMERINO
Montedison, Largo Donegani 1-2, Milan, Italy

and R. SCIAKY
Farmitalia, Viale Bezzi 24, Milan, Italy

1. INTRODUCTION The discovery in 1935 by Kochakian and Murlin that extracts from human male urine injected into dogs castrated and maintained on a constant diet produced, as well as the expected androgenic effect, a net retention of nitrogen, justified the suggestion that there are in male urine substances able to give a positive nitrogen balance. Later, after the isolation of testosterone by Laqueur (David et al., 1935) the same metabolic effect was observed with the pure hormone. Even earlier there was evidence of the presence of such a substance. Bogrov, in 1891, showed that an emulsion of rabbit testes caused a decrease in urea excretion in the urine of two subjects and Sacchi, in 1895, described a case of gigantism characterized by an enormous increase in muscular strength, weight and height now known to be caused by a marked increase in androgen production, of a boy affected by a Leydig cell tumor of the testes. After the isolation of the first androstane derivatives, chemical and biological studies led to a sound knowledge of the anabolic action of steroids and in 1946 Kochakian compiled the first exhaustive review. As a result of the great advances in the field of anabolic steroids in the 1950's numerous synthetic derivatives entered the market. In 1960 Camerino and Sala published a comprehensive review, followed in 1963 by a volume by Kriiskemper (English translation 1968) specifically devoted to the biochemistry and clinical applications and in 1966 by that of Overbeek on the chemistry and pharmacology of the anabolic steroids. A report on anabolism, growth and ageing has been contributed by Applezweig in his book "Steroid Drugs" (vol. I, 1962, vol. II, 1964). The term anabolic steroids applies to those steroids that, inter alia, promote the synthesis and storage of cytoplasmic protein and stimulate the growth of tissues in general. For human use, an anabolic steroid should not possess any androgenic action but no such substance has yet been discovered. Nevertheless, the classification of activities into androgenic and anabolic is of therapeutic convenience, the androgenic action being simply, in effect, an anabolizing effect localized in specific organs, i.e. those connected with sexual function (ventral prostate and seminal vesicles in rats). The term myotrophic effect, referring to the site of protein synthesis i.e. muscle, is often used instead of anabolic effect. The anabolic action of a steroid is usually measured in terms of the nitrogen balance--fundamental since it refers to the actual definition of an anabolic substance-and of elements such as potassium, calcium and phosphorus. Such experiments are time-consuming and technically difficult. Since the main effect of anabolic action is protein synthesis, an obvious possibility for determining the anabolic effect of a steroid is that of measuring the weight increase of certain muscles, after the administration of the substance. The relation between androgenic and anabolic action of a steroid is different in different animal species and
233

234

B. CAMERINO and R. SCIAKY

also in the various muscles. In fact workers in this field have found no direct relationship between the quantity of steroid administered and the increase in weight in most of the muscles studied. Although the use of the temporal muscle of the male guinea-pig has been proposed for detecting the anabolic effect, the method has not been accepted, not only for the reasons explained above but also because the temporal muscle seems to be related, in the guinea-pig, to the sexual function. In rats, some muscles, such as those of the head, the neck and the heart, are not androgen-dependent. One muscle, in particular, has been shown to be suitable for detecting the myotrophic effect viz. the so-called levator ani,* which becomes smaller after castration and grows after administration of androgens. Furthermore, the weight increase of the muscle following treatment with androgens is roughly proportional to the quantity of steroid administered. Despite the fact that some authors claim that the levator ani assay is not suitable for evaluating the anabolizing properties of a steroid, most research workers consider it to be the simplest and most rapid test for an initial laboratory screening. The method of Eisenberg and Gordan, later modified by Hershberger et al. (1953), uses castrated rats, beginning the treatment the day after castration and administering the steroid by injection or by gavage for 7 days. After sacrifice, the weight of the levator ani is taken as a measure of the myotrophic activity and that of the ventral prostate and of the seminal vesicles as an indication of the androgenic activity. The greater the ratio of the increase in weight of the levator ani to the increase in weight of the ventral prostate, between treated and non-treated animals, the greater is the myotrophic effect and the lesser the androgenic action. This value is called the myotrophic-androgenic ratio. The data on anabolic and androgenic activity vary greatly, depending on the method of determination used, the type and weight of the animal used and on various other factors. There is thus no practicable standard for determining the relationship between these factors. Hence, it is necessary, for purposes of comparison, to determine the activity of the products under test and of the standards, side by side in the same laboratory. There are numerous variations on this method but they are beyond the scope of this review. They are extensively described elsewhere (Suchowsky, 1964). The most significant data on the anabolic activity of steroids, however, are a positive nitrogen balance and retention of nitrogen, phosphorus, calcium and potassium. The nitrogen balance is calculated by the difference in quantity of nitrogen entering the body in the form of a standardized diet and that excreted in the urine and feces. The first fundamental research, that of Kochakian and Murlin (1935), showed that changes in the nitrogen balance were androgen-dependent and that a positive displacement was achieved by pure steroids as well as by urine extracts (Kochakian and Murlin, 1936; Kochakian, 1937). Later, many research workers noted that nitrogen retention was paralleled by a decrease in excretion of calcium, potassium, phosphorus, creatine and water. The concentrations of potassium and phosphorus, in particular, follow that of nitrogen. The increased binding capacity of the newly synthesized proteins contributes to the retention of water. The increase in body weight is directly proportional to the rise in the increased assimilation of nitrogen so that in clinical tests, it is often the only parameter for evaluating the anabolic effect of steroids. According to the most recent evidence, such a parameter depends on one of the following factors:
J

(1) a higher rate of protein synthesis (2) a lower rate of protein catabolism (3) a lower rate of breakdown of amino acids to urea.
*M. Levotorani, see footnote, p. 164, Pharmac. Therap. B, I: 2 (1975).

Structure and effectsof anabolic steroids

235

Bartlett (1953) has shown that the retention of nitrogen in dogs, caused by treatment with testosterone propionate, depends on both an increase in the synthesis of proteins and a decrease in the catabolism of amino acids. Stimulation of protein anabolism by anabolic steroids has been demonstrated in several animal types (Gordan et al., 1947; Rupp and Paschkis, 1953; Kochakian, 1946; Kochakian et al., 1950). The hypothesis that the anabolic steroids act in the same way as natural androgens is supported by the fact that anabolic drugs having a clinical application lead to the retention of nitrogen, phosphorus, calcium and potassium in the same way as natural androgens (Kriiskemper, 1968, p. 50).

2. GENERAL VIEW ON THE SYNTHETIC ANABOLIC STEROIDS The chief aim of research in this field has been that of so modifying the testosterone molecule as to obtain compounds still possessing anabolic activity but with the least possible androgenic effects. However, no anabolic steroid from which the androgenic activity is completely absent has yet been discovered. Other criteria that have guided the search for new anabolic compounds relate to methods of administration according to the demands of clinical practice. The first steroids used were active only by parenteral administration and hence presented difficulties in usage. However, the discovery that 17a-alkylation leads to products active on oral administration opened up the field and 17a-methyl-AL androstene-3]3,17/3-diol (methylandrostenediol) entered into clinical practice. With only a few exceptions, all the anabolic steroids at present in use orally are 17a -alkylated. Later, in order to avoid the necessity for repeated injections, the search began for steroids that would be absorbed slowly and so exert their action over a prolonged period. This was achieved by esterification, with long-chain fatty acids or with other acids having a large number of carbon atoms, of the 17/3-hydroxyl group which is always present in the steroids active as anabolic agents. The anabolically active steroids can be classified in two large groups: (I) androstane derivatives, (2) 19-nor-androstane derivatives (estranes). In each group are found the following classes of compounds: (a) Steroids having a hydroxyl radical, free or esterified, in the 17/3-position and a hydrogen atom in the 17a-position. The products of this group are active only on parenteral administration, the sole exception being methenolone acetate which is active in oral administration also. The type of acid used to esterify the 17/3-hydroxyl group determines the duration of the anabolic action i.e. short chain (Cr-C~) acids give rise to steroids which are short acting while those with long chains (C7-C~o) give long acting compounds. (b) Steroids having a 17fl-hydroxy group and a 17a-alkyl group (e.g. methyl or ethyl). These can be used for both oral and parenteral administration. Several authors have proposed classifications based on C~s and C,9 skeletons but the fact that these 17a-alkylated derivatives exist in the estrane and androstane series makes these classifications of little significance. Generally, an active derivative having a 17/3-hydroxyl group and a 17or-hydrogen becomes active for oral administration when alkylated in the 17or-position. These statements are now examined more closely, using as examples the more important anabolic steroids already on the market. The synthesis, some of the pharmacological properties and, when possible, the relationship between activity and structure, are discussed.

236

B. CAMERINOand R. SCIAKY 3. ANDROSTANE DERIVATIVES

As already mentioned, Kochakian (1946) studied the anabolic effect of natural androgens and confirmed that these substances bring about in animals and humans a series of modifications such as increase in weight, improved calcification of bone material, stimulation of appetite and increase in the sense of well-being. From this evidence stemmed the possibility of using testosterone and other natural androgens in the treatment of underweight children, of disturbances of calcium metabolism, of malnutrition and, later, of osteoporosis occurring after the administration of corticosteroids. OH

(I) Initially, testosterone (A'-androsten-17~l-ol-3-one) (I), the principal male hormone, was used. However, because of its masculinizing and antiestrogenic properties, this compound produces many undesirable effects, such as hairiness, acne, change in tone of the voice and libido, risk of masculinization of the fetus etc. In addition, testosterone has a short action time, repeated administrations being required. Esterification of the 17~-hydroxyl group is now the best method for prolonging the duration of action and it results in compounds of greater anabolic and androgenic activity. 3.1. 17ct-METHYL-AS-ANDROSTENE-3B,17fl-DIOL;METHYLANDROSTENEDIOL 17a-Methyl-ALandrosten-3/3,17/3-diol (II) was the first synthetic product which showed a differentiation between androgenic and anabolic activities. Ruzicka et al. in 1935 prepared this compound by the Grignard reaction of dehydroepiandrosterone (I) with methyl magnesium iodide, thus:
o

OH

CH}MII

HO~

O)

(ii)

With regard to androgenic activity in animals, Deanesly and Parkes (1936) found that methylandrostenediol was one-half to two-thirds as androgenic as testosterone or methyltestosterone; Gordan et al. (1950) reported the anabolic effects in animals. In man Gordan et el. (1951) described nitrogen retention and weight gain in patients suffering from osteoporosis. Other authors noted the same effects in administration of large daily dosages of methyl androstenediol to patients having a negative nitrogen balance (Homburger et al., 1953) and hypogonadism (Querido et al., 1952). Methylandrostenedioi has been reported by Warren and Hayes (1952) to have, in man, an anabolic activity equal to that of testosterone propionate. On the other hand, Partridge et al. (1953) demonstrated that the product possesses relatively weak anabolic activity. Furthermore, experiments on animals (Korner and Young, 1956) and clinical trials (Homburger et al., 1953) showed that it has considerable androgenic potency at doses effective in growth stimulation; there was thus no significant differentiation between the anabolic and androgenic effects. Among secondary effects, methylandrostenediol, like all other androgens, showed antiestrogenic activity (Payne et al., 1956).

