Cytotoxic effect of novel dehydroepiandrosterone derivatives on different cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2013.02.031

The aim of this study was to determine the cytotoxic effect of human cancer cells on three series of novel dehydroepiandrosterone derivatives containing triazole or pyrazole rings at C-17 and an ester moiety at C-3 of the androstane skeleton. The panel ca

Full text of DOI:10.1016/j.ejmech.2013.02.031

Accepted Manuscript
Cytotoxic effect of novel dehydroepiandrosterone derivatives on different cancer cell
lines
Mariana Garrido, Marisa Cabeza, Francisco Corté s, José Gutié rrez, Eugene Bratoeff
PII:
S0223-5234(13)00138-4
10.1016/j.ejmech.2013.02.031
EJMECH 6026
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2012
Revised Date: 18 February 2013
Accepted Date: 21 February 2013
Please cite this article as: M. Garrido, M. Cabeza, F. Corté s, J. Gutié rrez, E. Bratoeff, Cytotoxic effect of
novel dehydroepiandrosterone derivatives on different cancer cell lines, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.02.031.
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Graphical Abstract
Cytotoxic effect of novel steroids on cancer cell lines
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Highlights
1. Cytotoxic effect of three series of dehydroepiandrosterone derivatives was
determined
2. The following cancer cell lines were used in this study: PC-3, MCF-7 and SKLU-
1.
3. The steroids with triazol ring produced higher cytotoxicity than those with
pyrazol.
4. Electronegative groups in the ester moiety increased citotoxicity in cell lines.
5. Steroids 4a, 4e, 4i-4k, 5o and 5f exibited the highest citotoxic effect.t

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Cytotoxic effect of novel dehydroepiandrosterone derivatives on different
cancer cell lines
Mariana Garridoa, Marisa Cabezab, Francisco Cortésa, José Gutiérreza and Eugene
Bratoeffa.
Department of Pharmacy, Faculty of Chemistrya, National University of Mexico, Mexico,
D. F., Mexico.
Department of Biological Systems Metropolitan University-Xochimilcob, Mexico, D. F.,
Mexico.
Address correspondence to:
Marisa Cabeza Ph.D.
Departamento de Sistemas Biológicos.
Universidad Autónoma Metropolitana-Xochimilco.
Calzada del Hueso No. 1100.
México, D.F. C.P 04960, México.
Phone: (011-52-55) 5483-72-60.
FAX: (52-55) 5483-72-60.
E-mail: marisa@correo.xoc.uam.mx.

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Abstract
The aim of this study was to determine the cytotoxic effect of human cancer cells on
three series of novel dehydroepiandrosterone derivatives containing triazol or pyrazol rings
at C-17 and an ester moiety at C-3 of the androstane skeleton. The panel cancer cells used
in this study were the following: PC-3, MCF-7 and SKLU-1.
The results from this study indicated that the steroidal derivatives 4a-4e and 4f-4k
showed the highest cytotoxic potency. This difference in this activity could be attributed to
the ability of the triazole (three nitrogen atoms) to form stronger hydrogen bonds with the
active site of the cell as compared to the pyrazole group having two nitrogen atoms.
Compounds 4f-4k having an aromatic ester at C-3 showed an enhanced cytotoxic
activity as compared to their aliphatic counterpart’s 4a-4e. Apparently the electronegative
phenyl ring increased the polarity of the molecule, thus increasing the dipole-dipole
association of the steroidal molecule with the reactive site of the cell.

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Key words: Cancer cell lines, PC-3, MCF-7, SKLU-1, synthesis of dehydropiandrosterone
derivatives, cytotoxic effect, triazol ring, pyrazol ring.

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1. Introduction
Steroids are important physiological and pharmacological regulators of cell growth
and survival. There are several examples in the literature that indicate the use of natural and
synthetic steroids for the treatment of sex hormone-dependent cancer tumors, including
breast cancer [1), endometrial cancer [2), cervical cancer [3) and prostate cancer [4). These
hormone-dependent tumor cells can control tumor growth through hormone receptor
antagonists [1-4), whereas in sex hormone-independent organs, some synthetic steroids had
been previously described as inductors of apoptosis [5).
The pharmacological studies using synthetic steroids offer an important strategy for
tumor control growth and tumor cell apoptosis. In this respect, 7β-hydroxy-
epiandrosterone, a derivative of dehydroepiandrosterone at low doses induced anti-
proliferative effect in the mammary cancer cells MCF-7 [6).Others dehydroepiandrosterone
derivatives had been also reported having antitumor properties on ovarian cancer cells (ES-
2), human lung cells (A549) and liver carcinoma cell (HepG2) lines [7). These compounds
induce apoptosis in cancer cell lines. Mifepristone is another synthetic steroid capable of
inducing apoptosis in sex-hormone independent organ as receptor-positive gastric cancer
cell [5).
Heterocyclic compounds had been reported as anti-tumorigenic agents [8), therefore
the investigation of various heterocyclic derivatives has generated a great interest, since
most of them can interact with the steroidal receptors due to their similarity of shape. Njar
had demonstrated that dehydroepiandrosterone derivatives having an azole ring at C-17

