RELATIONSHIP BETWEEN PROGESTATIONAL ACTIVITY AND CHEMICAL STRUCTURE OF SYNTHETIC STEROIDS

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1 RELATIONSHIP BETWEEN PROGESTATIONAL ACTIVITY AND CHEMICAL STRUCTURE OF SYNTHETIC STEROIDS G. K. SUCHOWSKY and G. BALDRATTI Laboratori Ricerche Farmacologiche, Farmitalia, Milano, Italy (Received 5 October 963) SUMMARY Forty-two synthetic steroids have been studied for which certain relationships between chemical structure and progestational activity could be established. Thus, unsaturation and esterification in pregnane derivatives enhances progestational potency. Of the -dehydro derivatives, the 6\g=a\-CH3 and the 6\g=a\-fluorocompounds were the most active. Progestational potency on oral administration was increased in the -hydroxy derivatives. In the group with the :-dehydro configuration, the basic activity of the parent compound was increased only by the introduction of a 6\g=a\-chloroor fluoro group. The :6-dehydro derivative showed an increase in potency on oral administration of about the same extent on introduction of a 6-chloro, a 6-fluoro or a 6:6\g=a\-dimethylgroup. Only in the group of pregnane derivatives did changes of the parent molecule lead to a considerable increase in potency to inhibit ovulation. Since the maintenance of pregnancy in spayed animals is a parameter of progestational activity, it is felt that the results in animals are difficult to compare with those in female patients. introduction The purpose of this study was to relate chemical structure to biological activity in a group of synthetic steroids with progestational activity, and to discuss some compounds which have been chosen for clinical use during the past few years. The clinical use of progesterone is well established. The short duration of action on parenteral administration and the fact that progesterone is almost inactive when given orally led to the synthesis of numerous compounds with the classical progesta tional effect upon the maintenance of pregnancy in spayed animals. Later, a number of synthetic progestagens were found to exert specific activities different from those of their parent compounds. These different activities have been utilized after intensive experimental and clinical studies. We know today that the classical progestational effect, the maintenance of pregnancy, prevails in the derivatives of progesterone or 7a-hydroxyprogesterone. Most of the other synthetic steroids serve for the treat ment of miscellaneous gynaecological disorders and have recently also been used in the control of fertility.

2 MATERIALS AND METHODS (a) Progestational activity Groups of three to five immature female rabbits (weighing g.) were primed daily with oestradiol in aqueous solution (0-5 fig./val.) for 6 consecutive days. The progestagen treatment was begun on the 7th day. The progestagen was admi nistered in equal doses (total doses: 30,, 3,, 0-3, 0T, 0-03, 0-0 mg.) for 5 days. One day after the last administration the animals were killed, the uteri removed, fixed in % formalin and frozen sections prepared. Evaluation was carried out according to McPhail's (93) scale. (b) Pregnancy-maintaining effect in the rabbit When testing compounds with prolonged progestational effects, the following technique was used : adult female rabbits were mated, the day of copulation being designated day 0 of pregnancy. Five animals were used per group and spayed on day 7, or 2 of pregnancy. Three days before the operation the test compound was injected in a single dose. In testing steroids without a depot effect, adult rabbits were also used. Treatment was begun on the day before spaying on day 5 of pregnancy and the steroid ad ministered dailv throughout the rest of the pregnancy. (c) Pregnancy-maintaining effect in mice Five to eight pregnant mice were spayed on days 3-5 of pregnancy. The treat ment was begun on the day before the operation and was continued daily up to full term. (d) Pregnancy-maintaining effect in rats Adult female rats were mated. Copulation was detected by the presence of a vaginal plug or by finding spermatozoa in the vaginal smear. The day of copulation was designated day 0 of pregnancy. Five animals were used per group. The groups of rats were spayed on days 5, or 5 of pregnancy and killed 5, or 5 days later. Treatment was started 2 hr. before castration. (e) Ovulation-inhibiting effect in rats Two successive oestrous cycles were followed in normal, adult female rats. Animals with - to 5-day cycles were used and the treatment was started at the following metoestrus. The compounds were administered s.c, daily, on consecutive days. On the th day, one ovary, including the Fallopian tube, was removed. The tube was mounted in 0-9 % NaCl solution and studied under the microscope. When ovulation had occurred the tubal ova were easily seen. On the 5th day the animals were killed and the procedure was repeated with the remaining Fallopian tube. Ovulation was taken to be inhibited when no ova were detected. At least five animals were used for each dose.