Structure and effectsof anabolic steroids 3.2. 5a-ANDROSTANE-17~I-OL-3-ONE; ANDROSTANOLONE

237

One of the first modification of the testosterone molecule was the reduction of the double bond between carbon atoms 4 and 5 (Butenandt et al., 1935), in which two dihydro derivatives, the isomers 5a- and 5/3-androstane-17/3-ol-3-one, are formed. The first of these proved interesting from an anabolic point of view while the 5/3-isomer, like all other 5/3-derivatives, was practically devoid of activity. This is probably due to the different conformations of rings A and B in the two series

5a-series

50-series

Androstanolone has also been prepared by the oxidation of a 17-monoester of 5aandrostane-3/3,17/3-diol followed by hydrolysis (Ruzicka and K/igi, 1937; Ruzicka et al., 1941): OH OH OR

H R= C~

H or COCH2CH2COOH

In addition, it was shown that this steroid was derived, metabolically, from the liver (rat liver homogenates; Rubin and Dorfman, 1956). The myotrophic-androgenic index determined in the rat has been reported to be 0.4 by subcutaneous and 0.8 by oral administration (Junkmann and Suchowsky, 1962), [Testosterone = 1]. Barnes et al. (1954) reported the value of 1.7 by subcutaneous administration; testosterone propionate serving as index. Hence, the two types of activity were not well differentiated. The compound still possessed appreciable androgenic activity and differed little from testosterone. Although androstanolone has now been replaced by more recent products it is still used in clinical practice by intramuscular administration. It shows a good anabolic effect when used in the treatment of older people where the androgenic activity is not considered an undesirable side effect (Pearson et al., 1954; Watkin et al., 1955). It was shown that, in man, androstanolone was converted into 17-ketosteroids (Pearson and McGavack, 1954; Sendrail et al., 1958; Romani, 1957). It was found that, after the administration of 100mg of androstanolone, the daily excretion of 17ketosteroids rose to 20 mg (Romani, 1957). The in vitro experiments of Ryan (1960) showed that androstanolone was the only steroid not aromatized to estrogen by the enzyme system of the human placenta (Ryan, 1959). 3.3. I-METHYL-AI-5Ot-ANDROSTEN-17fl-OL-3-ONE (ACETATEAND ENANTHATE); METENOLONE(ACETATE AND ENANTHATE) From a large number of steroids alkylated in position 1 prepared by Neumann and Wiechert (1965) 1-methyl-A~-5a-androsten-17/i-ol-3-one acetate (metenolone acetate) was chosen for extensive experimentation and subsequently placed on the market. In the first syntheses reported (Wiechert and Kaspar, 1960; Popper and Wiechert, 1962) the addition of diazomethane to A'-5a-androsten-17/3-ol-3-one (I) led to the

238

B. CAMERINO and R. SCIAKY

pyrazoline (II); elimination of nitrogen (IIIa) followed by acetylation gave 1-methyl-A l5a-androsten-1713-ol-3-one acetate (IIIb). This reaction took place both by heating (II) at its melting point (ca 220C), and by treating it at room temperature with an acid adsorbent in carbon tetrachloride solution (Kaspar et al., 1961). The best yields (80 per cent) were obtained, however, by heating (II) with bases such as quinoline and aniline (Wiechert, 1962); the lot,2a-methylene compound (IV) was isolated as a by-product. OH OH OR

H (I)

H (II)

H IIIa: R = H

IIIb: R = COCH3 Oil

O~ i v

iv
H

(IV) To avoid the danger of using a large quantity of diazomethane, another synthesis, beginning with the acetate of (I), was devised. The addition of hypobromic acid to this compound by the action of N-bromo-succinimide gave the bromhydrin (V) from which the la-hydroxy derivative (VI) was obtained by catalytic dehalogenation; after ketalization of the 3-keto group to give compound (VII) followed by Jones' oxidation, the l-ketone (VIII) was obtained. The metenolone acetate IIIb (Schering, 1963; Wiechert and Goedicke, 1963) was then produced by the Grignard reaction followed by acid hydrolysis and acetylation. OCOCH~ OCOCH3

H
Vl, .

O12

(v)

v~o

H (VI:)

OCOCH3

OCOCH;

(D C H )M sB r (2) H + O) A~20

O (VIII)

(llIb)

Structure and effects of anabolic steroids

239

From the screening of a large series of androstane derivatives alkylated in C-I, the conclusion can be drawn that, in general, alkylation is accompanied by a decrease in activity of the compound on the prostate and seminal vesicles (Cekan and Pelc, 1966; Neumann and Wiechert, 1965). 1-MethyI-AI-5a-androsten-17/3-ol-3-one has been studied particularly as the acetate (metenolone acetate) for intramuscular administration and as the enanthate (metenolone enanthate) used as a depot for the compound. It was later found that in oral administration both metenolone alcohol (Kriiskemper and Breuer, 1962) and acetate (Weller, 1962) were active in man. Hence, 1-methyl-A ~5a-androsten-17/3-ol-3-one was the first anabolic steroid, not alkylated in the position 17a, found to be active in oral administration. Suchowsky and Junkmann (1961a) studied the product and reported that metenolone acetate had five times the myotrophic activity and one-tenth of the androgenic activity of testosterone propionate, determined in the rat. The myotrophic-androgenic index in the rat has been later reported to be 24.4 (Suchowsky and Junkmann, 1961b), and 16.0 subcutaneously and 13.0 orally (Junkmann and Suchowsky, 1962). This compound had little influence on the estrous cycle in female rats and depressed the estrogenic action of estradiol less effectively than did testosterone. Junkmann and Suchowsky (1962) demonstrated that metenolone acetate inhibited gonadotrophin secretion although not to the same extent as did other steroids. Clinical data demonstrated the retention of N, P, and Ca following the intramuscular administration of 10--40 mg of product. Also, favorable results have been obtained for the control of calcium excretion in patients treated with high doses of corticosteroids (Geyer and Jesserer, 1961). Experiments on castrated men carried out by Weller (1961) led to the conclusion that metenolone acetate produced nitrogen retention twice as high as that of testosterone propionate while the androgenic activity was a quarter to a sixth. Although it is not aikylated in the 17-position, metenolone acetate led to a slight retention of bromsulphalein in man (Kriiskemper, 1968, p. 171; Weller, 1962), however, it did not show any appreciable activity on the serum transaminases (Kriiskemper and Klesper, 1965). At therapeutic dosages, the acetate did not affect the excretion rate of 17-hydroxycorticosteroids (Breuer et al., 1962). As far as the metabolism of metenolone was concerned, Langecker (1962) found that the reduction of the double bond and the oxidation to 17-ketosteroid probably occurred, though to a much lesser extent than with testosterone. It was found that, in man, in 7-13 days, 21--47 per cent of the orally administered radioactive metenolone acetate was excreted in the urine and 14-22 per cent in the feces (Gerhards et al., 1965). The principal metabolite was 3-hydroxy-l-methylene-5a-androstan-17-one, isolated as its glucuronide; the formation of the exocyclic double bond may be explained by an enolization preceding the reduction of the keto group. It must thus be assumed that metenolone is conjugated as the 3-enol in the liver and that it is subsequently further metabolized as a conjugate. Many other 1-alkylated derivatives have been prepared (Neumann and Wiechert, 1965). It is interesting to note that the 19-nor analogue of metenolone acetate was almost devoid of activity. Metenolone enanthate is a long-acting derivative (as are all the 17-esters with a large number of carbon atoms). After a single injection of 10 mg in rats, its anabolic action persisted for 10 weeks although its androgenic effect disappeared much earlier (Suchowsky and Junkmann, 1961a). 3.4. 17a -METHYL- 17fl -HYDROXY-A1'%ANDROSTADIEN-3-ONE;
METHANDROSTENOLONE: METANDIENONE

Metandienone II was prepared by the introduction of a double bond between carbon atoms 1 and 2 of 17a-methyltestosterone (I) by the use of selenium dioxide in t-butanol containing acetic acid (Meystre et al., 1956) and microbiologically by the enzymatic dehydrogenation with fungi of the genus Didymella (Vischer et al., 1955).

240 O ~'-CH3 (I)

B.CAMERINO andR.SCIAKY OH O ~ J ~ (II)

OH "-CH~

Another method of synthesis using 2,3-dichloro-5,6-dicyanoquinone instead of selenium dioxide, has been more recently described by Balasubrahmanyam et al. (1963). The pharmacological properties of metandienone have been intensively studied by Desaulles et al. (1959). The anabolic action of this drug has been demonstrated by the increase in weight obtained in both young and adult rats treated with 1 mg/kg/day and by the retention of nitrogen observed in female adult rats which had received 2.5-10mg/kg of the compound. This action has been confirmed by Gordan (1957) using the levator ani test and by Burke and Liddle (1959) who, by making comparison with other steroids, have attributed to metandienone an anabolic activity six times as great as that of methyltestosterone. The anabolic action of this compound was exerted also in the presence of catabolizing agents such as the corticoids, the drug antagonizing both the true catabolic effect (Renzi and Chart, 1962) and the inhibitory action on the hypothalamic-pituitary axis. As far as other endocrinic effects were concerned, methandienone had smaller anti-gonadotrophic action than that of methyltestosterone (Desaulles et al., 1959; Desaulles, 1960) and hence had little effect on the estrous cycle. Like all the androstane derivatives it showed no progestational activity. In man, the anabolic activity of metandienone has been shown by an increase in body weight on administration of 50-200 mg (Raymondi and Clausi-Schettini, 1960) or of 10-25 rag/day (Pontiggia et al., 1960) and by the effect on the retention of nitrogen, accompanied by retention of phosphorus, potassium and calcium at dosages varying from 5 to 150mg (Almquist et al., 1961; Pontiggia et al., 1960; Schwarting, 1960; Schwarting and Neth, 1960; Werner et al., 1960). The anti-catabolic effect of this drug has been demonstrated in man by simultaneous treatment with prednisolone or dexamethasone. The metandienone reversed the balance of nitrogen, calcium, phosphorus and potassium from negative to positive. The very small androgenic effect exerted by this compound has permitted its use in pediatrics. Androgenic activity became evident only when it was used at 3 mg/kg doses (Bertolotti, 1960) which made the normal dose of 1 mg/kg quite acceptable (Ramenghi, 1960). Like other 17-alkylated steroids, when administered orally, metandienone can affect the liver function. In some patients treated with the compound there was a noticeable slowing down in the liberation of sulfobromphthalein from the liver (Wernze, 1960; Wernze and Kuschke, 1960; Wynn et al., 1961 ; Lucchelli, 1961); in others there was also the appearance of jaundice (Kaupp and Preston, 1962). Other authors, however, have reported normal excretion of phenol red in patients with limited kidney function (Werner et al., 1961). Also, normal histological patterns have been observed in biopsies of the liver of patients submitted to treatment with metandienone (Schaffner et al., 1959). Furthermore, the increased sensitivity to anticoagulants observed during therapy with metandienone (Py6r/il/i and Kekki, 1963) could be attributed to an impairment of the liver function. As far as aromatization to phenolic steroids is concerned, Lutzmann and Gerhards (1961) found that methandienone was not converted to estrogens by rat-liver slices. However, Breuer and Schikowski (1963) showed that aromatization was brought about by a placental enzyme system.

