Synthesis and cytotoxic effect of pregnenolone derivatives with one or two α,β-unsaturated carbonyls and an ester moiety at C-21 or C-3

Article abstract of DOI:10.1016/j.steroids.2018.01.004

Four series of pregnenolone derivatives having one or two α,β-unsaturated carbonyls and an ester moiety at C-21 or C-3 were synthetized to compare their cytotoxicity effect. The final compounds were evaluated on three human cancer cell lines: PC-3 (prostate cancer), MCF-7 (breast cancer), SKLU-1 (lung cancer) and a noncancerous cell line HGF (human gingival fibroblast). Two steroids with a 4-fluorinated benzoic acid ester at C-21 were the most active against lung cancer cell line with IC₅₀ of 13.1 ± 1.2 and 12.8 ± 0.5 μM and showed a low percentage of cytotoxicity for noncancerous cells (27.63 ± 2.3 and 18.39 ± 1.2% in the screening at 50 μM).

Full text of DOI:10.1016/j.steroids.2018.01.004

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxic effect of pregnenolone derivatives with one or two
α,β-unsaturated carbonyls and an ester moiety at C-21 or C-3
Alejandra Chávez-Riverosa,⁎, Abigail Cruz Noriegab, María Teresa Ramírez Apana,
Luis D. Mirandaa, Eugene Bratoeffb,1
a
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 Cd.Mx., Mexico
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de Mexico, 04510 Cd.Mx., Mexico
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synthesis
Pregnenolone derivatives
Cytotoxicity
SKLU-1
Four series of pregnenolone derivatives having one or two α,β-unsaturated carbonyls and an ester moiety at C-21
or C-3 were synthetized to compare their cytotoxicity effect. The final compounds were evaluated on three
human cancer cell lines: PC-3 (prostate cancer), MCF-7 (breast cancer), SKLU-1 (lung cancer) and a non-
cancerous cell line HGF (human gingival fibroblast). Two steroids with a 4-fluorinated benzoic acid ester at C-21
were the most active against lung cancer cell line with IC50 of 13.1
1.2 and 12.8
0.5 μM and showed a low
percentage of cytotoxicity for noncancerous cells (27.63 2.3 and 18.39
1.2% in the screening at 50 μM).
1. Introduction
by our group have displayed cytotoxicity in cancer cell lines and high
activity as inhibitors of 5α-reductase enzyme [18–21].
Cancer is still one of the major public health problem in the world,
resulting the leading cause of death. Breast cancer is the most common
cancer for women whereas prostate cancer is for men. Lung cancer is
the main cause of death for both gender, more than 50% of patients will
eventually die of the disease. These three malignancies have the highest
incidence and death rate [1,2]. For this reason, the search of new
treatment alternatives is crucial.
The structural modification of steroid molecules has demonstrated
to be a powerful tool to obtain compounds with a notable improvement
in their biological activities, especially for cancer treatment [3–9]. The
main characteristics of this kind of structures is their good bioavail-
ability, less toxicity and low vulnerability to multi-drug resistance
[10,11].
In this work, we present four series of derivatives: Steroids 10a-e
with two carbonyls at C-3 and C-6 conjugated with Δ4 double bond and
the ester attached at C-21; and compounds 11c-g containing just one
α,β-unsaturated carbonyl group at C-3 and the ester at C-21.
Derivatives 13a-e and 14a-e having one α,β-unsaturated carbonyl at C-
6 and an aliphatic ester at C-3 (Fig. 1). With this design we wanted to
make a comparison between the two extreme positions of the steroid;
first comparing the effect of the ester attached at C-21 or C-3 and on the
other hand the effect of the enone with one or two carbonyl groups.
This study is a preliminary approximation to the structure-activity re-
lationship between the pregnenolone derivatives and their cytotoxicity
on cancer cell lines.
Pregnenolone, a naturally occurring steroid, is a precursor to several
hormones including cortisone, estrogen, testosterone and progesterone
[12–14]. Different pregnenolone derivatives have been synthesized and
evaluated for various biological activities including as potential antic-
ancer agents [15–17].
Many of the natural or synthetic bioactive steroids have an α,β-
unsaturated carbonyl in their structure, such as testosterone, proges-
terone, finasteride, dexamethasone, cortisol etc. This conjugation, un-
doubtedly plays an important role in the union with active sites, con-
ferring them remarkable biological activities. We have designed
steroidal derivatives containing this scaffold, these compounds reported
2. Experimental
2.1. General
Reagents and solvents were purchased from commercial sources and
were used without further purification. Melting points were determined
on a Fisher Johns melting point apparatus and were uncorrected. Purity
of series 10, 11, 13 and 14 was obtained by HPLC on a Waters e2695
equipment using an Eclipse XDB-C18 5 μm 4.6 × 150 mm column at
238 nm. 1H and 13C NMR were taken on an Inova Varian 400 MHz.
Chemical shifts are given in ppm relative to that of TMS (δ = 0) in
⁎
Corresponding author.
E-mail address: chavez-riveros@iquimica.unam.mx (A. Chávez-Riveros).
This work is in memory of Professor Eugene Bratoeff.
1
https://doi.org/10.1016/j.steroids.2018.01.004
Received 12 October 2017; Received in revised form 29 December 2017; Accepted 16 January 2018
0039-128X/©2018PublishedbyElsevierInc.
Pleasecitethisarticleas:Chávez-Riveros,A.,Steroids(2018),https://doi.org/10.1016/j.steroids.2018.01.004

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
17), 72.49 (C-3), 120.47 (C-6), 141.91 (C-5), 204.89 (C-20). LRMS
FAB+: 445 [M+H]+
.
2.2.3. 16α,17α-Epoxy-21-hydroxy-20,20-dimethoxy-3β-
tertbutyldimethylsilyloxypregna-5-en-20-one (4)
Compound 3 (1 g, 2.25 mmol) was dissolved in 10 mL of di-
chloromethane; 20 mL of a methanolic solution of NaOH (1 g, 25 mmol)
and (diacetoxyiodo)benzene (1.2 g, 3.72 mmol) were added. The mix-
ture was stirred for 18 h. The solvent was evaporated in vacuo, water
was added (2 × 20 mL), and the precipitate was filtered and washed
with water (2 × 20 mL). The crude was purified on silica gel using 10%
ethyl acetate in hexane to give 0.74 g of the pure compound 4. Yield:
65%, mp 205–207 °C. IR (cm−1): 3596, 1666, 1070, 1033. 1H NMR
(CDCl3) δ: 0.05 (m, 6H, (CH3)2Si), 0.89 (s, 9H, (CH3)3CSi), 1.02 (s, 3H,
H-18), 1.26 (s, 3H, H-19), 2.2(m, 1H, H-16), 3.24 (m, 1H, H-3), 3.25 (m,
6H, CH3O–), 3.46 (s, 1H, –OH), 3.64 (d, J = 6 Hz, 1H, H-21), 5.31 (m,
1H, H-6). 13C NMR (CDCl3) δ: −4.96, 15.10 (C-18), 19.34 (C-19), 25.95
((CH3)3CSi), 27.12, 31.55 (C-Si), 32.60, 36.76, 36.98, 42.10, 43.71,
46.26, 49.63 (C-16), 50.34 (CH3OH–), 58.33, 62.64 (C-21), 69.65 (C-
17), 72.51 (C-3), 101.86 (C-20), 120.70 (C-6), 141.81 (C-5). LRMS
FAB+: 507 [M+H]+
.
Fig. 1. Position of the conjugated carbonyl group (red) and ester moiety (blue) in the
synthesized series. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
2.2.4. General methodology for the synthesis of compounds 6a-g
16α,17α-Epoxy-21-hydroxy-20,20-dimethoxy-3β-tert-butyldi-
methylsilyloxypregna-5-en-20-one 4 (1 eq.) was dissolved in CH2Cl2
(0.05 M). DCC (6.7 eq.), DMAP (6.7 eq.) and the corresponding car-
boxylic acid (6.7 eq.) were added. The resulting solution was stirred at
room temperature for 4 h. Ethyl acetate (100 mL) was added and the
precipitated dicyclohexyl urea was filtered. The organic phase was
washed with 10% aqueous hydrochloric acid (3 × 30 mL), 5% aqueous
sodium bicarbonate (3 × 30 mL) and water (3 × 30 mL), the solvent
was dried over anhydrous sodium sulfate and evaporated under vacuum
to get compound 5. The crude ester 5 was dissolved in acetone (0.1 M),
and to this solution hydrochloric acid (30%) was added drop by drop
and the mixture was stirred for 15 min, then water was added and the
precipitate was filtered and washed with water. The crude was purified
by column chromatography using 10% ethyl acetate in hexane to obtain
series 6a-g.
CDCl3. Low Resolution Mass Spectra (LRMS) were obtained with a
Thermo DFS spectrometer by direct infusion and using FAB+ ionization
mode and High Resolution Mass Spectra (HRMS) were recorded with an
AccuTOF LC equipment with an ionSense DART controller ionization
source. IR spectra were recorded on a PerkinElmer 200 spectrometer.