3 Progestational activity and chemical structure 6 (/) Inhibition of gonadotrophins in parabiotic rats Littermates (weighing 5-55 g.), male and female, were united abdominally as described by Bunster & Meyer (933) and the male castrated simultaneously. The test compounds were administered to the male partners daily for days. The animals were killed on the day after the last administration. The wet weight of the ovaries and uterus of each of the female partners was determined. Five pairs were used per dose. (g) Inhibition of the oestrous cycle in the rat After two normal oestrous cycles in adult female rats, the test compounds were administered once daily for consecutive days, beginning at the following metoestrus. Five animals were used for each dose and their vaginal smears were inspected daily. (h) Anti-oestrogenic effect in rats Adult female rats were spayed. One week after the operation the animal received a single s.c. injection of 25 ig. oestradiol- 7/?-undecylate. Three days after the injection the test compounds were administered s.c. in a single dose. Throughout the experiment vaginal smears were taken daily and classified according to Allen & Doisy (923). Five animals were used per dose. (i) Oestrogenic effect in rats Adult female rats were spayed. One week after the operation the test compound was administered in a single dose. Vaginal smears were taken 8 hr. after the injection. In groups (a) and (i) the minimal effective dose was used for classification. In groups (b) to (h), ED 50 was determined from the log dose-response curve. Since the mean values per group represent percentages, no confidence limits could be calculated. The mean error in these experiments was ± 20%. RESULTS AND DISCUSSION The steroids were studied for their progestational effect after s.c. and oral ad ministration (Table ). The activities of different steroids were compared in terms of their minimal effective dose, i.e. the dose which would induce a response of -5 in all three animals in the group or a response of 2 in one animal when classified according to McPhail's scale. Progesterone is inactive when given orally, only a few derivatives of androstane have been studied. Only two were found of clinical use, i.e. 7a-ethinyl-testosterone (Inhoffen & Hohlweg, 938) and 6a:2 -dimethyl- 7a-ethinyl-testosterone (David, Fellowes & Millson, 959). Since unsatisfactory results were obtained with derivatives of testosterone, a new group of substances was studied with the aim of finding more potent compounds which would produce fewer unwanted side effects, and which would be active when administered orally (see Table 2). A new group of steroids in which 3-keto configuration seems to play a secondary II-2

4 _ 62 G. K. Suchowsky and G. Baldratti i,,6,,,,,6,6,6,6,6 OH Table. C- <xf acl œch3 af acl CH3 F Cl CH, 6 ach«, ach3 ach3 ach, List of pregnane derivatives 7 Bibliography Junkmann & Witzel (957) Junkmann & Witzel (957) Turner (953) Junkmann & Witzel (957); Junkmann (959) Junkmann (959); Sciaky (96) Ringold & Rosenkranz (96) Babcock, Outsell, Herr, Hogg, Stucki, Barnes & Dulin (958); Greenblatt & Barfield (959); Sala, Camerino & Cavallero (958); Stucki (958); Junkmann (959); Ringold, Perez Ruelas, Batres & Djerassi (9596) Batres, Cardenas, Edwards, Moury, Mancera, Djerassi & Ringold (96); Bernstein, Canwall, Dusza & Joseph (96) Bowers, Ibanez & Ringold (959); Junk mann (959) Bowers et al. (959); Junkmann (959) Ringold et al. (9596) Bernstein et al. (96) Bowers et al. (959) Ringold, Batres, Edwards & Zderick (959 a) Ringold et al. (9596) Bernstein et al. (96) Bowers et al. (959) Ringold et al. (959 a) Iriarte & Franco (96) Baldratti & Sala (962); Camerino, Valcavi, Sala & Baldratti (962) ; Arcari, Baldratti & Sala (963) Table 2. Compound 7a-Methyl-9-nortestosterone 7œ-Ethyl-9-nortestosterone 7a-Ethinyl-9-nortestosterone 7a-Ethinyl-9-nortestosterone- 7/S-acetate 7a-Ethinyl-oestr-5()-en-7/?- ol-3-one lla.-alkyl-9-nortestoslerone derivatives Bibliography Djerassi, Miramontes, Rosenkranz & Sondheimer (95); Ferin (956, 957); Kotsalo (957); Miyake & Pincus (958); Junkmann (959); Moggian (959) Pincus, Chang, Hafez, Zarrow & Merril (956 a) ; Ferin (957); Saunders, Colton & Drill (957); Saunders & Drill (958); Greenblatt (958); Stucki (958); Junkmann (959) Djerassi et al. (95) ; Hertz, Tullner & Raffelt (95) ; Hertz, Wait & Thomas (956); Greenblatt (956, 958); Pincus et al. (956a); Pincus, Chang, Zarrow, Hafez & Merril (9566); Baquero (957); Ferin (957); Junkmann (959) Junkmann (959); Suchowsky (962) Colton (956); Greenblatt (956); Pincus et dl. (956a, 6); Colton, Nysted, Riegel & Raymond (957); Kupperman & Epstein (957); Edgren (958); Kistner (958) ; Miyake & Pincus (958) ; Saunders & Drill (958); Stucki (958)