Structure and effects of anabolic steroids

241

The 6/3-hydroxy derivative of metandienone, which has been found to be the major metabolite in man, was isolated together with another, unidentified, compound from the urine of a woman with advanced adenocarcinoma of the lung (Rongone and Segaloff, 1963).
3.5. 2-HYDROXYMETHYLENE170t -METHYL-17/3 -HYDROXY-50~-ANDROSTAN-3-ONE; OXYMETHOLONE

2-Hydroxymethylene- 17a-methyl- 17/3-hydroxy-5a-androstan-3-one (oxymetholone) (II) has been prepared by condensation of ethyl formate with 17a-methyl-17/3-hydroxy5a-androstan-3-one (I) in the presence of sodium methoxide or sodium hydride (Ringold et al., 1959) and also as an intermediate in the synthesis of stanozolol (Clinton et al., 1959). OH O ~ - - - C H ; OH [~~--CH;

H
(I)

H
(II)

In balance studies in rats, Arnold et al. (1963a) found that oxymetholone was 1] times more potent than 17a-methyltestosterone in inducing nitrogen retention while being only one-fifth as active as the standard androgenically. The myotrophic-androgenic index was 8.75. Other authors reported values of 10.0 (Desaulles, 1960), 4.7 (Camerino and Sala, 1960, p. 132), 6.0 (Junkmann and Suchowsky, 1962) and 5.0 (Baldratti and Arcari, 1961). Oxymetholone only slightly inhibited gonadotrophin secretion in animals (Desaulles, 1960; Junkmann and Suchowsky, 1962). Nitrogen retention in man was achieved by the oral administration of 5-30 mg of oxymetholone (Myerson, 1961). The anabolic action of the compound was confirmed by the fact that it counteracted the catabolic activity of administered prednisolone. It showed a low antiestrogenic activity at therapeutic doses (Suchowsky and Junkmann, 1962) but showed no gestagenic effect (Junkmann and Suchowsky, 1962). There are some reports on the favorable effect of oxymetholone on pediatric diseases such as premature dystrophy in infants. As in the case of other 17-alkylated steroids, oxymethoione administration led to increased sulfobromphthalein retention by the liver (Kriiskemper, 1968, p. 171). 3.6. 17ot-METHYL-17/3-HYDROXY-2-OXA-5Ot-ANDROSTAN-3-ONE, OXANDROLONE The synthesis of oxandrolone was reported in 1962 by Pappo and Jung. Oxidation of 17a-methyl-17/3-hydroxy-Al-5t~-androsten-3-one (I), at room temperature, with lead tetra-acetate in 90 per cent aqueous acetic acid, gave 17ot-methyl-17/3-hydroxy-l-oxo1,2-seco-A-nor-5o~-androstan-2-oic acid (II). This compound was also obtained by lead tetra-acetate cleavage of 17a-methyl-5ot-androstane-la,2a,17/3-triol-3-one (III) derived, in turn, by hydroxylation of (I) with potassium chlorate in the presence of catalytic amounts of osmium tetroxide in aqueous t-butyl alcohol. Sodium borohydride reduction of the aldehyde group of (II) gave the lactone 17o~-methyl-17/3-hydroxy-2oxa-5a-androstan-3-one (oxandrolone) (IV). Oxandrolone was found by oral administration, to be more active as an anabolic agent than 17a-methyl-17/3-hydroxy-5a-androstan-3-one, based on the nitrogen retention test and was practically devoid of androgenic properties. The pharmacological properties have been studied in castrated rats by Lennon and Saunders (1964). When compared, by oral administration with 17a-methyitestosterone, it was 3.22 times more active as a myotrophic agent and just under a quarter as active as an androgenic agent; the myotrophic-androgenic index resulting was thus 13.

242 OH ---CH,

B. CAMERINOand R. SCIAKY

OH 0~ v : v (1) ---CH~

OHC~'

OH
o.

(II)

H (Ill)

H (IV)

By intramuscular administration, myotrophic and androgenic activities were at a minimum compared with testosterone propionate, showing about 5 per cent and 2 per cent respectively of the activity of the standard. The fact that this product had a negligible effect on intramuscular administration while possessing considerable activity when administered orally, was surprising. Oxandrolone showed some activity in the inhibition of gonadotrophin secretion. In man, it proved to have an anabolic activity in oral administration equivalent to 6.3 times that of 17t~-methyl-testosterone (Fox et al., 1962). Nitrogen retention was evident at doses of 0.6 mg/day. H o w e v e r , Metcalf et al. (1964) recently reported that oxandrolone produced some degree of nitrogen retention with doses as low as 2.5 mg/day and further retention with doses increasing from 5 to 25 mg/day. Albanese et al. (1963) found oxandrolone to be twice as active as norethandrolone in stimulating a positive nitrogen balance in hospital patients. In mongoloid children, Ray et al. (1963) noted an increase in height with daily doses of 0.25 to 0.5 mg/kg of body weight without significant enhancement in bone age, thus confirming its low androgenic effect. 3.7. 2a, 17a -DIMETHYL-5t~ -ANDROSTAN-17/3 -OL-3,Y-AZINE; DIMETHAZINE Dimethazine (II) was synthesized by De Ruggieri et al. (1962; Ormonoterapia Richter, 1963) by condensation of 2a,17a-dimethyl-5a-androstan-17/3-ol-3-one (I) with its hydrazone derivative (De Ruggieri, 1962): OH ---CH,

H ~ C . ~ OH ---CH, H , C ~ ' O~Vi

H

_H --

N--N' CH~

v (I)

' H ~ C ..... "~ I u OH (II)

The oral anabolic activity of dimethazine was studied in rats by Bianco et al. (1962) at a total dose of 6 mg, using a modification of Metcalf and Broich's (1961) radiochemical method with 17a-methyltestosterone as standard. The specific activity of the ievator ani muscle when using dimethazine was 2.3 times

Structure and effects of anabolie steroids

243

that of untreated animals. No difference was noted between the untreated animals and those treated with 17a-methyltestosterone. Matscher et al. (1962) reported the activity in protein anabolism of dimethazine compared with 17a-methyltestosterone, oxymetholone, stanozolol and testosterone propionate. Tests were carried out on rats in various experimental situations. Weight gain with respect to the controls, a myotrophic effect and a nitrogen-retaining effect were always observed. The myotrophic-androgenic ratio was similar to that of the steroid pyrazoles and isoxazoles. These authors (Lupo et al., 1962) also concluded that at the given doses, dimethazine showed no estrogenic, progestational or cortical activity in rats and rabbits. Dorfman and Kincl (1963) found the anabolic activity to be 0.27, the androgenic activity 0.12 (seminal vesicles) and 0.06 (ventral prostate) times the activity shown by 17a-methyl-testosterone. Hence, dimethazine possesses slight anabolic activity but has almost no androgenic activity, this fact leading to the favorable myotrophic-androgenic index. Maggi (1964) reported favorable results on treatment with dimethazine in a group of 90 patients of both sexes affected by various morbid syndromes. In a group of fertile patients with normal ovarian cycle, treatment with dimethazine had no effect on cycle characteristics. Recently, 2a-methyl-5a-androstan-17~-ol-3,Y-azine capronate (dimethazine capronate) (Komeno, 1965) has been introduced into the market as a depot form having a long duration of action.
3.8. 170~-METHYL-17~-HYDROXY-50~-ANDROSTANE-[3,2-C]-PYRAZOLE; STANOZOLOL

The synthesis of stanozolol, the first member of a series of anabolically active steroids which had a heterocyclic ring fused to ring A of the steroid, was reported by Clinton et al. (1959, 1961). Condensation of ethyl formate with 17ot-methyl-1713hydroxy-5a-androstan-3-one (I) gave the 2-hydroxy-methylene derivative (II) from which, by the action of hydrazine hydrate in ethanol, stanozolol (III) was obtained: OH OH

O~ v i z i r (I)

(II)

OH ,

H N - , N ~ H
(III)

In the levator ani test in immature castrated male rats, stanozolol was twice as active as a myotrophic agent and one-third as active as an androgen, by oral administration, as 17ot-methyltestosterone (Potts et al., 1960). Stanozolol exerted its nitrogen-retaining action at doses of 0.4--6.4 mg in castrated rats (Beyler et al., 1961); multiple dose level assays in rats indicated that this compound was 35 times as active as an anabolic agent though only a quarter as active as 17a-methyltestosterone (Clinton et al., 1959) as an androgen. When administered parenterally, it was one-twentieth as anabolic and one-fortieth as androgenic as testosterone propionate (Arnold et al., 1959). Later (Arnold et al., 1963a), it was reported that stanozolol was about 10 times as active as 17c~-methyltestosterone in the nitrogen-retention test in rats; this was very much less than the 30 : 1 ratio previously reported. The myotrophic-androgenic index was reported to be 1.4 (Desaulles, 1960), 6.0 (Junkmann and Suchowsky, 1962), 6.1 (Camerino and Sala, 1960, p. 132), 10.6 (Baldratti and Arcari, 1961).

244

B. CAMERINOand R. SCIAKY

Stanozolol has been found to be moderately active in inhibiting secretion of gonadotrophin (Junkmann and Suchowsky, 1962). Early experiments on man confirmed the nitrogen-retaining activity of the product (Howard et al., 1959) and the fact that it was more anabolic than androgenic. In balance experiments with stanozolol being administered orally in man, nitrogen, phosphorus, potassium and calcium retention was observed (Howard and Furman, 1962). This compound has been found to be effective in the therapy of different kinds of inactivity osteoporosis shifting the calcium balance to a positive value (Hioco et al., 1964). In man given high doses of prednisolone, stanozolol showed an anti-catabolic effect (Albanese et al., 1964) antagonizing the catabolic action of corticosteroids. A decrease in gonadotrophin excretion was detected in man after administration of stanozolol (Howard and Furman, 1962). At therapeutic dosages an antiestrogenic action was observed (Junkmann and Suchowsky, 1962) but at these dose levels it did not show any gestagenic activity (Desaulles, 1960; Junkmann and Suchowsky, 1962). The compound was only slightly active as an estrus-inhibiting substance. With reference to liver toxicity in man, stanozolol increased sulfobromphthalein retention (Krfiskemper, 1968, p. 171). However, the effect brought about by this product was one of the slightest among the 17-alkylated synthetic anabolic agents. Stanozolol produced also an increase in serum transaminase at therapeutic dose levels. In man, some allergic symptoms, similar to those induced by other pyrazole derivatives, were observed, probably as a result of the presence of a heterocyclic ring in the molecule (Krfiskemper, 1968, p. 167). Slight modifications in the stanozolol molecule such as the introduction of double bonds had a great effect on the general activity pattern; the A'-dehydro derivative did not promote nitrogen retention, although it was myotrophic, weakly androgenic and estrogenic; the doubly unsaturated derivative, A"6, was typically estrogenic without anabolic or androgenic activity (Beyler et al., 1961). 3.9. 17a-METHYL-17~-HYDROXY-5Ot-ANDROSTAN=[3,2=C]=ISOXAZOLE; ANDROISOXAZOLE Androisoxazole has been synthesized by Italian workers (Marchetti and Donini, 1961) by condensation of 17o~-methyl-17//-hydroxy-5ot-androstan-3-one (I) with ethyl formate, giving the 2-hydroxymethylene derivative (II) which, by use of hydroxylamine produced a mixture of the two isoxazoles 17a-methyl-17fl-hydroxy-Sa-androstan-[3,2c]-isoxazole (III) and [2,3-d]-isoxazole (IV) which were difficult to separate. The ratio of the two isomers was greatly influenced by the type of solvent in which the reaction was carried out e.g. in pyridine, (III) was the predominant product. When the mixture was treated with sodium methoxide in methanol, (III) remained unaltered while (IV) was transformed into the 2t~-cyano-3-ketone (V), these compounds being easily separable. OH OH
, '

OH
,

(I)

~ OH -- C H, ' ~

(HI OH ---CH,

(m)

~
/ / C H ~ ' N ~ O ~ V H (IV)

(v)

Structure and effectsof anabolic steroids

245

The synthesis of (III) has also been reported by Manson et al. (1963) and by research workers at Syntex (Zderic et al., 1960). With regard to the pharmacological action of androisoxazole, nitrogen assays in rats have shown androisoxazole to be 9.7 times active as an anabolic agent than 17a-methyltestosterone and only about a quarter as active as an androgenic agent. The myotrophic-androgenic index was thus 40. When assayed for myotrophic effect androisoxazole was found to be twice as active as 17a-methyltestosterone as an anabolic agent and about a quarter as active as an androgenic agent. However, Junkmann and Suchowsky (1962) found that, in the rat, androisoxazole was only slightly more active than 17a-methyltestosterone as a myotrophic agent and reported a myotrophic-androgenic index of 1.7 on oral administration. Arnold et al. (1963b) estimated androisoxazole to be 1.55 times as anabolic and just over one-fifth as androgenic as 17a-methyl-testosterone, thus giving a therapeutic index of 7. As regard other endocrinological activities the product has been found to be 1/128 as active as progesterone as a gestagen and had practically no estrogenic activity (Donini and Montezemolo, 1961). Androisoxazole has been used clinically as an oral anabolic agent (Bertolotti and Lojodice, 1961; Antonini and Verdi, 1961; Sabato, 1961; Morellini et al., 1961). No effect has been observed on the excretion of 17-ketosteroids (Bertolotti and Lojodice, 1961). Increased retention of sulfobromphthalein has been recorded giving evidence of impaired liver function (Antonini and Verdi, 1961).

