Design, synthesis and biochemical studies of new 7α-allylandrostanes as aromatase inhibitors

Article abstract of DOI:10.1016/j.steroids.2013.02.016

Two series of derivatives of 7α-allylandrostenedione, namely its 3-deoxo and 1-ene analogs, were designed and synthesised and their biochemical activity towards aromatase evaluated. In each of these series, the C-17 carbonyl group was further replaced by the hydroxyl and acetoxyl groups. The attained data pointed out that the absence of the C-3 carbonyl group led to a slightly decrease in the inhibitory activity and the introduction of an additional double bond in C-1 revealed to be a very beneficial structural change in the studied compounds (compound 12, IC₅₀ = 0.47 μM, K_i = 45.00 nM). Furthermore, the relevance of the C-17 carbonyl group in the D-ring as a structural feature required to achieve maximum aromatase inhibitory activity is also observed for this set of derivatives.

Full text of DOI:10.1016/j.steroids.2013.02.016

Steroids 78 (2013) 662–669
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Design, synthesis and biochemical studies of new 7a-allylandrostanes as
aromatase inhibitors
Carla L. Varela , Cristina Amaral , Georgina Correia-da-Silva , Rui A. Carvalho , Natércia A. Teixeira b,c,
a
b,c
b,c
d
a
a,
⇑
a,
⇑
Saul C. Costa , Fernanda M.F. Roleira , Elisiário J. Tavares-da-Silva
a
CEF, Center for Pharmaceutical Studies, Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal
Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
Institute for Molecular and Cellular Biology (IBMC), University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of derivatives of 7a-allylandrostenedione, namely its 3-deoxo and 1-ene analogs, were
Received 14 December 2012
Received in revised form 17 February 2013
Accepted 26 February 2013
Available online 13 March 2013
designed and synthesised and their biochemical activity towards aromatase evaluated. In each of these
series, the C-17 carbonyl group was further replaced by the hydroxyl and acetoxyl groups. The attained
data pointed out that the absence of the C-3 carbonyl group led to a slightly decrease in the inhibitory
activity and the introduction of an additional double bond in C-1 revealed to be a very beneficial struc-
i
tural change in the studied compounds (compound 12, IC50 = 0.47 lM, K = 45.00 nM). Furthermore, the
Keywords:
Breast cancer
Synthetic steroids
relevance of the C-17 carbonyl group in the D-ring as a structural feature required to achieve maximum
aromatase inhibitory activity is also observed for this set of derivatives.
Ó 2013 Elsevier Inc. All rights reserved.
7a-Allylandrostanes
Aromatase inhibitors
Structure-activity relationships
1
. Introduction
Many breast tumors are dependent upon estrogens for their
quently the growth effects of estrogens on primary and metastatic
hormone responsive breast cancers [4].
The third-generation aromatase inhibitors (AIs) in clinical use,
such as the steroidal exemestane along with the non-steroidal
anastrazole and letrozole (Fig. 1), appear to be very potent with
greater target specificity, which allow circulating estrogens to fall
below detectable levels [5]. Despite their success, they still have
some disadvantages since they significantly increase musculoskel-
etal symptoms, osteopenia, osteoporosis and fracture rate [3]. In
addition, after some years of usage they can induce tumor resis-
tance. For this reason, it is important to seek for other potent and
specific molecules in order to increase the number of AIs available
to allow surpassing the side effects and the resistance phenomena.
In the present study, our focus is the design, synthesis and bio-
development and continued growth [1]. As a consequence, it might
be expected that estrogen deprivation will both prevent the
appearance of these tumors and cause regression of the established
ones [2]. This has been the rationale that supports the hormone
therapy of breast tumors.
The primary site of estrogen biosynthesis varies with the men-
opausal status. Thus, in premenopausal women the ovaries are the
major source of estrogen production whereas peripheral tissues
such as adipose tissue, brain, blood vessels, skin, bone, endome-
trium, and breast tissue are more important in postmenopausal pa-
tients [3]. Estrogens are the end-products in the sequence of
steroid biosynthesis being the last step catalyzed by the cyto-
chrome P-450 enzyme aromatase (CYP19). Aromatase is responsi-
ble for the conversion of androgen precursors (testosterone and
androstenedione) into the respective estrogens (estradiol and
estrone). For this reason, aromatase inhibition has been a useful
approach to selectively block the estrogen production and conse-
chemical evaluation of a new series of steroids, based in the 7a-
allylandrostenedione framework, containing extra modifications
in the A- and D-rings, in order to further study the structure–activ-
ity relationships (SAR) on AIs.
Brueggemeier and co-workers [6–9], have reported the synthe-
sis of many C-7-substituted androstanes as AIs, having the most
part of the substituents a phenyl moiety. Also, Labrie et al. [10],
have described a rather active AI with an allyl functional group
in the C-7
a position being this reference the only one found in
⇑
Corresponding authors. Tel.: +351 239 488 400; fax: +351 239 488 503.
E-mail addresses: froleira@ff.uc.pt (F.M.F. Roleira), etavares@ff.uc.pt (E.J. Ta-
the literature that describes this kind of substituent. In this man-
vares-da-Silva).
ner, the allyl group was not particularly studied. Recently, Ghosh
0
039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.02.016

C.L. Varela et al. / Steroids 78 (2013) 662–669
663
N
N
pounds was determined from the area peaks in the chromatogram
of the sample solution.
O
N
N
N
N
2.2. Chemistry
O
NC
CN
NC
CN
2.2.1. 3-Oxoandrost-4-en-17b-yl acetate (2)
To a solution of 1 (2.0 g, 6.93 mmol) in dry pyridine (48 mL),
acetic anhydride (7.9 mL, 83.85 mmol) was added and the reaction
was stirred for 21 h 30 min, at room temperature, until all the
starting material was consumed (TLC control). Dichloromethane
Exemestane
Anastrozole
Letrozole
Fig. 1. The aromatase inhibitors in clinical use.