2.2. Synthesis of series 10a-e and 11a-e
2.2.1. 16α,17α-Epoxy-3β-hydroxypregna-5-en-20-one (2)
The 16-dehydropregnenolone acetate (1 g, 2.8 mmol) was dissolved
in MeOH (66 mL) and a 4 N aqueous solution of NaOH (2 mL) was
added. To this mixture, a 30% hydrogen peroxide solution (4 mL) was
added drop by drop. The reaction was stirred at room temperature for
4 h. The solvent was evaporated, and cold water was added. The pre-
cipitate was filtered and washed with water (2 × 10 mL). The crude
was purified by recrystallization from methanol to afford 0.87 g of the
pure compound. Yield: 95%, mp 180–182 °C. IR (cm−1): 3454, 2936,
1692, 1642, 1042. 1H NMR (CDCl3) δ: 1.05 (s, 3H, H-18), 1.02 (s, 3H,
H-19), 2.2 (m, 2H, H-21), 2.3 (m, 1H, H-16), 3.5 (m, 1H, H-3), 3.68 (s,
1H, –OH), 5.33 (m, 1H, H-6). 13C NMR (CDCl3) δ: 15.16 (C-18), 19.30
(C-19), 20.01, 26.62, 27.51 (C-21), 29.23, 32.43, 32.51, 37.57, 42.06,
42.16, 45.31, 49.73, 60.49 (C-16), 60.61, 71.02 (C-17), 71.60 (C-3),
2.2.4.1. 21-Acetoxy-16α,17α-epoxy-3β-hydroxy-5-pregnen-20-one
(6a). Yield: 87%, mp 174–176 °C. IR (cm−1): 3566, 2943, 1754, 1380,
1058. 1H NMR (CDCl3) δ ppm 1.02 (s, 3H, H-18), 1.11 (s, 3H, H-19),
2.16 (s, 3H, –COOCH3), 3.52 (m, 1H, H-3), 3.79 (s, 1H, –OH), 4.62 (d,
J = 16 Hz, 1H, H-21), 4.70 (d, J = 16 Hz, 1H, H-21), 5.33 (s, 1H, H-6).
13C NMR (100 MHz, CDCl3) δ: 15.13 (C-18), 19.32 (C-19), 20.38
(CH3COO–), 27.48, 29.61, 31.50, 36.24, 36.32, 41.98, (C-4), 45.56,
45.71, 50.11, 61.55 (C-21), 65.05 (C-16), 70.56 (C-17), 71.58 (C-3),
120.89 (C-6), 141.11 (C-5), 170.41 (CH3COO–), 199.16 (C-20). LRMS
120.95 (C-6), 141.12 (C-5), 204.90 (C-20). LRMS FAB+: 331 [M+H]+
.
FAB+: 389 [M+H]+
.
2.2.2. 16α,17α-Epoxy-3β-tert-butyldimethylsilyloxypregna-5-en-20-one
(3)
2.2.4.2. 16α,17α-Epoxy-3β-hydroxy-21-propionoxy-5-pregnen-20-one
(6b). Yield: 80%, mp 186–187 °C. IR (cm−1): 3326, 2930, 1744, 1379,
1057. 1H NMR (CDCl3) δ: 1.03 (s, 3H, H-18), 1.12 (s, 3H, H-19), 2.28
(m, 3H, –COOCH2CH3), 2.24 (m, 2H, –COOCH2CH3), 3.49 (m, 1H, H-3),
3.8 (s, 1H, –OH), 4.63 (d, J = 20 Hz, 1H, H-21), 4.71 (d, J = 20 Hz, 1H,
H-21), 5.31 (s, H-6). 13C NMR (CDCl3) δ: 8.98 (–COOCH2CH3), 15.14
(C-18), 19.32 (C-19), 20.15 (–COOCH2CH3), 27.10, 29.71, 32.89,
35.70, 37.96, 42.35, 42.41 (C-4), 45.38, 45.61, 49.96, 61.54 (C-21),
65.78 (C-16), 70.57 (C-17), 71.58 (C-3), 120.91 (C-6), 141.10 (C-5),
Compound 2 (1 g, 3 mmol) was dissolved in 15 mL of DMF, tert-
butyldimethylsilyl chloride (0.76 g, 6.8 mmol) and imidazole (0.450 g,
5 mmol) were added to this solution. The mixture was stirred at room
temperature for 2 h. After completion of the reaction, the solvent was
evaporated and water was added. The precipitate was filtered and
washed with water (2 × 20 mL). The crude was purified by column
chromatography using a mixture of 20% ethyl acetate in hexane to give
1.3 g of the pure compound 3. Yield: 98%, mp 126–127 °C. IR (cm−1):
2929, 1698, 1659, 1083. 1H NMR (CDCl3) δ: 0.58 (m, 6H, (CH3)2Si),
0.89 (s, 9H, (CH3)3CSi), 1.0 (s, 3H, H-18), 1.04 (s, 3H, H-19), 2.3 (s, 3H,
H-21), 2.2(m, 1H, H-16), 3.4 (m, 1H, H-3), 5.24 (d, J = 4.9 Hz, 1H, H-
6). 13C NMR (CDCl3) δ: −0.51, 15.18 (C-18), 17.98, 19.38 (C-19),
21.51, 25.81, 25.93 ((CH3)3CSi), 27.50 (C-21), 30.02, 31.49 (C-Si),
32.04 37.48, 41.90, 43.26, 43.02, 45.92, 51.18, 60.52 (C-16), 71.07 (C-
173.88 (–COOCH2CH3), 199.26 (C-20). LRMS FAB+: 403 [M+H]+
.
2.2.4.3. 21-Benzoyloxy-16α,17α-epoxy-3β-hydroxy-5-pregnen-20-one
(6c). Yield: 82%, mp 202–203 °C. IR (cm−1): 3484, 3206, 1739, 1379,
1051. 1H NMR (CDCl3) δ: 1.02 (s, 3H, H-18), 1.13 (s, 3H, H-19), 3.52
(m, 1H, H-3), 3.87 (s, 1H, –OH), 4.86 (d, J = 16 Hz, 1H, H-21), 4.94 (d,
J = 16 Hz, 1H, H-21), 5.33 (s, 1H, H-6), 7.45 (t, J = 8 Hz, 2H, H-Ar),
2

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7.58 (t, J = 8 Hz, 1H, H-Ar), 8.08 (d, J = 8 Hz, 2H, H-Ar). 13C NMR
(CDCl3) δ: 15.15 (C-18), 19.30 (C-19), 20.03, 24.43, 27.86, 28.16,
29.63, 33.82, 37.30, 42.18 (C-4), 45.23, 50.11, 61.58 (C-21), 66.33 (C-
16), 70.03 (C-17), 71.58 (C-3), 120.88 (C-6), 126.40 (C-Ar), 128.40 (C-
Ar), 129.85 (C-Ar), 133.31 (C-Ar), 141.12 (C-5), 165.99(–COO) 198.94
5.35 (s, 1H, H-6), 6.74 (s, 1H, H-16). 13C NMR (CDCl3) δ: 15.83 (C-18),
19.28 (C-19), 20.53 (CH3COO–), 29.96, 31.29, 32.26, 34.27, 36.54, 37.09,
42.06, 46.99, 50.96, 56.23, 65.62 (C-21), 71.64 (C-3), 120.90 (C-6),
141.37 (C-5), 144.07 (C-16), 151.96 (C-17), 170.44 (CH3COO–), 190.54
(C-20). LRMS FAB+: 373 [M+H]+
.
(C-20). LRMS FAB+: 451 [M+H]+
.
2.2.5.2. 3β-Hydroxy-21-propanoyloxy-5,16-pregnadien-20-one
(7b). Yield: 60%, mp 183–185 °C. IR (cm−1): 3486, 2929, 1732, 1676.
1H NMR (CDCl3) δ: 0.93 (s, 3H, H-18), 1.03 (s, 3H, H-19), 1.18 (m, 3H,
–COOCH2CH3), 2.48 (m, 2H, –COOCH2CH3), 3.51 (m, 1H, H-3), 4.2 (s,
1H, –OH), 4.88 (d, J = 16 Hz, 1H, H-21), 5.03 (d, J = 16 Hz, 1H, H-21),
5.34 (s, 1H, H-6), 6.75 (s, 1H, H-16). 13C NMR (CDCl3) δ: 9.03
(COOCH2CH3), 15.84 (C-18), 19.29 (C-19), 20.88, 27.19
(CH3CH2COO–), 30.04, 31.96, 32.90, 33.83, 34.09, 42.38, 46.83,
50.46, 56.19, 65.49 (C-21), 71.67 (C-3), 120.91 (C-6), 141.37 (C-5),
143.92 (C-16), 152.02 (C-17), 173.86 (CH3CH2COO–), 190.69 (C-20).
2.2.4.4. 21-(p-Fluoro)benzoyloxy-16α,17α-epoxy-3β-hydroxy-5-pregnen-
20-one (6d). Yield: 82%, mp 210–212 °C. IR (cm−1): 3320, 3044, 2936,
1721, 1381, 1055. 1H NMR (CDCl3) δ: 1.02 (s, 3H, H-18), 1.13 (s, 3H,
H-19), 3.48 (m, 1H, H-3), 3.87 (s, 1H, –OH), 4.85 (d, J = 16 Hz, 1H, H-
21), 4.94 (d, J = 16 Hz, 1H, H-21), 5.33 (s, 1H, H-6), 7.12 (t, J = 12 Hz,
2H, H-Ar), 8.1 (t, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ: 15.17 (C-
18), 19.33 (C-19), 20.10, 27.89, 29.91, 31.84, 31.96, 37.26, 42.18 (C-
4), 42.35, 45.47, 50.09, 61.66 (C-21), 66.44 (C-16), 70.66 (C-17), 71.60
(C-3), 115.74 (C-Ar), 120.91 (C-6), 125.54 (C-Ar), 132.53 (C-Ar),
141.13 (C-5), 165.03 (–COO), 167.26 (C-Ar), 198.85 (C-20). LRMS
LRMS FAB+: 387 [M+H]+
.
FAB+: 469 [M+H]+
.
