Synthesis and biological activity of a new progestogen, 16-methylene- 17α-hydroxy-18-methyl-19-norpregn-4-ene-3,20-dione acetate

Article abstract of DOI:10.1016/S0039-128X(99)00109-9

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17α- hydroxy-18-methyl-19-norpregn-4-ene-3,20-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches. (C) 2000 Elsevier Science Inc.

Full text of DOI:10.1016/S0039-128X(99)00109-9

Steroids 65 (2000) 266–274
Synthesis and biological activity of a new progestogen, 16-methylene-
17␣-hydroxy-18-methyl-19-norpregn-4-ene-3,20-dione acetate
Zolta´n Tubaa,*, C. Wayne Bardinb, Anna Dancsia, Erzse´bet Francsics–Czinegea,
Csaba Molna´ra, Ja´nos Cso¨rgeia, George Falkayc, Samuel S. Koided, Narender Kumard,
Kalyan Sundaramd, Vilma Duka´t–Abro´ka, Ga´bor Balogha
aGedeon Richter Ltd., Department of Steroid Research, P.O. Box 27, 1475 Budapest, Hungary
bThyreos Corporation, 240 Dr. Martin Luther King Jr. Blvd, Newark, NJ 07102, USA
cDepartment of Pharmacodynamics, A. Szent–Gyo¨rgyi Medical University, Szeged, Eo¨tvo¨s u. 2., 6720, Hungary
dPopulation Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021, USA
Received 18 October 1999; received in revised form 24 November 1999; accepted 30 November 1999
Abstract
The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an
18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17␣-hydroxy-18-methyl-19-norpregn-4-ene-3,20-
dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel,
and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy
maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition
of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from
sustained release systems. 10 should be ideally suited for administration by implants or small skin patches. © 2000 Elsevier Science Inc.
All rights reserved.
Keywords: Progestin; 16-methylene-17␣-hydroxy-18-methyl-19-norpregn-4-ene-3; 20-dione acetate; Nestorone; Synthesis; Clauberg assay
1. Introduction
genic effects. The presence of an 18-methyl group in the
structure of the latter progestins (3, 4, 5, and 6) results in
increased progestogenic activity and reduced side effects
[2]. For instance, in laboratory bioassays, enhanced activity
was found for levonorgestrel (3) versus norethisterone (2)
with respect to inhibition of ovulation (7x) and endometrial
stimulation (21x) [3].
Several synthetic progestins in clinical use are derived
from hydroxyprogesterone, although many of them are ac-
tive only when given parenterally (hydroxy-progesterone
caproate, 7) (Fig. 2). Acylation of the 17␣-hydroxy group is
crucial in these types of progestins, for 17-hydroxy deriva-
tives otherwise lose much of their progestational activity
[4]. The 19-nor derivatives of hydroxyprogesterone are also
of great importance: Nomegestrol acetate [5] (8) is an orally
active progestin in clinical use, whereas Nestorone [6] (16-
methylene-17␣-hydroxy-19-norpregn-4-ene-3,20-dione ac-
etate, 9) must be administered parenterally due to its rapid
hepatic metabolism. The importance of the 16-methylene
substituent was demonstrated in the series of 17-hy-
Progestins act on the uterus to induce endometrial
changes characteristic of pregnancy and also serve to main-
tain pregnancy. The primary clinical use of progestins is in
contraception, specifically in the inhibition of ovulation and
transformation of the estrogen-primed endometrium [1].
The combined oral contraceptive has been one of the
cornerstones of fertility control for more than three decades.
The progestogens used in combined oral contraception are
all 19-nortestosterone derivatives (Fig. 1). According to
convention, norethynodrel (1) is considered the first gener-
ation, norethisteron (2) and levonorgestrel (3) second gen-
eration, and desogestrel (4), gestodene (5), and norgestimate
(6) third-generation progestogens. All of these compounds
lack the 19-methyl substitution and have a 17-ethynyl
group, which delays hepatic inactivation and reduces andro-
* Corresponding author. Tel.: ϩ36-1-431-5250; fax: ϩ36-1-260-4891.
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00109-9

Z. Tuba et al. / Steroids 65 (2000) 266–274
267
Fig. 1. 19-Nortestosterone derivatives.
droxyprogesterone derivatives where it enhanced progesta-
2. Experimental
tional activity far more than either 16␣-methyl or 16␤-
methyl substituents [7]. Nestorone is a potent progestin
without either androgenic or estrogenic activity [8]. Nestor-
one seems suitable for various controlled release dosage
formulations, such as subdermal implants, vaginal rings,
and transdermal products that are being investigated by The
Population Council [9,10]. Many of these dosage forms
make it possible to reduce the dose of progestin [11].