(
250 mL) was added and the organic layer was washed with 10%
NaHCO
(3 Â 150 mL), 10% HCl (3 Â 150 mL) and water
, filtered and concen-
and collaborators [11], have designed novel C-6-substituted 2-
alkynyloxy androstanes as potent AIs, where it was found that
the C-6 linear side chains protrude into the access channel cavity
immobilizing the catalytic residues. Based on this, the structure–
activity relationships of steroids containing C-6 or C-7 linear chains
deserve to be revisited and further studied. Further, evidence that
the aromatase inhibitory activity is similar when the same kind of
substituent is in position C-6 or C-7 of a given steroidal framework,
comes from previous reported works [9,12]. Keeping this in mind,
3
(3 Â 150 mL), dried over anhydrous MgSO
4
trated to dryness. Crystallization of the obtained residue from ethyl
acetate gave the pure compound 2 (1.92 g, 84%) as translucent
prism crystals: mp 141–142 °C (ethyl acetate); IR (NaCl plates,
CHCl
NMR (600 MHz, CDCl
2.03 (3H, s, CH COO), 4.58 (1H, dd, J17
17 -H), 5.71 (1H, s, 4-H); C NMR (150 MHz, CDCl
3
) 3018 (=CH), 1736 (C@O), 1675 (C@C), 1248 (C–O); 1
H
3
) d 0.82 (3H, s, 18-H
3
), 1.18 (3H, s, 19-H
= 9.0, J17 ,16b = 8.0,
) d 12.0 (C-
3
),
3
a
,16
a
a
1
3
a
3
we are interested in performing the synthesis of 7
a
-allylandros-
18), 17.4 (C-19), 20.5, 21.1, 23.4, 27.4, 31.4, 32.7, 33.9, 35.4, 35.7,
36.6, 38.6, 42.4, 50.2, 53.7, 82.4 (C-17), 123.9 (C-4), 170.9 (C-5),
171.1 (OC@O), 199.4 (C-3).
tenedione derivatives where two main structural features were
considered: the introduction of a double bond in C-1 and the
abstraction of the carbonyl group at C-3. The introduction of a dou-
ble bond in C-1 is based on the rationale that the A-ring planarity
in steroidal AIs is important for aromatase inhibition, as reported
in previous works [13–17], and also because it has been estab-
lished that the introduction of a double bond at C-1/C-2 of andro-
stenedione affords AIs that causes mechanism-based (suicide)
inactivation of aromatase [18–20]. In fact, the aromatization pro-
cess of androstenedione encompasses the C-1 and C-2 hydrogen
elimination [21]. There are also several studies revealing that, in
some AIs, the C-3 carbonyl group, present in the natural substrate
of the enzyme, is not essential for aromatase inhibition [13,22,23].
2.2.2. 3-Oxoandrosta-4,6-dien-17b-yl acetate (3)
To a solution of 2 (3.8 g, 11.50 mmol) in acetic acid (50 mL) and
toluene (10 mL), under nitrogen, chloranil (2.5 g, 10.17 mmol) was
added and the reaction mixture was refluxed for 3 h (UV spectro-
photometric control). After cooling, the mixture was extracted
with dichloromethane (3 Â 200 mL) and the organic layer was
sequentially washed with 2.5 N NaOH, 10% NaHCO
3
(3 Â 150 mL)
, the
and water (3 Â 100 mL). After drying over anhydrous MgSO
4
solvent was removed under reduced pressure yielding a crude
product which was purified by silica gel chromatography (petro-
leum ether 40–60 °C/diethyl ether) giving the pure compound 3
(2.2 g, 58%), as a yellow crystalline solid: mp 141–143 °C (ethyl
For this reason, 3-deoxy derivatives of 7a-allylandrostenedione
were synthesized and evaluated. Further, for each set of derivatives
it was studied the influence of the functional group at C-17 in aro-
matase inhibitory activity.
acetate/n-hexane); IR (NaCl plates, CHCl
(C@O), 1658 (C@C), 1617 (C@C), 1030 (C–O); H NMR (600 MHz,
CDCl ) d 0.88 (3H, s, 18-H ), 1.11 (3H, s, 19-H ), 2.05 (3H, s, CH3-
COO), 4.63 (1H, dd, J17 ,16b = 9.0, 17 -H), 5.67 (1H, s, 4-
H), 6.09 (1H, d, J6,7 = 10.4, 6-H), 6.12 (1H, dd, J7,6 = 10.4, J7,8 = 2.4,
3
) 3024 (=CH), 1734
1
3
3
3
a
,16a
= J17
a
a
2
. Experimental section
1
3
7
-H); C NMR (150 MHz, CDCl
21.1, 23.1, 27.4, 33.8, 33.9, 36.0, 36.5, 37.3, 43.4, 48.0, 50.6, 82.1
C-17), 123.8 (C-7), 128.2 (C-6), 139.9 (C-4), 163.4 (C-5), 171.1
(OC@O), 199.4 (C-3).
3
) d 11.9 (C-18), 16.3 (C-19), 20.2,
2.1. General
(
Reactions were controlled by thin layer chromatography (TLC)
using silica gel 60 F254 plates and the mobile phases used were
mixtures of petroleum ether 40–60 °C/ethyl acetate in different
proportions. Column chromatography was performed using silica
gel 60 (0.063–0.200 mm). Melting points (MPs) were determined
on a Reichert Thermopan hot block apparatus and were not cor-
rected. IR spectra were recorded on a Jasco 420 FT/IR spectrometer.
2.2.3. 7a-Allyl-3-oxoandrost-4-en-17b-yl acetate (4)
To a solution of 3 (0.55 g, 1.67 mmol) in dry dichloromethane
(50 mL), under magnetic stirring, nitrogen atmosphere and cooled
at À78 °C, titanium tetrachloride (3.0 mL, 27.29 mmol) was added.
Five min later,
a solution of allyltrimethylsilane (3.0 mL,
1
13
The H NMR and C NMR spectra were recorded at 600 MHz and
50 MHz, respectively on a Varian Unity 600. Chemical shifts were
18.6 mmol) in dry dichloromethane (5.0 mL) was carefully added
to the previous solution. The resulting mixture was stirred for
40 min and then allowed to warm to À30 °C. After that, water
was added, the mixture was extracted with dichloromethane
1
recorded in d values in parts per million (ppm) downfield from tet-
ramethylsilane as an internal standard. All J values are given in Hz.
