Synthesis of 6,19-cyclopregnanes. Constrained analogues of steroid hormones

Article abstract of DOI:10.1039/b706828j

A procedure for the synthesis of 6,19-cyclopregnanes is described involving an intramolecular alkylation reaction of Δ⁴-3-keto steroids with a 19-mesylate in the presence of KOH in isopropanol. Three 6,19-cyclopregnanes were prepared (4, 5, 9); in the rat, 6,19-cycloprogesterone (4) and its 21-hydroxy derivative 5 displaced [³H]-dexamethasone from glucocorticoid receptors, the former compound being more active. Both compounds did not compete with [³H]-aldosterone for kidney mineralocorticoid receptors nor with [³H]-R5020 for uterus progesterone receptors. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b706828j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of 6,19-cyclopregnanes. Constrained analogues of steroid
hormones†
Pablo H. Di Chenna,a Adriana S. Veleiro,a Juan M. Sonego,a Nora R. Ceballos,b M. Teresa Garland,c
Ricardo F. Baggiod and Gerardo Burton*a
Received 8th May 2007, Accepted 4th June 2007
First published as an Advance Article on the web 26th June 2007
DOI: 10.1039/b706828j
A procedure for the synthesis of 6,19-cyclopregnanes is described involving an intramolecular
alkylation reaction of D4-3-keto steroids with a 19-mesylate in the presence of KOH in isopropanol.
Three 6,19-cyclopregnanes were prepared (4, 5, 9); in the rat, 6,19-cycloprogesterone (4) and its
21-hydroxy derivative 5 displaced [3H]-dexamethasone from glucocorticoid receptors, the former
compound being more active. Both compounds did not compete with [3H]-aldosterone for kidney
mineralocorticoid receptors nor with [3H]-R5020 for uterus progesterone receptors.
Introduction
Glucocorticoids are a class of stress-induced, endogenously syn-
thesized steroid hormone molecules. Synthetic glucocorticoids
constitute an important group of compounds widely used for the
treatment of inflammatory disorders, autoimmunity and cancer.1
At the cellular level, they exert their activity upon binding to the
glucocorticoid receptor (GR), a member of a protein superfamily
of closely related intracellular receptors, which acts by activating
or repressing the expression of target genes.1–3 The structural sim-
ilarity among some of these receptors originates cross-reactions
and as a consequence, partial overlapping of biological and
pharmacological properties of the respective steroidal ligands.
Opposing conformational characteristics for glucocorticoids and
mineralocorticoids have been described by Weeks et al., who used
X ray diffraction to demonstrate that optimal glucocorticoid prop-
erties could be obtained with steroids exhibiting a twisted A ring
towards the alpha face of the steroid nucleus.4 Previous work from
our group has shown that 21-hydroxy-6,19-epoxyprogesterone (1)
Fig. 1 Examples of antiglucocorticoids (1–3) and structures of 6,19-cy-
is a highly selective antiglucocorticoid devoid of mineralocorticoid
and progestational activities (Fig. 1).5 In D4-steroids as 1, the
6,19-epoxy bridge bends the steroid skeleton at the A–B ring
junction stabilizing the quasi-cis conformation with an inverted
ring A 1b half chair, a structural characteristic also present in the
antiglucocorticoid and antiprogestagen RU-486 (mifepristone, 2).6
clopregnanes (4, 5).
The crystal structure of the glucocorticoid receptor ligand binding
domain in complex with RU-486, shows the latter molecule
with its ring A exaggeratedly bent towards the alpha face, in a
distorted conformation that closely matches that of compound 1.7
However, at variance with RU-486, which is a flexible molecule, 21-
hydroxy-6,19-epoxyprogesterone has a rigid structure that locks
the conformation of ring A. Replacement of oxygen with sulfur
in the 6,19-bridge results in a slightly more flexible, less bent
molecule, 6,19-sulfur-bridged pregnanes as in 21-hydroxy-6,19-
epithioprogesterone (3) have antiglucocorticoid activities similar
to 1, although with an increased potency and selectivity.8
In the search for new conformationally restricted analogues
with “specific” glucocorticoid or antiglucocorticoid activities, we
prepared 6,19-cycloprogesterone (4) and its 21-hydroxy derivative
5, anticipating that the increased tension introduced by the 4-
membered ring would result in more bent and rigid structures.