2.2.5.3. 21-Benzoyloxy-3β-hydroxy-5,16-pregnadien-20-one (7c). Yield:
61%, mp 163–165 °C. IR (cm−1): 3484, 2932, 1730, 1675. 1H NMR
(CDCl3) δ: 0.97 (s, 3H, H-18), 1.04 (s, 3H, H-19), 3.53 (m, 1H, H-3),
4.17 (s, 1H, –OH), 4.62 (d, J = 16 Hz, 1H, H-21), 5.13 (d, J = 16 Hz,
1H, H-21), 5.37 (s, 1H, H-6), 6.84 (s, 1H, H-16), 7.45 (t, J = 12 Hz, 2H,
H-Ar), 7.58 (t, J = 8 Hz, 1H, H-Ar), 8.11 (d, J = 12 Hz, 2H, H-Ar). 13C
NMR (CDCl3) δ: 15.94 (C-18), 19.35 (C-19), 20.08, 30.86, 32.04, 33.58,
34.16, 36.92, 37.78, 42.97, 46.22, 51.43, 56.25, 66.12 (C-21), 71.72
(C-3), 120.99 (C-6), 128.43 (C-Ar), 129.96 (C-Ar), 133.30 (C-Ar),
141.41 (C-5), 144.24 (C-16), 152.08 (C-17), 166.11 (–COO), 190.53
2.2.4.5. 21-(p-Chloro)benzoyloxy-16α,17α-epoxy-3β-hydroxyi-5-pregnen-
20-one (6e). Yield: 81%, mp 164–166 °C. IR (cm−1): 3366, 3210, 2927,
1733, 1380, 1067. 1H NMR (CDCl3) δ: 1.02 (s, 3H, H-18), 1.13 (s, 3H,
H-19), 3.52 (m, 1H, H-3), 3.87 (s, 1H, –OH), 4.84 (d, J = 20 Hz, 1H, H-
21), 4.94 (d, J = 16 Hz, 1H, H-21), 5.35 (s, 1H, H-6), 7.43 (d, J = 8 Hz,
2H, H-Ar), 8.1 (d, J = 12 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ: 15.15 (C-
18), 19.31 (C-19), 20.08, 27.98, 29.89, 33.75, 34.15, 42.23, 42.50 (C-
4), 45.32, 50.46, 61.62 (C-21), 66.48 (C-16), 70.65 (C-17), 71.61 (C-3),
120.90 (C-6), 127.77 (C-Ar), 128.77 (C-Ar), 131.24 (C-Ar), 139.82 (C-
Ar), 141.11 (C-5), 165.12 (–COO), 198.78 (C-20). LRMS FAB+: 485 [M
(C-20). LRMS FAB+: 435 [M+H]+
.
+H]+
.
2.2.5.4. 21-(p-Fluoro)benzoyloxy-3β-hydroxy-5,16-pregnadien-20-one
(7d). Yield: 69%, mp 280–282 °C. IR (cm−1): 3325, 2933, 1723, 1677.
1H NMR (CDCl3) δ: 0.95 (s, 3H, H-18), 1.03 (s, 3H, H-19), 3.51 (m, 1H,
H-3), 3.85 (s, 1H, –OH), 5.11 (d, J = 16 Hz, 1H, H-21), 5.25 (d,
J = 16 Hz, 1H, H-21), 5.34 (s, 1H, H-6), 6.82 (s, 1H, H-16), 7.11 (t,
J = 8 Hz, 2H, H-Ar), 8.11 (t, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ:
15.88 (C-18), 19.29 (C-19), 20.49, 30.18, 31.93, 33.52, 34.69, 36.87,
37.75, 42.16, 46.56, 50.06, 56.14, 66.09 (C-21), 71.68 (C-3), 115.67
(C-Ar), 120.93 (C-6), 125.70 (C-Ar), 132.45 (C-Ar), 141.34 (C-5),
144.26 (C-16), 152.00 (C-17), 165.07 (–COO), 167.21 (C-Ar) 190.31
2.2.4.6. 21-(p-Bromo)benzoyloxy-16α,17α-epoxy-3β-hydroxy-5-pregnen-
20-one (6f). Yield: 83%, mp 172–174 °C. IR (cm−1): 3567, 3322, 2926,
1754, 1380, 1058. 1H NMR (CDCl3) δ: 1.02 (s, 3H, H-18), 1.11 (s, 3H,
H-19), 3.52 (m, 1H, H-3), 3.78 (s, 1H, –OH), 4.61 (d, J = 16 Hz, 1H, H-
21), 4.69 (d, J = 20 Hz, 1H, H-21), 5.33 (s, 1H, H-6), 7.6 (d, J = 12 Hz,
2H, H-Ar), 7.95 (d, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ: 15.14 (C-
18), 19.33 (C-19), 20.06, 27.79, 29.86, 31.84, 31.99, 37.12, 42.20 (C-
4), 42.47, 45.38, 50.73, 61.55 (C-21), 65.92 (C-16), 70.58 (C-17), 71.65
(C-3), 120.94 (C-6), 128.62 (C-Ar), 131.61 (C-Ar), 131.80 (C-Ar),
141.12 (C-5), 170.37 (–COO), 199.12 (C-20). LRMS FAB+: 529 [M
(C-20). LRMS FAB+: 453 [M+H]+
.
+H]+
.
2.2.5.5. 21-(p-Chloro)benzoyloxy-3β-hydroxy-5,16-pregnadien-20-one
(7e). Yield: 60%, mp 168–170 °C. IR (cm−1): 3252, 2934, 1723, 1670.
1H NMR (CDCl3) δ: 0.96 (s, 3H, H-18), 1.04 (s, 3H, H-19), 3.53 (m, 1H,
H-3), 3.86 (s, 1H, –OH), 5.12 (d, J = 16 Hz, 1H, H-21), 5.26 (d,
J = 16 Hz, 1H, H-21), 5.37 (s, 1H, H-6), 6.83 (s, 1H, H-16), 7.43 (d,
J = 8 Hz, 2H, H-Ar), 8.04 (d, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ:
15.88 (C-18), 19.29 (C-19), 20.50, 30.19, 31.95, 33.63, 34.71, 36.92,
37.76, 42.18, 46.58, 50.10, 56.17, 66.15 (C-21), 71.68 (C-3), 120.92
(C-6), 127.93 (C-Ar), 128.74 (C-Ar), 131.29 (C-Ar), 139.73 (C-Ar),
141.35 (C-5), 144.28 (C-16), 152.01 (C-17), 165.19 (–COO), 190.19 (C-
2.2.4.7. 21-(p-Iodo)benzoyloxy-16α,17α-epoxy-3β-hydroxy-5-pregnen-
20-one (6g). Yield: 82%, mp 216–218 °C. IR (cm−1): 3474, 3324, 2931,
1721, 1379, 1056. 1H NMR (400 MHz, CDCl3) δ ppm 1.02 (s, 3H, H-18),
1.13 (s, 3H, H-19), 3.53 (m, 1H, H-3), 3.86 (s, 1H, –OH), 4.84 (d,
J = 16 Hz, 1H, H-21), 4.94 (d, J = 16 Hz, 1H, H-21), 5.34 (s, 1H, H-6),
7.8 (m, 4H, H-Ar). 13C NMR (100 MHz, CDCl3) δ ppm 15.17 (C-18),
19.34 (C-19), 20.13, 27.86, 30.02, 31.96, 42.21 (C-4), 42.57, 45.61,
50.03, 58.13, 61.64 (C-21), 66.51 (C-16), 70.68 (C-17), 71.65 (C-3),
101.25 (C-Ar), 120.93 (C-6), 128.83 (C-Ar), 131.26 (C-Ar), 137.81 (C-
Ar), 141.13 (C-5), 165.54 (–COO), 198.75 (C-20). LRMS FAB+: 577 [M
20). LRMS FAB+: 469 [M+H]+
.
+H]+
.
2.2.5. General methodology for the synthesis of compounds 7a-g
Compound 6 (1 eq.) was dissolved in acetone (0.02 M), CrCl2
(5.25 eq.) and acetic acid were added (2%) and the mixture was stirred
at room temperature for 45 min. Ice and water (100 mL) were then
added and the precipitate was filtered. The crude was purified on silica
gel using 10% ethyl acetate in hexane.
2.2.5.6. 21-(p-Bromo)benzoyloxy-3β-hydroxy-5,16-pregnadien-20-one
(7f). Yield, 63%, mp 157–159 °C. IR (cm−1): 3466, 2930, 1729, 1677.
1H NMR (CDCl3) δ: 0.94 (s, 3H, H-18), 1.05 (s, 3H, H-19), 3.51 (m, 1H,
H-3), 3.78 (s, 1H, –OH), 4.88 (d, J = 16 Hz, 1H, H-21), 5.03 (d,
J = 16 Hz, 1H, H-21), 5.35 (s, 1H, H-6), 6.75 (s, 1H, H-16), 7.6 (d,
J = 8 Hz, 2H, H-Ar), 7.95 (d, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ:
15.86 (C-18), 19.31 (C-19), 20.61, 30.21, 32.05, 33.53, 34.85, 36.83,
37.82, 42.23, 46.70, 50.22, 56.33, 65.65 (C-21), 71.69 (C-3), 120.94
(C-6), 127.51 (C-Ar), 129.51 (C-Ar), 131.11 (C-Ar), 132.04 (C-.Ar),
141.39 (C-5), 144.03 (C-16), 152.02 (C-17), 170.43 (–COO), 190.55 (C-
2.2.5.1. 21-Acetoxy-3β-hydroxy-5,16-pregnadien-20-one (7a). Yield: 65%,
mp 156–157 °C. IR (cm−1): 3464, 2935, 1729, 1677. 1H NMR (CDCl3) δ:
0.93 (s, 3H, H-18), 1.03 (s, 3H, H-19), 2.16 (s, 3H, –COOCH3), 3.51 (m,
1H, H-3), 4.87 (d, J = 16 Hz, 1H, H-21), 5.01 (d, J = 16 Hz, 1H, H-21),
20). LRMS FAB+: 513 [M+H]+
.