5 Progestational activity and chemical structure 63 part in the progestational activity is of interest, namely the 7a-ethyl-oestr--enllß-ol, the 7a-ethinyl-oestr--en-7/?-ol (de Winter, Siegmann & Szpilfogel, 959; Junkmann & Suchowsky, 962; Suchowsky, 962) and the 7a-allyl-oestr--enllß-ol (de Winter et al. 959 ; Overbeek, 959 ; Overbeek & Madjerek, 960; Madjerek, de Visser, van der Vies & Overbeek, 960; Suchowsky, 962). The pregnancy-maintaining effect has been studied in compounds which are already employed in clinical work. The steroids investigated can be divided into three main groups according to their chemical structure, i.e. pregnane derivatives, androstane derivatives and 7a-alkyl-l9-nortestosterone derivatives. To discuss the pregnancy-maintaining effect of some of these compounds more effectively, it seems justifiable to compare the results of different authors in different species obtained by different methods (Table 3). Table 3. Compound Pregnane derivatives: Progesterone 7a-Acetoxy-progesterone 7a-Hydroxyprogesterone-7a-caproate -Hydroxy-7a-acetoxy-progesterone 7a-Acetoxy-progesterone-3-cyclopentylenol-ether 6a-Methyl-7a-acetoxy-progesterone Progesterone 7a-Acetoxy-progesterone 7a-Hydroxyprogesterone-7a-caproate 6a-Methyl-7a-acetoxy-progesterone Progesterone 6a-Methyl-7<x-acetoxy-progesterone Androstane derivatives : 6a:2-Dimethyl-7a-ethinyl-testosterone 7a-Alkyl-9-nortestosterone derivatives : 7a-Methyl- 9-nortestosterone 7a-Ethyl-9-nortestosterone 7a-Ethinyl-9-nortestosterone 7a- Allyl-oestr--en-l 7/?-ol 7a Methyl-9-nortestosterone 7a-EthinyI-9-nortestosterone Potency of progesterone, androstane and derivatives of nortestosterone Animal Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Mouse Mouse Mouse ED50(mg.) Day of castration s.c. per os 5 7,, ,, 5 8 7, 5,, 5 5,, 5 8 5,, ,, 5 8 5,, 5 5,, 5,, (-0) (25-0) (20-0) (io'o) -6 (-0) (-0) Bibliography Elton & Edgren (958) Sala, Baldratti & Arcari (959) Falconi, Gardi, Bruni & Ercoli (96) Falconi et al. (96) Suchowsky & Junkmann (958)* Baldratti & Sala (962) Falconi et al. (96) Baldratti (96, unpublished results) Sala et al. (959) Falconi et al. (96) Baldratti & Sala (962) Alexander, Frazer & Lee (955) Suchowsky & Junkmann (957) Stucki (958) Marois (960) Lerner, Brennan, Yacas, De Phillipo & Borman (962) Suchowsky (962, unpublished results) Suchowsky & Junkmann (957) Stucki(958) -0 Madjerek, de Visser, Overbeek (960) 0-55 Lerner et al. (962) 0-2 Suchowsky (963) Hall (957) Sala et al. (959) Sala et al. (959) Madjerek et al. (960) (-0) -0 (20-0) (ÍO'O) (-0) 0 60 van der Vies & Madjerek et al. (960) Suchowsky (963) Stucki(958) Suchowski (963) Lerner et al. (962) Madjerek et al. (960) Suchowsky & Junkmann (957) Suchowsky (963) Madjerek et al. (960) Suchowsky (963) Sala et al. (959) Sala et al. (959) Single dose/kg. body weight, given 3 days before castration.