3,10. 17ot-METHYL-17fl-HYDROXY-5a-ANDROSTANE-[2,3-C]-FURAZANE,FURAZABOL, ANDROFURAZANOL

The synthesis of furazabol was reported in 1965 by Shimizu et al. (1965) and Ohta et al. (1965). Reaction of 17a-methyl-17~-hydroxy-5ot-androstan-3-one (I) with t-butyl nitrite in the presence of potassium t-butoxide or hydrochloric acid gave 2hydroximino-17a-methyl-17/3-hydroxy-5a-androstan-3-one (II) which on treatment with hydroxylamine hydrochloride and pyridine gave the dioxime (III). Alternatively, (III) was prepared by the formation of the dioxime of 17ot-methyl-17/3hydroxy-Sa-androstan-2,3-dione (IV) obtained by base-catalyzed air oxidation of (I) according to Camerino et al. (1961a, b, 1962). The dioxime (III), was cyclized by warming with potassium hydroxide in ethylene glycol, producing 17t~-methyl-17/3-hydroxy-5a-androstane-[2,3-c]-furazane (V). OH OH ~-'-CH,

---~--CH~.~
o

HON~~,,,~I l
H

OH

(I)

H (II)

0 /N~
H

1 OH ~---CH3
H (IV)
JPTB Vo]. I,No. 2---G

j/ OH ~---CH~

,v,

tt (III)

246

B.C A M E R I N O andR.SCIAKY

Independently the synthesis of (V) via (I) and (IV) was reported by Havranek et al. (1966). Pharmacological studies have been reported by Kasahara et al. (1965). Orally, furazabol was 2.7-3.3 times as myotrophic and 0.73-0.94 as androgenic as 17amethyltestosterone, thus giving a myotrophic-androgenic index of 3.5-3.7. In nitrogen retention studies, furazabol was reported to be 29 times as effective as 17a-methyltestosterone with regards to the 'total nitrogen retained'. In the chick's comb assay for evaluation of the androgenic activity, furazabol was one fourth as active as testosterone propionate by inunction and half as active when compared subcutaneously. Furazabol was also able to prevent the adrenal atrophy in cortisone-treated rats and to antagonize the inhibition of growth due to the corticoid. It did not show estrogenic activity up to 10 mg/kg in castrated female rats (Kasahara et al., 1966) and was only 0.036 as active as progesterone in the Clauberg test. Of the furazabol derivatives that have been prepared, the 4,5-unsaturated derivative showed more or less the same activity as the parent compound (Kasahara et al., 1965); this is very strange since the 4,5 unsaturated derivative of stanozolol has practically no activity (Beyler et al., 1961).

3.11. 4 = C H L O R O = A 4 = A N D R O S T E N 1 7 / 3 O L 3 = O N ACETATE; E 4 C H L O R O T E S T O S T E R O N E ACETATE; C L O S T E B O L

In the course of extensive research in the laboratories of Farmitalia, on androstanes substituted in the 4-position, the 4-halogen derivatives of testosterone were prepared. Of these, the 4-chloro derivative proved to be the most interesting and, in fact, entered readily into clinical practice (Steranabol, Farmitalia). By treatment of testosterone (I) with alkaline hydrogen peroxide, a mixture of the epoxides, a (IIa) and/3 (IIIa) which could be easily separated by fractional crystallization, were obtained (Camerino et al., 1956). The configuration of the epoxide group was assigned on the basis of optical rotation studies and confirmed by LiAIH4 reduction which gave respectively the 5a- and 5/3-hydroxy derivatives (Camerino and Cattapan, 1958). The /3-epoxide (Ilia), after acetylation and treatment with dry HCI in glacial acetic acid, gave clostebol (IV), while the a-isomer (IIb) produced 4/3chloroandrostane-5a,17/3-diol-3-one-17-acetate (V) which, on prolonged heating, was transformed into (IV): OR OCOCH3

. )~ (

~ ' ~ ' ~ I J

(III)

Co)(a) RR .~--COcH,HCI OR ~

(IV) CH3

(I)

~
(II)

CI

(V)

Independently, Ringold et al. (1956) performed a similar series of reactions obtaining the unacetylated 4-chlorotestosterone.

Structure and effectsof anabolic steroids

247

Japanese research workers (Mukava, 1960) prepared closteboi (IV) and 4chlorotestosterone propionate by chlorination, with isocyanuric chloride in acetone--acetic acid, of the corresponding esters of testosterone. The chlorination of testosterone esters has also been described by Kirk et al. (1956) using a reaction medium of ethyl ether/propionic acid. A 4~ 5~-dichloro derivative (VI) was obtained, from which, by use of bases such as pyridine, the 4-chloro-A'-3-ketone was derived.

(I)

cI (Vl)

(IV)

From the preliminary screening of a series of 4-substituted androstane derivatives it was evident that clostebol, which was active in subcutaneous administration, possessed high myotrophic activity in the castrated rat but low androgenic activity (Sala and Baldratti, 1957). A more exhaustive study at various dose levels (Sala et al., 1956) showed that this product was approximately 0.7 as active as testosterone propionate as an anabolic agent while only 0.14 as active as an androgenic agent. Hence, if the anabolic-androgenic index of testosterone propionate is taken as l, that of clostebol is 5 (Sala et al., 1956). Other authors, adopting different methods, have reported therapeutic indices of 2.8 (Desaulles, 1960) and 11.0 (Suchowsky and Junkmann, 1961b). Sala et al. (1957b) reported that, in nitrogen balance experiments, in doses of 1 mg per day for 7 days in castrated male rats, clostebol acted similarly to testosterone propionate. Because this retention of nitrogen was accompanied also by retention of potassium and phosphorus, Sala et al. claimed that clostebol did, in fact, favor intracellular protein synthesis. The anabolic effect of this compound has also been demonstrated in conditions of altered carbohydrate metabolism. In alloxan diabetic rats, Cavallero and Malandra (1956) found that 100-500 mg daily doses of clostebol caused a definite retention of nitrogen and a favorable effect on body weight, without marked changes in carbohydrate metabolism. In rats treated with high doses of hydrocortisone, the simultaneous administration of clostebol (Baldratti et al., 1957; Camanni et al., 1958) was shown to prevent adrenal hypotrophy and to increase body weight, thus opposing the catabolic (or anti-anabolic) effect of the corticoids. Clini et al. (1958) found that in parabiotic castrated rats clostebol did not inhibit the rise in gonadotrophins which followed castration, at least at dosages at which testosterone exerted its maximum effect. Also, as regards the action on the estrous cycle, whereas testosterone propionate and all anabolically active 19-norsteroids had a marked activity, 4-chlorotestosterone was only slightly active in inhibiting estrus (Krtiskemper, 1968, p. 101). In clinical trials in man, clostebol showed a definite retention of nitrogen and phosphorus (Bekaert et al., 1958; Adezati et al., 1958; Amerio et al., 1958). In the case of renal insufficiency, Amerio et al. (1958) found, besides the retention of nitrogen, a decrease in the excretion of phosphorus. A positive displacement in the nitrogen balance has been observed in patients subjected to surgery (Petrucci and Belelli, 1958; Fior, 1958). The anabolic action of clostebol has also been observed in specific regions of the body such as the bone system, where an improvement of the calcium balance in osteoporosis (Sala et al., 1959; Pozzi and Salvi, 1959) and of prolonged use of corticoids, (Sala et al., 1957a) has been recorded. Similar improvements have been noted in anorexia nervosa (Bekaert et al., 1958), in senile distrophies (Mars, 1958) and in degenerative changes in the hepatic parenchyma e.g. in cirrhosis of the liver (Grassi and Cagianelli, 1959; Zannini, 1961) or viral hepatitis (Tolentino, 1966).

248

B. CAMERINO and R. SCIAKY

In pediatrics, clostebol showed a positive influence on the growth rate, on the weight gain in premature dystrophic infants and on the hypo-evolution of first infancy (Toniolo and Gualandi, 1958; Lagonigro and Pontonieri, 1959; Sereni et al., 1957; Petrocini and Bullio, 1958). At therapeutic doses, the acetate did not change the rate of excretion of 17hydroxycorticosteroids (Jacono et al., 1959) and had no activity as a gestagen, in contrast with many steroids of the 19-nor series (Sala et al., 1956; Desaulles, 1960). So far as possible secondary effects are concerned, treatment for several weeks with clostebol of patients with severely reduced kidney function, did not change the phenol red excretion, inulin clearance or renal plasma loss (Amerio et al., 1958). In contrast to 17tx-alkylated steroids (in cross-comparison experiments with testosterone propionate) clostebol after prolonged use, did not increase the retention of sulfobromphthalein (Marzullo and Squadrini, 1958; Wernze, 1960; Miiting, 1964). As far as the metabolism is concerned, the presence of the chlorine atom in position 4 limited the oxidation of the 17/3-alcohol to ketone (Sala and Castegnaro, 1958); in fact, incubation of clostebol with guinea pig liver resulted in a smaller yield of 4-chloro-A'androstene-3,17-dione than that of A'-androstene-3,17-dione given by testosterone. It follows that clostebol did not increase the daily excretion of total 17-ketosteroids in normal subjects. A very small increase was observed in ovaro-adrenolectomized women (Molinatti et al., 1961). The principal metabolite isolated from the urine of adrenolectomized women treated with 100 rag/day of clostebol, was 4-chloro-3~,-hydroxy-A'-androsten-17-one (Castegnaro and Sala, 1961). It is interesting to note that, in the case of testosterone, no metabolite analogous to that above was isolated (i.e. A'-androsten-3-ol-17-one) while in the case under examination no 17-ketosteroid both saturated and chlorinated was observed. It was therefore evident that the introduction of a chlorine atom into position 4 of the testosterone molecule, greatly influenced the metabolic path. In man, administration of the product did not lead to modification in the excretion of 17-ketosteroids (Jacono et al., 1959).
3.12. 4-CHLORO-17ot-METHYL-17~-HYDROXY-A~"t-ANDROSTADIEN-3-ONE; 4-CHLORO-A tDEHYDRO- 17Ct -METHYLTESTOSTERONE

4-Chioro-17a-methyl-17/3-hydroxy-At"-androstadien-3-one (IIl) has been prepared by Schubert et al. (1965) by two methods: (a) by SeO2 dehydrogenation of 4-chloro17a-methyl-17/3-hydroxy-A4-androsten-3-one (I) and (b) by chlorination in C~HsN-CCI, of 17o~-methyl-17/3-hydroxy-At"-androstadien-3-one (II). OH OH OH

~., ~ O / ~ x , . ~

~ Cb~

o" (II)

CI

(I)

CI

(llI)

This compound has also been prepared by microbiological dehydrogenation of (I) with Alcaligenes faecalis and Fusarium c a u c a s i u m (Wix et al., 1965). The biological properties were studied by D6rner (1965) on castrated male rats using A'-dehydro-17ot-methyltestosterone (metandienone) as standard. It was found to have 52 per cent of the anabolic, 15-23 per cent of the androgenic and 20 per cent of the gonadotrophic effect on the standard. According to D6rner et al. (1963), it is not metabolized to estrogens in endocrinologically healthy males. Of the analogues of the compound later studied, the 11/3-hydroxy derivative proved interesting, showing 8.7 times the anabolic and 1.1 times the androgenic activity of methandienone (Hiittenrauch and Schubert, 1966).