The importance of the presence of the 18-methyl group
in Nestorone inspired us to synthesize a new representative
of the 19-nor-hydroxyprogesterones, 16-methylene-17␣-
acetoxy-18-methyl-19-norpregn-4-ene-3,20-one (10). In the
present communication, we describe the synthesis of 10 and
its in vivo biologic activity relative to 3, 9, and progesterone
(11).
¨
Melting points were determined on a BUCHI-510 apparatus
and are uncorrected. The NMR spectra were recorded on
either a Varian UnityINOVA-300 or a Varian UnityINOVA-
500 spectrometer, using either CDCl3 or DMSO-d6 as a
solvent. Infrared spectra were recorded on a Perkin–Elmer
Spectrum 1000 FT-IR instrument with a resolution of 4 cm,
using a KBr matrix. Electron ionization mass spectral anal-
yses were carried out on a VG-TRIO-2 quadrupol mass
spectrometer at 70 eV, with direct introduction, and at an
ion source temperature of 250°C. The ‘fast atom bombard-
ment’ (FAB) experiments were performed with a Finnigan
MAT 95SQ hybrid tandem mass spectrometer. TLC analy-
ses were carried out on silica gel 60 F254 aluminum sheets
(20 ϫ 20 cm with 250-␮m layer).
Fig. 2. Hydroxyprogesterone derivatives.

268
Z. Tuba et al. / Steroids 65 (2000) 266–274
All reactions were carried out under a nitrogen atmo-
was cooled to Ϫ60°C, and the solution of 14 (20 g, 0.064
mol) in tetrahydrofuran (200 ml) was added. After stirring
under nitrogen at Ϫ60°C for 2.5 h, the reaction mixture was
poured into water (2 l) and extracted with ethyl acetate (3 ϫ
500 ml). The combined ethyl acetate layer was washed with
water and then brine, dried over anhydrous sodium sulfate,
concentrated to a volume of 50 ml in vacuo, and cooled to
0°C. After filtration, the product was dried at 50°C to give
17 (15 g, 72%). m.p.: 150–152°C. IR: 3575, 3297,1648,
1622, 1455, 1386, 1236, 1200, 1050, 911, 851, 667, 635,
519 cm. 1H NMR {300 MHz, CDCl3(TMS), ␦(ppm)}: 0.98
(3H,t,18-Me), 2.59 (1H,s,ϵCH), 3.57 (3H,s,OMe), 5.13 &
5.43 (2H,m & m, ϭ CH2), 5.22 (1H,d,H-4), 5.32 (1H,m,H-
6). MS: 338 (m/z) (Mϩ). Analysis calculated for C23H30O2:
C 81.61, H 8.93; Found: C 81.79, H 9.01.
sphere. Most chemicals and solvents were analytical grade
and used without further purification. Phenylsulfenyl chlo-
ride was prepared from thiophenol with chlorine gas, as
previously described [12].
2.1. 3-Methoxy-13␤-ethyl-gona-3,5-dien-17-one (13)
Trimethyl orthoformate (60 ml, 0.55 mol) and a solution
of concentrated sulfuric acid (0.12 ml) in tetrahydrofuran
(11 ml) were added to a solution of 13␤-ethyl-gon-4-ene-
3,17-dione 12 (100 g, 0.35 mol) in tetrahydrofuran (500 ml)
at room temperature. The mixture was stirred for 2 h. After
adding pyridine (37 ml), the mixture was concentrated to a
volume of 260 ml in vacuo, then diluted with a mixture of
petroleum ether and diethyl ether (2:1, 75 ml). The suspen-
sion was cooled to 0°C, and the crystals were filtered,
washed with a mixture of petroleum ether and diethyl ether,
and then dried at 50°C to obtain 13 (94.4 g, 90% yield).
m.p.: 171–172°C. IR: 1729, 1653, 1627, 1454, 1384, 1236,
1166, 856 cm. 1H NMR {300 MHz, CDCl3(TMS),
␦(ppm)}: 0.79 (3H,t,18-Me), 3.57 (3H,s,OMe), 5.23
(1H,d,H-4), 5.33 (1H,m,H-6). MS: (m/z) 300 (Mϩ). Analy-
sis calculated for C20H28O2: C 79.95, H 9.40; Found: C
80.09, H 9.31.