Mass spectra ESI and LC–MS were obtained with a mass spectrom-
eter QIT-MS Thermo Finningan, model LCQ Advantage MAX cou-
(3 Â 100 mL) and the organic layer was washed with 10% NaHCO
(2 Â 100 mL) and water (3 Â 100 mL), dried over anhydrous Na2-
SO , filtered and concentrated under reduced pressure yielding
3
pled to
a
Liquid Chromatography Equipment of High
4
Performance Thermo Finningan. Testosterone (1) was purchased
from Pharmacia & Upjahn Company, Kalamazoo, Michigan (USA).
Reagents and solvents were used as obtained from suppliers with-
out further purification, with exception to dichloromethane, which
an oily product (0.674 g). Purification by silica gel flash column
chromatography (petroleum ether 40–60 °C/diethyl ether) affor-
ded the pure compound 4 (0.35 g, 56%): mp 150–152 °C (petro-
leum ether 40–60 °C/diethyl ether); IR (NaCl plates, CHCl
3
) 3012
2
was dried through reflux and distilled from CaH [24].
(=CH), 1745 (C@O ester), 1722 (C@O), 1663 (C@C), 1041 (C–O);
1
All SAR compounds possess a purity superior to 98%. The purity
was checked by HPLC with a C18-reversed phase column and
water/acetonitrile 40:60 as solvent. The purity of individual com-
H NMR (600 MHz, CDCl
), 2.04 (3H, s, CH COO), 4.61 (1H, dd, J17
-H), 4.98 (1H, ddd, CH@CH ), 5.02 (1H, ddd, CH@CH
2
3
) d 0.84 (3H, s, 18-H
3
), 1.20 (3H, s, 19-
= 8.9, J17 ,16b = 8.2,
), 5.64
H
3
3
a,16a
a
17a
2

6
64
C.L. Varela et al. / Steroids 78 (2013) 662–669
2
2
200
000
[
AI] = 0 µM
AI] = 0.1 µM
AI] = 0.4 µM
AI] = 0.8 µM
[
[
1800
1600
[
1
1
1
400
200
000
8
6
4
2
00
00
00
00
0
-
0.2
-0.15
-0.1
-0.05
0
0.05
0.1
1
/[S] (nM)
Fig. 2. Lineweaver–Burk plot for inhibitor 10 at different concentrations (0, 0.1, 0.4 and 0.8 lM), with androstenedione as substrate at 10, 20 and 30 nM. Each point
represents the mean of three independent experiments, performed in triplicate.
(
1H, m, CH@CH
2
), 5.71 (1H, s, 4-H); 13C NMR (150 MHz, CDCl
1.9 (C-18), 17.9 (C-19), 20.7, 21.1, 22.8, 27.3, 30.2, 33.9, 35.9,
3
) d
(C@O), 1669 (C@C); 1H NMR (600 MHz, CDCl
H3), 1.22 (3H, s, 19-H3), 5.01 (1H, ddd, CH@CH
CH@CH ), 5.67 (1H, m, CH@CH ), 5.73 (1H, s, 4-H); C NMR
(150 MHz, CDCl ) d 13.5 (C-18), 17.9 (C-19), 20.5, 21.3, 30.2, 31.1,
33.9, 35.4, 35.6, 35.9, 36.0, 38.1, 38.7, 46.7, 47.1, 47.5, 117.1 (C-
2), 126.4 (C-4), 136.4 (C-21), 168.5 (C-5), 198.9 (C-3), 220.0 (C-
3
) d 0.92 (3H, s, 18-
), 5.05 (1H, ddd,
1
3
2
3
2
13
6.0, 36.1, 36.4, 38.3, 38.6, 42.5, 45.9, 46.9, 82.4 (C-17), 116.8 (C-
4), 126.2 (C-4), 136.8 (C-23), 169.2 (C-5), 171.1 (C@O), 199.1 (C-
); MS (ESI) m/z 369.53 ([MÀH] , 100%).
2
2
3
+
2
+
2
.2.4. 7
To a mixture of dioxane/water (85:15) (50 mL) with 2% NaOH
10 mL), compound 4 (0.62 g, 1.67 mmol) was added and the
a-Allyl-3-oxoandrost-4-en-17b-ol (5)
17); MS (ESI) m/z 325.13 ([MÀH] , 100%).
(
2.2.6. 7 -Allylandrost-4-en-17-one (9)
a
resulting solution was stirred overnight, at room temperature,
after which chilled water was added leading to the formation of
a precipitate. This precipitate was separate by filtration, dissolved
in ethyl acetate/dichloromethane (4:1), and the resulting solution
Jones Reagent (1.1 mL); compound 8 (134.7 mg, 0.43 mmol);
acetone/dioxane (60:10) (23 mL); the solid residue obtained
(129.3 mg) was purified by a silica gel column chromatography
(n-hexane/ethyl acetate (99:01)) affording the pure compound 9
(105.6 mg, 79%) as a white crystalline solid: mp 139–142 °C (n-
4
was dried over anhydrous MgSO and concentrated under reduced
pressure yielding the pure compound 5 (0.57 g, 96%) as a white so-
lid: mp 216–218 °C (ethyl acetate/dichloromethane); IR (NaCl
3
hexane/ethyl acetate); IR (NaCl plates, CHCl ) 3082 (=CH), 1734
1
(C@O), 1640 (C@C); H NMR (600 MHz, CDCl
), 1.05 (3H, s, 19-H ), 2.44 (1H, ddd, J16b,16
= 1.3, 16b-H), 4.99 (2H, m, CH@CH
= 2.2, J4,3b = 2.2, 4-H), 5.72 (1H, m, CH@CH
) d 13.5 (C-18), 19.2, 19.9 (C-19), 20.7, 21.4, 25.7,
3
) d 0.89 (3H, s, 18-
plates, CHCl
3
) 3421 (OH), 1722 (C@O), 1645 (C@C), 1061 (C–O);
H NMR (600 MHz, DMSO-d ) d 0.69 (3H, s, 18-H ), 1.16 (3H, s,
= 8.5, J17
-H), 4.47 (1H, d, J17b-OH,17 = 4.8, 17b-OH), 4.94 (1H, ddd,
), 5.56 (1H, s, 4-H), 5.72 (1H, m,
); C NMR (150 MHz, DMSO-d ) d 11.0 (C-18), 17.4 (C-
H
3
3
a
= 18.9, J16b,15b = 8.5,
1
6
3
J
J
16b,15
a
2
), 5.28 (1H, dd,
13
1
3
9-H ), 3.46 (1H, ddd, J17
a,16a
a
,16b = 8.5, J17
a
,17b-OH = 4.8,
4,3a
2
);
C NMR
1
7a
a
(150 MHz, CDCl
3
CH@CH
CH@CH
2
2
), 5.00 (1H, m, CH@CH
2
29.5, 31.4, 35.2, 35.7, 35.7, 37.2, 37.9, 38.3, 46.9, 47.3, 47.7, 115.8
1
3
6
(C-22), 122.2 (C-4), 138.0 (C-21), 140.5 (C-5), 221.1 (C@O); MS
+
1
4
2
9), 20.4, 22.3, 29.6, 29.8, 33.6, 35.2, 35.5, 35.7, 36.0, 37.8, 38.2,
(ESI) m/z 311.39 ([MÀH] , 100%).