Structure and effectsof anabolicsteroids

249

3.13. 17a-METHYL-4,17/3-DIHYDROXy-A'-ANDROSTEN-3-ONE,4-HYDROXY17tx-METHYLTESTOSTERONE: OXYMESTERONE During extensive research in the laboratories of Farmitalia, it was found that some derivatives of 4-substituted androstanes possessed interesting anabolic properties. Of these, the 4-hydroxy derivative of 17a-methyltestosterone (oxymesterone) proved to be particularly active. Oxymesterone (IV) has been synthesized in many ways. Hydrogen peroxide epoxidation of 17a-methyltestosterone (I) gave a mixture of the 4r,,5a- and the 4/3,5/3epoxides (II) which on acid hydrolysis produced a mixture of 4,5-diols (III). Direct dehydration by acid or alkali led to oxymesterone (IV). A mixture of diols (III) was also obtained by O~O4 hydroxylation of (I). OH O~ - ' - C H ~ O ~ OH ...... CH, ~,

(I) OH O ~ "'-CH~

, ~--CH,
OH
(IV)

(II)

OH
(III)

Oxymesterone was also obtained by treatment of the mixture of the 4,5-epoxides in t-butanol with quaternary bases (Camerino et al., 1961a) or by alkaline air oxidation of 17a-methyl-5/3-androstan-17/3-ol-3-one (V) produced in turn by catalytic reduction of (I) (Camerino et ai., 1961b, 1962).
OH OH

---CH,

-..... CH~

(I)

_,

0 "/ ~

(v)

(Iv)

Baldratti et al. (1959) studied the biological properties of the compound in animals. In the levator ani test, by oral administration, it was 3.3 times as active as 17a-methyltestosterone and only 0.48 as androgenic, hence giving a myotrophic-androgenic index of 6.9. Doses of 1 mg/day in castrated male rats fed with a fixed diet induced nitrogen retention. Nitrogen retention has also been observed by Chiappino (1960) in alloxan-diabetic rats treated orally with oxymesterone. Since a contemporaneous reduction in urinary glucose was observed, a decrease in gluconeogenesis from proteins was suggested. In male mice treated with prednisolone, oxymesterone largely abolished the inhibitory effect 6f the corticoid (Barbera et al., 1962). As regard other h~rmonal action, the product did not show any progestational effects according to the Clauberg test in rabbits (Baldratti et al., 1959) and demonstrated only a slight androgenic effect.

250

B. CAMERINO and R. SCIAKY

Oxymesterone has been extensively studied in man. Sala et al. (1960) and Sala (1960), treated adults daily with doses of 20-40 mg, and showed that it possessed a good anabolic effect, as shown by a positive nitrogen balance, by retention of calcium and phosphorus and by an increase in body weight. At this dose level no adverse androgenic effect was noted. In patients treated with dexamethasone and triamcinolone, oxymesterone counteracted the catabolic effect of the corticoids (Sala et al., 1960). Its anabolic activity was also shown in patients with various metabolic disorders (Benedetti et al., 1961; Fossati, 1963; Guidi et al., 1964; Quaini and Valobra, 1963) and in pediatric patients (Rainero, 1960; Scarzeila, 1960; D'Urso et al., 1965). In a comparative study with norethandrolone and metandienone, Colombo (1962) found that oxymesterone caused the least increase in sulfobromphthalein retention and hence concluded that, at therapeutic doses this compound did not initiate hepatic insufficiency. With regard to its metabolism, Molinatti et al. (1960) found that, unlike testosterone, oxymesterone did not give rise to 17-ketosteroids or to compounds which could be classified chemically as estrogens. 3.14. 17t~ -METHYL-A :"-ANDROSTADIENE-4,17/3-DIOL-3-ONE. 17a-Methyl-A L'-androstadiene-4,1713-diol-3-one (III) is a derivative of oxymesterone (I) and has been synthesized by Camerino and Sciaky (1965) by bromination of (I) followed by dehydrobromination with lithium carbonate and lithium chloride in dimethylformamide:
OH OH

o
(l) (If)

OH

OH
(Ill)

The biological activity of this compound has been studied by Baldratti and Arcari (1967). By oral administration in the levator ani test it was shown to be 4 times more active than 17a-methyltestosterone as an anabolic agent while presenting only half the androgenic activity. The low androgenic activity was confirmed by application on a chicken's comb. The product showed an antiestrogenic effect but did not change the normal estrous cycle pattern at doses at which 17a-methyltestosterone completely abolished any cyclical activity. It was much less active as an antigonadotrophic agent than oxymesterone, from which it is distinguished only by the presence of a double bond in position 1,2. It had no estrogenic and corticoidal activity. 3.15. 7a, 17a -DIMETHYL-17/3 -HYDROXy-A4-ANDROSTEN-3-ONE; 7a, 17ot-DIMETHYLTESTOSTERONE, BOLASTERONE

The synthesis of bolasterone was 1,6-Addition of methylmagnesium androstadien-3-one (I) (prepared in methyltestosterone) gave a mixture of

reported by Campbell and Babcock (1959). bromide to 17a-methyl-17/3-hydroxy-A"'6turn by chloranil dehydrcagenation of 17athe 7a- (II) and 7/3-methyl (III) derivatives of

Structure and effects of anabolic steroids

251

17a -methyltestosterone, the former of which, due to the backside attack of the reagent, predominated. Separation of the two isomers being difficult, the mixture was treated with chloranil, the 7/3-epimer giving the 6-dehydro derivative while the 7o~-isomer remained unchanged. This mixture was separated by chromatography: OH OH

O~
(Ill)

OH

' ' C H '

(1)

(II)

Another synthesis was later reported by the same authors (Campbell and Babcock, 1965). The 1,6-addition of methylmagnesium bromide to A~-dehydrotestosterone (IV) led to 7a-methyltestosterone. Oxidation of the 17/3-alcohol to (VI), followed by protection of the unsaturated ketone as the pyrrolidinyl enamine (VII) and the Grignard reaction followed by hydrolysis of the protective group, furnished (II). OH OH 0

(IV) o

(V) OH

(VI)

) (II)

(VII)

(VIII)

Stucki et al. (1965) evaluated the activity, on oral administration, of the compound and found it to be over 13 times as active as 17a-methyltestosterone in the levator ani assay for myotrophic activity while having only 3 times the androgenic activity, as indicated by the seminal vesicles assay. Hence it had a myotrophic-androgenic ratio of 4.3. In a series of carefully controlled metabolic balance studies (Stucki et al., 1965) bolasterone was found to be 6.6 times as efficient as 17o~-methyltestosterone in inducing nitrogen retention in the monkey, i.e. it showed an activity of the same order of that of fluoxymesterone. It was also reported to be effective in very low doses in man (Campbell et al., 1963). Arnold et al. (1963c) found that bolasterone was 4.2 as effective as 17amethyltestosterone in increasing nitrogen retention in castrated male rats over a five day period and 1.3 times as androgenic as 17a-methyltestosterone according to the ventral prostate assay. The anabolic-androgenic ratio was thus 3.2. Bolasterone led to increased sulfobromphthalein retention in man (Kriiskemper, 1968, p. 171). The 19-nor analogue of bolasterone has been synthesized (Campbell et al., 1963) and was found, when assayed orally in the rat, to have about 18 times the androgenic activity of 17ot-methyltestosterone (seminal vesicles assay) and 41 times the myotrophic activity (levator ani assay). Later, this compound was compared with fluoxymesterone (Lyster and Duncan, 1963). In twin-double crossover metabolic studies in the monkey, measuring the

252

B. CAMERINOand R. SCIAKY

nitrogen and potassium balances and the gain in body weight, 7,-,,17ot-dimethyl-19nortestosterone was found to be 14.4 times as active as fluoxymesterone. 3.16. 1a,7a =BIS(ACETYLTHIO)= 170t=METHYL= 17~ =HYDROXY-A4-ANDROSTEN=3=ONE; TIOMESTERONE In the course of studies carried out in the Merck laboratories on steroids substituted in rings A and B by sulfur-containing groups, Kriimer et al. (1963) obtained tiomesterone (II) by addition of mercaptoacetic acid to 17o~-methyl-17/3-hydroxy-A l"4'6androstatrien-3-one (I): OH 0~ ' C H ; OH C~--CH,

(I)

(II)

The addition takes place from the less hindered side of the molecule and hence gives the 1a , 7 a - d e r i v a t i v e . Kraft and Briikner (1964) have studied the activity on oral administration in the castrated male rat. Tiomesterone was 4.56 times as active as an anabolic agent and 0.61 as an androgen as 17ot-methyltestosterone; thus the therapeutic index was 7.5. Later, Kraft and Kieser (1964) studied the product more thoroughly, investigating the possible side effects. At the usual doses, it was completely devoid of progestational and estrogenic activity and almost completely devoid of antiestrogenic effects. In patients treated with 20-25 mg of prednisolone (Weller, 1964) or prednisoione sodium-succinate (Cesnik and Fink, 1964), tiomesterone has shown good anticatabolic activity, counteracting the catabolic action of the corticoid. Among side effects, Kriiskemper (1968, p. 171) found that tiomesterone led to increased sulfobromphthalein retention in man. However, compared with other steroids tested, this compound produced quantitatively one of the smallest effects, together with stanozolol and metenolone.

3.17. 17or-METHYL=11fl, 17g =DIHYDROXY=9a=FLUORO-A4=ANDROSTEN-3-ONE; FLUOXYMESTERONE While making a wide study in this field of anabolic steroids, Lyster et al. (1956) noted that the l l/3-hydroxy and l l-keto derivatives of 17a-methyltestosterone were only slightly less potent as androgenic agents but more active as myotrophic agents, than the parent compound. Working on the assumption that, as in the case of corticosteroids, the introduction of a fluorine atom in position 9or of these 11-oxygenated derivatives might increase the biological activity or at least result in a differentiation between the two properties, they synthesized 17a-methyl-ll/3,17/3-dihydroxy-9a-fluoro-A'-androsten3-one (fluoxymesterone) (Herr et al., 1956). The starting point of the synthesis was l la-hydroxy-17a-methyltestosterone (I), prepared by microbiological hydroxylation of 17ot-methyltestosterone (Eppstein et al., 1954). By means of the 1lt~-tosylate (II), (1) was converted to the Az ' ' dehydro derivative (III). Following the classical series of reactions of Fried and Sabo (1953, 1954, 1957), the 9,11 bromhydrin (IV) was subjected to alkaline cyclization to give the epoxide (V). Treatment of this product with hydrofluoric acid gave fluoxymesterone (VI). The corresponding l l-keto derivative (VII) was prepared from (VI) by oxidation with chromic acid in acetic acid:

Structure and effectsof anabolic steroids OH OH OH

253

o CHoFiv
(I) (II) OH

(III) OH

~ - - - C H ~

~ - - - C H ~

O -I ~

O "f ~
0v) OH

(v) OH

0 -/

0 -t

~ "

(Vl)

(VII)