6 6 G. K. Suchowsky and G. Baldratti Most of the compounds described in this paper have also been studied for their inhibitory effect upon the central control mechanisms. In Table only compounds already discussed in the previous section are considered. If they are contrasted with the parent compound or with each other the test methods employed demonstrate clearly the different effects of the compounds used as progestagens at the present time. Table. II. Androstane derivatives : Testosterone 7a-Ethinyl-testosterone 6a:2 -Dimethyl-7a-ethinyl-tes tosterone Inhibition of central control mechanisms Parabiosis x s.c. < 0-07 < 0-07 < 0-35 < III. 7-Alkyl-9-nortestosterone derivatives: 9-Nortestosterone 7a-Methyl-9-nortestosterone a-Ethyl-9-nortestosterone a-Ethinyl-9-nortestosterone a-Ethinyl-9-nortestosterone-7/5- -3 acetate 7a-Ethinyl-oestr-5( ) -en- Iß ol-3-one 7a-Ethinyl-oestr--en-7/?-ol a-Allyl-oestr--en-7/?-ol 0-07 I Relative potency Compound I. Pregnane derivatives: Progesterone 7a-Acetoxy-progesterone 7a-Hydroxyprogesterone-7acaproate 7a-Acetoxy-progesterone-3-cyclopentyl-enol-ether 6-Chloro-6-dehydro-7a-acetoxyprogesterone 6a-Methyl-7a-acetoxy-progesterone 6:6a-Dimethyl-6-dehydro-7aacetoxy-progesterone Antioestrous cycle x s.c. < 003 < 0-03 < Antiovulation x s.c. < 0-0 < < 0-00 < Antioestrogen X s.c M.E.D. (mg.) oestrogenic act. X B.c. The dose-range used in these experiments is as follows:, 3,, 0-3, 0-, 0-03, 00 mg./day s.c The 7a-hydroxy-5a-pregnane-3:20-dione (Table 5) was practically inactive on s.c. and oral application. The introduction of a Ax-double bond led to a com pound active by both methods of administration. The A-analogue was markedly less active, whereas the A>-compound retained the activity of the A-steroid and increased the oral activity about three times. The A>6-double bonds led again to loss of activity. The most favourable effect was seen in the 9-nor-A-configuration on s.c. administration; however, the compound was inactive when given orally. When the influence of C7 acetylation on the corresponding 7a-OH compound was considered (Table 6) it was evident that the progestational activity on s.c. and -0

7 Progestational activity and chemical structure 65 Table 5. Unsaturation and progestational activity Clauberg assay (minimal effective dose in mg.) Compound s.c. per os 7a-Hydroxy-5a-pregnane-3:20-dione 7a-Hydroxy-5a-pregn-l-ene-3:20-dione ^ 3-0 ^ a-Hydroxy-pregn--ene-3:20-dione S -0 > a-Hydroxy-pregna-l:-diene-3:20-dione ^ 3-0 ^ -0 7a-Hydroxy-pregna-:6-diene-3:20-dione > -0 > a-Hydroxy-9-norpregn--ene-3:20-dione ^ -0 > -0 Table 6. Effects of esterification at C7 Relative progestational potency -7a OH -7a-acetate* Compound s.c. per os s.c. per os 7a-Hydroxy-5a-pregnane-3:20-dione > 30 > ( mg.) (30 mg.) 7a-Hydroxy-5a-pregn-l-ene-3:20-dione a-Hydroxy-pregn--ene-3:20-dione a-Hydroxy-pregna-l:-diene-3:20-dione a-Hydroxy-pregna-:6-diene-3:20-dione 300 > 0 7a-Hydroxy-9-norpregn--ene-3:20-dione 300 > 0 * The ratio is based on the value for the corresponding 7a-OH compound, which is equal to unity. oral administration was considerably increased. Thus, contrary to the effect of the A-derivative, the A-derivative was thirty times more potent on s.c. administra tion, and three hundred times more potent on oral administration. The A>-compound increased the s.c. and oral potency about equally. The A'6-configuration seemed to be the most active. In 7a-acetoxy-progesterone (Table 7) the introduction of a 2a-CH3 or 6a-CH3 group diminished both the s.c. and oral potencies, whereas the 60C-CH3 configuration led to a compound three times more potent on s.c, and ten times more potent on oral administration. The introduction of a 6a-chloro group did not alter the basic potency. A 6a-fluoro group increased both the s.c. and the oral potencies three and ten times respectively. The introduction of oxygen at C slightly decreased the s.c. potency of the parent compound but increased the oral potency five times. Similar results were obtained with 7a-acetoxy-l-dehydro-progesterone, except that the introduction of a 6a-CH3 group did not influence the s.c. potency and diminished the oral potency to one-third. The 6a-CH3 derivative was a third less active than the parent compound by both methods of administration. The 6a-chloro and fluoro derivatives were the most active compounds in this group. The introduction of a 6-chloro group into 7a-acetoxy-6-dehydro-progesterone enhanced the potency on both s.c. and oral administration three and ten times respectively. The same effect was evident in the 6-fluoro derivative. Introduction of a 6-CH3 group decreased the s.c. potency and did not influence the oral activity; however, a 6a-CH3 group increased the oral potency. The compound with two methyl groups (C6 and C6) had the same s.c. potency as the parent compound but was ten times more active orally.

