Synthesis, structutre and biological activity of substituted [16α,17α]cyclopropapregn-4-ene-3,20-diones

Article abstract of DOI:10.1007/s11172-013-0208-9

Aryldiazomethanes generated by vacuum thermolysis of arenecarbaldehyde tosylhydrazone sodium salts react stereospecifically with 16-dehydropregnenolone acetate. Subsequent de- composition of the pyrazoline adducts yielded hitherto unknown substituted [16α

Full text of DOI:10.1007/s11172-013-0208-9

Russian Chemical Bulletin, International Edition, Vol. 62, No. 6, pp. 1450—1453, June, 2013
1450
Synthesis, structutre and biological activity of substituted
[16,17]cyclopropapregnꢀ4ꢀeneꢀ3,20ꢀdiones
I. S. Levina,a L. E. Kulikova,a E. V. Shulishov,a Yu. V. Tomilov,a and A. N. Smirnovb†
aN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 6390. Eꢀmail: li@ioc.ac.ru
bM. V. Lomonosov Moscow State University, Faculty of Biology,
1/12 Leninskie Gory, 119992 Moscow, Russian Federation
Aryldiazomethanes generated by vacuum thermolysis of arenecarbaldehyde tosylhydrazone
sodium salts react stereospecifically with 16ꢀdehydropregnenolone acetate. Subsequent deꢀ
composition of the pyrazoline adducts yielded hitherto unknown substituted [16,17]ꢀ
cyclopropaprogesterones bearing the aryl substituents at the position 3´. Interaction of such
cyclopropaprogesterones with progesterone receptor from rat uterine cytosol was studied.
Key words: synthesis, steroids, progesterone, pentaranes, receptor, cyclopropanes, 1,3ꢀdiꢀ
polar cycloaddition, Xꢀray diffraction.
Progestins (progesterone analogs) are the synthetic horꢀ
mones obtained by chemical transformations of the natuꢀ
ral steroid skeleton.1 This approach served for the syntheꢀ
sis of a large variety of highly active progestins widely used
in medicine to treat different gynecological diseases, for
hormonal contraception, and in hormone replacement
therapy. The effective use of progestins is mainly associatꢀ
ed with their ability to bind with the progesterone recepꢀ
tors (PR). Selective progesterone receptor ligands are also
used in the treatment of hormoneꢀdependent cancer.1
Among synthetic progestins we synthesized, a series of
biologically active progesterone analogs bearing the D´
carbocycle fused with the D ring, namely, [16,17]ꢀ
cycloalkaprogesterons (pregnaꢀD´3—6ꢀpentaranes) are of
special interest.2,3 Compared to natural hormone, these
compounds show the minimal geometric distortion of the
steroid skeleton and have more hydrophobic molecular
surface allowing interaction with PR of experimental aniꢀ
mals and humans with an affinity comparable with progesꢀ
terone affinity as well as a symbasis between binding of
some of these steroidꢀPR complexes (formed by hydrogen
bonding and hydrophobic interactions) with hormone
response elements in DNA and progestational effect
in vivo.2,3. The study of the relative binding affinity of the
steroidꢀreceptor complexes of D´6ꢀ and D´3ꢀpentaranes
for hormone response elements in DNA revealed (1) comꢀ
plete symbasis of these values for progesterone, D´6ꢀpentꢀ
arane and its close homologs and (2) the absence of this
symbasis for D´3ꢀpentarane.4 This fact can be explained
suggesting the different tertiary conformations of the steꢀ
roidꢀPR complexes of D´6ꢀ and D´3ꢀpentaranes with the
same receptor. To confirm this assumption, we syntheꢀ
sized a series of substituted [16,17]cyclopropaprogestꢀ
erones5 and studied they binding with PR. The only known
pentaranes bearing an additional threeꢀmembered cycle
D´ are [16,17]cyclopropaprogesterone exhibiting high
progestational activity2—4 and two its spiroꢀanalogs,5,6
which biological properties were not studied.
In the framework of our research on the pentarane
structure—biological activity relationship, we report hereꢀ
in the synthesis and structural study of novel substituted
[16,17]cyclopropaproꢀ
gesterones, 3´ꢀphenylꢀ
[16,17]cyclopropaꢀ
pregnꢀ4ꢀeneꢀ3,20ꢀdione
(1a) and 3´ꢀ(4ꢀfluoroꢀ
phenyl)[16,17]cycloꢀ
propapregnꢀ4ꢀeneꢀ3,20ꢀ
dione (1b). The binding of these steroids and specially
prepared compounds 4—6 (Table 1) with the uterine cytoꢀ
sol progesterone receptors were also studied.