aDepartamento de Qu´ımica Orga´nica and UMYMFOR (CONICET-
FCEN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Pabello´n 2, Ciudad Universitaria, C1428EGA Buenos Aires, Ar-
gentina. E-mail: burton@qo.fcen.uba.ar; Fax: +54 11 4576-3385; Tel: +54
11 4576-3385
bDepartamento de Biodiversidad y Biolog´ıa Experimental, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello´n 2,
Ciudad Universitaria, C1428EGA Buenos Aires, Argentina
cDepartamento de F´ısica, Facultad de Ciencias F´ısicas y Matema´ticas,
Universidad de Chile y CIMAT, Av. Blanco Encalada 2008, Casilla 487–3,
Santiago, Chile
dDepartamento de F´ısica, Comisio´n Nacional de Energ´ıa Ato´mica, Av. Gral.
Paz 1499, B1650 San Mart´ın, Pcia. de Buenos Aires, Argentina
† Electronic supplementary information (ESI) available: Synthetic proce-
dure and characterization of compound 6. See DOI: 10.1039/b706828j
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Table 1 Formation of 6,19-cyclopregnanes from 19-mesyloxy-D4-3-
ketopregnanes
Entry Steroid Base (equiv.)
Time/h Product (yield%)a
1
2
3
4
5
6
6
Na2S–isopropanol (6)
Na2S–DMF (15)
KOH–isopropanol (5)
KOH–isopropanol (5)
KOH–isopropanol (5)
8
24
3
1.5
2
7 (30)
7 (20)
7 (53)
4 (74)
5 (60)
6
10
11
a Yields correspond to isolated products purified by flash chromatography
on silica gel (ethyl acetate–hexane).
Results and discussion
Chemistry
The strategy followed to obtain 6,19-cyclosteroids was based on
our initial observation, while attempting to prepare an 11,19-
epithiopregnane by reaction of dimesylate 6 with sodium sulfide
in i-PrOH or DMF leading to 6,19-cyclopregnane 7 in low yield
(Scheme 1; Table 1, entries 1 and 2). The 6,19-cyclopregnane 7
was formed, presumably, by the intramolecular alkylation of the
thermodynamically controlled enolate derived from the D4-3-keto
moiety with the 19-mesylate. Use of KOH in i-PrOH as a base
led to shorter reaction times and an increased yield of cyclized
product (Table 1, entry 3).
Scheme 2 Reagents and conditions: (a) MsCl, py, 3 h, 0 ◦C; (b) KOH,
i-PrOH, 90 min, 25 ◦C.
Scheme 1 Reagents and conditions: see Table 1.
1
In the H NMR of 7, the absence of the low field AB quartet
characteristic of the 19-CH2OMs, indicated the loss of the 19-
mesylate group of compound 6. This was in agreement with the
presence of a singlet at d 3.01 corresponding to only one mesylate
group (11-OMs). In the COSY 45 spectrum, the signal at d 3.22
assigned to H-6 showed correlation peaks with resonances at d
1.55 (H-19b), 2.13 (H-7b) and 2.54 (H-19a). Highly diagnostic
for this structure were the correlations observed in the COSY LR
spectrum which revealed long-range connectivities between H-4
(d 5.65) and the hydrogens at positions 6 (d 3.22), 19a (d 2.54) and
2a (d 2.29). The HSQC spectra showed correlation of the carbon
at d 32.3 (C-19) with H-19a (d 2.54) and H-19b (d 1.55) and of the
carbon at d 43.1 (C-6) with H-6 (d 3.22).
Fig. 2 Displacement ellipsoid diagrams (30% probability level) for
both independent moieties of 6,19-cycloprogesterone (4), showing the
numbering scheme used.