2.4. 16-Methylene-17␤-hydroxy-18-methyl-19-nor-17␣-
pregn-4-en-3,20-dione (17)
A solution of concentrated sulfuric acid (9.9 ml, 0.185
mol) and yellow mercury(II) oxide (2.1 g, 0.008 mol) in
water (60 ml) was added to a solution of 16 (60 g, 0.185
mol) in acetone (300 ml). The reaction mixture was heated
to 50–55°C and stirred for 1 h. After adding 30 g of celite
and stirring for an additional 30 min, the celite was filtered
off, and the filtrate was poured into water (2.4 l) and stirred
for another 15 min. The precipitate was collected, washed
with water (300 ml), and dried at 60°C. The crude product
(59 g) was dissolved in methylene chloride (590 ml) con-
taining acetic acid (3 ml), then stirred with zinc powder (0.6
g) for 30 min. Subsequently, celite (20 g) was added, and
the mixture was filtered and evaporated in vacuo. The res-
idue was crystallized from acetone yielding 17 (48.5 g,
80%). m.p.: 125–130°C. IR: 3500, 2934, 2867, 1706, 1664,
1614, 1435, 1350, 1258, 1092, 995, 894, 605 cm. 1H NMR
{300 MHz, CDCl3(TMS), ␦(ppm)}: 1.04 (3H,t,18-Me),
2.25 (3H,s,17-CO-CH3), 5.18 & 5.19 (2H,m & m, ϭ CH2),
5.84 (1H,m,H-4). MS: (m/z) 342 (Mϩ). Analysis calculated
for C22H30O3: C 77.15, H 8.83; Found: C 77.02, H 8.89.
2.2. 3-Methoxy-16-methylene-13␤-ethyl-gona-3,5-dien-17-
one (14)
Dimethyl oxalate (55.5 ml, 0.47 mol) and a 28% solution
of sodium methoxide in methanol (117 ml, 0.6 mol) were
added to a solution of 13 (75 g, 0.25 mol) in tetrahydrofuran
(950 ml). The reaction mixture was stirred for 30 min at
room temperature, then acetic acid (9 ml), triethylamine (47
ml), methanol (180 ml), and 35% aqueous formaldehyde
solution (30 ml, 0.35 mol) were added. After stirring was
continued at room temperature for an additional 5 h, the
mixture was added to a 5% solution of sodium chloride and
sodium hydrogen carbonate (19 l) and stirred for 1 h. The
precipitate was filtered off, washed several times with wa-
ter, and dried at 50°C temperature to give 14 (74.8 g,
95.9%). m.p.: 175–180°C. IR: 1717, 1640, 1625, 1451,
2.5. 16-Methylene-17␤-hydroxy-18-methyl-19-norpregn-4-
en-3,20-dione (17) in a direct way from 14
1
1384, 1234, 1165, 1039, 974, 930, 852, 629, 588 cm. H
A mixture of n-hexane (160 ml) and tetrahydrofuran
(400 ml) was cooled to Ϫ60°C, then a 1.7 mol/l solution of
tertiary buthyl lithium in n-hexane (400 ml, 0.68 mol) was
added within 30 min. Ethyl vinyl ether (104 ml, 1.08 mol)
was added to the mixture at Ϫ60°C. The reaction mixture
was allowed to warm up to 0°C, stirred for 10 min, and then
cooled again. A solution of 14 (40 g, 0.128 mol) in tetra-
hydrofuran (800 ml) was added dropwise at Ϫ60°C over 30
min. After stirring was continued for another 30 min, a 10%
aqueous solution of ammonium chloride (400 ml) was
added dropwise to the reaction mixture. The aqueous layer
was separated and washed with ethyl acetate (200 ml).
Concentrated hydrochloric acid (15 ml) in water (400 ml)
NMR {300 MHz, CDCl3(TMS), ␦(ppm)}: 0.79 (3H,t,18-
Me), 3.58 (3H,s,OMe), 5.23 (1H,d,H-4), 5.33 (1H,m,H-6),
5.34 & 6.06 (2H,m & m, ϭ CH2). MS: (m/z) 312 (Mϩ).
Analysis calculated for C21H28O2: C 80.72, H 9.03; Found:
C 80.59, H 9.10.
2.3. 3-Methoxy-16-methylene-17␤-hydroxy-18-methyl-19-
nor-17␣-pregn-3,5-diene-20-yne (17)
Lithium acetylide solution, which was prepared by con-
ducting acetylene to a 15% solution of n-buthyl lithium in
hexane (200 ml, 0.47 mol) and tetrahydrofuran (800 ml),

Z. Tuba et al. / Steroids 65 (2000) 266–274
269
was added to the combined organic phase, and the reaction
mixture was stirred at room temperature for 8 h. After
separation, the aqueous phase was extracted with ethyl ac-
etate (200 ml). The combined organic layer was washed
with water and then brine, dried over anhydrous sodium
sulfate, concentrated to a volume of 80 ml in vacuo and
diluted with n-hexane (60 ml). The suspension was stirred at
0°C for 1 h, and then the crystals were filtered off and dried
at 50°C to give 17 (31.6 g, 72%). m.p.: 127–131°C. The
structure is identical with the one described above. Analysis
calculated for C22H30O3: C 77.15, H 8.83; Found: C 77.09,
H 8.79.
calculated for C22H30O3: C 77.15, H 8.83; Found: C 77.26,
H 8.74.