3

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
2.2.5.7. 21-(p-Iodo)benzoyloxy-3β-hydroxy-5,16-pregnadien-20-one
(7g). Yield: 57%, mp 195–197 °C. IR (cm−1): 3476, 2932, 1735, 1677.
1H NMR (CDCl3) δ: 0.96 (s, 3H, H-18), 1.04 (s, 3H, H-19), 3.53 (m, 1H,
H-3), 3.85 (s, 1H, –OH), 5.12 (d, J = 16 Hz, 1H, H-21), 5.25 (d,
J = 16 Hz, 1H, H-21), 5.77 (s, 1H, H-6), 6.82 (s, 1H, H-16), 7.8 (m,
4H, H-Ar). 13C NMR (CDCl3) δ: 15.17 (C-18), 19.31 (C-19), 30.15,
32.20, 33.76, 34.65, 36.71, 37.74, 42.19, 45.95, 50.21, 56.28, 66.19 (C-
21), 71.72 (C-3), 101.15 (C-Ar), 120.95 (C-6), 129.00 (C-Ar), 131.33
(C-Ar), 137.77 (C-Ar), 141.37 (C-5), 144.25 (C-16), 152.06 (C-17),
16), 151.81 (C-17), 165.08 (COO–), 167.22 (C-Ar), 198.8 (C-20). LRMS
FAB+: 469 [M+H]+
.
2.2.7. General methodology for the synthesis of compounds 9a-d
Compound 8 (1 eq.) was dissolved in acetone (0.1 M). An aqueous
solution (1.8 M) of chromium trioxide was added dropwise at 0 °C for
10 min. The resulting mixture was allowed to warm up to room tem-
perature and after 30 min the same amount of chromium trioxide was
added in the same manner. The mixture was diluted with cold water
(75 mL) and the precipitate was filtered and dried. The product was
recrystallized from methanol.
165.59 (–COO), 190.17 (C-20). LRMS FAB+: 561 [M+H]+
.
2.2.6. General methodology for the synthesis of compounds 8a-d
Compound 7 (1 eq.) and m-chloroperbenzoic acid (3 eq.) were dis-
solved in dichloromethane (0.07 M), the reaction was stirred at room
temperature for 45 min. After this, a saturated aqueous solution of so-
dium bicarbonate (30 mL) containing sodium bisulfite (0.3 g) was
added. The reaction mixture was extracted with dichloromethane
(3 × 30 mL); the organic phase was washed with water (3 × 30 mL),
dried over sodium sulfate, and the solvent was removed under vacuum.
The product was recrystallized from methanol.
2.2.7.1. 21-Acetoxy-5α-hydroxy-16-pregnen-3,6,20-trione (9a). Yield: 82%,
mp 218–220 °C. IR (cm−1): 3346, 2943, 1711, 1681. 1H NMR (CDCl3) δ:
0.89 (s, 3H, H-18), 1.0 (s, 3H, H-19), 2.14 (s, 3H, –COOCH3), 2.84 (d,
J = 16 Hz, 1H, H-4), 2.87 (d, J = 16 Hz, 1H, H-4), 3.78 (s, 1H, –OH), 4.85
(d, J = 16 Hz, 1H, H-21), 4.99 (d, J = 16 Hz, 1H, H-21), 6.71 (s, 1H, H-16).
13C NMR (CDCl3) δ: 13.75 (C-18), 15.88 (C-19), 20.48 (CH3COO–), 22.31,
31.97, 32.18, 34.16, 35.77, 37.86, 42.09, 43.67, 45.21, 45.79, 55.89, 65.58
(C-21), 82.69 (C-5), 143.44 (C-16), 151.57 (C-17), 170.44 (CH3COO–),
190.44 (C-20), 210.2 (C-3), 211.13 (C-6). LRMS FAB+: 403 [M+H]+
.
2.2.6.1. 21-Acetoxy-5α,6α-epoxy-3β-hydroxy-16-pregnen-20-one
(8a). Yield: 78%, mp 191–195 °C. IR (cm−1): 3446, 2936, 1749, 1677,
1076. 1H NMR (CDCl3) δ: 0.84 (s, 3H, H-18), 1.06 (s, 3H, H-19), 2.14 (s,
3H, –COOCH3), 2.89 (d, J = 4 Hz, 1H, H-6), 3.45 (s, 1H, –OH), 3.86 (m,
1H, H-3), 4.8 (d, J = 16 Hz, 1H, H-21), 4.99 (d, J = 16 Hz, 1H, H-21),
6.69 (s, 1H, H-16). 13C NMR (CDCl3) δ: 15.74 (C-18), 15.88 (C-19),
20.30 (CH3COO–), 27.86, 27.95, 31.15, 32.26, 32.45, 33.90, 35.13,
39.97, 42.36, 46.77, 55.82, 58.75 (C-6), 65.57 (C-21), 65.8 (C-5), 68.59
(C-3), 143.7 (C-16), 151.78 (C-17), 170.38 (CH3COO–), 190.48 (C-20).
2.2.7.2. 5α-Hydroxy-21-propanoyloxy-16-pregnen-3,6,20-trione
(9b). Yield: 76%, mp 288–190 °C. IR (cm−1): 3327, 2943, 1750, 1678.
1H NMR (CDCl3) δ: 0.86 (s, 3H, H-18), 0.97 (s, 3H, H-19), 1.12 (m, 3H,
–COOCH2CH3), 2.4 (m, 2H, –COOCH2CH3), 2.8 (d, J = 16 Hz, 1H, H-4),
2.87 (d, J = 16 Hz, 1H, H-4), 3.75 (s, 1H, –OH), 4.86 (d, J = 16 Hz, 1H,
H-21), 4.98 (d, J = 16 Hz, 1H, H-21), 6.67 (s, 1H, H-16). 13C NMR
(CDCl3) δ: 9.95 (COOCH2CH3), 12.80 (C-18), 14.93 (C-19), 23.75,
23.80, 26.19 (CH3 CH2COO–), 31.24, 32.24, 34.22, 35.89, 37.93,
42.26, 43.74, 46.11, 55.90, 64.47 (C-21), 81.79 (C-5), 142.30 (C-16),
150.67 (C-17), 172.80 (CH3CH2COO–), 189.59 (C-20), 209.05 (C-3),
LRMS FAB+: 389 [M+H]+
.
2.2.6.2. 5α,6α-Epoxy-3β-hydroxy-21-propanoyloxy-16-pregnen-20-one
(8b). Yield: 70%, mp 219–221 °C. IR (cm−1): 3493, 2929, 1729, 1679,
1056. 1H NMR (CDCl3) δ: 0.86 (s, 3H, H-18), 1.08 (s, 3H, H-19), 1.17
(m, 3H, –COOCH2CH3), 2.47 (m, 2H, –COOCH2CH3), 2.91 (d, J = 4 Hz,
1H, H-6), 3.47 (s, 1H, –OH), 3.89 (m, 1H, H-3), 4.84 (d, J = 16 Hz, 1H,
H-21), 5.03 (d, J = 16 Hz, 1H, H-21), 6.72 (s, 1H, H-16). 13C NMR
(CDCl3) δ ppm 9.02 (COOCH2CH3), 15.77 (C-18), 15.90 (C-19), 20.36,
27.18 (CH3 CH2COO–), 27.90, 27.99, 31.18, 32.33, 32.45, 34.26,
35.25, 39.98, 42.70, 55.99, 58.80 (C-6), 65.47 (C-21), 65.80 (C-5),
68.60 (C-3), 143.63 (C-16), 151.84 (C-17), 173.85 (CH3CH2COO–),
209.94 (C-6). LRMS FAB+: 417 [M+H]+
.
2.2.7.3. 21-Benzoyloxy-5α-hydroxy-16-pregnen-3,6,20-trione
(9c). Yield: 78%, mp 203–205 °C. IR (cm−1): 3324, 2928, 1711, 1679.
1H NMR (CDCl3) δ: 0.92 (s, 3H, H-18), 1.25 (s, 3H, H-19), 2.88 (s, 2H,
H-4), 3.6 (s, 1H, –OH), 5.13 (d, J = 16 Hz, 1H, H-21), 5.27 (d,
J = 16 Hz, 1H, H-21), 6.83 (s, 1H, H-16), 7.46 (t, J = 8 Hz, 2H, H-
Ar), 7.5 (m, 1H, H-Ar), 8.1 (d, J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ:
14.05 (C-18), 15.95 (C-19), 23.89, 29.22, 29.78, 33.80, 35.66, 36.79,
38.45, 41.88, 44.36, 47.69. 68.15 (C-21), 82.72 (C-5), 128.41 (C-Ar),
128.78 (C-Ar), 129.91 (C-Ar), 130.88 (C-Ar), 132.41 (C-Ar), 133.31 (C-
Ar), 143.54 (C-16), 151.66 (C-17), 167.7 (–COO), 190.38 (C-20),
190.66 (C-20). LRMS FAB+: 403 [M+H]+
.