2.4, 45.5, 46.4, 79.8 (C-17), 116.1 (C-22), 125.2 (C-4), 137.4 (C-
+
1), 169.2 (C-5), 197.5 (C-3); MS (ESI) m/z 327.31 ([MÀH] , 100%).
2.2.7. 7a-Allylandrosta-1,4-diene-3,17-dione (12)
Jones Reagent (0.3 mL); compound 10 (25.1 mg, 0.077 mmol);
acetone/dioxane (60:10) (10 mL); the solid residue obtained
(27.5 mg) was purified by a silica gel column chromatography (n-
hexane/ethyl acetate (70:30)) affording the pure compound 12
(22.1 mg, 88%) as a white crystalline solid: mp 205–207 °C (n-hex-
2
(
3
5
.2.4.1. General procedure to obtain 7
6), 7 -allylandrost-4-en-17-one (9) and 7
,17-dione (12). Jones Reagent was added dropwise to solutions of
, 8 or 10 in acetone/dioxane (60:10), at 0 °C, with magnetic stir-
a
-allylandrost-4-ene-3,17-dione
a
a
-allylandrosta-1,4-diene-
ring, until persistent brown coloration was obtained. The excess
of oxidant agent was destroyed by addition of 2-propanol until a
greenish colour was obtained and the reaction mixture was poured
in water (150 mL), extracted with ethyl acetate (3 Â 100 mL) and
ane/ethyl acetate); IR (NaCl plates, CHCl
3
) 3076 (=CH), 1737 (C@O),
1661 (C@C), 1617 (C@C); H NMR (600 MHz, CDCl ) d 0.95 (3H, s,
), 5.01 (1H, ddd, CH@CH ), 5.08 (1H,
), 6.06 (1H, dd, J4,2 = 1.8,
1
3
18-H
3
), 1.26 (3H, s, 19-H
3
2
ddd, CH@CH
2
), 5.67 (1H, m, CH@CH
2
the organic layer washed with 10% NaHCO
3
(2 Â 100 mL) and
, filtered and con-
4,6a
J = 1.7, 4-H), 6.24 (1H, dd, J2,1 = 10.1, J2,4 = 1.8, 2-H), 7.05 (1H,
1
3
water (2 Â 100 mL), dried over anhydrous MgSO
4
d, J1,2 = 10.1, 1-H); C NMR (150 MHz, CDCl
(C-19), 21.4, 21.7, 29.4, 30.9, 35.2, 35.3, 36.7, 37.7, 43.4, 44.9,
6.3, 47.6, 117.2 (C-22), 126.5 (C-4), 127.6 (C-2), 136.1 (C-21),
3
) d 13.5 (C-18), 19.0
centrated to dryness.
4
2
.2.5. 7
a
-Allylandrost-4-ene-3,17-dione (6)
154.9 (C-1), 165.2 (C-5), 185.6 (C-3), 219.4 (C-17); MS (ESI) m/z
323.39 ([MÀH] , 100%).
+
Jones Reagent (2.6 mL); compound 5 (0.53 g, 1.61 mmol); ace-
tone/dioxane (60:10) (70 mL); the residue obtained was insolubi-
lized in diisopropylether giving the pure compound 6 (0.386 g,
2.2.8. 7a-Allylandrost-4-en-17b-yl acetate (7)
7
3%) as a white crystalline compound: mp 218–221 °C (n-hex-
Sodium borohydride (202.0 mg, 5.33 mmol) was added in small
ane/ethyl acetate); IR (NaCl plates, CHCl ) 3076 (=CH), 1734
3
proportions to a stirred and cooled mixture of trifluoracetic acid

C.L. Varela et al. / Steroids 78 (2013) 662–669
665
2
2
1
1
1
1
1
200
000
800
600
400
200
000
temperature, overnight. The reaction mixture was then cooled at
À10 °C and 3 N HCl was added until pH 5–7. Ethanol was evapo-
rated under vacuum and the oily residue obtained was poured in
water (50 mL) and extracted with ethyl acetate (3 Â 50 mL). The
organic layer was washed with water (3 Â 100 mL), dried over
[
S] = 10 nM
S] = 20 nM
S] = 30 nM
[
[
4
anhydrous MgSO , filtered and concentrated to dryness giving a so-
lid residue (18.2 mg). This residue was washed with chilled n-hex-
ane leading to the pure compound 8 (4.4 mg, 29%) as a white solid:
mp 140–143 °C (ethyl acetate); IR (NaCl plates, CHCl
3
3
) 3297 (OH),
076 (=CH), 1640 (C@C), 1059 (C–O); H NMR (600 MHz, CDCl ) d
.77 (3H, s, 18-H ), 1.04 (3H, s, 19-H ), 3.64 (1H, dd, J17 = 8.6,
,16b = 8.6, 17 -H), 4.96 (2H, m, CH@CH ), 5.25 (1H, dd,
= 2.3, J4,3b = 2.3, 4-H), 5.70 (1H, m, CH@CH
) d 10.9 (C-18), 19.3, 19.9 (C-19), 21.1, 22.8, 25.8,
9.5, 30.4, 35.3, 36.2, 36.6, 37.2, 37.9, 38.8, 42.9, 46.3, 47.3, 82.0
(C-17), 115.5 (C-22), 121.8 (C-4), 138.5 (C-21), 141.1 (C-5); MS
8
6
4
2
00
00
00
00
0
1
3
0
3
3
a,16a
J
J
17
a
a
2
13
4,3
a
2
);
C NMR
(150 MHz, CDCl
3
-
700-600-500-400-300-200-100
0
100 200 300 400 500 600 700 800 900
2
[
AI] (nM)
+
(
ESI) m/z 313.49 ([MÀH] , 100%).