2.8. 16-Methylene-17␣-hydroxy-18-methyl-19-norpregn-4-
ene-3,20-dione acetate (10)
A 70% perchloric acid solution (16 ml, 0.18 mol) was
added to a solution of 19 (41 g, 0.12 mol) in acetic acid (200
ml) at 15–20°C. Acetic anhydride (64 ml, 0.66 mol) was
added dropwise, and the mixture was stirred vigorously for
10 min at 15–20°C. Subsequently, water (400 ml) was
added, and the precipitate was filtered, washed several times
with water, and dried at 60°C. The crude product was
recrystallized from a mixture of acetone and n-hexane, and
10 (36.8 g, 80%) was obtained as white crystals. m.p.:
2.6. 16-Phenylsulfinylmethyl-18-methyl-19-norpregna-
4,16-diene-3,20-dione (18)
1
154–155°C. IR: 1740, 1716, 1663, 1616, 1239, 979. H
NMR {300 MHz, CDCl3(TMS), ␦(ppm)}: 0.70 (3H,t,18-
Me), 2.05 (3H,s,17-O-CO-CH3), 2.21 (3H,s,17-CO-CH3),
5.45 & 5.56 (2H,m & m, ϭ CH2), 5.85 (1H,m,H-4). MS:
(m/z) 384 (Mϩ). Analysis calculated for C24H32O4: C 74.96,
H 8.39; Found: 75.01, H 8.31.
A solution of 17 (32.8 g, 0.1 mol) and triethylamine
(55.7 ml) in methylene chloride (328 ml) was cooled to
Ϫ50°C, and a solution of phenylsulfenyl chloride (28.8 g,
0.2 mol) in carbon tetrachloride (82 ml) at the same tem-
perature was added and stirred for 10 min. Methanol (65 ml)
and a 2 N hydrochloric acid solution (130 ml) were added to
the reaction mixture, which was allowed to warm up to
room temperature. The methylene chloride phase was sep-
arated, washed with water (300 ml), dried over anhydrous
sodium sulfate, and concentrated in vacuo. The residue was
crystallized from a mixture of acetone and diisopropyl ether,
the suspension was cooled to Ϫ5°C and the crystals were
filtered. After drying at 60°C, 18 (30 g, 68.8%) was ob-
tained (as a mixture of isomers). m.p.: 146–150°C. IR:
1668, 1642, 1612, 1581, 1048, 757, 690 cm. 1H NMR {300
MHz, CDCl3(TMS), ␦(ppm), 2:1 ratio mixture of two iso-
mers (major/minor)}: 0.71/0.80 (3H,t,18-Me), 2.20/2.19
(3H,s,17-CO-CH3), 3.86 & 4.18/3.94 (2H,m,S-CH2), 5.85
(1H,m,H-4), 7.40–7.72 (5H,m,Ar). MS: (m/z) 451 (Mϩ).
Analysis calculated for C28H35O4S: C 74.46, H 7.81, S 7.10;
Found: C 74.59, H 7.92, S 7.22.
2.9. Biologic evaluation
2.9.1. Clauberg test
2.9.1.1. Animals and reagents. Immature female New Zea-
land rabbits (700–800 g) were obtained from Mareland
Farms (Hewitt, NJ, USA). All steroids were dissolved in
ethanol and diluted to the appropriate concentrations with
corn oil. 17␤-Estradiol (Sigma no. E4260) was prepared at
a concentration of 5 ␮g/0.2 ml in corn oil. The suspension
was stirred for 2 h at room temperature with a magnetic
stirrer.
2.9.1.2. Samples. Solutions of 3 and 9 were prepared at the
following concentrations: 100, 30, 10, 3, 1, and 0.3 ␮g in
0.2 ml of corn oil. The concentration of 10 was expanded to
include 10, 3, 1, 0.5, and 0.3 ␮g in 0.2 ml of corn oil.
Solutions of 11 were prepared in the following concentra-
tions: 1 mg, 500 ␮g, 300 ␮g, 150 ␮g, 100 ␮g, and 50 ␮g in
0.2 ml of corn oil. Bouin’s solution (Sigma Chemical Co.,
St. Louis, MO, USA) was used as tissue fixative.