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inhibited the proliferation of cancer cell lines [9). On the other hand, similar compounds
synthesized by our group having an ester moiety at C-3 improved the lipophilicity and
exhibited an ability to penetrate the cell membrane thus showing an enhanced biological
effect [10).
The results from these investigations inspired us to synthesize several new
dehydroepiandosterone derivatives containing heterocyclic function at C-17 and an
aliphatic or an aromatic ester moiety at C-3 and test them as antitumoral agents.
In the present study three series of novel dehydroepiandrosterone derivatives were
synthesized; having a triazol or pyrazole function at C-17 and an ester moiety at C-3.
Series 1:
16-formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5,16-diene-3β-yl-cyclopropanoate 4a; 16-
formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-,16 –diene-3β-yl-cyclobutanoate 4b; 16-
formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-cyclopentanoate 4c; 16-
formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-cyclohexanoate 4d; 16-
formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl- cycloheptanoate 4e.
Series 2:
16-formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-p-methoxybenzoate 4f;
16-formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-p-methylbenzoate 4g;16-
formyl-17-(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-benzoate 4h; 16-formyl-17-
(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-p-trifluorobenzoate 4i; 16-formyl-17-

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(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-p-cyanobenzoate 4j; 16-formyl-17-
(1H-1, 2, 4-triazole-1-yl)androsta-5, 16-diene-3β-yl-nitrobenzoate 4k.
Series 3:
16-formyl-17-(1H-pyrazole-1-yl)androsta-5, 16-diene-3β-yl-p-methoxybenzoate 5f; 16-
formyl-17-(1H-pyrazole-1-yl)androsta-5, 16-diene-3β-yl-p-methylbenzoate 5g; 16-formyl-
17-(1H-pyrazole-1-yl)androsta-5, 16-diene-3β-yl-benzoate 5h; 16-formyl-17-(1H-pyrazole-
1-yl)androsta-5, 16-diene-3β-yl-p-trifluoromethylbenzoate 5i ;16-formyl-17-(1H-pyrazole-
1-yl)androsta-5, 16-diene-3β-yl-p-cyanobenzoate 5j;
yl)androsta-5, 16-diene-3β-yl-p-nitrobenzoate
yl)androsta-5, 16-diene-3β-yl-p-fluorobenzoate 5l
16-formyl-17-(1H-pyrazole-1-
5k;16-formyl-17-(1H-pyrazole-1-
;
16-formyl-17-(1H-pyrazole-1-
yl)androsta-5, 16-diene-3β-yl-p-chlorobenzoate 5m; 16-formyl-17-(1H-pyrazole-1-
yl)androsta-5, 16-diene-3β-yl-p-bromobenzoate 5n; 16-formyl-17-(1H-pyrazole-1-
yl)androsta-5, 16-diene-3β-yl-p-iodobenzoate 5o and 16-formyl-3-hydroxyandrosta-5, 16-
diene-(1H-1, 2, 4-pyrazole-1-yl) 6.
These compounds were evaluated in vitro, in order to determine its cytotoxic effect
in a panel of human cancer cells. The panel used in this study was the following: PC-3
(human prostate cancer cell line), MCF-7 (human breast cancer cell line) and SKLU-1
(human lung cancer cell line).
Ketoconazole was used as a reference compound for the in vitro assays. This
compound is an antimycotic drug that had previously been described as an agent that
inhibits cytochrome P450 enzymes, which are required for the synthesis of androgens [11).

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Ketoconazole had shown cytotoxic activity in prostate cancer cell lines in the in-vitro
studies [11,12). Furthermore, it had previously been demonstrated that at low-dose, this
compound can be used for the treatment of prostate cancer which has failed androgen
deprivation therapy and whose cytotoxic chemotherapy would have a significant impact on
the quality of life [11).