Compounds 1a and 1b were synthesized in four steps
from 16ꢀdehydropregnenolone acetate (DPA) (Scheme 1).
1,3ꢀDipolar cycloaddition of DPA to phenylꢀ and 4ꢀfluoꢀ
rophenyldiazomethanes generated by vacuum thermolysis
of sodium salts of benzaldehyde tosylhydrazone and
4ꢀfluorobenzaldehyde tosylhydrazone7 proceeds regioꢀ and
stereospecifically to give the corresponding 3´ꢀarylꢀsubꢀ
stituted [17,16ꢀc]pyrazolines 2a and 2b. Pyrolysis of
compounds 2a,b was carried out in a melt at 180 C and
a vacuum of 20 Torr. Under these conditions, decomposiꢀ
tion of pyrazolines occurs with high conversion (>95%)
and is accompanied by partial resinification of the reacꢀ
tion mixture. Phenylꢀsubstituted cyclopropasteroids 3a and
† Deceased.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1449—1453, June, 2013.
1066ꢀ5285/13/6206ꢀ1450 © 2013 Springer Science+Business Media, Inc.

Cyclopropapregnenediones
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 6, June, 2013
1451
Table 1. Relative binding affinity (RBA) values of studied compounds for progesterone receptor (PR) from rat uterus
Entry
Compound
D´ (16,17)
RBA (%)
1
2
3
4
Progesterone
—
CH—Ph
CH—C6H4ꢀFꢀ4
CH2
100
1.8
2.2
936
3´ꢀPhenyl[16,17]cyclopropapregnꢀ4ꢀeneꢀ3,20ꢀdione (1a)
3´ꢀ(4ꢀFluorophenyl)[16,17]cyclopropapregnꢀ4ꢀeneꢀ3,20ꢀdione (1b)
16,17ꢀCyclopropaprogesterone (4)
5
6
[16,17](Spiro[2.2]penta)pregnꢀ4ꢀeneꢀ3,20ꢀdione (5)
122
4.3
4´,4´ꢀDimethyl[16,17](spiro[2.2]penta)pregnꢀ4ꢀeneꢀ3,20ꢀdione4 (6)
3b (see Scheme 1) were isolated by silica gel column
chromatography following crystallization from hexane—diꢀ
ethyl ether (isolated yields of 90—95%). Removal of the
3ꢀacetate group of compounds 3a and 3b and subsequent
Oppenauer oxidation of the resulted 3ꢀhydroxy derivaꢀ
tives leads to target compounds 1a and 1b in ~80% yields.
The structures of compounds synthesized are confirmed
data indicate that the introduction of the cyclopropane
fragment into the progesterone 16,17 position (comꢀ
pound 4) does not noticeably affect the affinity for PR.
Besides, the affinity of spiropentaprogesterone 5 for PR
slightly exceeds the affinity of [16,17]cyclopropaproꢀ
gesterone 4 due apparently to additional hydrophobic bindꢀ
ing with the corresponding hormoneꢀbinding pocket of
PR, which implies an extra space for this substituent. Howꢀ
ever, the further increase in the volume of the molecule by
introduction of two methyl groups (compound 6) dramatꢀ
ically decreases the affinity. In the case of compounds 1a
and 1b, the equatorial 3´ꢀaryl substituent of the cycloproꢀ
pane ring (roughly in the plane of the steroid skeleton)
almost prevents binding of the steroid with PR. It can be
assumed that in the case of compounds 1a and 1b, the size
of the aryl substituent at the D´ ring exceeds the size of the
cavity of the ligandꢀbinding site of PR thereby sterically
hindering ligandꢀreceptor binding.
1
by microanalysis data, mass spectroscopy, and H NMR
spectroscopy. The Xꢀray analysis reveals the cis orientaꢀ
tion of the phenyl group of the fused cyclopropane moiety
and 17ꢀacetyl group of the steroid skeleton (Fig. 1).
