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Orally active steroidal ovulation inhibitors in the adult estrus rabbit
521
oRtXLTX ACTIVE SlEFiOW
OWLATION
INRIBITORSIN!I'READUI3ESIRUSRABBIl! Fred A. Kincl and Ralph I. Dorfman SyntexResearchIaboratories, Mexico,D.F., Mexico and WorcesterFoundationfor ExperimentalBiology Shrewsbury,Massachusetts
ReceivedOctober23, 1363
Orallyadministered1$3-norprogesterone derivativesand selected s-deoxysteroidshave been studiedin a rabbitanti-ovulation test. Compared to norethisterone the s-deoxysteroidslynestrenol, allyestrenol and ethylestrenolwere300, 33 and lOC$ effective, Removalof carbon19 from the progesteroneand l'J+acetoxyplcogesterone resultedin ten to twenty-foldgains in activity. Two 6,lkMimethylated C21 steroidsalso exhibitedrelativelyhigh anti-ovulatory activity. A previouspublicationfrom these laboratories has consideredthe anti-ovulatory activityof 36 steroidsadministeredsubcutaneously to mated estrusrabbitsand the anti-ovulatory activityof 37 steroidsadmzkktered by gavageto the same test aMmall. The presentreport ineludesstructureactititystudiessmonS certainlg-norprogesterone &?rivativesand selected34eozg steroids.
The method,reportedin detailp?eviously&,consistedin the administrationof the test compcundby gavageto adult estrusrabbits which were bred 18 to 24 hours later. The end-pointwas the number of
STEROIDS
522
2:5
ovulation points in the ovaries 18 to 24 hours after mating. The assays were performed by Dr. Elva Shipley at the Endocrine Laboratories,Mdison, Wisconsin, and the precision of the assay was indicated by the X value of 0.24.
RESULTS AND CONCLUSIONS
Table 1 presents a swmnary of the anti-ovulatoryactivity of norethisterone (l~c+ethynyl-l7~-hydroxy-l~-norandrost-4-en-3-one) when administered by gavage. The relative potency of the steroids studied in this paper are expressed in terms of the standard, norethisterone.
TABLE1 THE ANTI-OVUMTION ACTIVITY OF NORETHISTEZONEADMINISTERED BY GAVAGE
Norethisterone mg. - Dose,--0
0.312 0.625 1.25 2.5
-P-P--
No. of Rabbits 26 11 11 17 3 -
Mean Ovulation 5 S. E. (Range) 5.2 6.2 1.6 2.1
+ 0.3 + 0.4 +- 0.9 + 0.6 0 -
(0-10) (4- 8) (o- 7) (o- 6)
An oxygen function is not a necessary requirement for anti-ovulatory activity (Table 2).
Thus, the steroids lynestrenol (17c+ethynyl-
estr-4-en-17!3-ol), allylestrenol (17cd-allylestr-4-en-17~3-ol), and ethylestrenol (17a-ethylestr-4-en-17g-ol), all lack an oxygen function at carbon three, but had relative potencies compared to norethisterone (100) of 300, 33, ma 100, respectively. The change of 17a-ethynyl to the l‘i'a-vinyl or l'j'a-methyl group in the norethisteronemolecule resulted
Nov. 1963
523
STEROIDS
in compounds with 40 and lOO$ of the anti-ovulatory activity respectively. Reduction of the a
4
double bond to the 5a-dehydro derivative led to a loss
of 88% of the activity (Table 2).