Under similar cyclization conditions, mesylates 10 and 11
afforded cyclosteroids 4 and 5 respectively (Scheme 2; Table 1,
entries 4 and 5). Mesylates were obtained by treatment of the
corresponding 19-hydroxy steroids (8 and 9) with methanesul-
fonyl chloride in pyridine. Spectral data (1D and 2D NMR,
HRFABMS) of compounds 4 and 5 were in agreement with the
proposed structures. The structure of 4 was further confirmed by
single crystal X-ray diffraction (Fig. 2). 6,19-Cycloprogesterone
(4) crystallized in two well defined conformations with ring A as a
1b envelope, and differing slightly in the degree of bending at the
A–B ring junction. Interestingly, these two conformations were
almost identical to those observed for 6,19-epoxyprogesterone8
but at variance with this compound, AM1 calculations predicted
the more bent structure of 4 to be more stable.
Receptor binding studies
Fig. 3 shows the binding properties of 6,19-cycloprogesterone (4)
and its 21-hydroxy derivative 5, to the glucocorticoid receptor from
2454 | Org. Biomol. Chem., 2007, 5, 2453–2457
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structure factors) for the structure reported in this paper has
been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC 622907.‡ Vacuum
liquid chromatography (VLC) and column flash chromatography
were carried out on Kieselgel 60-G (Merck) and Kieselgel S 0.040–
0.063 mm respectively. TLC analysis was performed on silica gel
60 F254 (0.2 mm thick). The homogeneity of all compounds was
confirmed by thin layer chromatography. Solvents were evaporated
at reduced pressure and ca. 40–50 ◦C. 19-Hydroxyprogesterone
(8) and 19-hydroxy-21-acetyloxyprogesterone (9) were obtained
from pregnenolone acetate and 21-acetyloxypregnenolone re-
spectively (Steraloids Inc.), following essentially the procedures
15
described by Kirk et al.
Mesylate 6 was obtained from
Fig. 3 [3H]-dexamethasone (Dex) displacement assays on glucocorticoid
receptor from rat liver. Competition was measured by displacement of
5 nM [3H]-dexamethasone with unlabelled competitors: dexamethasone
(Dex), compounds 4 and 5. Data for 21-hydroxy-6,19-epoxyprogesterone
(1) is included for comparison purposes. Each treatment was done in
triplicate. Means SD from a representative experiment (n = 3) are shown.
11a-hydroxyprogesterone via 11a-acetyloxy-6,19-epoxyprogester-
one16 (see ESI†). In all cases, iodobenzene diacetate (DIB)
was used instead of lead tetraacetate for functionalization of
C-19.17
11-Methanesulfonyloxy-6,19-cyclopregn-4-ene-3,20-dione 7
Mesylate 6 (0.025 g, 0.05 mmol) was dissolved in a solution of
KOH (0.014 g, 0.25 mmol) in isopropanol (2.0 cm3). The reaction
mixture was stirred at room temperature for 90 min and then
concentrated to a third of its volume. Water was added to the
residue and then extracted with dichloromethane. The organic
layer was washed with water, dried with sodium sulfate and the
solvent evaporated under vacuum. The resulting solid was purified