[16,17](Spiro[2.2])penta)pregnꢀ4ꢀeneꢀ3,20ꢀdione 5
was synthesized in four steps analogously to 4´,4´ꢀdimethꢀ
yl[16,17](spiro[2.2]penta)pregnꢀ4eneꢀ3,20ꢀdione (6)5
by 1,3ꢀcycloaddition of DPA to diazacyclopropane genꢀ
erated by thermolysis of NꢀcyclopropylꢀNꢀnitrosourea.
To estimate the effect of the substituents at the posiꢀ
tion 16,17 of the progesterone skeleton on binding with
PR, the competition experiments were performed (disꢀ
placement of [3H]progesterone from the protein complexꢀ
es).8 The relative binding affinity (RBA) values of the synꢀ
thesized compounds relative to binding affinity of natural
progesterone (RBA = 100%) are given in Table 1. These
In summary, we like to note that, 16,17ꢀsubstituted
cyclopropaprogesterones 1a,b—6 exhibit significant cytotoxic
effect against HeLa cell line (human cervical cancer). The
results of this study and a suggested mechanism of growth inꢀ
hibition of cancer cell are going to be published elsewhere.
Scheme 1
R = H (a), F (b)
Reagents and conditions: i. ArCH=NN(Na)Ts, 175 C, 0.05 Torr, MeOH—CH2Cl2 ; ii. 180—200 C, 20 Torr; iii. KOH—MeOH;
iv. Al(OPri)3, cyclohexanone, PhMe, reflux, 2.5 h.

1452
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 6, June, 2013
Levina et al.
3ꢀAcetoxyꢀ4´,5´ꢀdihydroꢀ5´ꢀphenylpregnꢀ5ꢀeno[17,16ꢀc]ꢀ
pyrazolꢀ20ꢀone (2a) was synthesized from benzaldehyde tosylꢀ
hydrazone sodium salt7 (1.1 g, 3.6 mmol) and DPA (1.0 g, 2.8
mmol). The yields of pyrazoline 2a was 1.1 g (95%), crystalline
compound, m.p. 174.5 C (methanol—dichloromethane). 1H
NMR, : 0.85 (s, 3 H, 18ꢀMe); 1.05 (s, 3 H, 19ꢀMe); 2.05
(s, 3 H, acetate); 2.60 (s, 3 H, 21ꢀMe); 2.95 (dt, 1 H, H(5´)); 4.60
(m, 1 H, H(3)); 5.30 (m, 1 H, H(6)); 6.99 (dd, 2 H, Ph); 7.30
(m, 3 H, Ph). MS, m/z (Irel (%)): 446 [M – N2]+ (26); 403 [M – N2,
Ac]+ (13); 386 [M – CHPh]+ (14); 371 [M – CHPh, CH3]+
(35); 343 [M – CHPh, CH3, N2]+ (78).
C(2)
C(3)
O(1)
C(21)
C(11)
C(1)
C(10)
C(9)
C(12)
C(13)
C(20)
O(2)
C(19)
C(17)
C(18)
C(4)
C(24)
C(5)
C(25)
C(26)
C(22)
C(14)
C(15)
C(8)
C(16)
C(23)
C(6)
C(7)
C(27)
C(28)
3ꢀAcetoxyꢀ4´,5´ꢀdihydroꢀ5´ꢀ(4ꢀfluorophenyl)pregnꢀ5ꢀenoꢀ
[17,16ꢀc]pyrazolꢀ20ꢀone (2b) was synthesized from 4ꢀfluoroꢀ
benzaldehyde tosylhydrazone sodium salt7 (0.52 g, 1.7 mmol)
and DPA (0.37 g, 1.0 mmol). The yield of pyrazoline 2b was 0.46
g (95%), colorless crystals, m.p. 166 C (decomp.). 1H NMR, :
0.88 (s, 3 H, 18ꢀMe); 1.05 (s, 3 H, 19ꢀMe); 2.05 (s, 3 H, acetꢀ
ate); 2.58 (s, 3 H, 21ꢀMe); 2.95 (dt, 1 H, H(5´)); 4.60 (m, 1 H,
H(3)); 5.35 (m, 1 H, H(6)); 6.99 (m, 2 H, Ar); 7.15 (m, 2 H, Ar).
MS, m/z (Irel (%)): 464 [M – N2]+ (36); 449 [M – N2, Me]+ (7);
421 [M – N2, Me, Ac]+ (5).