TABLE 2 TRJzREzA!rIvEFOl!Ex!Y0Fl+NOR STEROIDS ADMINISTERED BY GAVAGE: Dosage Range Steroid
Rabbits
Qynestrenol Ethylestrenol 17(MSSthy1-1gnortestosterone 17cx-Vinyl-?+ -nortestosterone Allylestrenol 17cx-Ethynyl-17@-hydroxy-5cr_estran-3-one Norethynodrel 17cM%hynyl-5a-estrane-38,178-diOl
Relative FMency Norethisterone= 100
:,'
0.05 0.1
- 7.5 - 6.4
100 100
52
0.1
- 6.4
40
17 16
0.312 - 7.5 0.5 - 5.0
33 I2
41 11
0.312 - 2.5 1.25 -15.0
< 25 <4 ---_
The removal of carbon 19 from at least two C21 steroids resulted in an important gain in oral anti-ovulatoryactivity (Table 3). The gain from progesterone to lg-norprogesteronewas of the order of 10 fold from 0.17 to 2s of the standard, norethisterone. From 17cl-acetoxyprogesterone the gain was from < 1 to 25s of the to 17cx-acetoxy-lg-norprogesterone, standard. The two 6, 16-dimethylatedprogesterone derivatives are examples of compounds which, though lacking a 17a-acetoxy gr;roup, are orally active. A dose of 600 me:of progesteronewas needed to produce significant inhibition of ovulation, when given orally'. Significant inhibition was achieved with 0.3 mg of both @,l&~-dimethylprogesterone and 6,16~~-
STEROIDS
524
Steroid ~-----------=----_-~-. Progesterone 17cx-Acetoxyprogesterone 19"Norprogesterone 17cx-Acetoxy-lg6,l~-R~et~l-~6-norprogesterone -dehydroprogesterone 6@,lkx-Dimethyl proaesterone I__------_-
20 21
Dosage Range Relative Potency Norethisterone = 100 mg* ------II-c-_~---cI --.-.O.l?l < 1.01 1.25 - 30.0 2.0 0.2 - 7.5 25.0
13
0.3
- 10.0
20.0
26
0.1
-
20.0
No. of Rabbits
5.0
__----___f.l--_-
___--.-__-
l Kin&L and Dorfman (1963)
THE:RELA!3!IVE l?WJ%NCYOF MISCEWOUS
STEROIDS ADMTNISI'EREDBY GAVAGE
z--
Steroid---
A6-Dehydroretroprogesterone 3@-Fluoro-17cx-acetoxypregn-5-en-20-one 17cx-Acetoqypregn-5-en-20-one l'j"c-Acetoxypregn_3,5j-dien-20-one Gxymetholone
*
No. of Rabbits
Dosage Range mg* . ..-L----~--
19
0.5 - l.p.0
30 *
16
0.25-
10.0
10
14
1.0 - 30.0
40
15
0.1 -
50
18
0.1 - 10.0
B-._-__-__I-
5.0
Relative Potenw Norethisterone= 100 - ~~~-.-.---1
c:5
--
Complete suppression could not be obtained even at doses 10 times the mW.mum effective doses.
Nov. 1963
STEROIDS
525
-dimethyl-6-dehydroprogesterone.These compounds had shallow dose response curves and complete suppression of ovulation was not achieved even at high doses, some 15 to 30 times the minimal effective doses. Table 4 records the comparative activity of various miscellaneous steroids. The steroid A6-dehydroretroprogesteronevaried in its relative potency. At 1~
doses, it was some 3CY$as active as norethisterone,but
with increased doses no complete suppression could be obtained. Oxymetholone (2-hydro~ethylene-l7~-meth~l-178-h?rdroyy-5a-androstan-3-one) gave no positive responses and under the conditions of the study and dosages employed, oxymetholonewas less than 5s as active as the standard, if active at all. Shifting of the double bond from the 4-5 position to the 5-6 position, and elimination of the 3-oxygen function in 17cx-acetoxyprogesterone, produced a compound some 40 times as active as the standard (Tables 3 and 4).
If only the double bond is shifted from the 4 to the 5 position, an .
increase in relative potency, compared to norethisterone,of 3s1 to 4077 was observed. The doubly unsaturated 3-deoxy steroid, 17cx-acetox&q)regn-3,5-dien-20-onewas highly active, being about one-half as active as the was 10% as active standard. The 3S-fluoro-17cx-acetoxypregn-5-en-20-one as norethisterone.
REFXRENCES 1.