by flash chromatography (silica gel, EtOAc–hexane 7 : 3) to give
compound 7 (0.011 g, 53%) as a white solid: mp 145–146 ◦C (from
methanol); mmax (KBr) (cm−1) 2939, 2876, 1701, 1655, 1339, 1173,
914; dH(500.13 MHz, CDCl3; Me4Si) 5.65 (1 H, s, 4-H), 5.10 (1 H,
m, 11b-H), 3.22 (1 H, t, J 5.4, 6-H), 3.01 (3 H, s, 11-OMs), 2.85
(1 H, dd, J 12.0 and 4.9, 12b-H), 2.55 (1 H, m, 1a-H), 2.56 (1 H,
t, J 9.2, 17-H), 2.54 (1 H, d, J 9.8, 19a-H), 2.45 (1 H, ddd, J 17.5,
14.5 and 4.3, 2b-H), 2.29 (1 H, dt, J 17.5 and 2.9, 2a-H), 2.21 (1 H,
m, 16b-H), 2.13 (1 H, m, 7b-H), 2.13 (3 H, s, 21-H), 2.12 (1 H,
m, 9-H), 2.08 (1 H, m, 8-H), 1.85 (1 H, dd, J 14.4 and 4.3, 1b-H),
1.80 (1 H, m, 16a-H), 1.74 (1 H, t, J 11.0, 12a-H), 1.68 (1 H, m,
15a-H), 1.59 (1 H, m, 7a-H), 1.55 (1 H, dd, J 9.8 and 5.4, 19b-H),
1.47 (1 H, m, 14-H), 1.29 (1 H, m, 15b-H), 0.78 (3 H, s, 18-H);
dC(125.77 MHz, CDCl3; Me4Si) 208.0 (C-20), 199.0 (C-3), 178.6
(C-5), 112.2 (C-4), 79.5 (C-11), 62.3 (C-17), 53.7 (C-14), 52.2 (C-9),
50.3 (C-10), 46.9 (C-12), 45.0 (C-13), 43.1 (C-6), 39.8 (11-OMs),
34.6 (C-2), 32.6 (C-7), 32.3 (C-19), 32.0 (C-1), 31.5 (C-8), 31.2
(C-21), 23.3 (C-16), 23.0 (C-15), 14.7 (C-18); m/z (EI) 406 (M+,
1), 311 (3), 310 (11), 267 (7), 43 (100); m/z (HRFAB) 407.1872
(M+ + H) C22H31O5S requires 407.1892.
rat liver compared to 21-hydroxy-6,19-epoxyprogesterone (1). No
binding was observed in competition assays with [3H]-aldosterone
for the mineralocorticoid receptor (from rat kidney) nor with
[3H]-R5020 for the progesterone receptor (from rat uterus).9,10
These results show that the selectivity towards the glucocorticoid
receptor associated with the bent structure with an inverted A ring
half chair is maintained.
Most interesting is the fact that while the 21-deoxy analogue of
1 (6,19-epoxyprogesterone) has no affinity for the glucocorticoid
receptor,5 the non-hydroxylated analogue 4 has higher activity
than its 21-hydroxy counterpart 5. This activity increase should
originate in the structural characteristics of the 6,19-cyclo bridge
(smaller steric bulk, increased bending of ring A) and not in
mere changes in lipophilicity or hydrogen bond acceptor capacity
associated with the removal of the oxygen bridging atom, as
replacing the oxygen bridge in 6,19-epoxyprogesterone by a
methylene group (e.g. 6,19-methanoprogesterone) did not increase
the affinity for the glucocorticoid receptor.11
Experimental
Melting points were taken on a Fisher–Johns apparatus and are
uncorrected. IR spectra were recorded in thin films using KBr
1
disks on a Nicolet Magna IR 550 FT-IR spectrophotometer. H
and 13C NMR spectra were recorded on a Bruker Avance II 500 at
500.13 and 125.77 MHz in deuterochloroform. Chemical shifts are
given in ppm downfield from TMS as internal standard, J values
are given in Hz. Multiplicity determinations and 2D spectra were
obtained using standard Bruker software. EIMS were collected
on a VG Trio-2 mass spectrometer at 70 eV by direct inlet,
HRFABMS were measured on a VG-ZAB mass spectrometer.