2.2.6.3. 21-Benzoyloxy-5α,6α-epoxy-3β-hydroxy-16-pregnen-20-one
(8c). Yield: 75%, mp 194–197 °C. IR (cm−1): 3423, 2928, 1721, 1624,
1087. 1H NMR (CDCl3) δ: 0.92 (s, 3H, H-18), 1.12 (s, 3H, H-19), 2.95
(d, J = 4 Hz, 1H, H-6), 3.49 (s, 1H, –OH), 3.92 (m, 1H, H-3), 5.13 (d,
J = 16 Hz, 1H, H-21), 5.3 (d, J = 16 Hz, 1H, H-21), 6.84 (s, 1H, H-16),
7.48 (t, J = 12 Hz, 2H, H-Ar), 7.61 (t, J = 12 Hz, 1H, H-Ar), 8.13 (d,
J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ: 15.86 (C-18), 15.94 (C-19),
20.42, 28.24, 28.36, 31.47, 32.12, 32.45, 34.66, 35.51, 39.79, 42.56,
46.87, 55.97, 58.90 (C-6), 65.97 (C-21), 66.10 (C-5), 68.56 (C-3),
128.43 (C-Ar), 129.93 (C-Ar), 133.32 (C-Ar), 144.01 (C-16), 151.82 (C-
210.22 (C-3), 210.9 (C-6). LRMS FAB+: 465 [M+H]+
.
2.2.7.4. 21-(p-Fluoro)benzoyloxy-5α-hydroxy-16-pregnen-3,6,20-trione
(9d). Yield: 86%, mp 135–136 °C. IR (cm−1): 3332, 2950, 1710, 1685.
1H NMR (CDCl3) δ: 0.95 (s, 3H, H-18), 1.04 (s, 3H, H-19), 2.8 (m, 2H,
H-4), 3.89 (s, 1H, –OH), 5.12 (d, J = 16 Hz, 1H, H-21), 5.26 (d,
J = 16 Hz, 1H, H-21), 6.81 (s, 1H, H-16), 7.13 (t, J = 8 Hz, 2H, H-
Ar), 8.12 (m, 2H, H-Ar). 13C NMR (100 MHz, CDCl3) δ ppm 13.82 (C-
18), 15.98 (C-19), 20.40, 27.99, 31.78, 32.56, 34.47, 35.79, 41.86,
42.78, 44.46, 47.80, 55.74, 66.10 (C-21), 82.77 (C-5), 115.52 (C-Ar),
125.68 (C-Ar), 132.59 (C-Ar), 143.62 (C-16), 151.68 (C-17), 165.09
(–COO), 167.28 (C-Ar), 190.26 (C-20), 210.18 (C-3), 210.87 (C-6).
17), 166.08 (COO–), 190.48 (C-20). LRMS FAB+: 451 [M+H]+
.
2.2.6.4. 21-(p-Fluoro)benzoyloxy-5α,6α-epoxy-3β-hydroxy-16-pregnen-
20-one (8d). Yield: 77%, mp 213–215 °C. IR (cm−1): 3517, 2930, 1718,
1678, 1087. 1H NMR (CDCl3) δ: 0.89 (s, 3H, H-18), 1.1 (s, 3H, H-19),
2.93 (d, J = 4 Hz, 1H, H-6), 3.84 (s, 1H, –OH), 3.91 (m, 1H, H-3), 5.09
(d, J = 16 Hz, 1H, H-21), 5.27 (d, J = 16 Hz, 1H, H-21), 6.8 (s, 1H, H-
16), 7.12 (t, J = 8 Hz, 2H, H-Ar), 8.11 (m, 2H, H-Ar). 13C NMR (CDCl3)
δ: 15.14 (C-18), 15.91 (C-19), 20.16, 28.11, 28.25, 31.76, 32.05, 32.36,
34.46, 35.32, 39.68, 42.47, 46.76 58.80 (C-6), 65.87 (C-21), 66.08 (C-
5), 68.60 (C-3), 115.47 (C-Ar), 125.71 (C-Ar), 132.56 (C-Ar), 143.97 (C-
LRMS FAB+: 483 [M+H]+
.
2.2.8. General methodology for the synthesis of compounds 10a-d
Compound 9 (1 eq.) was dissolved in dichloromethane (0.25 M),
0.004 eq. of pyridine was added and the solution was cooled to 0 °C.
Thionyl chloride (0.0015 eq.) was added dropwise under nitrogen at-
mosphere and the resulting solution was stirred at room temperature
for 2 h. Iced water (100 mL) was added and the solution was extracted
three times with ethyl acetate (25 mL). The organic layer was washed
4

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
with 10% aqueous hydrochloric acid (25 mL), 5% aqueous sodium bi-
carbonate (25 mL), and water (25 mL). The organic layer was dried over
sodium sulfate and the solvent was evaporated under vacuum. The
product was purified by column chromatography using a mixture of
20% ethyl acetate in hexane.
433.2372 found 433.2372.
2.2.9.2. 21-(p-Fluoro)benzoyloxypregna-4,16-dien-3,20-dione
(11d). Yield: 61%, purity 98%, mp 148–150 °C. IR (cm−1): 2929,
1728, 1678. 1H NMR (CDCl3) δ: 0.87 (s, 3H, H-18), 1.15 (s, 3H, H-19),
5.15 (d, J = 16 Hz, 1H, H-21), 5.3 (d, J = 16 Hz, 1H, H-21), 5.67 (s, 1H,
H-4), 6.63 (s, 1H, H-16), 7.3 (m, 2H, H-Ar), 8.1 (m, 2H, H-Ar). 13C NMR
(CDCl3) δ ppm 14.78 (C-18), 19.73 (C-19), 26.16, 28.85, 28.97, 31.96,
32.31, 32.48, 34.44, 38.05, 48.12, 53.19, 54.93, 67.15 (C-21), 114.45
(C-Ar), 122.97 (C-4), 127.78 (C-Ar), 129.86 (C-Ar), 131.45 (C-Ar),
143.08 (C-16), 154.14 (C-17), 166.74 (COO–), 169.91 (C-Ar), 170.10
(C-5), 195.69 (C-20), 198.51 (C-3). HRMS [M+H]+ cal for C28H32FO4
451.2285 found 451.2298.
2.2.8.1. 21-Acetoxypregna-4,16-dien-3,6,20-trione (10a). Yield: 87%, purity
98%, mp 215–217 °C. IR (cm−1): 2942, 1745, 1677. 1H NMR (CDCl3) δ:
0.98 (s, 3H, H-18), 1.21 (s, 3H, H-19), 2.19 (s, 3H, –COOCH3), 4.9 (d,
J = 16 Hz, 1H, H-21), 5.3 (d, J = 16 Hz, 1H, H-21), 6.2 (s, 1H, H-4), 6.76
(s, 1H, H-16). 13C NMR (CDCl3) δ: 15.84 (C-18), 17.45 (C-19), 20.60
(CH3COO–), 31.96, 33.83, 35.71, 39.89, 36.26, 36.55, 51.49, 56.17, 65.57
(C-21), 125.81 (C-4), 143.16 (C-16), 151.37 (C-17), 160.41 (C-5), 170.34
(CH3COO–), 190.30 (C-20), 199.18 (C-3), 201.21 (C-6). HRMS [M+H]+
cal for C23H29O5 385.2015 found 385.2023.
2.2.9.3. 21-(p-Chloro)benzoyloxypregna-4,16-dien-3,20-dione
(11e). Yield: 65%, purity 98%, mp 157–159 °C. IR (cm−1): 2931, 1727,
1676. 1H NMR (CDCl3) δ: 0.89 (s, 3H, H-18), 1.15 (s, 3H, H-19), 5.04
(d, J = 16 Hz, 1H, H-21), 5.2 (d, J = 16 Hz, 1H, H-21), 5.67 (s, 1H, H-
4), 6.76 (s, 1H, H-16), 7.36 (d, J = 8 Hz, 2H, H-Ar), 7.98 (d, J = 8 Hz,
2H, H-Ar). 13C NMR (CDCl3) δ: 14.99 (C-18), 19.73 (C-19), 26.12,
28.46, 28.55, 31.50, 32.05, 32.21, 34.19, 38.00, 48.10, 53.06, 67.16 (C-
21), 123.04 (C-4), 127.77 (C-Ar), 129.85 (C-Ar), 130.29 (C-Ar), 142.91
(C-Ar), 143.22 (C-16), 150.80 (C-17), 169.60 (COO–), 170.60 (C-5),
196.30 (C-20), 199.10 (C-3). HRMS [M+H]+ cal for C28H32ClO4
467.1989 found 467.1995.
2.2.8.2. 21-Propanoyloxypregna-4,16-dien-3,6,20-trione (10b). Yield: 83%,
purity 99%, mp 212–215 °C. IR (cm−1): 2940, 1743, 1671. 1H NMR
(CDCl3) δ: 0.97 (s, 3H, H-18), 1.0 (s, 3H, H-19), 1.12 (m, 3H,
–COOCH2CH3), 2.4 (m, 2H, –COOCH2CH3), 4.8 (d, J = 16 Hz, 1H, H-
21), 5.2 (d, J = 16 Hz, 1H, H-21), 6.2 (s, 1H, H-4), 6.67 (s, 1H, H-16). 13
C
NMR (CDCl3) δ: 9.95 (COOCH2CH3), 14.90 (C-18), 16.11 (C-19), 26.26
(CH3 CH2COO–), 31.88, 33.71, 35.46, 39.86, 36.22, 36.30, 51.27, 56.03,
64.34 (C-21), 125.56 (C-4), 142.12 (C-16), 150.67 (C-17), 160.20 (C-5),
172.85 (CH3CH2COO–), 189.72 (C-20), 199.11 (C-3), 201.06 (C-6). HRMS
[M+H]+ cal for C24H31O5 399.2172 found 399.2181.