Biological tests showed that, in fact, the introduction of a fluorine atom in position 9a did increase, notably, both the myotrophic and the androgenic activity. No difference was observed between the 1l-keto and 11/3-hydroxy derivatives. Fluoxymesterone, in immature castrated rats, showed an androgenic activity 9.5 times greater than that of 17a-methyltestosterone and an anabolic effect 20 times greater (Lyster et al., 1956) resulting in a myotrophic-androgenic index of 2.1. The therapeutic index greater than 1 indicated a differentiation between the anabolic and androgenic activity. However, both values are very high, thus rendering the compound of special interest as an androgen. The anabolic activity of fluoxymesterone has been confirmed in the castrated female monkey, by lessening the loss of nitrogen at doses of 0.05-0.4 mg/kg (Stucki et aL, 1960). Its strong androgenic activity has been demonstrated also by the masculinization of the fetuses of rats and rabbits whose mothers were treated during pregnancy (Jost,

1958). Fluoxymesterone has been shown to have little effecton inhibitionof gonadotrophin secretion (Junkmann and Suchowsky, 1962) and little antiestrogenic activity (Suchowsky and Junkmann, 1962). Fluoxymesterone has been used in clinicalpractice as an anabolic agent but, as a consequence of its high androgenic activity,mainly as a substitute for testosterone (Bartter, 1957). In women with metastaticbreast carcinoma, dailydoses of 10-15 mg resultedin signs of androgenicity (Field, 1957). However, according to Kennedy (1958), the extent of masculinization, contrary to
observations made in laboratory animals, was less than that with testosterone propionate. The effect on the liver, measured by increase in retention of sulfobromphthalein (Marquardt et al., 1961; Wernze, 1960; Wernze and Kuschke, 1960) was one of the highest among the many steroids examined. Bartter (1957) claimed that fluoxymesterone was not metabolized to 17-ketosteroids. 3.18. AI'4-ANDROSTADIEN-17fl-OL-3-ONE-17-(CYCLOPENT-I'-ENYL)ETHER;QUINBOLONE Quinbolone was synthesized by Ercoli et al. (1962) during work on increasing the oral activity of steroids by converting the keto groups into enol ethers.

254

B. CAMERINOand R. SCIAKY

By heating A~"-androstadien-17/3-ol-3-one (I) with cyclopentanone diethylacetate without solvent or in benzene, the corresponding 17-(cyclopenten-I'-enyl) ether (quinbolone, II) was formed directly:

OH

(I)

(II)

The biological properties have been studied by Ercoli and Falconi (1963). It was compared to metandienone from which it differs only in the group responsible for the oral activity. Even though it is not 17a-alkylated it showed, in the levator ani and ventral prostate tests in castrated rats, an oral anabolic activity which, for prolonged treatment, was almost the same as that of metandienone. However, the androgenic activity was less, so that the myotrophic-androgenic index was more favorable. Inhibition of the pituitary, as shown by the parabiotic tests, was practically equal to that of the reference compound. At doses that were practically inactive from an androgenic point of view, quinbolone was able to counteract the anti-growth effect of prednisolone (Galletti and Bruni, 1963).

4. 19-NORSTEROIDS 4.1. 19-NORTESTOSTERONE;NANDROLONE Biological activity in 19-norandrostane derivatives was first noted by Dirscherl (Dirscherl et al., 1936) who hydrogenated the benzene ring and carbonyl group of estrone thus isolating weakly androgenic compounds. From both the chemical and the biological point of view, interest in 19-norsteroids rose when Birch discovered a new and efficient method for synthesizing 19-nortestosterone. During work on 'Reduction by Dissolving Metals', Birch and Mukherji (1949) reported the reduction of the glyceryl ether of estradiol with sodium or potassium and ethanol in liquid ammonia. The enol ether (II) which was not isolated was subjected to gentle acid hydrolysis giving a /3,y-unsaturated ketone (III) which was later isomerized (Birch, 1950) to 19-nortestosterone (IV) (nandrolone).This compound has a new asymmetric OH OH

0) OH

0D OH

(Ill)

(IV)

Structure and effectsof anabolic steroids

255

centre at C-10, introduced during the synthesis, but the H-substituent has the same /3-configuration as the methyl group of testosterone, as later shown by optical rotatory dispersion studies by Djerassi et al. (1956). Wilds and Nelson (1953a) modified the procedure by using the more easily prepared methyl ether instead of the glyceryl ether, lithium instead of sodium or potassium, a co-solvent and by adding the alcohol at the end of the reaction. By this method it was easily possible to obtain a good yield of nandrolone (Wilds and Nelson, 1953b) and at the same time it opened the way to the preparation of a large number of analogues. More recently, Heusler et al. (1962), Heusler and Kalvoda (1964), and Bowers et al. (1962) have devised a procedure for the preparation of 19-norsteroids using as starting material the more readily available compounds of the androstane series instead of the costly estrogens. Dehydroisoandrosterone acetate (V) on treatment with calcium hypochloride led to the 5a-chloro-6/3-hydr~lxy derivative (VI) which was treated with lead tetra-acetate in tetrahydrofuran in the presence of iodine to give the 6/3 -19-oxide (VII). This intermediate has been the gateway to many compounds: O 0

C H ; C O 0 ~ (V)

C H ~ C O 0 ~ (Vl) o

CH;CO0~

CI vii)

Hydrolysis of the 3-acetate group, oxidation of the alcohol to the ketone (IX) and dehydrohalogenation with base gave 6/3,19-oxido-A'-androstene-3,17-dione (X):

O HO
, O

CI (VIII)

CI (IX)

(X)

Treatment with zinc in isopropanol opened the epoxide ring, giving 19-hydroxy-A'androstene-3,17-dione (XI). Chromic acid oxidation of (XI) furnished the acid (XII) which was readily decarboxylated by heating in pyridine, at the same time bringing about isomerization of the double bond to the 5(10) position (XIII). Finally, protection of the 3-keto group as the dimethylacetal, followed by reduction of the 17-ketone and acid hydrolysis of the acetal, resulted in nandrolone (IV) (Ueberwasser et al., 1963): O O O

(X)

'

0~ ~

v (XI)

0~ ~

v (Xll) (XIII)

256
o

B. CAMERINOand R. SCIAKY OH
OH

' c.CHio
(XIV)

, CH ~ O ~ - - - - - - ~ CH~O v v (XV)

(IV)

By using suitable transformations on the various intermediates a wide range of derivatives have been prepared (Heusler and Kalvoda, 1964, note 19). Recently, nandrolone has become available by total synthesis either by direct formation (Velluz et al., 1965) or by dearomatization of synthetic estrone (Douglas et al., 1963). In the initial biological tests, nandrolone was found to be weakly androgenic and was considered, above all, as a starting material for the preparat:on of derivatives. However, in 1953, Hershberger et al. showed that the compound possessed myotrophic activity equal to that of testosterone and confirmed the low androgenic activity. The ratio of the increase in weight of the levator ani to that of the ventral prostate was found to be 0.3 for testosterone and 1 for nandrolone. According to Barnes et al. (1954), nandrolone was two-thirds as anabolic and 0.07 as androgenic as testosterone propionate when tested in rats, thus giving an anabolic-androgenic index of 9.4. Hence, it can be concluded that substitution of the C-19 methyl group by a hydrogen atom in testosterone changes appreciably the ratio between anabolic and androgenic activity. Analogous results have been obtained by Holtkamp et al. (1955) who found that, to produce the same effect on the levator ani muscle as I 1 mg/kg of testosterone, 7 mg/kg of nandrolone were necessary. On oral administration, nandrolone showed about one-tenth of the activity produced on subcutaneous administration. However, it was still active orally in castrated male rats at a dose level of 60 mg/kg/day. In prepuberal boys (Gordan, 1957) and in women (Baker et al., 1955; Ferin, 1955) nandrolone gave evidence of some androgenic stimulation. Certain 17-esters of nandrolone are much more clinically interesting than the compound itself e.g. the phenylpropionate, cyclopentylpropionate and the decanoate, and the compound itself is no longer used in clinical practice.

4.2. A4-ESTREN-17~-OL-3-ONE-PHENYLPROPIONATE; 19-NORTESTOSTERONE PHENYLPROPIONATE:NANDROLONEPHENPROPIONATE

Nandrolone phenpropionate was first prepared in the Organon Laboratories by esterification of nandrolone with phenylpropionic acid chloride (Organon, 1959).

OCOCH.CH2~

o~

"

The biological properties of this compound were investigated by Overbeek and De Visser (1957) who reported that, in the rat, it was 4 times more active as a myotrophic agent than the corresponding testosterone ester, while showing only one-half of the androgenic activity. Other authors have reported a therapeutic index of 2.4 (Suchowsky and Junkmann, 1961b). An increase in body weight of rats treated with nandrolone phenpropionate has been noted by Rinne and N~ifitiinen (1958a).

Structure and effects of anabolic steroids

257

Calcium retention brought about by this compound has been demonstrated in the bone of growing male rats by a rapid increase in weight of the femur without increase in length of the bone (Kriiskemper, 1968, p. 69). Overbeek and De Visser (1957) showed that nandrolone phenpropionate counteracted the catabolic effect induced by hydrocortisone in rats, by observing that it prevented weight loss. This anticatabolic effect has also been noted by Rinne and N~i~t~inen (1958b) and by Laron et al. (1963). 19-Nortestosterone phenylpropionate has shown some side effects, for example, a strong inhibition of gonadotrophin secretion (Desaulles et al., 1959) and antiestrogenic activity evident at doses sufficient to produce anabolic activity (Suchowsky and Junkmann, 1962). Derivatives of nandrolone show (as do all 19-norsteroids) considerable progestational activity, a generalization valid also for nandrolone phenpropionate (Overbeek and De Visser, 1957). This product has been widely used in clinical practice and at the moment is one of the most requested anabolic compounds. Nitrogen and calcium retention on intramuscular administration in man have been reported (Huguenard and Godard, 1959). In patients treated with corticoids, nandrolone phenpropionate showed a definite anticatabolic activity as measured by a positive shift in the nitrogen, calcium and phosphorus balances (Montuschi, 1959). In studies on the dynamics of protein metabolism (Torizuka et al., 1963) it was shown that nandrolone phenpropionate, like other anabolic agents, stimulated protein synthesis rather than inhibiting the degradation. Good results have been obtained in cases of endocrine or presenile osteoporosis, in which treatment with the product led to nitrogen and calcium retention (Nowakowsky, 1962; Hernberg, 1960; Li~vre and Camus, 1961). Nandrolone phenpropionate has been used with some success in pediatrics, inducing gain in weight and accelerating the growth of premature dystrophic infants and chronically sick older children (Ungari and Benedetti, 1958; Ungari and Rossoni, 1958; Hugon, 1959). Nandrolone phenpropionate, like all esters of nandrolone, other than 17o~-alkylated derivatives, has practically no side effect on the liver. A decrease in the excretion of gonadotrophins has been observed in man following the administration of nandrolone phenpropionate (Berczeller and Kupperman, 1960). At therapeutic doses the product had no effect on the rate of excretion of 17-hydroxycorticosteroids (Berczeller and Kupperman, 1960). A surprising observation was that of Ryan (1960) that nandrolone was aromatized to the extent of 44 per cent (in terms of testosterone) by the aromatizing enzyme system (Ryan, 1959) of the human placenta. Experiments of Breuer (1962) in vivo showed that esters of nandrolone after intramuscular injection in healthy male subjects increased estrogen secretion by 0.02-0.07 per cent. With regard to metabolism, Engel et al. (1958), after administering nandrolone to a postmenopausal breast cancer patient, isolated nandrolone and 19-nor5/3-androstan-3a-ol-17-one. 4.3. A4-ESTREN17/]-OL-3-ONE-CYCLOPENTYLPROPIONATE; 19-NORTESTOSTERONECYCLOPENTYLPROP1ONATE;NANDROLONECYPIONATE Although it was one of the first esters of nandrolone to be studied, the cyclopentylpropionate o ~ O C H 2 C H 2 - - ~

258

B. CAMERINO and R. SCIAKY

was used clinically for only a short time and it has now been abandoned almost completely. Nandrolone cypionate was studied by Barnes et al. (1954) who found that, in comparison with testosterone propionate, it had twice the effect on the levator ani but only one-fifth of the androgenicity, giving a myotrophic-androgenic index of 9.6. The same authors later reported a therapeutic index of 10.7 (Barnes et al., 1954), while others have given a value of 0.65 (Sala et al., 1956). Net nitrogen retention was observed by Stafford et al. (1954) by measuring nitrogen retention in force-fed castrated male rats. They reported the nitrogen retention to be 0.5-2 times that produced by testosterone propionate. A significant anabolic effect in man was reported by Foreman et al. (1955) and by Hollifield et al. (1956), as shown by increased retention of nitrogen, potassium and phosphorus and by an increase in body weight. In comparison with testosterone, nandrolone cypionate, in clinical assays with eunuchoid males and menopausal females, showed a more favorable anabolic-androgenic ratio. In women, side effects such as amenorrhea, metrorrhagia and acne have been noted, showing that it has strong progestational activity and considerable androgenicity. 4.4.