2.7. 16-Methylene-17␣-hydroxy-18-methyl-19-norpregn-4-
ene-3,20-dione (19)
In a temperature of 20–25°C, 18 (30 g, 0.069 mol) was
suspended in methanol (150 ml). Triethylamine (5.8 ml,
0.041 mol) and trimethyl phosphite (32.6 ml, 0.276 mol)
were added, and the reaction mixture was stirred at 66°C for
8 h. The progress of the reaction was monitored by TLC to
check for the disappearance of starting material. After com-
pleting the reaction, the mixture was cooled to room tem-
perature and poured into a 10% sodium chloride solution (3
l). The resulting crystals were filtered off, washed with
water, and dried at 60°C to give 19 (20.5 g, 90.5%). m.p.:
222–226°C. IR: 3457, 1705, 1657, 1608, 1210, 900 cm. 1H
NMR {300 MHz, CDCl3(TMS), ␦(ppm)}: 0.68 (3H,t,18-
Me), 2.38 (3H,s,17-CO-CH3), 5.09 & 5.28 (2H,m & m, ϭ
CH2), 5.84 (1H,m,H-4). MS: (m/z) 342 (Mϩ). Analysis
2.9.1.3. Methods. On the first day of the experiment, all
rabbits were weighed, and their body weights were re-
corded. For six consecutive days, each rabbit was injected
subcutaneously (s.c.) with 5 ␮g/day of estradiol to prime the
uterus. The injection site was the hindquarters or the back of
the neck. Subsequently, the estradiol-primed rabbits were
divided into groups of five or six rabbits each. Animals in
each group were injected s.c. with progestin for 5 consec-
utive days. The negative control group animals were in-
jected with 200 ␮l of corn oil. On the 12th day, the animals
were sacrificed by asphyxiation with CO2. The body
weights were recorded, and the uteri were excised. Excess

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Z. Tuba et al. / Steroids 65 (2000) 266–274
fat was removed, and the uterine weights were recorded.
Each uterus was fixed in Bouin’s solution. The tissues were
embedded in paraffin, sectioned, and stained with hematox-
ylin and eosin. Three sections of each uterine horn were
prepared. The slides were used to determine the McPhail
Index of endometrial growth.
from dry ice. The number of embryos in the oviduct was
counted and recorded.
3. Results
3.1. Chemistry
2.10. Determination of binding affinities to progesterone
receptors (PR)
Unlike Merck’s synthetic approach [13] for the prepara-
tion of Nestorone starting from pregnadienolone acetate and
completing the synthesis with removal of the 19-methyl
group, our strategy was based on the introduction of the
16-methylene group and pregnane side chain into the D-ring
of 13-ethyl-gon-4-ene-3,17-dione (12, Scheme 1).
Selective introduction of the methylene group into posi-
tion 16, utilizing the presence of the 17-oxo group, required
protection of the 3-oxo substitution. By using sulfuric acid
as a catalyst, 12 was reacted with trimethyl-orthoformate to
yield 13 (THF, RT, 90% yield). Following Beal’s method
[14] for introducing a methylene substituent into position 2
of the steroid skeleton by retro-Claisen condensation, the
16-methylene group was built-in via a methoxy-oxalyl in-
termediate by reacting 13 first with dimethyl oxalate in the
presence of sodium methoxide and subsequently with aque-
ous formaldehyde solution and triethylamine (THF, RT,
95% yield) to form 14.
Two different pathways were applied successfully to
synthesize the 17-isopregnan side chain. In the first
method, 15 was prepared with lithium acetylide formed
in situ from n-butyl lithium and acetylene (THF, 0°C,
72% yield). Addition of water to the ethynyl group was
carried out in the presence of mercury (II) oxide and
sulfuric acid (acetone, 50°C) [15]. The strong acidic
medium catalyzed hydrolysis of the 3-methyl enolether,
affording 17 (80% yield). In the second method, 14 was
reacted with ethoxyvinyl lithium prepared in situ from
ethyl vinyl ether and t-butyl lithium (THF, Ϫ60°C, 72%
yield). The yield of 17 was better than that in the first
method as both enolether groups in the non-isolated in-
termediate (16) were hydrolyzed in the presence of hy-
drochloric acid. Although the direct route via 16 provided
a better yield, the hazard involved in the use of t-butyl
lithium is a disadvantage; consequently, the first method
via 15 is preferred when scaling up the process.
Ovariectomized rats were treated s.c. with estradiol, 1.0
␮g/day for 3 days. They were killed on Day 4. A cytosolic
fraction of the uterine homogenate was prepared and used
for binding studies. By using [3H]promegestone as a tracer,
displacement curves were generated with the progestins,
and ED50 values were calculated. Levonorgestrel was as-
signed a value of 100 for expressing the relative binding
affinities.
2.11. Maintenance of pregnancy in rats
2.11.1. Animals and reagents
Young adult female Sprague–Dawley rats (approx. 200–
250 g) were purchased from Charles River (Wilmington,
MA, USA). An estrone suspension (Sigma no. E-9750) was
prepared at a concentration of 1 ␮g/0.1 ml in corn oil. Either
Metophane (methoxyflurane), obtained from Miles Inc.