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2. Results and discussion
2.1 Chemistry
The synthetic pathways for the preparation of 4a-4e, 4f-4k and 5f-5o are outlined in
figures 1, 2 and 3 respectively. Commercially available dehydroepiandrosterone (1) was
treated
with
the
corresponding
acid,
p-dimethylaminopyridine
and
dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 2 h.
After the usual work-up, the desired esters were obtained with a yield of 90-95%.
The following step in this synthesis involved the Vilsmeyer-Haack reaction. The
ester derivatives 2a-2p were refluxed with phosphorus oxychloride in DMF and chloroform
for 5 h. After the usual work-up, the desired 16-formyl derivatives 3a-3p (Figures 1, 2 and
3) were obtained with a yield of 70-93%.
The final compounds 4a-4e, 4f-4k (triazole ring at C-17) and 5f-5o (pyrazole ring at
C-17), were prepared using the corresponding steroidal derivative (3a-3p), potassium
carbonate or cesium carbonate (Figure 1, 2 and 3) in DMF; each mixture was warmed at
50 °C for 3 h. The yields of the final compounds in the three series were between 60-70%.
The desired compounds and intermediates were characterized by IR, H1-NMR, C13-NMR
and mass spectrometry.
2.2. Biological activity
The effect of the synthesized compounds (4a-4e, 4f-4k and 5f-5o) on the growth of
three cancer cell lines: PC-3 (human prostate cancer cell line), MCF-7 (human breast
cancer cell line) and SKLU-1 (human lung cancer cell line) was determined using SRB

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assay method. The values of the cell growth inhibition are shown in the Table 1. These data
showed that the cyclopropane ring present in the ester moiety of compound 4a (series 1)
produced a higher citotoxicity as compared to compounds 4b-4e having 4-7 C-atoms in
their ring, and a triazole ring at C-17. Steroid 4e inhibited the cell growth, albeit in a lower
percentage as compared to 4a (Table 1). These results suggested that the size of the ring of
the ester moiety could be related to the cytotoxic activity. The cyclopropane ring is a
strained, planar and very reactive moiety and could form much easier a complex with the
reactive sites present in the cell. Probably, the highest cytotoxicity of steroid 4a could be
explained on the ground of this phenomenon.
The activity of compound 4e (series 1) could be explained by considering the
conformation of the cycloheptane ring. It is a well known fact that the twisted chair
conformation has a low energy and as result of this, this conformation could interact with
the active sites of the cell and produce a cytotoxic effect. The replacement of the
cycloalkane ring in series 1 with a benzene group in the ester moiety increased the co
planarity of the molecule and thus enhanced the cytotoxic activity; this fact was evident
when the cytotoxic activity of 4d from series 1, and 4h from series 2 were compared.
Compound 4d (series 1) has a puckered nonplanar cyclohexane ring and failed to exhibit a
cytotoxic activity. On the other hand, compound 4h having a planar benzene ring, did
exhibit a moderate cytotoxic activity (See Table 1).
Ketoconazole showed also a cytotoxic activity in all three cancer lines studied;
however, its effect was similar to that observed for 4a on PC-3 cell line. Nevertheles,
ketoconazole exhibited a lower percentage of inhibition of citotoxicity on SKLU-1 cells
than steroid 4a (Table 1).

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Compounds 4i and 4j (Series 2) exhibited a higher cytotoxic activity than steroid 4h
(non substituted benzene ring) and ketoconazole in all cell lines studied (Table 1). The
difference between 4h and the other steroids from series 2 is the substitution pattern of the
benzene ring at C-3. The introduction of polar groups in this position increases the polarity
of the molecule (stronger dipole-dipole interaction with the active site on the cell) and this
phenomenon increased their cytotoxic activity (4i and 4j). However, the stable ether group
in 4f, is very hydrophobic and did not increase the cytotoxic effect in PC-3, MCF-7 and
SKLU-1 cell lines, as compared to that of 4h. Steroid 4k increased the cytotoxic activity as
compared to 4h, since the nitro group increased the hydrogen bonding association. On the
other hand the stable methyl group in C-4 of the benzene ring in the ester moiety (steroid
4g) slightly increased the cytotoxic activity of MCF-7 and SKLU-1 as compared to that of
4h (Table 1). The overall, data indicated that an increase of the polarity of the molecule
increases the cytotoxic activity
The activity of the compounds of series 3 on PC-3, MCF-7, and SKLU-1 cancer cell
lines is also exhibited in Table 1. These results showed that none esterified-pyrazole
derivative 6 with a free OH group in C-3 is an active steroid that produces a higher
cytotoxic effect in PC-3 and SKLU-1 cancer cell lines. Thus, the citotoxicity of this
compound was linked to the presence of the free OH group. This functional group allowed
strong hydrogen bonds formation of the steroidal compound with polar active groups in the
cells and as a result of this, a stronger cytotoxic effect was observed.
Apparently the methyl group in C-4 of the ester moiety of compound 5g did not
induce cytotoxicity in the studied cancer cell lines as compared to the ether function of
compound 5f .The non polar methyl group could thwart a binding interaction of the steroid