A4-ESTREN-17/3-OL-3-ONE-DECANOATE; 19-NORTESTOSTERONE DECANOATE; NANDROLONEDECANOATE

OCO(CH2)sCH;

Nandrolone decanoate was studied by De Visser and Overbeek (1960)

The principal characteristic of this long-chain ester of nandrolone was that it showed marked anabolic activity, prolonged over a period of time, a property already noted in other long chain esters. The anabolic activity was much more pronounced than the androgenic, gonadinhibiting and antiestrogenic properties. However, at high dose levels, when the anabolic effect approached its maximum, the other effects were more marked (De Visser and Overbeek, 1960). Doses of 50 mg every 18--24 days in man led to nitrogen and calcium retention (Van Wayjen, 1960). Good clinical results have been obtained in cases of senile osteoporosis (Nowakowsky, 1962). During treatment with corticosteroids the product counteracted the catabolic effect of corticoids, as shown by the lower calcium excretion (Van Wayjen and Buyze, 1962). 4.5. 17a -ETHYL-A'-ESTREN17/3-OL-3-ONE; 17a -ETHYL-19-NORTESTOSTERONE; NORETHAN-

DROLONE
Norethandrolone (II), was prepared by Colton et al. (1957) by two different methods. The first involved reduction of the triple bond of 17a-ethynyl-19-nortestosterone (I) previously prepared by Djerassi et al. (1954): OH O ~ ...... C~CH

OH O ~ ---C2H~

(I)

(ll)

By the other method, the 3-methyl ether of estrone (III) was ethynylated to give the 3-methyl ether of 17a-ethynylestradiol (IV). Catalytic reduction of the double bond

Structure and effects of anabolic steroids

259

followed by the Birch reduction, as modified by Wild and Nelson (1953a, b), led to the de-aromatization of ring A, giving compound (VI), which by action of aqueous hydrochloride acid in methanol gave norethandrolone (II): o OH

(III)

(IV)

OH

OH

OH

(V)

(VI)

(II)

Norethandrolone was studied by Saunders and Drill (1956, 1957) in the levator ani test in rats. These workers found that it was as active as testosterone propionate as a myotrophic agent while having one-sixteenth the androgenic activity, leading to a myotrophic-androgenic index of about 15. Other workers have studied this product; reported values of the therapeutic index are 2.0 (Sala et al., 1956), 2.2 (Suchowsky and Junkmann, 1961b), 3.0 (Overbeek et al., 1962) 1.8 (Junkmann and Suchowsky, 1962) and 3.5 (Baldratti and Arcari, 1961). Later, Saunders and Drill (1958) reported that, when administered intramuscularly to castrated male rats, norethandrolone had about the same activity as testosterone propionate in respect of nitrogen retention, while in oral administration it showed the same activity as 17tx-methyltestosterone. Aschkenasy (1959) confirmed that in rats it induced nitrogen retention on both oral and subcutaneous administration. Norethandrolone was more active than the homologous 17o~-methyl derivative in oral administration but in intramuscular injection they were almost equally active (Saunders and Drill, 1956). The product was highly active as a progestational (Saunders et al., 1957), deciduomatogenic (Pincus et al., 1956), antiestrogenic (Edgren and Calhoun, 1957; Richter, 1958; Dorfman et al., 1961; Edgren, 1957) and antigonadotrophic agent (Goldman et al., ! 957). Norethandrolone interfered with the reproductive process, inhibiting ovulation in rabbits, having antifertility activity in rats (Pincus et al., 1956) and inhibiting spontaneous estrus in adult female rats (Richter, 1958). Norethandrolone has been widely studied in man. At doses of 50-100 mg in oral or intramuscular administration it produced retention of nitrogen, phosphorus, potassium and calcium (McSwiney and Prunty, 1957; Spencer et al., 1957; Prunty et al., 1958; Woodford-Williams and Webster, 1958; Chalmers et al. 1959). In underweight patients, at a daily dosage of 50 mg it caused a significant gain in body weight and nitrogen retention without any marked androgenic effect (Woodford-Williams and Webster, 1958). An anticatabolic effect in patients treated with prednisolone has been observed after administration of norethandrolone (BrSchner-Mortensen et al., 1959). It also counteracted the excretion of calcium induced by the corticoids (Harris et al., 1960; Br6chner-Mortensen et al., 1959). McCracken and Parson (1958) and Gjeirup and Thaysen (1958) showed that norethandrolone had a favorable therapeutic effect in some cases of chronic renal failure by inhibiting protein catabolism, as shown by the sharp decrease of non-protein nitrogen in the blood. Skeletal maturation in children was not accelerated by administering norethandrolone, proving that it did not exert any appreciable androgenic effect (Geller, 1964).

260

B. CAMERINOand R. SCIAKY

It did, however, show other endocrine side effects. It has been shown to have some progestational activity in man at therapeutic dose levels (Ferin, 1957). A decrease in the excretion of gonadotrophins after its administration has been reported (Broussolle et al., 1960; Feldman and Carter, 1960; Leach et at., 1958; Epstein et al., 1957), showing its effect on the hypothalamic-pituitary system. Many other side effects induced by norethandrolone can be explained in terms of the estrogenic activity of the product e.g. transformation of the vaginal epithelium in menopausal women (Goldfarb et al., 1958) and exacerbation of metastatic mammary carcinoma accompanied by hypercalciuria (Emerson et al., 1961). According to recent studies norethandrolone lowered the rate of secretion or the biosynthesis of glucocorticoids (Schriefers et al., 1965; Vermeulen, 1964; Muller et al., 1960). The former hypothesis seems the most reliable since at therapeutic doses the level of 17-hydroxycorticosteroids in the blood was not affected by norethandrolone (Feldman and Carter, 1960; Carter et al., 1958), while the urinary excretion was reduced (Harris et al., 1960; Brooks and Prunty, 1957; Brendler and Winkler, 1959; Brooks, 1956; Dorfman, 1954). Of the 17a-alkylated steroids norethandrolone was among those which produced the greatest retention of sulfobromphthalein in the liver (Marquardt et al., 1961; Marks et al., 1961; Kory et al., 1957; Watson et al., 1959; Heaney and Whedon, 1958; Dowben, 1958; Kriiskemper, 1968, p. 172), thus showing a liver damaging effect. This was confirmed by the appearance of several cases of jaundice, some fatal, in patients treated with the compound (Plum and Dunning, 1958; Dunning, 1958; Schaffner et al., 1959; Gilbert et al., 1963). Various homologues of norethandrolone have been synthesized. The A~~ analogue had no anabolic activity (Drill and Riegel, 1958). The 17a-methyl homologue possessed good anabolic and androgenic properties (Saunders and Drill, 1956) but had such a high progestational activity (Saunders et al., 1957; Peters et al., 1957) as to prohibit its use as an anabolic product and was used instead as a progestational agent. Substitution of the 17a-ethyl group by propyl, allyl and methallyl resulted in decreased myotrophic activity when tested in castrated rats (Drill and Riegel, 1958). 4.6. 17ot-ETHYL-A4-ESTREN - 17fl-OL; ETHYLESTRENOL;3-DESOXY-19-NORTESTOSTERONE Ethylestrenol has been prepared, along with other analogues, by De Winter et al. (1959) during a study of the relationship between structure and activity of 19norsteroids. Reaction of ethane-l,2-dithiol with nandrolone (I) produced the 3-thioketal (II) which was reduced with sodium in liquid ammonia to give A4-estren - 17/3-ol (III). Oxidation of (III) with chromic acid in acetone led to A'-estren-17-one (IV), a useful intermediate in the preparation of 17a-alkylated derivatives. Ethyl-estrenol (V) was prepared from (IV) by the Grignard reaction with ethyl-magnesium halide. OH OH OH

(I) O

(11) OH

(i1I)

(w)

(v)

Structure and effects of anabolic steroids

261

The biological properties of ethylestrenol have been studied in the rat by Overbeek et al. (1962). Among the many analogous compounds prepared, the 17a-ethyl derivative showed the most interesting properties. In the levator ani assay it revealed considerable anabolic activity and a favorable 'Q quotient' (according to Overbeek)about 19 times that of 17a-methyltestosterone. For comparison, Overbeek determined this 'Q quotient' for norethandrolone and stanozolol to be 3.1 and 3.8 respectively (Overbeek, 1966, p. 41). These results have been substantially confirmed by Cerquiglini and Marchetti (1961), the only variation being the use of the ventral prostate instead of the seminal vesicles for determination of the androgenic activity. Hence, the 'Q quotient' was 33 when compared by oral administration with 17a-methyitestosterone. Junkmann and Suchowsky (1962) reported a therapeutic index of 3.7. Later, Arnold et al. (1963b) investigated the relative, oral, anabolic-to-androgenic activity ratios of ethylestrenol in comparison with androisoxazole and testosterone. The anabolic effect was evaluated by nitrogen balance studies and the androgenic effect on the basis of the increase in weight of the ventral prostate gland in a 10-day assay. Under these conditions, the ratios of their anabolic to androgenic activities with respect to 17a-methyltestosterone, were: ethylestrenol (1.7/0.21) 8.1; androisoxazole (1.55/0.22) 7.0; testosterone (0.38/0.57) 0.67. Ethylestrenol may be viewed as being closely related to norethandrolone which was found to have an anabolic-androgenic ratio of (3.9/0.21) 20. These relative differentiations between the anabolic and androgenic activities in ethylestrenol and norethandrolone differ from those found by Overbeek et al. (1962). In another publication, Overbeek et al. (1961) reported that ethylestrenol counteracted the negative influence of hydrocortisone on rat body weight, thus showing its anti-catabolic activity. The product has been shown to be as active, as a progestagen, as norethandrolone but since it is 4 times more anabolic the fact is not of great importance in therapeutic application. This gestagenic activity has been confirmed by Junkmann and Suchowsky (1962), who observed that the compound showed progestational activity at therapeutic doses. Ethylestrenol was also found to be highly active in inhibiting the release of gonadotrophins in animals (Junkmann and Suchowsky, 1962). In addition, it was noted that the administration of ethyl-estrenol lowered or inhibited the biosynthesis of glucocorticoids. (Vermeulen, 1964). Other authors found also that, at therapeutic doses, it did not change the rate of excretion of 17hydroxycorticosteroids (Van Vaerenbergh et al., 1961). Ethylestrenol has been widely used in clinical practice. Net retention of calcium, phosphorus and nitrogen have been reported (Van Wayjen and Buyze, 1962; Nowakowski, 1962) at doses of 5 rag/day in man. An anticatabolic effect has been observed in patients treated with dexamethasone on oral administration of 8 mg ethylestrenol (Ruchelman and Ford, 1963), the product thus counteracting the catabolic effect of the corticoid. It also had a positive effect on the excretion of calcium (Nowakowski, 1962). Ethylestrenol has also been used in pediatrics (Anderson, 1962). One side effect caused by administration of the product is metrorrhagia in menopause, evidence of progestational activity (Uhry and Cohen, 1962). Of the many analogues and homologues of ethylestrenol that have been prepared, 17a-ethynyl and 17a-allyl-A'-estren-17/3-ol have shown high progestational activity and are in use in clinical practice (De Winter et al., 1959). 4.7. 4,1713-DIHYDROXY-A4-ESTREN-3-ONE17-CYCLOPENTYLPROPlONATE; 4-HYDROXY-19NORTESTOSTERONE-17-CYCLOPENTYLPROPIONATE; OXABOLONECYPIONATE Oxabolone cypionate was synthesized by Camerino (1962) during a search for androstane and estrane derivatives with potential anabolic activity. Nandrolone (I) was treated with alkaline hydrogen peroxide to give a mixture of 4,5-epoxides(II). This
JPTB Vol. I, No. 2--H