Single crystal X-ray measurements were performed on a Bruker
SMART CCD diffractometer, with graphite monochromated
Mo Ka radiation. The structure was solved by direct methods
with SHELXS9712 and refined by full matrix least squares in
F2 using SHELXL97.13 Hydrogen atoms were idealized at their
19-Methanesulfonyloxypregn-4-ene-3,20-dione 10
Methanesulfonyl chloride (0.191 g, 1.67 mmol) was added to a
stirred solution of 19-hydroxyprogesterone 8 (0.100 g, 0.30 mmol)
in dry pyridine (3.0 cm3) at 0 ◦C under nitrogen. After 3 hours, the
reaction mixture was acidified with 1 N HCl, and extracted with
dichloromethane. The organic layer was washed with 5% aqueous
NaHCO3 and water, dried with sodium sulfate, and the solvent
was evaporated. The residue was purified by flash chromatography
˚
˚
˚
expected positions (C–H: 0.93 A; C–H2: 0.97 A; C–H3: 0.96 A)
and allowed to ride. Molecular plots were drawn with XP, in
the SHELXTL-NT package.14 Crystallographic data (excluding
‡ CCDC reference number 622907. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b706828j
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The Royal Society of Chemistry 2007
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©

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(ethyl acetate–hexane 7 : 3) to give 10 (0.102 g, 84%) as a white
solid: mp 137–138 ◦C (from hexane–EtOAc); (found: C, 64.7; H,
7.9. C22H32O5S requires C, 64.7; H 7.9%); mmax(KBr)/cm−1 2941,
1699, 1668, 1355, 1174 and 957; dH(500.13 MHz, CDCl3; Me4Si)
5.97 (1 H, s, 4-H), 4.60 (1 H, d, J 9.8, 19a-H), 4.39 (1 H, d, J 9.8,
19b-H), 3.00 (3 H, s, 19-OMs), 2.12 (3 H, s, 21-H), 0.69 (3 H, s,
18-H); dC(125.77 MHz, CDCl3; Me4Si) 208.8 (C-20), 198.5 (C-3),
163.8 (C-5), 127.1 (C-4), 70.6 (C-19), 63.1 (C-17), 56.4 (C-14), 53.7
(C-9), 43.6 (C-13), 41.8 (C-10), 38.7 (C-12), 37.5 (19-OMs), 36.0
(C-8), 34.2 (C-2), 32.8 (C-6 or C-1), 32.7 (C-1 or C-6), 31.9 (C-7),
31.3 (C-21), 24.0 (C-15), 22.7 (C-16), 21.5 (C-11), 13.3 (C-18); m/z
(EI) 408 (M+, 21), 312 (12), 269 (5), 241 (2), 43 (100).
dH(500.13 MHz, CDCl3; Me4Si) 5.95 (1 H, s, 4-H), 4.70 (1 H,
d, J 16.8, 21a-H), 4.59 (1 H, dd, J 9.8 and 0.7, 19a-H), 4.52 (1 H,
d, J 16.8, 21b-H), 4.38 (1 H, d, J 9.8, 19b-H), 3.00 (3 H, s, 19-
OMs), 2.17 (3 H, s, 21-H), 0.72 (3 H, s, 18-H); dC(125.77 MHz,
CDCl3; Me4Si) 203.3 (C-20), 198.6 (C-3), 170.3 (CH3COO), 163.7
(C-5), 127.3 (C-4), 70.4 (C-19), 69.0 (C-21), 58.9 (C-17), 56.4 (C-
14), 53.7 (C-9), 44.5 (C-13), 41.8 (C-10), 38.5 (C-12), 36.1 (C-8),
34.4 (C-1), 32.9 (C-6 and C-2), 32.0 (C-7), 24.3 (C-15), 22.8 (C-16),
21.5 (C-11), 13.3 (C-18); m/z (EI) 466 (M+, 4), 370 (4), 269 (19),
251 (5), 43 (100).
21-Hydroxy-6,19-cyclopregn-4-ene-3,20-dione 5
Cyclopregnane 5 was obtained from mesylate 11 (0.233 g,
0.5 mmol) following the procedure described for compound 7.
The resulting solid was purified by flash chromatography (silica
gel, EtOAc–hexane 7 : 3) to give 5 (0.099 g, 60%) as a white solid,
6,19-Cyclopregn-4-ene-3,20-dione 4
Cyclopregnane 4 was obtained from mesylate 10 (0.021 g,
0.05 mmol), following the procedure described for compound 7.