Fig. 3. Dixon plot for inhibitor 10 at different concentrations (0, 0.1, 0.4 and
.8 M), with androstenedione at 10, 20 and 30 nM, to determine the apparent
inhibition constant (K ). Each point represents the mean of three independent
experiments, performed in triplicate.
0
l
i
2
.2.10. 7
To a stirred solution of 5 (538.7 mg, 1.64 mmol) in toluene
55 mL), DDQ (645.0 mg, 2.84 mmol) and benzoic acid (215.0 mg,
.76 mmol) were added. After 18 h at reflux, an additional amount
a-Allyl-3-oxoandrosta-1,4-dien-17b-ol (10)
(
1
(
1.24 mL), glacial acetic acid (1.24 mL) and acetonitrile (1.24 mL)
followed by a solution of 4 (400.2 mg, 1.08 mmol) in dry dichloro-
methane (20 mL). The temperature was raised up at room temper-
ature and the reaction was stirred under nitrogen, until all the
starting material had been consumed (1 h 30 min, TLC control).
The reaction mixture was neutralized with a solution of 10%
of DDQ (250 mg, 1.10 mmol) and benzoic acid (150 mg, 1.23 mmol)
was added and the reaction proceeded for more 16 h. The reaction
mixture was cooled at room temperature, filtered and the resulting
filtrate was evaporated giving an oily residue, which was dissolved
with dichloromethane and then mixed with silica gel. This mixture
was filtered through basic alumina and washed with petroleum
ether 40–60 °C/ethyl acetate (8:2) (250 mL). Evaporation of the fil-
trate afforded a crystalline residue which was purified by silica gel
column chromatography (toluene/diethyl ether (65:35)) affording
the pure compound 10 (268 mg, 50%) as a pure solid: mp 175–
NaHCO
ganic layer was washed with water (3 Â 100 mL), dried over anhy-
drous MgSO , filtered and concentrated to dryness giving a white
3
, extracted with dichloromethane (4 Â 100 mL) and the or-
4
solid residue (380.8 mg). This residue was separated by silica gel
column chromatography (n-hexane/ethyl acetate (99:1)) affording
2
93.8 mg of a fraction containing compound 7, which was further
1
3
3
78 °C (petroleum ether 40–60 °C/toluene); IR (NaCl plates, CHCl )
purified by crystallization from ethanol/water giving the pure 7
1
378 (OH), 3018 (=CH), 1652 (C@C), 1053 (C–O); H NMR
) d 0.83 (3H, s, 18-H ), 1.24 (3H, s, 19-H ), 3.65
= 8.6, J17 ,16b = 8.6, 17 -H), 5.00 (1H, ddd, CH@CH ),
.05 (1H, ddd, CH@CH ), 5.64 (1H, m, CH@CH ), 6.06 (1H, dd,
= 1.7, 4-H), 6.23 (1H, dd, J2,1 = 10.1, J2,4 = 1.8, 2-H),
(
(
1
(
26.9 mg) as clear and bright needle-like crystals: mp 129–130 °C
ethanol/water); IR (NaCl plates, CHCl ) 3076 (=CH), 1728 (C@O),
) d 0.81 (3H, s, 18-H ), 1.03
COO), 4.59 (1H, dd, J17 = 8.5,
), 5.25 (1H, dd,
(
(
600 MHz, CDCl
1H, dd, J17
3
3
3
3
a
,16a
a
a
2
1
041 (C–O); H NMR (600 MHz, CDCl
3
3
5
2
2
3H, s, 19-H
3
), 2.04 (3H, s, CH
3
a,16a
J4,2 = 1.8, J4,6
a
J
J
17
a
,16b = 8.5, 17
a
-H), 4.96 (2H, m, CH@CH
2
13
7
1
4
2
3
.06 (1H, d, J1,2 = 10.1, 1-H); C NMR (150 MHz, CDCl ) d 10.9,
1
3
4,3
a
= 2.3, J4,3b = 2.3, 4-H), 5.69 (1H, m, CH@CH
150 MHz, CDCl ) d 11.8 (C-18), 19.2, 19.9 (C-19), 20.9, 21.2, 22.9,
5.8, 27.4, 29.5, 35.3, 36.2, 36.7, 37.1, 37.9, 38.5, 42.6, 46.1, 47.2,
2
);
C NMR
9.1, 22.2, 22.9, 29.4, 30.2, 35.4, 36.1, 37.5, 38.2, 43.1, 43.6, 45.2,
5.9, 81.4 (C-17), 116.9 (C-22), 126.3 (C-4), 127.5 (C-2), 136.7 (C-
(
3
2
8
1
1), 155.6 (C-1), 166.1 (C-5), 185.9 (C-3); MS (ESI) m/z 325.39
2.8 (C-17), 115.9 (C-24), 121.9 (C-4), 138.4 (C-23), 140.9 (C-5),
+
(
[MÀH] , 100%).
+
71.2 (C@O); MS (ESI) m/z 355.49 ([MÀH] , 100%).
2.2.9. 7a-Allylandrost-4-en-17b-ol (8)
2.2.11. 7a-Allyl-3-oxoandrosta-1,4-dien-17b-yl acetate (11)
Compound 7 (223.2 mg, 0.63 mmol) was added to a mixture of
To a solution of 10 (50.0 mg, 0.15 mmol) in dry pyridine
(4.0 mL), acetic anhydride (0.2 mL, 1.79 mmol) was added and
the reaction was stirred for 33 h 15 min at room temperature, until
dioxane/water (85:15) (18 mL) with 2% NaOH (3.6 mL), at room
temperature, and the reaction mixture was stirred until total trans-
formation of the starting material (48 h, TLC control) being after
this time neutralized with a solution of 5% HCl. The dioxane was
evaporated under vacuum leading to a residue that was dissolved
in ethyl acetate (50 mL), diluted with water (100 mL) and extracted
with ethyl acetate (3 Â 100 mL). The organic layer was washed
Table 1
a
Aromatase inhibition of A-, B- and D-ring modified steroids.