2.2.8.3. 21-Benzoyloxypregna-4,16-dien-3,6,20-trione (10c). Yield: 83%,
purity 99%, mp 165–167 °C. IR (cm−1): 2930, 1721, 1683. 1H NMR
(CDCl3) δ: 1.13 (s, 3H, H-18), 1.18 (s, 3H, H-19), 5.08 (d, J = 16 Hz, 1H, H-
21), 5.2 (d, J = 16 Hz, 1H, H-21), 6.13 (s, 1H, H-4), 6.78 (s, 1H, H-16), 7.36
(s, 2H, H-Ar), 7.52 (m, 1H, H-Ar), 8.05 (s, 2H, H-Ar). 13C NMR (CDCl3) δ:
14.89 (C-18), 16.45 (C-19), 19.99, 29.77, 31.47, 32.39, 33.19, 34.22, 37.68,
38.52, 50.08, 54.48, 67.14 (C-21), 124.80 (C-4), 127.43 (C-Ar), 128.89 (C-
Ar), 129.81 (C-Ar), 132.36 (C-Ar), 142.37 (C-16), 150.39 (C-17), 159.51 (C-
5), 165.01 (COO–), 189.33 (C-20), 198.34 (C-3), 200.41 (C-6). HRMS [M
+H]+ cal for C28H31O5 447.2171 found 447.2183.
2.2.9.4. 21-(p-Bromo)benzoyloxypregna-4,16-dien-3,20-dione
(11f). Yield: 60%, purity 99%, mp 161–163 °C. IR (cm−1): 2929, 1727,
1677. 1H NMR (CDCl3) δ ppm 0.94 (s, 3H, H-18), 1.16 (s, 3H, H-19), 5.1
(d, J = 16 Hz, 1H, H-21), 5.25 (d, J = 16 Hz, 1H, H-21), 5.67 (s, 1H, H-
4), 6.77 (s, 1H, H-16), 7.6 (d, J = 8 Hz, 2H, H-Ar), 7.7 (d, J = 8 Hz, 2H,
H-Ar). 13C NMR (CDCl3) δ: 15.10 (C-18), 19.91 (C-19), 26.79, 28.66,
28.79, 31.99, 32.27, 34.61, 38.56, 48.19, 48.30, 53.29, 67.5 (C-21),
124.15 (C-4), 127.18 (C-Ar), 129.09 (C-Ar), 130.17 (C-Ar), 132.44 (C-
Ar), 144.32 (C-16), 153.95 (C-17), 166.38 (COO–), 170.39 (C-5),
196.40 (C-20), 199.12 (C-3). HRMS [M+H]+ cal for C28H32BrO4
511.1484 found 511.1495.
2.2.8.4. 21-(p-Fluoro)benzoyloxypregna-4,16-dien-3,6,20-trione
(10d). The characterization of this compound was already reported in
our previous work [20].
2.2.9.5. 21-(p-Iodo)benzoyloxypregna-4,16-dien-3,20-dione
(11g). Yield: 58%, purity 99%, mp 186–188 °C. IR (cm−1): 2932, 1729,
1675. 1H NMR (CDCl3) δ: 0.97 (s, 3H, H-18), 1.15 (s, 3H, H-19), 5.03
(d, J = 16 Hz, 1H, H-21), 5.19 (d, J = 16 Hz, 1H, H-21), 5.66 (s, 1H, H-
4), 6.74 (s, 1H, H-16), 7.8 (d, J = 8 Hz, 2H, H-Ar), 7.88 (d, J = 8 Hz,
2H, H-Ar). 13C NMR (CDCl3) δ: 14.90 (C-18), 19.60 (C-19), 26.15,
28.33, 28.58, 31.03, 32.45, 34.89, 38.63, 48.59, 48.96, 53.54, 67.00 (C-
21), 98.50 (C-Ar), 124.10 (C-4), 129.34 (C-Ar), 130.96 (C-Ar), 137.55
(C-Ar), 144.07 (C-16), 154.11 (C-17), 167.32 (COO–), 169.96 (C-5),
197.27 (C-20), 199.09 (C-3). HRMS [M+H]+ cal for C28H32IO4
559.1345 found 559.1353.
2.2.9. General methodology for the synthesis of compounds 11c-g
Compound 7 (1 eq.) was dissolved in toluene (0.025 M), N-methyl-
4-piperidone (20 eq.) were added. After the addition, 25% of toluene
were distilled with a Dean-Stark system to eliminate the water of the
reaction. Aluminium isopropoxide (5 eq.) was added and other 25% of
toluene were distilled. The reaction was stirred under reflux for 18 h.
The mixture was dissolved with 50 mL of ethyl acetate and 30 mL of a
15% aqueous solution of HCl were added, the reaction was stirred for
30 min and the organic layer was separated and washed with a satu-
rated solution of sodium bicarbonate (3 × 30 mL) and water
(3 × 30 mL). The organic layer was evaporated and the crude was
purified on silica gel using 10% ethyl acetate in hexane getting yields
from 58 to 65%.
2.3. General synthesis of compounds 13a-e and 14a-e
The synthesis and spectroscopy data of compounds 12, 13a, 13b
and 14a-d were published in our previous work [19,22–24], and just
the purity and HRMS is reported here. The Steglich reaction was done
following the general procedure: the steroid 12 (1 eq.), DCC (3 eq.),
DMAP (3 eq.) and the corresponding carboxylic acid (3 eq.) were dis-
solved in CH2Cl2 (0.09 M) and stirred for 4 h. Chilled ethyl acetate
(100 mL) was added and the precipitated dicyclohexyl urea was fil-
tered. The organic layer was washed three times with 10% aqueous
hydrochloric acid (50 mL), 5% aqueous sodium bicarbonate
(3 × 50 mL) and water (3 × 50 mL). The solution was dried over an-
hydrous sodium sulfate and the crude was purified by recrystallization
from methanol.
2.2.9.1. 21-Benzoyloxypregna-4,16-dien-3,20-dione (11c). Yield: 63%,
purity 99%, mp 192–194 °C. IR (cm−1): 2953, 1727, 1672. 1H NMR
(CDCl3) δ: 0.98 (s, 3H, H-18), 1.21 (s, 3H, H-19), 5.11 (d, J = 16 Hz,
1H, H-21), 5.27 (d, J = 16 Hz, 1H, H-21), 5.73 (s, 1H, H-4), 6.83 (s, 1H,
H-16), 7.4 (t, J = 8 Hz, 2H, H-Ar), 7.57 (t, J = 8 Hz, 1H, H-Ar), 8.09 (t,
J = 8 Hz, 2H, H-Ar). 13C NMR (CDCl3) δ: 15.96 (C-18), 20.71 (C-19),
27.38, 31.82, 32.40, 33.19, 33.98, 24.10, 35.66, 38.99, 48.72, 53.95,
55.70, 66.03 (C-21), 124.03 (C-4), 128.38 (C-Ar), 129.89 (C-Ar),
133.26 (C-Ar), 143.74 (C-16), 151.35 (C-17), 166.01 (COO–), 170.65
(C-5), 190.38 (C-20), 199.39 (C-3). HRMS [M+H]+ cal for C28H33O4
5

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
2.3.1. 3β-Acetoxypregna-4,16-dien-6,20-dione (13a)
Yield 81%, purity 98%, HRMS [M+H]+ cal for C23H31O4 371.2222
found 371.2227.
(C-17), 175.67 (C]O del éster), 196.53 (C-20), 201.44 (C-6). HRMS [M
+H]+ cal for C29H41O4 453.3004 found 4453.3013.
3. Results and discussion
2.3.2. 3β-Propanoyloxypregna-4,16-dien-6,20-dione (13b)
Yield 75%, purity 98%, HRMS [M+H]+ cal for C24H33O4 385.2379
found 385.2374.
3.1. Chemistry
The commercially available 16-dehydropregnenolone acetate 1 was
the starting material for two synthetic routes. To prepare the first series
with an α,β-unsaturated dicarbonyl group, compound 2 was synthe-
sized from the starting material 1 using hydrogen peroxide under basic
condition to form the 16α,17α-epoxy steroid 3. The protection of the
alcohol 3 with tert-butyldimethylsilyl chloride followed by oxidation of
C-21 using (diacetoxyiodo)benzene afforded 4 [25]. The alcohol 4 was
esterified using the corresponding aliphatic or aromatic carboxylic acid
to get 5a-g. The deprotection of the alcohol and the carbonyl groups of
molecules 5a-g were performed under acid conditions to obtain deri-
vatives 6a-g. In order to regenerate the double bond at C-16, the ep-
oxide was reduced using CrCl2 [26] to give compounds 7a-g. Alter-
natively, compound 7a could be synthesized from a 21-iodo derivative
[27]. For the series 10a-e the double bond at C-5 of steroids 7a-e were
reacted with m-chloroperoxybenzoic acid to afford the 5α,6α-epox-
ycompounds 8a-e. Oxidation of the epoxide 8a-e with chromic acid
yielded the 3,6-diketo-5α-dialcohol 9a-e. Dehydration of the hydroxyl
group at C-4 in 9a-e with thionyl chloride afforded the α,β-unsaturated
dicarbonyl analogs 10a-e with global yields from 10 to 16% (Scheme
1). The series 11c-g were prepared using the Oppenauer reaction from
the alcohol derivatives 7c-g (Scheme 1). The desired molecules were
obtained with global yields of 14–18%.
2.3.3. 3β-Butanoyloxypregna-4,16-dien-6,20-dione (13c)
Yield: 73%, purity 99%, mp 142–144 °C. IR (cm−1): 1732, 1688,
1656. 1H NMR (CDCl3) δ: 0.9 (m, 3H, CH3 ester), 0.92 (s, 3H, H-18),
1.3(s, 3H, H-19), 2.2(s, 3H, H-21), 5.3(m, 1H, H-3), 6.0 (s, 1H, H-4),
6.7(m, 1H, H-16). 13C NMR (CDCl3) δ: 13.62 (CH3 ester), 15.75 (C-18),
18.92, 19.59 (C-19), 20.11, 23.70, 27.08, 31.77, 32.03, 33.48, 36.24,
38.22, 38.41, 45.98, 46.13, 51.50, 56.07 (C-21), 68.93 (C-3), 129.27
(C-4), 143.74 (C-16), 147.78 (C-5), 154.89 (C-17), 173.26 (C]O del
ester), 196.53 (C-20), 201.78 (C-6). HRMS [M+H]+ cal for C25H35O4
399.2535 found 399.2519.