(Shawnee Mission, KS, USA) or a 1:10 mixture of Ketaset
(100 mg/ml), obtained from Aveco Co., Inc. (Fort Dodge,
IA, USA), and Rompun (20 mg/ml xylazine), obtained from
Miles Inc. (Shawnee Mission, KS, USA) was used as the
anesthetic agent. Surgical suture (3.0 silk) was obtained
from Ethicon Inc. (Sommerville, NJ, USA). Prepodyne
Scrub was obtained from West Chemical Products, Inc.
(New York, NY, USA). Wound clips (9 mm) were pur-
chased from Clay Adams (Parsippany, NJ, USA).
2.11.2. Samples
All progestin solutions were prepared in a volume of 0.2 ml
of corn oil. The amounts of progestins were 0.01, 0.03, 0.1, and
0.3 mg for 10; 0.01, 0.03, 0.1, 0.3, 1, and 3 mg for 9; 0.3, and
0.5 mg for 3; and 0.3, 1, 3, 5, 8, and 10 mg for 11.
2.11.3. Methods
Each female rat was placed with a male breeder rat in a
suspended cage in the morning. The next morning, the
lining paper underneath each cage was examined for vaginal
plugs. The presence of a vaginal plug was recorded as Day
1 of pregnancy. The pregnant rats were separated from the
males, and their ovaries were removed on the 8th day of
pregnancy. From the day of ovariectomy up to the 21st day
of pregnancy, each animal received daily 1 ␮g injection of
estrone and various doses of the gestagens administered s.c.
The rats in the negative control group received 1 ␮g of
estrone in corn oil without gestagen. On the 22nd day, the
animals were sacrificed by asphyxiation with CO2 sublimed
The key step of our synthetic approach was the isomer-
ization of the isopregnane side chain by the interconversion
of allylic sulfenates and sulfoxides in a [2,3] sigmatropic
rearrangement [16–20]. Thus, 17 was reacted with a solu-
tion of benzenesulfenyl chloride in carbon tetrachloride in
the presence of triethylamin, and the immediate [2,3] sig-
matropic rearrangement afforded a 16-phenylsulfinylmethyl
derivative (18) as a mixture of diastereomers (methylene chlo-
ride, Ϫ50°C, 69% yield). Diastereoisomerism is attributed to
the asymmetry of the sulfur atom in the sulfoxide group that
provided the diastereomers in a ratio of 7:3. Rearrangement of
the 16-phenylsulfinylmethyl group was carried out in the pres-

Z. Tuba et al. / Steroids 65 (2000) 266–274
271
Scheme 1. Synthesis of 16-methylene-17␣-hydroxy-18-methyl-19-norpregn-4-ene-3,20-dione acetate.
ence of trimethyl phosphite to form 19 with stereospecificity
(methanol, 60°C, 91% yield). The steric bulk of the angular
ethyl group at position 13 forced this step to occur predomi-
nantly on the ␣ face of the steroid skeleton.
was used as the catalyst and acetic acid was used as solvent
(RT, 85%) [21].
3.2. Progestational assays
The final step of the synthesis was the acetylation of the
tertiary 17␣-hydroxy group with acetic anhydride. Due to
steric hindrance of the 17␣-hydroxy group, perchloric acid
Progestogenic activities of 10 were determined using the
most reliable bioassays, Clauberg test, and maintenance of

272
Z. Tuba et al. / Steroids 65 (2000) 266–274
Table 1
Progestagenic activity of the compounds tested in the rabbit (Clauberg assay)
Compunt
Dose
(␮g/rabbit/day)
Number of
animals tested
Mean body
wt. gain (g)
Uterine wt. (g)
Uterine cross-sect.