3ꢀAcetoxyꢀ3´ꢀphenyl[16,17]cyclopropapregnꢀ5ꢀenꢀ20ꢀ
one (3a). Pyrazoline 2a (0.52 g, 1.1 mmol) was slowly heated at
180 C and a pressure of 15 Torr. At melting temperature, foamꢀ
ing was observed. After gas evolution ceased and cooling of the
reaction mixture, cyclopropane 3a was obtained in the yield
of 0.44 g (90%), m.p. 209—210 C (from acetone—hexane).
Found (%): C, 80.23; H, 8.56. C30H38O3. Calculated (%):
C, 80.68; H, 8.58. 1H NMR, : 0.87 (s, 3 H, 18ꢀMe); 1.08 (s, 3 H,
19ꢀMe); 1.70 (s, 3 H, 21ꢀMe); 2.05 (s, 3 H, acetate); 2.71 (dt, 1 H,
H(5´)); 4,62 (m, 1 H, H(3)); 5.40 (m, 1 H, H(6)); 7.05—7.30
(m, 5 H, Ph). MS, m/z (Irel (%)): 446 [M]+ (20); 431 [M – CH3]+
(0.1); 403 [M – Ac]+ (2.5); 386 [M – CHPh]+ (8).
3ꢀAcetoxyꢀ3´ꢀ(4ꢀfluorophenyl)[16,17]cyclopropaꢀ
pregnꢀ5ꢀenꢀ20ꢀone (3b) was obtained by pyrolysis of pyrazoline 2b
(0.27 g, 0.57 mmol) in the yield of 0.253 g (96%), white powder.
1H NMR, : 0.88 (s, 3 H, 18ꢀMe); 1.02 (s, 3 H, 19ꢀMe); 1.68
(s, 3 H, 21ꢀMe); 2.03 (s, 3 H, acetate); 2.69 (dt, 1 H, H(5´)); 4.62
(m, 1 H, H(3)); 5.40 (m, 1 H, H(6)); 6.85—7.10 (m, 4 H, Ar).
MS, m/z (Irel (%)): 464 [M]+ (55); 390 [M – Ac, Me]+ (25).
3´ꢀPhenyl[16,17]cyclopropapregnꢀ4ꢀeneꢀ3,20ꢀdione (1a).
A suspension of acetate 3a (0.30 g, 0.67 mmol) in MeOH (55 mL)
and KOH (86.5 mg, 1.54 mmol) in H2O (0.5 mL) was stirred for
3 h at 40 C. The reaction mixture was neutralized with 10%
HCl, poured into iceꢀwater (100 mL), and the colorless precipiꢀ
tate formed was filtered off. Chromatographically homogeneous
3ꢀhydroxy derivative (0.25 g, 92%) obtained after drying in air
was subjected to Oppenauer oxidation by refluxing with Al(PriO)3
(325 mg) and cyclohexanone (4 mL) in toluene (30 mL) for 3 h.
The reaction mixture was acidified with diluted AcOH, the orꢀ
ganic layer was separated, washed to neutral, dried with anꢀ
hydrous MgSO4, and concentrated in vacuo. Purification of the
oily residue by column chromatography (elution with petroleum
ether—acetone, 96 : 4  90 : 10) afforded conjugated ketone 1a
in the yield of 0.20 g (80%), m.p. 249—251 C (from diethyl
ether—hexane). Found (%): C, 83.59; H, 8.59. C28H34O2. Calꢀ
culated (%): C, 83.54; H, 8.51. MS, m/z (Irel (%): 402 [M]+ (65),
387 [M – 15]+ (10), 359 [M – 43]+ (35). []D +119.4 (c 1.30).
1H NMR, : 0.96 (s, 3 H, Me(18)); 1.23 (s, 3 H, Me(19)); 1.75
(s, 3 H, Me(21)); 2.73 (d, 1 H, H(2´), J = 4.32 Hz); 5.74 (s, 1 H,
H(4)); 7.05—7.30 (Harom).
Fig. 1. Crystal structure of 3´ꢀphenyl[16,17]cyclopropaꢀ
pregnꢀ4ꢀeneꢀ3,20ꢀdione (1a).
Experimental
Melting points were determined on a Boetius apparatus.
1H NMR spectra were run on a Bruker AMꢀ300 spectrometer
(Germany) on working frequency of 300.13 MHz in CDCl3 at 30 C.