2.3.4. 3β-Pentanoyloxypregna-4,16-dien-6,20-dione (13d)
Yield: 69%, purity 99%, mp 125–127 °C. IR (cm−1): 1735, 1688,
1656. 1H NMR (CDCl3) δ: 0.9 (m, 3H, CH3 ester), 0.92 (s, 3H, H-18),
1.3(s, 3H, H-19), 2.2(s, 3H, H-21), 5.3(m, 1H, H-3), 6.0 (s, 1H, H-4),
6.7(m, 1H, H-16). 13C NMR (CDCl3) δ: 13.71 (CH3 ester), 14.11, 15.80
(C-18), 19.64 (C-19), 20.16, 22.15, 24.33, 27.02 (C-21), 31.96, 32.36,
38.48, 41.96, 45.88, 48.77, 48.98, 51.87, 46.20, 60.44, 68.98 (C-3),
129.33 (C-4), 143.77 (C-16), 147.81 (C-5), 154.93 (C-17), 173.50 (C]
O ester), 196.59 (C-20), 201.78 (C-6). HRMS [M+H]+ cal for C26H37O4
413.2691 found 413.2697.
We reported the methodology and spectroscopy data to obtain de-
rivative 12 in our previous work [19]. The esters at C-3 in compounds
13a-e and 14a-e were synthesized from alcohol 12 using the Steglich
reaction [28] to afford two groups of derivatives, one with a different
size of lineal chain substituents obtained with global yields of 31–50%
and the other with alicyclic substituents with global yields ranging from
29 to 32% (Scheme 2).
2.3.5. 3β-Hexanoyloxypregna-4,16-dien-6,20-dione (13e)
Yield: 77%, purity 98%, mp 105–106 °C. IR (cm−1): 1726, 1688,
1664. 1H NMR (CDCl3) δ: 0.9 (m, 3H, CH3 del ester), 0.95 (s, 3H, H-18),
1.1(s, 3H, H-19), 2.2(s, 3H, H-21), 5.3(m, 1H, H-3), 6.0 (s, 1H, H-4),
6.7(m, 1H, H-16). 13C NMR (CDCl3) δ: 13.87 (CH3 ester), 15.76 (C-18),
19.59 (C-19), 20.23, 22.05, 22.19, 24.11, 24.16, 27.08 (C-21), 31.77,
31.89, 32.06, 34.40, 38.06, 38.21, 46.20, 50.60, 50.73, 56.27, 68.94 (C-
3), 129.29 (C-4), 143.74 (C-16), 147.77 (C-5), 154.89 (C-17), 173.46
(C]O ester), 196.54 (C-20), 201.73 (C-6). HRMS [M+H]+ cal for
3.2. Cytotoxic effect
C
27H39O4 427.2848 found 427.2853.
We evaluated the cytotoxic effect of series 10a-d, 11c-g, 13a-e and
14a-e, in a panel of three human cancer cell lines (PC-3, prostatic
adenocarcinoma; MCF-7, mammary adenocarcinoma and SKLU-1, lung
adenocarcinoma) and one noncancerous cell line (HGF, human gingival
fibroblast). The values of the primary screening are showed in Table 1.
Etoposide, a glycoside of podophyllotoxin was used as a reference drug.
For molecules with an ester moiety at C-21 (10a-d and 11c-g),
derivatives 10d and 11d with an aromatic ester having a fluor at para
position, showed high cytotoxic activity on the three cancer cell lines
and a low cytotoxicity on the noncancerous cell line, 11d had even a
lower inhibition percentage than that of the etoposide on HGF.
Steroids 13a-e with an aliphatic ester at C-3 showed a higher in-
hibition percentage in the three cancer cell lines when the side chain
has even number of carbons (13a, 13c, 13e) as compare to those with
odd number (13b and 13d). Besides, when the aliphatic ester is at-
tached at C-21 (10a and 10b) the same phenomenon is observed, this
behavior might be due to the specific orientation of the aliphatic chain
at the active site. In the case of compounds 14a-e, just 14b (with a
cyclobutyl ester) showed good cytotoxicity, especially on SKLU-1 cell
line.
2.3.6. 3β-Cyclopropanecarbonyloxypregna-4,16-dien-6,20-dione (14a)
Yield 65%, purity 98%, HRMS [M+H]+ cal for C25H33O4 397.2378
found 397.2386.
2.3.7. 3β-Cyclobutanecarbonyloxypregna-4,16-dien-6,20-dione (14b)
Yield 67%, purity 99%. HRMS [M+H]+ cal for C26H35O4 411.2535
found 411.2531.
2.3.8. 3β-Cyclopentanecarbonyloxypregna-4,16-dien-6,20-dione (14c)
Yield 70%, purity 99%. HRMS [M+H]+ cal for C27H37O4 425.2691
found 425.2691.
2.3.9. 3β-Cyclohexanecarbonyloxypregna-4,16-dien-6,20-dione (14d)
Yield 71%, purity 99%. HRMS [M+H]+ cal for C28H39O4 439.2848
found 4439.2845.
2.3.10. 3β-Cycloheptanecarbonyloxypregna-4,16-dien-6,20-dione (14e)
Yield: 61%, purity 99%, mp 134–137 °C. IR (cm−1): 1727, 1693,
1652. 1H NMR (CDCl3) δ: 0.93 (s, 3H, H-18), 1.02(s, 3H, H-19), 2.19 (s,
3H, H-21), 2.27 (m, 1H, CH ester), 5.33(m, 1H, H-3), 6.06 (s, 1H, H-4),
6.70(m, 1H, H-16). 13C NMR (CDCl3) δ: 14.54 (C-18), 19.65 (C-19),
20.19, 23.86, 27.56, 28.98 (C-21), 31.79, 33.95, 34.04, 38.14, 38.39,
43.24, 43.46, 44.60, 46.39 (CH ester), 51.27, 51.53, 53.85, 56.48,
57.83, 68.65 (C-3), 129.55 (C-4), 143.74 (C-16), 147.32 (C-5), 154.89
Compounds 10a, 10d, 11d, 13e and 14b, were chosen to determine
their IC50 on the lung cancer cell line and derivatives 10d and 11d have
the lower IC50 (Table 2). This results might be due to fluorine electro-
static interactions [29] but other mechanisms could be implicated.
From this study, we can infer that the steroids with an ester moiety
at C-21 present more cytotoxicity on the lung cancer cell line than that
6

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of compounds 10a-d and 11c-g.
Scheme 2. Synthesis of compounds 13a-e and 14a-e.
7

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
Table 1
Preliminary cytotoxicity screening of series 10a–d, 11c–g, 13a–e and 14a–e, at 50 µM.
Structure
Etoposide
Compound (50 µM)
PC-3
MCF-7
SKLU-1
HGF
44.2
49.8
21.5
57.5
98.1
1.2a
2.9
0.9
3.4
1.9
73.3
75.3
56.6
40.2
92.2
1.0a
2.5
2.8
3.7
2.5
83.5
94.2
63.3
67.7
100
1.1a
0.6
4.8
4.8
24.21
12.2
25.6
21.62
27.63
1.1
3.8
1.8
2.4
10a (R1 = CH3)
10b (R1 = C2H5)
10c (R1 = C6H5)
10d (R1 = 4-F-C6H5)
2.3
11c (R2 = C6H5)
19.0
92.0
55.9
50.0
57.1
1.8
4.3
3.0
7.0
7.4
25.9
89.1
43.9
65.7
95.5
3.6
4.9
5.8
6.1
2.0
27.6
98.1
63.7
51.8
68.9
3.6
1.9
1.9
7.1
4.4
NCb
18.39
12.8
NCb
11d (R2 = 4-F-C6H5)
11e (R2 = 4-Cl-C6H5)
11f (R2 = 4-Br-C6H5)
11g (R2 = 4-I-C6H5)
1.2
1.2
11.04
1.9
13a (R3 = CH3)
13b (R3 = C2H5)
13c (R3 = C3H7)
13d (R3 = C4H9)
78.1
59.7
82.7
NCb
71.8
3.3
2.4
4.9
50.6
39.5
66.5
16.9
68.9
3.4
2.5
3.0
2.4
1.8
87.14
71.5
98.4
17.2
99.3
5.1
1.9
1.5
4.9
0.4
12.75
12.6
29.72
18.31
15.3
5.8
2.2
2.6
0.8
2.5
13e (R3 = C5H11
)
4.7
14a (R4 = C3H5)
19.7
97.3
52.8
68.7
72.0
4.1
2.6
2.9
2.2
9.4
15.6
90.9
64.9
76.2
87.8
3.7
3.4
7.7
6.2
5.7
8.8
1.5
14.85
66.45
18.3
14.04
13.47
3.8
3.2
3.4
3.3
2.2
14b (R4 = C4H7)
14c (R4 = C5H9)
14d (R4 = C6H11
100
76.4
85.2
78.9
4.3
2
5.6
)
)
14e (R4 = C7H13
a
These values were obtained with a concentration of 25 µM.
NC = No cytotoxic.
b
α,β-unsaturated carbonyls at C-3 and C-6 (10a-d). The final derivatives
were evaluated in a panel of three cancer cell lines, the results showed
that the compounds with a 4-fluorinated benzoic acid ester moiety (10d
and 11d) were the most active steroids against lung cancer cell line
Table 2
IC50 values for the most active compounds in SKLU-1
cancer cell line.