diameter (mm)
McPhail Indexa
10
0.3
0.5
1
3
10
0.3
1
3
10
30
100
3
10
30
100
50
100
150
300
500
1000
0
6
5
5
6
6
6
6
12
40
6
6
6
6
6
33
5
5
477 Ϯ 99
366 Ϯ 144
481 Ϯ 86
447 Ϯ 107
521 Ϯ 55
490 Ϯ 43
465 Ϯ 28
417 Ϯ 74
402 Ϯ 139
417 Ϯ 81
389 Ϯ 37
360 Ϯ 80
314 Ϯ 143
336 Ϯ 116
420 Ϯ 74
436 Ϯ 30
378 Ϯ 187
411 Ϯ 135
332 Ϯ 52
382 Ϯ 34
369 Ϯ 81
408 Ϯ 145
1.95 Ϯ 0.38*
1.96 Ϯ 0.49*
3.2 Ϯ 0.26
3.3 Ϯ 0.45*
4.6
4.0
5.6
7.5
8.6
2.0
6.2
8.8
8.8
10.0
10.5
3.2
5.2
8.0
7.6
3.3
4.5
5.6
5.4
6.3
7.4
2.2
3.84 Ϯ 1.44***
5.22 Ϯ 1.07***
6.42 Ϯ 1.31***
1.52 Ϯ 0.37
2.95 Ϯ 0.46***
4.34 Ϯ 0.81***
5.00 Ϯ 0.97***
4.03 Ϯ 0.48***
4.60 Ϯ 1.21***
1.58 Ϯ 0.32
2.16 Ϯ 0.90**
3.24 Ϯ 0.56***
3.97 Ϯ 0.56***
1.96 Ϯ 1.16
2.12 Ϯ 0.66**
2.36 Ϯ 0.77***
3.05 Ϯ 1.10***
3.46 Ϯ 0.62***
4.27 Ϯ 0.85***
1.38 Ϯ 0.50
4.1 Ϯ 0.65***
4.8 Ϯ 0.42***
5.4 Ϯ 0.38***
2.66 Ϯ 0.26
3.50 Ϯ 0.45***
4.58 Ϯ 0.36***
4.98 Ϯ 0.64***
4.50 Ϯ 0.32***
4.92 Ϯ 0.80***
2.83 Ϯ 0.26
3.33 Ϯ 0.41**
4.00 Ϯ 0***
4.39 Ϯ 0.43***
3.3 Ϯ 0.76
3.6 Ϯ 0.42*
3.9 Ϯ 0.55*
9
3
11
5
12
12
12
34
3.8 Ϯ 0.5*
4.0 Ϯ 0.33**
4.6 Ϯ 0.29**
2.81 Ϯ 0.49
Control
aMcPhail Index, uterine estrogenic/progestogenic activity.
*P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001.
pregnancy test. The potency of 10 was compared to those of
3, 9, and 11.
3.2.2. Maintenance of pregnancy test [24,25]
Progestogen is required for maintaining pregnancy in rats.
After removal of ovaries from pregnant rats, pregnancies can
be maintained by administration of progestogen. In this bio-
logic test, the ovaries of pregnant rats were removed on the 8th
day following implantation of the fertilized zygotes. Progesto-
gen was administered daily, together with estrone, from the 8th
to the 21st day to maintain pregnancy. The presence and
number of embryos in the uterus were assessed on the 22nd
day. The aim of this experiment was to determine the mini-
mum effective doses of the gestagens tested required to main-
tain developing embryos in the pregnant rat (Table 3). The
minimum effective doses required to maintain pregnancy in
100% of treated animals were as follows (mg/rat/day): 0.03 for
10, 0.3 for 9, 0.3 for 3, and 5.0 for 11.
3.2.1. Clauberg test
The Clauberg test is a bioassay used to measure the
progestogenic activity of compounds by administration to
immature female rabbits primed with 17␤-estradiol [22].
The test progestin induces a change in the endometrium
from the proliferative to the secretory stage. The extent of
change in the endometrium is measured by the McPhail
Index [23]. In this assay estradiol was administered initially
to prime the uterus. Varying doses of progestins were then
administered. Compounds 3, 9, and 11, known progestins,
were used as positive controls for comparison of potency.
Estradiol-primed rabbits treated with corn oil were used as
negative controls. The aim of the study was to determine the
dose of 10 having half maximum progestogenic activity
(ED50 value) and to compare its potency to those of 9, 3, and
11. The body and uterine weights and McPhail indices for
the treated and control animals are shown in Table 1. The
minimum effective doses (EDmin) required to induce an
increase in uterine weight and the McPhail index are shown
in Table 2. Based on the plot of uterine weights and McPhail
indices, the approximate doses that elicited a half–maxi-
mum response (ED50) were determined and are shown in
Table 2. These values are approximations since the dose-
response curves do not have the same slope. Table 2 also
shows that the RBA of 10 for the progesterone receptor was
3ϫ greater than those for Nestorone and levonorgestrel.
Table 2
The minimum effective doses and ED50s of the tested compounds
required to increase the uterine weight and the McPhail indices
Compounds
Increase of uterine
weight
Increase of the
McPhail index
Edmin
*
ED50
*
EDmin
*
ED50*
10
9
0.3
1.0
1.0
1.0
0.3
1.0
1.0
1.0
10.0
100.0
3
11
10.0
100.0
20.0
200.0
30.0
50.0
*(␮g/rabbit/day).