262

B. CAMERINOand R. SCIAKY

mixture was treated with 3-cyclopentyl-propionic acid chloride to give the 17-ester (III), which on treatment with sulfuric acid in acetic acid gave rise to a rearrangement already studied by Camerino et al. (1956) producing oxabolone cypionate (IV). OH OH

(I) o~COCH.,CH2--~

(If) OCOCH~CH2(~

OH (III) (iv) This product proved to be intensely myotrophic more so than the corresponding ester of testosterone and of the 19-methylated homologue, 4-hydroxytestosterone. Its chief characteristic was its prolonged anabolic activity. This was studied by the levator ani test in immature castrated male rats by Sala and Baldratti (1963) and Baldratti et al. (1963). With a single injection of 0.5-2.0 mg the product showed an intense myotrophic activity lasting for 28 days; the androgenic activity was weak and tended to disappear earlier. Comparison with testosterone cypionate showed clearly that with oxabolone cypionate there was a differentiation between the anabolic and androgenic effects both as regards the intensity and the duration of action. In fact, while the weight of the ventral prostate in rats treated with testosterone cypionate continued to increase until 14 days after treatment and then decreased slowly, in rats treated with oxabolone cypionate the weight of the ventral prostate decreased after the second day and was negligible by the 28th day. In female rats, treatment with the product led to an increase in body weight. There was practically no progestational and estrogenic activity. Oxabolone cypionate has been subjected to wide clinical experimentation. Luchmann et al. (1966) studied the effect of oxabolone cypionate administered intramuscularly in single doses of 50-75 mg. It induced retention of 1-4 g/day of nitrogen. After total administration of 75 mg the anabolic effect was maximum in the most underweight patients. No androgenic or estrogenic side effects were noted. Luppino (1965) in a series of experiments on patients affected by various diseases (chronic hepatopathy, osteoporosis, chronic nephropathy, hyperthyroidism), by controlling the body weight and the nitrogen, calcium and phosphorus balances, concluded that the product had an intense protracted anabolic action which was evident in protein and mineral metabolism. Oxabolone cypionate has also been used in pediatrics. Scarzella (1963) administered it to children in first and second infancy and reported that the best results were obtained for growth retardation, hypoevolution, in atrepsics and for convalescence from infectious diseases. The work of Quazzi (1964) showed that the optimal dose for children was 0.5 mg/kg of body weight injected intramuscularly every 5 days; the androgenic action was negligible even on prolonged treatment. From data collected from aldolase and serum transaminase determinations, it was concluded that oxabolone cypionate did not have a damaging effect on the liver.

Structure and effects of anabolic steroids

263

4.8. 4-CHLORO-17/3-HYDROxy-A4-ESTREN-3-ONE ACETATE; 4-CHLORO-I9-NORTESTOSTERONE ACETATE; NORCLOSTEBOI.

4-Chloro-19-nortestosterone acetate has been synthesized by Camerino et al. (1956). Nandrolone (I) was treated with alkaline hydrogen peroxide to give a mixture of the 4a,5a- and 4/3,5/3-epoxides (II). Acetylation gave a mixture of the 17-acetates (III) which, by action of hydrochloric acid in acetic acid, gave 4-chloro-19-nortestosterone acetate (IV), norclostebol.
OH OH OCOCH~

(I)

(I|) OCOCH,

(lid

CI

(IV)

The biological properties were studied by Sala et al. (1956). Judged by the ievator ani test, the product had a myotrophic activity equivalent to that of testosterone propionate, while its androgenic activity, as shown by the ventral prostate test, was only 0.02. Similar results were obtained by Noack et al. (1963). The anabolic activ!ty has been confirmed by the demonstration of a positive nitrogen balance (Sala et a l . , ' i 9 5 7 b ) accompanied by retention of phosphorus and potassium. Clostebol did not promote corticoidal, estrogenic or progestational activity (Sala et al., 1956). It has been studied in man, by Albanese et al. (1965), who found that a single intramuscular dose of 5-20mg produced a marked increase in nitrogen retention, lasting for two weeks.
4.9. 17a-METHYL-A4"9'tt-ESTRATRIEN-17fl-OL-3-ONE; METRIBOLONE

Metribolone was prepared by Velluz et al. (1963) from an intermediate obtained by total synthesis. Compound (I), treated with acetic anhydride and toluene-p-sulfonic acid, gave the enolacetate (II), which, by bromination and subsequent dehydrobromination gave the a,/3-3,,~-unsaturated ketone (IV). Alkaline treatment of (IV) gave the triene (VI), reaction of which with hydroxylamine led to the mixture of syn- and anti-isomers (VII). Oxidation, Grignard reagent treatment and hydrolysis of the oximino group with pyruvic acid resulted in metribolone (X):
OCOC,H~ OCOC,.H~

/t
CI (I) OCOCH; (ll)

OCOC~H~

(III)

264

B. CAMERINOand R. SClAKY OCOC~H~ OCOC~H,

'C I ~
(IV)

(v) OR (a) R = COC6H, R=H oil

(b)
(Vl)

'H
o OH O~ -

N ~ (vID OH C H ~

(VIII)

(IX)

(X)

Metribolone, following oral administration to castrated rats, was 50 times as androgenic as 17a-methyltestosterone (Velluz et al., 1963). This was confirmed by Feyel-Cabanes (1963, 1965), who found that metribolone had 60 times the androgenic activity of 17a-methyltestosterone and 100 times that of 19-nor-17amethyltestosterone. At dosages resulting in the same androgenic activity the anabolic effect of metribolone was 100 times that of 19-nor-17a-methyltestosterone and 300 times that of 17a-methyltestosterone. The compound is thus both an anabolic and an androgenic agent, very active when given orally. Its estrogenic activity was 10 times that of 19-nor-17a-methyltestosterone and 300 times that of 17a-methyltestosterone. It was also shown to be a progestational agent and to inhibit the secretion of gonadotrophins. The observed differentiation between the androgenic and the anabolizing effects, was doubtless due to a certain estrogen-androgen antagonism which partially masked the androgenic effect on the male sexual receptors, leaving the anabolizing effect. Kriiskemper et al. (1967) used metribolone in man and found that it exhibited high anabolic activity in adults when given orally in daily doses from 0.5 to 2.0 mg. Administration in such doses greatly reduced the excretion of 17-ketosteroids and 17-hydroxycorticoids. 4.10. DL-13/3,17a-DIETHYL-17il3-HYDROXY-A'-GONEN-3-ONE; NORBOLETONE Norboletone was the first member of a new group of anabolic compounds of the gonane class in which the methyl group at C-13 is replaced by an ethyl group. It was prepared by total synthesis (the commercial form is racemic) as a result of a study on the total synthesis of steroids at the Wyeth Laboratories (Smith et al., 1963, 1964). Base-catalyzed condensation of a mixture of ketones (I) and (II) with 2-ethylcyclopentane-l,3-dione (III) gave the trione (IV) which was cyclodehydrated with toluene-p.sulfonic acid or polyphosphoric acid in benzene to give the gonapentaene (V). Catalytic hydrogenation of (V) gave the gonatetraene (VI). Isomerization of (VI) to the A~t'-isomer (VII) was performed with methanolic hydrochloric acid, reduction with 10 per cent palladized charcoal in ethanol giving 13/3-ethyl-3methoxy-A~'3~~-gonatrien-17-one (VIII), a homologue of estrone. On reaction of (VIII) with lithium acetylide in dimethylformamide-ethylene diamine the 17a-ethynyl compound (IX) was produced. This was followed by catalytic reduction using palladized

Structure and effects of anabolic steroids

265

calcium carbonate in benzene to give the 17a-ethyl derivative (X). Birch reduction and hydrolysis, then gave the dl- 13/3,17a -diethyl- 17/3-hydroxy-A4-gonen-3-one (norbolethone). NEh 0 and/or H~CO (I) H , C O ~ (II) + O ~''H~ (III) O

O" H~CO (IV) H,CO (V)

H~CO (Vl)

H3CO ~

v (VII) O~___C~_~_CH

H~CO (VIII) OH

H3CO" ~

V (IX) 0~.

---C.,H~

--C~H5

H~CO" ~

v (X)

O~ ~

V (Xl)

Initial biological results (Smith et al., 1964) using the Hershberger test showed norboletone to be 4 times as potent as a myotrophic agent as the corresponding estrene i.e. 13-CH3, and to have an anabolic-androgenic ratio of about 3. Norboletone is a racemic form but it has been resolved, allowing evaluation of the separate isomers. It has been observed that the l-form is completely inactive while the d-form, having the absolute configuration of natural steroids, shows twice the activity of the racemate, the same activity being achieved with half the dose (Edgren et al., 1963). Edgren (1963) made a more detailed study of norboletone, comparing it with other steroids. Myotrophic and androgenic potencies were evaluated using the Hershberger test. The usual anabolic studies were supplemented by studies of the acceleration of body growth in young intact male rats. Of all the compounds examined norbolethone was the most potent as a myotrophic agent. It also accelerated body growth at a dose of 300 ~g, lower than that required for any of the other compounds. Androgenicity was low and it therefore showed a marked separation between anabolic and androgenic effects.

266

B. CAMERINO and R. SCIAKY

It proved to be about 3 times more potent than testosterone propionate as a myotrophic agent on subcutaneous administration and only 13 per cent as androgenic, the myotrophic-androgenic ratio thus being - 2 0 . On oral administration, Tomarelli and Bernhardt (1964) found that the activity of norboletone was 16.3 times that of 17a-methyltestosterone. On subcutaneous administration, it was found to be 6 times more active than by oral administration. Furthermore, these workers found that in castrated male rats, doses of 0.05-1.0 mg caused a significant retention of nitrogen. As for hormonal effects, norboletone showed considerable progestational- and gonadotrophin-inhibiting activity (Overbeek, 1966, p.

51).
Greenblatt et al. (1964) measured the anabolic activity in man. As would be expected for a 17a-alkylated steroid, it led to increased sulfobromphthalein retention in man (Zimmerman, 1963). Many analogues of these 13-ethyl steroids have been prepared by Smith et al. (1964) but none has been found to be more interesting than norboletone. More recently, Hiraga et al. (1965) reported the synthesis of 13/3-iso-propyi-17aethyl-17/3-hydroxy-A'-gonen-3-one but rather surprisingly, this product showed no anabolic or androgenic activity.

ACKNOWLEDGMENTS We wish to express our warm appreciation and our grateful thanks to Drs. G. Baldratti, A. Consonni, L. Giglio and I. Vessey for their substantial help and generous contribution.

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