The resulting solid was purified by flash chromatography (EtOAc–
hexane 7 : 3) to give 4 (0.012 g, 74%) as white crystals: mp 108–
110 ◦C (from dioxane), mmax(KBr)/cm−1 2945, 2862, 1701, 1662,
1356, 1190; dH(500.13 MHz, CDCl3; Me4Si) 5.59 (1 H, s, 4-H),
3.20 (1 H, t, J 5.7, 6-H), 2.55 (1 H, t, J 6.8, 17-H), 2.45 (1 H,
d, J 9.2, 19a-H), 2.26 (2 H, m, 2-H), 2.19 (1 H, m, 16b-H), 2.15
(1 H, m, 1a-H), 2.12 (1 H, m, 12b-H), 2.12 (3 H, s, 21-H), 2.09
(1 H, dd, J 12.8 and 6.5, 7b-H), 1.94 (1 H, m, 8-H), 1.85 (1 H,
m, 1b-H), 1.80 (1 H, m, 9-H), 1.73 (1 H, m, 11a-H), 1.69 (1 H,
m, 16a-H), 1.65 (1 H, m, 15a-H), 1.55 (1 H, t, J 12.8, 7a-H), 1.52
(1 H, m, 11b-H), 1.52 (1 H, t, J 11.0, 12a-H), 1.45 (1 H, dd, J
9.2 and 5.7, 19b-H), 1.39 (1 H, m, 14-H), 1.28 (1 H, m, 15b-H),
0.70 (3 H, s, 18-H); dC(125.77 MHz, CDCl3; Me4Si) 209.2 (C-20),
199.0 (C-3), 180.9 (C-5), 110.9 (C-4), 63.2 (C-17), 55.1 (C-14), 50.1
(C-10), 49.5 (C-9), 45.5 (C-13), 43.5 (C-6), 39.2 (C-12), 34.1 (C-2),
34.0 (C-8), 33.5 (C-7), 31.4 (C-21), 31.2 (C-19), 30.7 (C-1), 25.2
(C-11), 23.6 (C-15), 23.2 (C-16), 14.1 (C-18); m/z (EI) 312 (M+,
32), 297 (3), 269 (20), 251 (5), 227 (16), 43 (100); m/z (HRFAB)
313.2128 (M+ + H) C21H29O2 requires 313.2168.
◦
mp 153–154 C (from methanol); mmax (KBr)/(cm−1) 2926, 2856,
1701, 1659, 1100; dH(500.13 MHz, CDCl3; Me4Si) 5.60 (1 H, s,
4-H), 4.23 (1 H, d, J 19.1 Hz, 21-H), 4.17 (1 H, d, J 19.1, 21-H),
3.27 (1 H, bs, OH), 3.22 (1 H, t, J 5.7 Hz, 6-H), 2.50 (1 H, t, J 9.2,
17-H), 2.45 (1 H, d, J 9.5, 19a-H), 2.28 (2 H, m, 2-H), 2.25 (1 H,
m, 16b-H), 2.16 (1 H, m, 1a-H), 2.12 (1 H, m, 7b-H), 2.01 (1 H,
m, 12b-H), 1.97 (1 H, m, 8-H), 1.86 (1 H, m, 1b-H), 1.81 (1 H,
m, 16a-H), 1.80 (1 H, m, 9-H), 1.73 (1 H, m, 11a-H), 1.71 (1 H,
m, 15a-H), 1.57 (1 H, m, 7a-H), 1.53 (1 H, m, 11b-H), 1.47 (1 H,
m, 19b-H), 1.46 (1 H, m, 12a-H), 1.40 (1 H, m, 14-H), 1.38 (1 H,
m, 15b-H), 0.76 (3 H, s, 18-H); dC(125.77 MHz, CDCl3; Me4Si)
210.1 (C-20), 199.1 (C-3), 180.8 (C-5), 110.9 (C-4), 69.3 (C-21),
58.6 (C-17), 55.1 (C-14), 50.0 (C-10), 49.3 (C-9), 46.2 (C-13), 43.4
(C-6), 38.8 (C-12), 34.0 (C-2), 33.9 (C-8), 33.4 (C-7), 31.1 (C-19),
30.6 (C-1), 25.1 (C-11), 23.6 (C-15), 23.2 (C-16), 14.2 (C-18); m/z
(EI) 328 (M+, 30), 297 (33), 269 (42), 251 (10), 241 (6), 227 (15),
41 (100); m/z (HRFAB) 329.2117 (M+ + H) C21H29O3 requires
329.2130.