Compounds
Aromatase inhibition (%) ± SEM
4
5
6
7
8
9
1
11
12
10.12 ± 0.74
24.74 ± 1.56
83.14 ± 1.95
31.33 ± 2.24
52.44 ± 3.85
84.29 ± 3.23
87.94 ± 3.04
13.01 ± 0.35
94.49 ± 1.04
99.21 ± 0.04
with water (3 Â 100 mL), dried over anhydrous MgSO
and concentrated to dryness giving white solid residue
209.3 mg). A portion of this residue was separated by silica gel
4
, filtered
a
(
column chromatography (n-hexane/diethyl ether (80:20)) afford-
ing compound 8 in mixture with some impurities.
A pure sample of 8 was obtained from 9 by the following
procedure:
0
Formestane
To a stirred solution of 9 (15.3 mg, 0.049 mmol) in ethanol
(
0
3.0 mL), under nitrogen, at À10 °C, sodium borohydride (0.6 mg,
.016 mmol) was added. The mixture was stirred for 1 h and after
two subsequent additions of sodium borohydride (0.6 mg,
.016 mmol; 0.7 mg, 0.018 mmol), the reaction was left, at room
a
Concentrations of 40 nM [1b-3H] androstenedione, 20
placental microsomes, 2 M of the compounds and 15 min incubation were used.
Results represent the mean ± S.E.M. of three different experiments, performed in
triplicate. Formestane at 0.5 M was used as reference compound.
lg protein from human
l
l
0

6
66
C.L. Varela et al. / Steroids 78 (2013) 662–669
OH
O
1
i
OCOCH3
O
2
ii
OCOCH3
O
3
iii
OCOCH
3
OCOCH3
vi
OCOCH3
O
O
O
O
O
1
1
1
1
0
2
4
5
7
8
9
x
iv
v
vii
OH
OH
OH
ix
v
v
viii
O
O
O
O
6
Scheme 1. Synthesis of aromatase inhibitors from testosterone. Reagents and conditions: (i) (CH
18 °C, 3 h; (iii) TiCl , allyltrimethylsilane, dry dichloromethane, À78 °C, 40 min; (iv) 2% NaOH, dioxane/water, rt, overnight; (v) Jones Reagent, acetone/dioxane, 0 °C; (vi)
CF COOH, CH COOH, CH CN, NaBH , dry dichloromethane, rt, 1 h 30 min; (vii) 2% NaOH, dioxane/water, rt, 48 h; (viii) NaBH
, ethanol, À10 to 5 °C, overnight; (ix) DDQ,
benzoic acid, toluene, 118 °C, 34 h; (x) (CH CO O, dry pyridine, rt, 33 h 15 min.
3 2
CO) O, dry pyridine, rt, 21 h 30 min; (ii) chloranil, acetic acid, toluene,
1
4
3
3
3
4
4
3
2 2
)
all the starting material had been consumed (TLC control). Dichlo-
romethane (100 mL) was added and the organic layer was washed
hospital, human placenta were placed in cold 67 mM potassium
phosphate buffer (pH 7.4) containing 1% KCl. The cotyledon tissue
was separated and homogenized in a Polytron homogenizer with
67 mM potassium phosphate buffer (pH 7.4) containing 0.25 M su-
crose and 0.5 mM dithiothreitol (DTT, 1:1, w/v). The homogenate
was centrifuged at 5000g for 30 min and the supernatant was cen-
trifuged at 20,000g for 30 min and after, at 54,000g for 45 min to
yield the microsomal pellet. After ultra-centrifugation, the micro-
somes were washed and ressuspended in 67 mM potassium phos-
phate buffer (pH 7.4) containing 0.25 M sucrose, 20% glycerol, and
0.5 mM DTT and stored at À80 °C. All the procedures were carried
out at 0–5 °C. Using bovine serum albumin (BSA) as a standard, the
protein content was calculated by the Bio-Rad protein assay (Bio-
Rad Labs, Munich, Germany).
with 10% NaHCO
3
(2 Â 100 mL), 10% HCl (2 Â 100 mL) and water
(
2 Â 100 mL), dried over anhydrous MgSO
4
, filtered and concen-
trated to dryness. The resulting residue was crystalized from ethyl
acetate/n-hexane giving the pure compound 11 (29.6 mg, 54%) as
white needles: mp 155–159 °C (ethyl acetate/n-hexane); IR (NaCl
3
plates, CHCl ) 3076 (=CH), 1731 (C@O), 1661 (C@C), 1617 (C@C),
1
1
(
042 (C–O); H NMR (600 MHz, CDCl
3H, s, 19-H ), 2.04 (3H, s, CH COO), 4.59 (1H, dd, J17
,16b = 8.5, 17 -H), 5.01 (1H, ddd, CH@CH ), 5.05 (1H, ddd,
CH@CH ), 5.64 (1H, m, CH@CH ), 6.04 (1H, dd, 4,2 = 1.6,
= 1.5, 4-H), 6.23 (1H, dd, J2,1 = 10.1, J2,4 = 1.6, 2-H), 7.05 (1H,
3
) d 0.87 (3H, s, 18-H
3
), 1.24
= 8.5,
3
3
a,16a
J
17
a
a
2
2
2
J
J
4,6a
1
3
d, J1,2 = 10.1, 1-H); C NMR (150 MHz, CDCl
3
) d 11.9 (C-18), 19.2
(
C-19), 21.1, 22.1, 23.1, 27.3, 29.4, 35.4, 36.3, 37.5, 38.0, 42.7,
4
2
3
3.5, 45.0, 45.7, 82.2 (C-17), 117.1 (C-24), 126.4 (C-4), 127.6 (C-
2
.3.2. Aromatase assay procedure
By measuring the tritiated H O released from [1b- H] andro-
2
), 136.5 (C-23), 155.4 (C-1), 165.9 (C-5), 171.0 (C@O), 185.8 (C-
3
+
); MS (ESI) m/z 367.49 ([MÀH] , 100%).
stenedione during the aromatization process, the aromatase activ-
ity was measured in human placental microsomes according to
Thompson and Siiteri [21] and Heidrich et al. [25] method with
modifications [13]. The [1b- H] androstenedione was purchased
from PerkinElmer Life Sciences (Boston, MA, USA).