Z. Tuba et al. / Steroids 65 (2000) 266–274
273
Table 3
Maintenance of pregnancy in ovariectomized rats by various gestagens and estrone
Compound
tested
Dose (mg/rat/day)
Number of
animals tested
Number of pregnant
rats
Number of embryos
(mean)
Pregnancy maintained
(%)
10
0.01
0.03
0.1
0.3
0.01
0.03
0.1
0.3
1
5
6
5
5
5
5
6
19
5
4
5
6
5
5
5
6
6
5
4
6
4
5
0
2
3
19
5
4
5
6
0
2
3
6
6
5
10.5 Ϯ 3.9
13.2 Ϯ 2.5
9.0 Ϯ 3.4
15.4 Ϯ 3.5
0
11.5 Ϯ 3.5
12.3 Ϯ 1.5
11.4 Ϯ 3.5
12.0 Ϯ 2.0
12.7 Ϯ 2.1
13.8 Ϯ 3.1
14.0 Ϯ 2.5
0
10 Ϯ 7.1
8.0 Ϯ 1.0
12.2 Ϯ 4.3
1.6 Ϯ 1.9
14.8 Ϯ 2.3
0
80
100
80
100
0
9
40
50
100
100
100
100
100
0
40
60
100
100
100
0
3
3
0.3
0.5
0.3
1
3
5
8
10
0
11
Control
19
0
n ϭ number of rats in separate group.
4. Discussion
The pharmacological profile of Nestorone, a 19-norproges-
terone derivative, has identified this progestin to be highly
selective based on its binding to PR, minimal binding to an-
drogen receptor (AR), and its potent progestational activity.
The possible adverse effects of some progestins are attributed
to their androgenic effect on lipid metabolism [28]. The selec-
tivity index, based on binding affinity to PR and AR, was
found to be highest for Nestorone, followed by keto-deso-
gestrel and levonorgestrel [29]. The progestational activities of
second- and third-generation progestins were shown to be
markedly increased by addition of an 18-methyl group. Thus,
we were interested to examine the influence of 18-methyl
substitution of Nestorone on binding to PR and progestational
activity. As with other progestins, the 18-methyl-Nestorone
derivative was found to be 3 to 10 times more potent than
Nestorone. Part of the reason for the increased progestational
activity of 10 was because of its higher RBA to PR than that
of Nestorone. Binding affinities of Nestorone and 10 to AR
were compared to that of testosterone. Both Nestorone and 10
showed negligible binding to AR (data not shown). Thus, the
selectivity index, which is the ratio of progesterone to andro-
gen receptor binding affinities [30], will be greater for 10 than
that for Nestorone.
Synthetic progestins, commonly employed in oral contra-
ceptives, have the disadvantage of exerting undesirable effects
on the liver. Non-oral routes, such as subdermal implants,
vaginal rings, and skin patches, using new progestins, are being
developed for long-term treatment and to minimize adverse
effects [10,11,26]. A new progestin, 10, was synthesized, and
its biologic activity was compared with Nestorone, levonorg-
estrel, and progesterone using PR binding assays, the Clauberg
test in rabbits, and the pregnancy maintenance test in the rat.
These studies, as summarized in Table 4, show that 10 is three
to ten times more potent than Nestorone, which is in-turn more
potent than levonorgestrel. These progestins were compared by
parenteral route of administration as the intended route of
administration and also because both Nestorone and proges-
terone are not orally active [6,27]. The oral activity of 10 is not
known.
Table 4
Comparison of activities of various gestagens in separate tests
Test
Order of activities and effective doses of gestagens
RBA to PR*
(rat uterus):
10
Ͼ Nestorone Ͼ Levonorgestrel Ͼ Progesterone
107 100 22
Ͼ Nesterone Ͼ Levonorgestrel Ͼ Progesterone
1 ␮g 3 ␮g 100 ␮g
Ͼ Nesterone Ͼ Levonorgestrel Ͼ Progesterone
0.3 mg 1.0 mg
Several structural modifications/substitutions of progester-
one and/or 19-nortestosterone have been shown to increase
binding affinity to PR. Thus, the introduction of a 17␣-ethynyl
or methyl substituent to the 19-nortestosterone led to a signif-
icant increase in binding to PR, whereas decreasing binding to
AR [31]. In addition, introduction of an 18-methyl group to
norethisterone led to a significant increase in PR binding [32].
Similarly, structural modification of 19-norprogesterone, such
as substitution at the 6 or 16 positions, showed a significant
355
/
/
/
Clauberg**:
10
0.3 ␮g /
/
/
Maintenance of 10
pregnancy:
0.03 mg / 0.3 mg
/
/
*Relative binding affinity (RBA) to rat PR was based on IC50 calculated
from the displacement curves.
**The minimum effective doses for the Clauberg test, which are not
identical to the doses required to get full effect.

274
Z. Tuba et al. / Steroids 65 (2000) 266–274
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