Crystallographic data and data collection parameters. Col-
orless prismatic crystals recrystallized from dioxane. C21H28O2,
M = 312.43, monoclinic, space group P21 (no 4); cell constants
Binding studies
Male Sprague Dawley rats, 21 days old, were adrenalectomized
48 h prior to experiments and maintained on Purina chow (diet 1),
saline and fresh water ad libitum. Animals were sacrificed and bled
by heart puncture. Liver was used as a source of glucocorticoid
receptors. Tissue was homogenized with two volumes of TEGM
buffer at pH 7.4 (0.1 M Tris-HCl, 10 mM EDTA, 10 mM
b-mercaptoethanol, 20 mM Na2MoO4, 25% glycerol, 0.1 mM
PMSF, 2.0 IU cm−3 aprotinin and 1 mg leupeptin (R buffer).
The cytosolic fraction was prepared from the homogenate by
differential centrifugation. After sedimentation of the nuclear
fraction at 800 × g for 10 min, the cytosolic fraction was
separated from mitochondria and microsomes by centrifugation at
105000 × g for 60 min. All steps were carried out at 4 ◦C. Binding
was assayed in triplicate employing 400–600 lg cytosolic proteins
and 1.2 nM [3H]-dexamethasone in TEGM buffer. Binding
was obtained by the displacement of [3H]-dexamethasone with
different concentrations of unlabelled dexamethasone (5, 50 and
5000 nM), or the corresponding competitor (1, 4 and 5). All
the incubations were carried out in a final volume of 0.5 cm3
˚
˚
˚
a = 11.3312(12) A, b = 11.4371(12) A, c = 14.5628(16) A; b =
◦
3
ꢀ
−3
˚
111.863(2) ; V = 1751.5(3) A , Dc (Z = 4, Z = 2) = 1.185 g cm ;
l = 0.074 mm−1; crystal dimensions 0.26 × 0.18 × 0.16 mm,
reflections measured: 13024, reflections unique (Friedel pairs
merged): 4172, reflections observed (I > 2r(I)): 2950; R = 0.057
2
and Rw = 0.090. The structure is made up of two independent
moieties (of the same chirality) in the asymmetric unit. The
X-ray analysis only established the relative stereochemistry of this
all-light-atom compound.‡
21-Acetyloxy-19-methanesulfonyloxypregn-4-ene-3,20-dione 11
Mesylate 11 was obtained from 21-acetyloxy-19-hydroxy-
progesterone 9 (0.150 g, 0.386 mmol) and methanesulfonyl chlo-
ride (0.220 g, 1.93 mmol) following the procedure described for
compound 10. The product was purified by flash chromatography
(ethyl acetate–hexane 6 : 4) to give 11 (0.158 g, 88%) as a white
solid, mp 161–162 ◦C (from hexane–ethyl acetate); (found C
62.1, H 7.6. C24H34O7S requires C 61. 8, H 7.3%); mmax/(KBr)
(cm−1) 2939, 2878, 1749, 1724, 1356, 1234, 1175, 959, 833;
◦
at 4 C. After equilibrium was reached, unbound [3H]-steroids
were removed from the incubation with an equal volume of
2456 | Org. Biomol. Chem., 2007, 5, 2453–2457
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charcoal–dextran (2 : 0.2%) in PBS, pH 7.4 during 20 min and
subsequent centrifugation. Bound radioactivity was determined
by scintillation counting.
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Acknowledgements
This work was supported by grants from Agencia Nacional de
Promocio´n Cient´ıfica y Tecnolo´gica (PICT-10962), Universidad
de Buenos Aires, and CONICET (Argentina). MTG and RFB
thank the Spanish Research Council (CSIC) for providing a free-
of-charge license to the CSD system and CONICYT-FONDAP
11980002 for the purchase of the diffractometer and detector.
13 G. M. Sheldrick, SHELXL-97. Program for Structure Refinement,
University of Go¨ttingen, Germany, 1997.
14 SHELXTL-NT (Version 6.10) for Windows NT, Bruker AXS Inc.,
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