2
2
.3. Biochemistry
3
.3.1. Preparation of placental microsomes
The human placental microsomes were obtained as described
previously by our group [13,15]. After delivery from a local
The steroidal AIs tested were dissolved in DMSO and diluted in
67 mM potassium phosphate buffer (pH 7.4) before assay. To

C.L. Varela et al. / Steroids 78 (2013) 662–669
667
Table 2
IC50 and kinetic studies of the most potent inhibitors.
Compounds
IC50
(l
M)a
Type of inhibition
Kinetic Studiesb
Vm (mol/min/ug prot)
K
i
(nM)
Real affinity
(K /K ) (nM)
m
i
6
9
1
1
0.59
0.75
0.45
0.47
0.04
Competitive
Competitive
Competitive
Competitive
–
0.009 ± 0.001
0.017 ± 0.003
0.004 ± 0.002
0.006 ± 0.001
–
80.00
60.00
65.00
45.00
–
0.458 ± 0.042
0.014 ± 0.003
0.835 ± 0.041
0.926 ± 0.055
–
0
2
Formestane
a
Concentrations of 100 nM [1b-3H] androstenedione, 20
were used.
lg protein from human placental microsomes, different concentrations of the compounds and 15 min incubation
b
Concentrations of 10, 20 and 30 nM [1b-3H] androstenedione, 20
l
g protein from human placental microsomes, different concentrations of the compounds and 5 min
incubation were used. Apparent inhibition constants (K
i
) were obtained by Dixon Plot. Inhibition type was based on analysis of Lineweaver–Burk plot. The experiments were
done in triplicate in three independent experiments.
determine the percentage of aromatase inhibition as a screening
submitted to base-catalysed hydrolysis giving a crude product
which, after column chromatography, allowed obtaining alcohol
8, with some remaining inseparable impurities. Oxidation of the
impure compound 8 with Jones reagent gave rise, after column
chromatography, to the pure ketone 9, in 79% yield. Compound 8
was then further reobtained in its pure form as a white solid, in
29% yield, by reduction of ketone 9 with sodium borohydride in
ethanol [29].
assay, for the reaction mixture was used 20
tein, 40 nM of [1b- H] androstenedione (1
l
g of microsomal pro-
3
lCi) and 2 M of each
l
aromatase inhibitor in a final reaction volume of 1 mL. To deter-
3
mine the IC50 it was used 100 nM (1
one and different concentrations of the inhibitors (0.0125–5
The aromatase reaction was initiated by the addition of 150
l
Ci) of [1b- H] androstenedi-
lM).
lM
of reduced nicotinamide adenine dinucleotide phosphate (NADPH),
and incubations were performed in a shaking water bath at 37 °C
for 15 min. For the kinetic studies were used different concentra-
Aiming to prepare the D1-derivative 10 (Scheme 1), compound
5 was treated with 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ)
and benzoic acid in refluxing toluene [30]. After several filtrations
and purification by silica gel column chromatography, the alcohol
10 was isolated in 50% yield. Compound 10 was then treated with
acetic anhydride in pyridine giving a crude product which, after
ethyl acetate/n-hexane crystallization, led to acetate 11. Jones oxi-
dation of 10 led, after column chromatography purification, to the
pure ketone 12, in 88% yield.
3
tions of [1b- H] androstenedione (10–40 nM) and inhibitors
(
0–0.8
aromatization rate was used 5 min of incubation. The aromatase
reactions were stopped by addition of 250 L of 20% trichloroacetic
lM) and to minimize the time-dependent loss of the initial
l
acid (TCA) and the mixture was transferred to microcentrifuge
tubes containing a charcoal–dextran pellet, vortexed and incu-
bated during 1 h. After incubation and centrifugation at 14,000g
for 10 min, the supernatants were transferred to new charcoal-
dextran pellets and incubated for 10 min. Following a new
centrifugation cycle, the supernatant containing the tritiated water
product was mixed with a liquid scintillation cocktail from ICN
Radiochemicals (Irvine, CA, USA) and counted in a liquid scintilla-
tion counter (LS-6500, Beckman Coulter, Inc., Fullerton, CA). All
experiments were carried out in triplicate. As a reference com-
3.2. Biochemistry
In a series of A-, B- and D-ring modified steroids it was evalu-
ated the aromatase activity in human placental microsomes using
a radiometric assay [21]. Firstly, it was done a screening assay by
determining the percentage of aromatase inhibition (%) for all the
pound it was used formestane at 0.5 lM.
studied compounds at 2 lM (Table 1). Four of the evaluated ste-
roids presented an inhibitory aromatase activity higher than 80%.
The aromatase inhibitor 4-hydroxyandrostenedione (formestane)
3
. Results
.1. Chemistry
The reaction of testosterone 1 with acetic anhydride yielded its
at 0.5
was determined for the four most potent steroids, 6, 9, 10 and 12
Table 2). For these steroids, it was also performed kinetic studies
to characterize the type of binding to the active site of aromatase
and the apparent inhibition constant (K ), using different concen-
lM was used as a reference compound. Secondly, the IC50
3
(
i
acetate 2, in 84% yield (Scheme 1). Dehydrogenation of 2 with
chloranil was undertaken in a dry environment, leading to a solid
residue [26]. This residue was then purified by column chromatog-
raphy affording the dienone 3, in 58% yield. Subsequently, a Saku-
rai reaction was performed in 3 with allyltrimethylsilane and
titanium tetrachloride, in order to introduce the allyl group at
trations of inhibitor and substrate. As shown by the Lineweaver–
Burk plot in Fig. 2, the new compounds revealed to be competitive
inhibitors and by the Dixon plots it was characterized the enzyme
affinity (Fig. 3). Our results showed that the most potent AIs are
compounds 10 and 12, being 12 the most effective with higher
affinity to the enzyme aromatase.
the C-7
tained which was purified over column chromatography allowing
the isolation of the 7 -allyl derivative 4, in 56% yield. Compound
was next submitted to alkaline hydrolysis giving the alcohol 5,
a position [10,27]. After work-up, an oily crude was ob-
a
4. Discussion
4
in 96% yield. Compound 5 was subsequently oxidized using Jones
Reagent leading, after insolubilization in diisopropylether, to the
ketone 6, in 73% yield.