The chemical shifts are given in the  scale relative to the residual
solvent signal ( 7.27). Mass spectra (EI, 70 eV, direct inlet, 100 C)
were recorded on a Finnigan MAT INCOS 50 instrument. Eleꢀ
mental analyses were performed on a Perkin—Elmer 2400 Series
II C,H,N elemental analyzer. TLC was performed on Silica gel
60 F254 preꢀcoated plates (Merck), elution with hexane—aceꢀ
tone or hexane—diethyl ether. The spots of the compounds were
visualized by spraying with 1% Ce(SO4)2 in 10% aqueous H2SO4
followed by heating. The specific rotations were measured on
a PU 07 polarimeter (Russia) in CH2Cl2 at 27 C. Column chroꢀ
matography (Kieselgel 60, 0.063—0.100 m (Merck), a comꢀ
pound—sorbent ratio of 1 : 40) was used for product isolation.
Commercial tosylhydrazide, benzaldehyde, and 4ꢀfluorobenzꢀ
aldehyde (Acros) were used as purchased. 16ꢀDehydropregnꢀ
enolone acetate (DPA) is available from Sigma. The solvents
were purified prior to use according to the standard procedures.
The organic extracts were washed to neutral, dried with
MgSO4, and concentrated in vacuo.
Singleꢀcrystal Xꢀray diffraction of compound 1a was performꢀ
ed on a Bruker 1K SMART CCD automated diffractometer (MoꢀK
radiation). Crystals are monoclinic, at 120 K a = 9.1768(13) Å, b =
= 6.6126 (9) Å, c = 17.943(3) Å,  = 96.500(3), space group P21.
Crystal structure was calculated using SHELXTL PLUS program
package.9 The experimental details are given in Table 2. The atomꢀ
ic coordinates and complete crystallographic data were depositꢀ
ed with the Cambridge Structural Database (CCDC 930437).
1,3ꢀDipolar cycloaddition of aryldiazomethanes to 16ꢀdeꢀ
hydropregnenolone acetate (general procedure). The 30 mL oneꢀ
neck flask charged with sodium salt of the corresponding benzꢀ
aldehyde tosylhydrazone was connected to a cold trap containꢀ
ing a preliminary prepared solution of DPA in methanol—
dichloromethane (1 : 1, 6 mL). The trap was cooled with liquid
nitrogen and the system was carefully evacuated (5•10–2 mbar),
then the thermolysis was started by slow rising the bath temperaꢀ
ture to 175 C. After completion of pyrolysis (1—1.5 h) and
thawing, the content of the trap was stirred, and the obtained red
solution was kept in a freezer at –18 C for 12—15 h. After
decolorization of the reaction mixture and formation of the white
precipitate, cycloadduct was filtered off and crystallized from
acetone—petroleum ether.

Cyclopropapregnenediones
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 6, June, 2013
1453
3´ꢀ(4ꢀFluorophenyl)[16,17]cyclopropapregnꢀ4ꢀeneꢀ3,20ꢀ
dione (1b) was synthesized as described for 1a. From acetate 3b
(0.25 g, 0.54 mmol), 3ꢀhydroxy derivative (0.205 g, 90%) was
obtained, which was further subjected to Oppenauer oxidation
to give conjugated ketone 1b in the yield of 0.166 g (82%), m.p.
211—213 C (from diethyl ether—hexane). Found (%): C, 79.78;
H, 8.14. C28H33FO2. Calculated (%): C, 79.97; H, 7.91. MS,
m/z (Irel (%)): 420 [M]+ (53), 377 [M – 43]+ (30). []D +121.6
(c 1.30). 1H NMR, : 0.92 (s, 3 H, 18ꢀMe); 1.20 (s, 3 H, 19ꢀMe);
1.75 (s, 3 H, 21ꢀMe); 2.72 (d, 1 H, H(2´), J = 4.32 Hz); 5.74
(s, 1 H, H(4)); 6.88—7.10 (Harom).
Table 2. Crystallographic characteristics, details of Xꢀray experꢀ
iment and refinement statistics for compound 1a
Parameter
Value
Molecular formula
Molecular weight
C28H34O2
402.55
T/K
120(2)
/Å
Crystal system
Space group
0.71073
Monoclinic
P21
a/Å
b/Å
c/Å
/deg
9.1768(13)
6.6126(9)
17.943(3)
90
96.500(3)
90
1081.8(3)
2
1.236
[16,17](Spiro[2.2]penta)pregnꢀ4ꢀeneꢀ3,20ꢀdione (5).