Kincl, F. A. and Dorfman, R. I., ACTA ERDOCRINOL. 42, Suppl. 73 (1963).
oRtXLTX ACTIVE SlEFiOW
OWLATION
INRIBITORSIN!I'READUI3ESIRUSRABBIl! Fred A. Kincl and Ralph I. Dorfman SyntexResearchIaboratories, Mexico,D.F., Mexico and WorcesterFoundationfor ExperimentalBiology Shrewsbury,Massachusetts
ReceivedOctober23, 1363
Orallyadministered1$3-norprogesterone derivativesand selected s-deoxysteroidshave been studiedin a rabbitanti-ovulation test. Compared to norethisterone the s-deoxysteroidslynestrenol, allyestrenol and ethylestrenolwere300, 33 and lOC$ effective, Removalof carbon19 from the progesteroneand l'J+acetoxyplcogesterone resultedin ten to twenty-foldgains in activity. Two 6,lkMimethylated C21 steroidsalso exhibitedrelativelyhigh anti-ovulatory activity. A previouspublicationfrom these laboratories has consideredthe anti-ovulatory activityof 36 steroidsadministeredsubcutaneously to mated estrusrabbitsand the anti-ovulatory activityof 37 steroidsadmzkktered by gavageto the same test aMmall. The presentreport ineludesstructureactititystudiessmonS certainlg-norprogesterone &?rivativesand selected34eozg steroids.
The method,reportedin detailp?eviously&,consistedin the administrationof the test compcundby gavageto adult estrusrabbits which were bred 18 to 24 hours later. The end-pointwas the number of
STEROIDS
522
2:5
ovulation points in the ovaries 18 to 24 hours after mating. The assays were performed by Dr. Elva Shipley at the Endocrine Laboratories,Mdison, Wisconsin, and the precision of the assay was indicated by the X value of 0.24.
RESULTS AND CONCLUSIONS
Table 1 presents a swmnary of the anti-ovulatoryactivity of norethisterone (l~c+ethynyl-l7~-hydroxy-l~-norandrost-4-en-3-one) when administered by gavage. The relative potency of the steroids studied in this paper are expressed in terms of the standard, norethisterone.
TABLE1 THE ANTI-OVUMTION ACTIVITY OF NORETHISTEZONEADMINISTERED BY GAVAGE
Norethisterone mg. - Dose,--0
0.312 0.625 1.25 2.5
-P-P--
No. of Rabbits 26 11 11 17 3 -
Mean Ovulation 5 S. E. (Range) 5.2 6.2 1.6 2.1
+ 0.3 + 0.4 +- 0.9 + 0.6 0 -
(0-10) (4- 8) (o- 7) (o- 6)
An oxygen function is not a necessary requirement for anti-ovulatory activity (Table 2).
Thus, the steroids lynestrenol (17c+ethynyl-
estr-4-en-17!3-ol), allylestrenol (17cd-allylestr-4-en-17~3-ol), and ethylestrenol (17a-ethylestr-4-en-17g-ol), all lack an oxygen function at carbon three, but had relative potencies compared to norethisterone (100) of 300, 33, ma 100, respectively. The change of 17a-ethynyl to the l‘i'a-vinyl or l'j'a-methyl group in the norethisteronemolecule resulted
Nov. 1963
523
STEROIDS
in compounds with 40 and lOO$ of the anti-ovulatory activity respectively. Reduction of the a
4
double bond to the 5a-dehydro derivative led to a loss
of 88% of the activity (Table 2).