Compound
IC50 (µM)
with an IC50 of 13.1
1.2 and 12.8
0.5 µM respectively and were
10a
10d
11d
13e
14b
Etoposide
18.4
13.1
12.8
24.2
17.4
4.1
0.5
less toxic than etoposide on the healthy cell line.
1.2
0.5
2.1
0.5
Acknowledgments
0.6
The authors thanks Dirección General de Asuntos del Personal
Académico-UNAM (DGAPA) for funding granted through the project
IN211312 (UNAM) and Consejo Nacional de Ciencia y Tecnología
(CONACyT) for the financial support granted to project CB 2011/
165049. Alejandra Chávez-Riveros also acknowledge CONACyT for the
fellowship awarded (No. 240059).
We also thank Eddy Ivanhoe Jiménez Gutiérrez, Davir González
Calderón, Bindu Santhamma and Guillermina Yazmín Arellano Salazar.
For their comments.
with the ester at C-3, the number of enones did not seem to be relevant
for the activity on the three cancer cell lines, but derivatives with an
α,β-unsaturated carbonyl at C-3 (11c-g) were less toxic for the non-
cancerous cell line than the compounds with α,β-unsaturated dicarbo-
nyls at C-3 and C-6 (10a-d). Regarding the aliphatic ester derivatives,
compound 13e just had an IC50 of 24.2
2.1 µM. These results will
enable us to improve the design of new cytotoxic molecules, knowing
that the best position for a lipophilic moiety is at C-21 and that the
toxicity on noncancerous cell lines could be determine by the number of
conjugations.
In conclusions, the synthesis of three series of steroids with one or
more conjugated carbonyls and an ester moiety at C-21 or C-3 were
developed. These preliminary biological results showed that the
number of conjugated carbonyl groups of these series of compounds did
not determine the cytotoxicity on the three cancer cell lines studied, but
molecules with just one α,β-unsaturated carbonyl at C-3 (11c-g) were
less toxic for the noncancerous cell line than the compounds with two
References
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, Cancer J. Clin. 66 (2016) 7–30.
[2] M. Malvezzi, P. Bertuccio, T. Rosso, M. Rota, F. Levi, C. La Vecchia, E. Negri,
European cancer mortality predicitons for the year 2015; does lung cancer have the
highest death rate in EU for women? Ann. Oncol. 26 (2015) 779–786.
[3] R. Bansal, P.C. Acharya, Man-made cytotoxic steroids: exemplary agents for cancer
therapy, Chem. Rev. 114 (2014) 6986–7005.
[4] A. Gupta, B.S. Kumar, A.S. Negi, Current status on development of steroids as
cancer agents, J. Steroid Biochem. Mol. Biol. 137 (2013) 242–270.
[5] M.I. Choudhary, M.S. Alam, Atta-ur-Rahma, S. Yousuf, Y.-C. Wu, A.S. Lin,
F. Shaheen, Pregnenolone derivatives as potential anticancer agents, Steroids 76
8

A. Chávez-Riveros et al.
Steroidsxxx(xxxx)xxx–xxx
derivatives of pregnenolone and related ether analogues, Med. Chem. Res. 25
(2016) 310–321.
(2011) 1554–1559.
[6] F. Cortés-Benítez, M. Cabeza, M.T. Ramírez-Apan, B. Alvarez-Manríque, E. Bratoeff,
Synthesis of 17β-N-arylcarbomoylandrost-4-en3-one derivatives and their anti-
proliferative effect on human androgen-sensitive LNCaP cell line, Eur. J. Med.
Chem. 121 (2016) 737–746.
[7] É. Frank, G. Schneider, Stereoselective synthesis of novel dispiro oxindole pyrro-
lothiazole-androsterone hybrids, J. Steroid Biochem. Mol. Biol. 137 (2013)
301–315.
[18] V. Pérez, M. Cabeza, E. Bratoeff, I. Heuze, M. Sánchez, E. Ramírez, E. Naranjo, New
5α-reductase inhibitors: in vitro and in vivo effects, Steroids 70 (2005) 217–224.
[19] A. Chávez-Riveros, M. Garrido, M.T. Ramírez-Apan, A. Zambrano, M. Díaz,
E. Bratoeff, Synthesis and cytotoxic effect on cancer cell lines and macrophages on
novel progesterone derivatives having an ester or a carbamate function at C-3 and
C-17, Eur. J. Med. Chem. 82 (2014) 498–505.
[8] M.M. Rafat, Y.M. Abdo, A.M. Abeer, Cytotoxicity and anti-proliferative properties
of heterocyclic compounds derived from progesterone, Anti-Cancer Agents Med.
Chem. 16 (2016) 1043–1054.
[9] M.A. Tantawy, M.S. Nafie, G.A. Elmegeed, I.A.I. Ali, Auspicious role of the steroidal
heterocyclic derivatives as a platform for anti-cancer drugs, Bioorg. Chem. 73
(2017) 128–146.
[10] A.H. Banday, B.P. Mir, I.H. Lone, H.M. Kumar, Studies on novel D-ring substituted
steroidal pyrazolines as potential anticancer agents, Steroids 77 (2010) 805–809.
[11] R.M. Mohareb, F. Al-Omran, Reaction of pregnenolone with cyanoacetylhydrazine:
novel synthesis of hydrazine-hydrazone, pyrazole, pyridine, thiazole, thiophene
derivatives and their cytotoxicity evaluations, Steroids 77 (2012) 1551–1559.
[12] R. Morfin, G. Courchay, Pregnenolone and dehydroepiandrosterone as precursors of
native 7-hydroxylated metabolites which increase the immune response in mice, J.
Steroid Biochem. Mol. Biol. 50 (1994) 91–100.
[20] A. Chávez-Riveros, E. Bratoeff, Y. Heuze, J. Soriano, I. Moreno, A. Sánchez-
Marquez, M. Cabeza, Synthesis and identification of pregnenolone derivatives as
inhibitors of isozymes of 5α-reductase, Arch. Pharm. 348 (2015) 1–9.
[21] J.J. Naveja, F. Cortés-Benítez, E. Bratoeff, J.L. Medina-Franco, Activity landscape
analysis of 5α-reductase inhibitors, Mol. Divers. 30 (2016) 771–780.
[22] M. Cabeza, E. Bratoeff, E. Ramírez, I. Heuze, S. Recillas, H. Berrios, A. Cruz,
O. Cabrera, O. Cabrera, V. Perez, Biological activity of novel progesterone deriva-
tives having a bulky ester side chains at C-3, Steroids 73 (2008) 838–843.
[23] M. Cabeza, M. Heuze, E. Sánchez, E. Bratoeff, A. Ramírez, A. Rojas, Relative binding
affinity of novel steroids to androgen receptors in hamster prostate, J. Enzyme
Inhib. Med. Chem. 20 (2005) 357–364.
[24] E. Bratoeff, M. Cabeza, V. Pérez-Ornelas, S. Rencillas, I. Heuze, In vivo and in vitro
effect of novel 4,16-pregnadien-6,20-dione derivatives, as 5α-reductase inhibitors,
J. Steroid Biochem. Mol. Biol. 111 (2008) 275–281.
[13] A.H. Playne, R.B. Jaffe, Androgen formation from pregnenolone sulfate by the
human fetal ovary, J. Clin. Endocrinol. Metab. 39 (1974) 300–304.
[14] I.J. Testas, Z.Y. Hu, E.E. Baulieuf, P. Robel, Neurosteroids: biosynthesis of preg-
nenolone and progesterone in primary cultures of rat glia cells, Endocrinology 125
(1989) 2083–2091.
[25] A. Kamernitzky, A. Turuta, T. Fadeeva, Z. Istomina, Use of diacetoxyphenyliodine
for α-hydroxylation of 20-oxosteroids fused with heterocycles in position 16α, 17α,
Synthesis 3 (1985) 326–328.
[26] W. Cole, P. Julian, Sterols. XIV. Reduction of epoxy ketones by chromous salts, J.
Org. Chem. 19 (1954) 131–138.
[15] G. Szalóki, A. Pantzou, K.C. Prousis, O. Mavrofrydi, P. Papazafiri,
T. Calogeropoulou, Design and synthesis of 21-alkynylary pregnenolone derivatives
and evaluation of their anticancer activity, Bioorg. Med. Chem. 22 (2014)
6980–6988.
[16] A.H. Banday, S.M.M. Akram, S.A. Shameem, Benzylidine pregnenolones and their
oximes as potential anticancer agents: synthesis and biological evaluation, Steroids
84 (2014) 64–69.
[27] F.K. Yoshimoto, H.M. Peng, H. Zhang, S.M. Anderson, R.J. Auchus, Epoxidation
activities of human Cytochromes P450c17 and P450c21, Biochemistry 53 (2014)
7531–7540.
[28] B. Neises, W. Steglich, Simple method for the esterification of carboxylic acids,
Angew. Chem. Int. Ed. 17 (1978) 522–524.
[29] J.A. Olsen, D.W. Banner, P. Seiler, B. Wagner, T. Tschopp, U. Obst-Sander,
M. Kansy, K. Müller, F. Diederich, Fluorine interactions at the thrombin active site:
protein backbone fragments H-Cα-C=O comprise a favorable C-F environment and
interactions of C-F with electrophiles, ChemBioChem 5 (2004) 666–675.
[17] N.A. Al-Masoudi, N.A. Abdul-Rida, R.A. Kadhim, S.J. Krug, M. Engel, B.A. Saeed,
Synthesis and CYP17α hydroxylase inhibition activity of new 3α and 3β-ester
9