8 66 G. K. Suchowsky and G. Baldratti Table 7. Effects of alkyl or halogen substitution, or introduction of oxygen function Relative progestational potency* an additional 7a- 2a- 6a- 6a- 6:6a- 6a- 6a- Compound Ace- CH3 CH3 CH3 CH3 F Cl -OH täte 7a-Hydroxy-pregn--ene-3:20-dione 7a-Hydroxy-pregna-l:-diene- 3:20-dione 7a-Hydroxy-pregna-:6-diene- 3:20-dione s.c. os s.c. os s.c. os t 3t 3 * The ratio is based on the corresponding 7a-acetate value, which is equal to unity, f The fluoro or chloro group is not in a-position. In the group of alkylated testosterone derivatives (Table 8), the parent compound testosterone was weakly active when administered s.c, and did not lead to a complete progestational stimulation of the endometrium. The 7a-ethinyl derivative was at least three times more potent subcutaneously than testosterone. The oral potency ofthe two compounds cannot be compared. Adequate doses ( mg.) led to a fully deve loped progestational endometrium. This compound has lost its clinical interest due to its androgenic side-effect. The other representative of this group, 6œ:2-dimethyl- 7a-ethinyl-testosterone, was about ten times more active than testosterone when given s.c, and about ten times more active when given orally than the 7a-ethinyl derivative. However, this steroid still remains in the class of weakly active progestagens and, due to the fact that it is derived from testosterone, a slight andro genic side-effect might be expected in the female rat foetus. Table 8. Potency of alkylated testosterone derivatives Clauberg assay (minimal effective dose in mg.) Compound s.c. per os Testosterone 3-0 7a-Ethinyl-testosterone a:2-Dimethyl-7a-ethinyl-testosterone Nortestosterone (Table 9), is much more active by both methods of administra tion than testosterone. The introduction of a 7a-alkyl group increased both the s.c. and oral potencies. Thus the 7a-methyl compound was fifty times more active, s.c, and about one hundred times more potent when given orally. The 7a-ethyl compound is one hundred times more active s.c. than the parent compound, and as active as the methyl-derivative when given orally. The 7a-ethinyl steroid has much the same activity as the methyl compound ; acetylation at C7 increases the potency by both methods of administration. The last compound of this group was 7a-ethinyl-oestr- 5()-en-7/?-ol-3 one. Its subcutaneous potency was three times that of the parent compound and the oral activity was ten times greater. These compounds do not exert a complete progestational transformation in the endometrium (Klopper, 962).

9 Progestational activity and chemical structure 67 Table Q. lla-alkyl-9-nortestosterone derivatives Relative progestational potency _A_ Compound s.c. per os 9-Nortestosterone 7a-Methyl-l 9-nortestosterone a-Ethyl-9-nortestosterone 0 0 7a-Ethinyl-9-nortestosterone a-Ethinyl-9-nortestosterone-7/?-acetate a-Ethinyl-oestr-5()-en-7/?-ol-3-one 3 Most of the 9-nortestosterone derivatives had androgenic side effects as measured by the weight increase of the ventral prostate, the seminal vesicles and the preputial glands in castrated male rats. All compounds exerted a virilizing effect upon the female rat foetus, although the androgenic effect was more or less dissociated from the progestational effect. For some of the compounds, observations have been published on mothers treated during pregnancy who delivered female babies with various signs of virilization. However, some of these compounds are still employed for the treatment of habitual or threatened abortion. They are also used in some gynaecological disorders. Table shows that none of the 3-deoxo compounds is as active as the 3-keto analogue. There is one exception : the 7a-ethyl-derivative has the same progestational potency on oral administration as its corresponding 3-keto steroid. Table. 3-Deoxo-lla-alkyl-l9-nortestosterone derivatives Relative progestational potency Corresponding 3-keto compound,-*-, Compound s.c. per os s.c. per os 7a-Ethyl-oestr--en-7/?-ol -3 7a-Ethinyl-oestr--en-7/?-ol a-Allyl-oestr--en-7/?-ol ]* * * Calculated from the data published by Drill & Riegel (958). According to our results (Table 3) and those of other authors, progesterone main tains pregnancy in all mammalian species so far studied. Differences between the published results may be due to the fact that the animals were spayed at different stages of pregnancy, or that the treatment was given for a few days only, or for the entire period of pregnancy. These results show also that species differences exist and that there is no parallelism with the progestational activity in the Clauberg assay. Suchowsky (963) found that compounds active in the rat inhibit the pituitary. The pregnancy-maintaining activity of progesterone can be enhanced by adding oestrogens (Stucki & Forbes, 960; Baldratti, unpublished observations; Suchowsky, 963), or can be maintained by a combination of compounds which are inactive when given alone. However, so far no attempt to imitate physiological conditions by