TABLE 2 TRJzREzA!rIvEFOl!Ex!Y0Fl+NOR STEROIDS ADMINISTERED BY GAVAGE: Dosage Range Steroid
Rabbits
Qynestrenol Ethylestrenol 17(MSSthy1-1gnortestosterone 17cx-Vinyl-?+ -nortestosterone Allylestrenol 17cx-Ethynyl-17@-hydroxy-5cr_estran-3-one Norethynodrel 17cM%hynyl-5a-estrane-38,178-diOl
Relative FMency Norethisterone= 100
:,'
0.05 0.1
- 7.5 - 6.4
100 100
52
0.1
- 6.4
40
17 16
0.312 - 7.5 0.5 - 5.0
33 I2
41 11
0.312 - 2.5 1.25 -15.0
< 25 <4 ---_
The removal of carbon 19 from at least two C21 steroids resulted in an important gain in oral anti-ovulatoryactivity (Table 3). The gain from progesterone to lg-norprogesteronewas of the order of 10 fold from 0.17 to 2s of the standard, norethisterone. From 17cl-acetoxyprogesterone the gain was from < 1 to 25s of the to 17cx-acetoxy-lg-norprogesterone, standard. The two 6, 16-dimethylatedprogesterone derivatives are examples of compounds which, though lacking a 17a-acetoxy gr;roup, are orally active. A dose of 600 me:of progesteronewas needed to produce significant inhibition of ovulation, when given orally'. Significant inhibition was achieved with 0.3 mg of both @,l&~-dimethylprogesterone and 6,16~~-
STEROIDS
524
Steroid ~-----------=----_-~-. Progesterone 17cx-Acetoxyprogesterone 19"Norprogesterone 17cx-Acetoxy-lg6,l~-R~et~l-~6-norprogesterone -dehydroprogesterone 6@,lkx-Dimethyl proaesterone I__------_-
20 21
Dosage Range Relative Potency Norethisterone = 100 mg* ------II-c-_~---cI --.-.O.l?l < 1.01 1.25 - 30.0 2.0 0.2 - 7.5 25.0
13
0.3
- 10.0
20.0
26
0.1
-
20.0
No. of Rabbits
5.0
__----___f.l--_-
___--.-__-
l Kin&L and Dorfman (1963)
THE:RELA!3!IVE l?WJ%NCYOF MISCEWOUS
STEROIDS ADMTNISI'EREDBY GAVAGE
z--
Steroid---
A6-Dehydroretroprogesterone 3@-Fluoro-17cx-acetoxypregn-5-en-20-one 17cx-Acetoqypregn-5-en-20-one l'j"c-Acetoxypregn_3,5j-dien-20-one Gxymetholone
*
No. of Rabbits
Dosage Range mg* . ..-L----~--
19
0.5 - l.p.0
30 *
16
0.25-
10.0
10
14
1.0 - 30.0
40
15
0.1 -
50
18
0.1 - 10.0
B-._-__-__I-
5.0
Relative Potenw Norethisterone= 100 - ~~~-.-.---1
c:5
--
Complete suppression could not be obtained even at doses 10 times the mW.mum effective doses.
Nov. 1963
STEROIDS
525
-dimethyl-6-dehydroprogesterone.These compounds had shallow dose response curves and complete suppression of ovulation was not achieved even at high doses, some 15 to 30 times the minimal effective doses. Table 4 records the comparative activity of various miscellaneous steroids. The steroid A6-dehydroretroprogesteronevaried in its relative potency. At 1~
doses, it was some 3CY$as active as norethisterone,but
with increased doses no complete suppression could be obtained. Oxymetholone (2-hydro~ethylene-l7~-meth~l-178-h?rdroyy-5a-androstan-3-one) gave no positive responses and under the conditions of the study and dosages employed, oxymetholonewas less than 5s as active as the standard, if active at all. Shifting of the double bond from the 4-5 position to the 5-6 position, and elimination of the 3-oxygen function in 17cx-acetoxyprogesterone, produced a compound some 40 times as active as the standard (Tables 3 and 4).
If only the double bond is shifted from the 4 to the 5 position, an .
increase in relative potency, compared to norethisterone,of 3s1 to 4077 was observed. The doubly unsaturated 3-deoxy steroid, 17cx-acetox&q)regn-3,5-dien-20-onewas highly active, being about one-half as active as the was 10% as active standard. The 3S-fluoro-17cx-acetoxypregn-5-en-20-one as norethisterone.
REFXRENCES 1.
Kincl, F. A. and Dorfman, R. I., ACTA ERDOCRINOL. 42, Suppl. 73 (1963).