In order to obtain the 3-deoxo derivative 7 (Scheme 1), the reac-
tion of 4 with a mixture of sodium borohydride in trifluoracetic
acid, glacial acetic acid and acetonitrile was performed in a con-
trolled environment [28] giving, after column chromatography,
the almost pure compound 7. An analytical sample of 7 was ob-
tained by ethanol/water crystallization. This compound was then
As referred in the introduction, many studies suggest that the
presence of alkyl or phenyl groups in the C-6 or C-7 positions of
the steroidal androstane scaffold is beneficial for achieving aroma-
tase inhibitory activity [6–12,22]. This is because it was believed
that a hydrophobic binding pocket of the enzyme aromatase would
interact with these groups leading to a more effective ligand-en-
zyme interaction. Surprisingly, when, recently, the 3D structure
of aromatase in a complex with its substrate androstenedione
was elucidated [31], no particular binding pocket near the C-6

6
68
C.L. Varela et al. / Steroids 78 (2013) 662–669
and C-7 region of androstenedione was found. However, a shallow
hydrophobic crevice was observed to accommodate the extra C-6-
methylidene group of exemestane (Fig. 1), when its molecule was
built into the active site of aromatase. In a more recent work,
Ghosh and collaborators [11], determined the crystal structures
of inhibited complexes of aromatase with exemestane and two
other C-6-substituted 2-alkynyloxy compounds. It was found that,
the linear C-6-alkynyloxy side chains protrude into the access
channel cavity immobilizing the catalytic residues, resulting in
very potent inhibition. This means that the hydrophobic binding
pocket of aromatase can possibly be the access channel to the en-
zyme. Taking this in consideration along with the evidence that the
aromatase inhibitory activity is similar when the same substituent
is in position C-6 or C-7, of a given steroidal framework [9,12], we
allowing the establishment of hydrogen bonds with aromatase
active site residues, and also because the A-ring planarity is in-
1
creased, by the presence of the
D
double bond. In fact, the A-ring
planarity seems to assume an important role since the same type
of substitution in 6 to give 5 produces a dramatic decrease in the
aromatase inhibition (Table 1). The substitution of the C-17 car-
bonyl group of 12 by an acetate group as in 11, decreases the aro-
matase inhibitory activity (Table 1) even more than that observed
in the 3-deoxo derivatives series. This dramatic decrease was also
observed when substituting the C-17 carbonyl group of 6 by an
acetate group as in 4. This can be due to stereochemical conflicts
with aromatase active site, because of the larger size of inhibitors
4 and 11 when compared with 7.
In summary, several 7a-allyl derivatives of androstenedione
have decided to synthesize C-7
one by introducing an allyl group in its C-7 position (Scheme 1) and
obtaining compound 6, which is a strong aromatase inhibitor
a
-allyl derivatives of androstenedi-
were prepared and found to be strong aromatase inhibitors. Con-
cerning its SAR, the presence of a double bond in C-1 as in 10
and 12, which increases the planarity of the steroidal A-ring, is
beneficial for aromatase inhibition. The abstraction of the C-3 car-
bonyl group did not particularly favour the aromatase inhibitory
activity, in some of these compounds, being the introduction of a
double bond in C-1 more beneficial for aromatase inhibition than
the C-3 carbonyl abstraction. The substitution of the C-17 carbonyl
group by a hydroxyl group usually decreases the aromatase inhibi-
tion, except if a double bond in C-1 is present as in 10. The substi-
tution of the C-17 carbonyl group by an acetate group dramatically
decreases the aromatase inhibitory activity. This effect seems to be
lower when the length of the steroid molecule is also smaller as in
7.
(
IC50 = 0.59
by Labrie et al. [10]. From the 7
two main chemical modifications were further performed. One of
them consisted in C-3 decarbonylation in order to obtain 7
l
M, K
i
= 80.00 nM) (Tables 1 and 2) as before described
a
-allylandrostenedione scaffold,
a
-
allyl-3-deoxo-4-androstenes. Actually, it is known from previous
works [13,22,23], that the C-3 carbonyl group is not essential for
aromatase inhibition, since the C-17 carbonyl group remains in
the molecule [14]. In fact, aromatase establishes two hydrogen
bonds with the carbonyl groups at C-3 and C-17 [31] of its sub-
strate androstenedione and, at least one of them must be present
in the steroid molecule in order to bind the enzyme.
Other modification was the introduction of an additional double
bond at C-1 position aiming to obtain 7
a
-allyl-3-oxo-1,4-androsta-
Acknowledgments
dienes, since it is also known that the increase in A-ring planarity
and electronic density are very beneficial for aromatase inhibitory
activity [13–17]. In addition, for each set of derivatives it was stud-
ied the influence of several functional groups at C-17.
The authors are grateful to FCT (Fundação para a Ciência e Tecn-
ologia) for financial support and for the PhD grants for Carla Varela
and Cristina Amaral (SFRH/BD/44872/2008 and SFRH/BD/48190/
2008, respectively). We also acknowledge the ‘‘Rede Nacional de
RMN’’ (REDE/1517/RMN/2005) for access to the facilities. This
work was funded by FEDER Funds through the Operational Com-
petitiveness Program-COMPETE and by National Funds through
FCT under the project FCOMP-01-0124-FEDER-020970 (PTDC/
QUI-BIQ/120319/2010).
Regarding the 7
observed that the 3-deoxo analogue 9 of compound 6 is slightly
less potent (IC50 = 0.75 M) (Table 2) than 6 showing that, in this
a-allyl-3-deoxo-4-androstenes 7, 8 and 9, it was
l
case, the C-3 carbonyl group is important for aromatase inhibitory
activity. When one substitutes the C-17 carbonyl group of 9 by a
hydroxyl group as in 8, the activity decreased, as expected (Ta-
ble 1). In this case, no C-3 or C-17 carbonyl groups are present,
being therefore difficult the binding of the inhibitor to the enzyme
active site. Substituting the C-17 carbonyl group of 9 by an acetate
group as in 7, the aromatase inhibitory activity decreased even
more (Table 1). In fact, the volume of the aromatase active site is
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.
3
short (400 Å ) [31], and bulky substituents in the androstane
0
2.016.
framework are not allowed if one aims to obtain strong aromatase
inhibitors.
References
Concerning the 7
2, it was observed that the
IC50 = 0.47 M, K = 45.00 nM) with great affinity for the enzyme
a-allyl-3-oxo-1,4-androstadienes 10, 11 and
1
1
(
D
analogue 12 is a strong AI
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i
1
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at C-7 position, instead of the shorter one (methylene group) at
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