3ꢀAcetoxyꢀ4´,5´ꢀdihydroꢀ3´ꢀcyclopropapregnꢀ5ꢀeno[17,16ꢀc]ꢀ
pyrazolꢀ20ꢀone (m.p. 126—128 C) synthesized by 1,3ꢀdipolar
cycloaddition according to the general procedure was subjected
to pyrolysis at 180—200 C. Column chromatography afforded
3ꢀacetoxy[16,17](spiro[2.2]penta)pregnꢀ5ꢀenꢀ20ꢀone aceꢀ
tate. Cleavage of the acetate group and subsequent oxidation as
described above furnished compound 5, m.p. 148—149 C (from
hexane—acetone) (cf. Ref. 6: 142—145 C). 1H NMR,
: 0.99 (s, 3 H, Me(18)); 1.19 (s, 3 H, Me(19)); 1.96 (s, 3 H,
Me(21)); 5.72 (s, 1 H, H(4)).
/deg
/deg
V/Å3
Z
dcalc/g cm–3
Absorption coefficient, /mm–1
F(000)
0.075
436
Crystal size/mm
Scanning range /deg
hkl Ranges
0.55×0.40×0.25
2.23—27.00
–11  h  11, –8  k  8,
–22  l  22
10296
Biological experiments. The ligandꢀreceptor binding was esꢀ
timated as earlier described.8 The experiments were carried out
with sexually mature virgin female rats of a mixed population
with the body mass of 200—250 g. For 4 days, animals daily
received intramuscular injections of estradiol (10 g) in propylꢀ
ene glycol (200 L) and were decapitated on the 5th day. The
uteri were minced and homogenized in a glass homogenizer for
5 min at 0—4 C in a buffer solution containing 10 mM TirsꢀHCl
(pH 7.5), 10 mM KCl, 1 mM EDTA, 0.5 mM phenylmethylsulꢀ
fonyl fluoride (PMSF), 1 mM dithiothreitol, 10% glycerol at
a tissue—buffer ratio of 1 : 6. The homogenate was centrifuged
(50000 g) for 1 h at 4 C. Supernatant (cytosol) with protein
concentration of 4—6 mg mL–1 was used immediately .
Number of reflections measured
reflections independent reflections
Rint
Number of refined parameters
Number of reflections with I  2(I)
R1, wR2
R Factors on all reflections
R1, wR2
GOOF
2540
0.0278
271
2242
0.0400, 0.0898
0.0459, 0.0931
1.021
0.191/–0.161
Residual electron density
max/min, e Å–3
Steroids. Progesterone and hydrocortisone (Sigma, USA),
[1,2,6,7ꢀ3H]progesterone with a specific radioactivity of
86 Ci mmol–1 (St. Petersburg) were used.
Relative binding affinity (RBA) of the steroids for PR were
measured using [3H]progesterone in the presence of hydrocortiꢀ
sone (3 mol L–1), which was added to block possible interacꢀ
tion of [3H]progesterone with blood transcortin. Uterine cytosol
(100 L) was incubated for 20 h at 0—4 C with a steroid mixꢀ
ture in the buffer (100 L) containing [3H]ꢀlabeled ligand
((60—80)•103 dpm, final concentration of 3—6 nmol L–1) and
unlabeled competitor (final concentrations from 0 to 10 mol L–1).
The free and proteinꢀbound ligands were separated by incubaꢀ
tion with 2% suspension (100 L) of Norit A (Serva, Germany)
coated with Dextraneꢀ70 (Fluka, Switzerland) for 5 min at
0—4 C. After centrifugation for 5 min at 3000 g, the aliquots
(250 mL) were taken and radioactivity was measured. The incuꢀ
bation was carried out in quartz tubes coated with BSA. Radioꢀ
activity were measured using dioxane scintillator at the counting
efficiency of 20%. The protein content was determined using
Coumassie blue.10 From 3 to 4 independent experiments were
performed in duplicate. The equilibrium dissociation constants
(Kd) were determined by adjustment of the Kd and Bmax parameꢀ
ters providing the minimum deviation of the experimental data
from "one protein—two ligands" kinetic model. The dimenꢀ
sionless RBA values were calculated as a ratio of the Kd values
for progesterone and the ligand under study obtained in
the independent experiments, the calculated RBA values were
averaged.
References
1. F. J. Zeelen, Medicinal Chemistry of Steroids, Elsevier, Amꢀ
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Received December 25, 2012;
in revised form March, 19 2013