10 68 G. K. Suchowsky and G. Baldratti varying the amount of oestrogen administered during the course of pregnancy seems to have been made. Compounds which are metabolized to oestrogens (likel7cc-ethinyl- 9-nortestosterone) have a deleterious effect on the foetus but this effect can be compensated for by combination with a compound (7a-hydroxyprogesterone-7acaproate) which is inactive in the rat (Suchowsky, 963). The absence of an inhibitory effect of 7<x-hydroxyprogesterone-7a-caproate on the central control mechanisms can probably be avoided by replacement with 7a-ethinyl-9-nortestosterone. The results shown in Table are concerned with the interference by steroids with the central control mechanisms. The results in group I refer to progesterone. It has been known for some time that progesterone exerts a central effect: appropriate amounts of this hormone block ovulation, inhibit gonadotrophin secretion and inter fere with the cyclic changes of the vaginal epithelium. When the central-inhibitory properties of the synthetic compounds are compared, parallelism between the three parameters mentioned was observed (Table ). Introduction of a hydroxy group at C7 and esterification led to compounds which were practically devoid of any central effect but an additional 6a-CH3 group increased the potency of the parent compound considerably. Introduction of a double bond at C6 and a halogen or additional methyl group increased the potency ofthe parent compound to a moderate degree. The anti-oestrogenic activity reflects the progestational potency combined with a direct anti-hormonal effect. The oestrogenic activity of group I was negligible. None of the compounds derived from testosterone (group II) was more potent than its parent compound. The parent compound of group III was 9-nortestosterone. There was a very good parallelism between the different compounds with regard to the potencies studied. Alkylation at C7 by itself did not increase the potency of the parent compound. Acetylation at C7, in 7a-ethinyl-9-nortestosterone, produced a moderate increase in potency. Absence of the 3-keto group decreased the inhibitory activity considerably. A slight decrease was caused by the shift of the A-double bond to A5(). It must be emphasized that 7<x-ethinyl-l 9-nortestosterone, its acetate and 7a-ethuryl-oestr-5()-en-7/?-ol-3-one possess intrinsic oestrogenic activity, due to the endogenous metabolism of these steroids to oestrogens, or to contamination with oestrogens. That oestrogens by themselves are the most potent inhibitors of the central control mechanism is well established; one may, therefore, presume that the inhibitory activity of the former compounds is partly due to the oestrogenic side-effect, which enhances this activity. REFERENCES Alexander, D. P., Frazer, J. F. D. & Lee, J. (955). The effect of steroids on the maintenance of pregnancy in the spayed rat. J. Physiol. (Lond.), 30, 8. Allen, E. & Doisy, E. A. (923). An ovarian hormone. Preliminary report on its localization, extraction and partial purification, and action on test animals. J. Amer. med. Ass. 8, 89. Arcari, G., Baldratti, G. & Sala, G. (963). Progestational activity of -hydroxy-7a-acetoxyprogesterone. Nature (Lond.), 97, 292. Babcock, F. C, Gutsell, E. S., Herr, M. E., Hogg, J., Stucki, J. C, Barnes, L. E. & Dulin, W. E. (958). 6a-Methyl-7a-hydroxyprogesterone-7-aeylates: a new class of potent progestins. J. Amer. chem. Soc. 80, 290. Baldratti, G. & Sala, G. (962). Pregnancy-maintaining activity of -hydroxy-7a-acetoxyprogesterone. Ada endocr. (Kbh.), 3, suppl. 67. Baquero, R. E. (957). Una nueva sustancia progestacional activa por via oral: la Noretisterona. Rev. colomb. endocr., 209.

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