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with the active sites in the cancer cell lines; on the other hand the methoxy group in 5f
forms hydrogen bonds with the active sites in the cells and this phenomenon explains very
well the high cytotoxic effect of compound 5f.
Compounds 5l, 5m and 5n, all containing a halogen atom, fluorine, chlorine and
bromine respectively, showed a low cytotoxic activity in all cancer lines tested. On the
other hand, compound 5o having an iodine atom in the ester group, surprisingly showed a
much higher activity than the other halogen containing compounds. Compound 5o has a
much higher Log P (partition coefficient) (higher lipophilicity) and can penetrate much
easier the cell membrane and as a result of this, it exhibited an enhanced cytotoxic activity.
Steroid 5k having a nitro group showed a low cytotoxicity. Although compounds 5k and 4k
are very similar (both having a nitro groups), steroid 4k having a triazole group in C-17 has
a much higher cytotoxic activity than 5k with a pyrazole group in C-17. This difference in
the biological activity of both compounds can be attributed to the nature of the azole groups
in C-17. Compounds 4k, having a triazole group at C-17 could form easier hydrogen bonds
with the binding sites of the cells as compared to the pyrazole ring at the same position in
5k and as a result of this steroid 5k has a much lower biological activity.
Previously it had been reported that 7β-hydroxy-epiandrosterone (17β-OH-EpiA) is
an endogenous androgenic derivative of dehydroepiandrosterone that decreases the
synthesis of DGE2, a prostaglandin that induces an inflammatory effect [13]. On the other
hand 17-β estradiol produces an anti-inflammatory effect notably via NFκB, chemokine
production [14] and also via a decrease of PGE2 synthesis [15, 16]. These responses are
mediated through an estrogen receptor (ER).

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Furthermore other studies demonstrated that 17β-OH EpiA exerted an anti-
estrogenic effect in MCF-7 cancer cell line associated with cell proliferation inhibition and
cell cycle arrest. These data indicated that this dehydroepiandrosterone derivative is capable
of interacting with the ER and also with the membrane GPR30 receptors thus producing an
antiproliferative effect [6]. The overall data suggested that steroids 4a, 4i-5f reported in this
study could act trough an ER and GPR30 receptors and as a result of this an
antiproloferative effect was observed in MCF7 cell line [18].
On the other hand, previously it had been reported that the prostate cancer cell line
PC3 expresses ERβ1 or ERβ2 [17]. ERβ1 represses the expression of bone metastasis
regulator genes in PC3, whereas ERβ2 increases the expression of the genes that regulate
this metastasis [17]. Since 17β-OH EpiA exerts its action trough an ER, steroids 4a, 4i-4k
and 6 reported in this study could inhibit the proliferation of this PC3 cell via the ERβ1.
Earlier it had been reported that some transcription factors as hepatocyte nuclear
factor 3β(HNF3β) and CCAAT/enhancer binding protein α (C/EBPα) are strongly
expressed in the lung, and both are tumor suppressors in lung cancer [18]. This effect is
different between both genders [19]. On the other hand, recently it had been demonstrated
that lung adenocarcinomas express ERα and β. These receptors might be coupled to G-
protein (GPER/GPR30) whose expression is increased in lung cancer cells as well as in
tumors when compared to normal lung [20]. The overall data from this study suggested that
the novel dehydroepiandrosterone derivatives reported in this study 4a, 4e, 4i-4K, 5f, 5o
and 6 could induce an apoptotic effect via the activation of the ER in SKLU-1 cell line.

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Conclusion
On the ground of the biological results, described in this paper, we can infer that the
steroidal compounds having a triazole ring at C-17 showed a much higher cytotoxic activity
as compared to compounds 5f-5o having a pyrazole functional group at the same position.
Apparently, this difference can be explained on the base that the triazole ring having three
nitrogen atoms can form much easier hydrogen bonds with the reactive site of the cell as
compared to that of the pyrazole ring (two nitrogen atoms in the ring) at the same position.
It is important to note that compound 6 having a free hydroxyl group at C-3 and a pyrazole
ring at C-17 showed a very high cytotoxic activity. This is probably due to the ability of the
hydroxyl group to form hydrogen bonds with the reactive site of the cell.
Compounds 4f-4k having an aromatic ester at C-3 showed an enhanced cytotoxic
activity as compared to their aliphatic counterpart’s 4a-4e. On the other hand, the aromatic
esters having an electronegative group in the phenyl ring of the ester moiety (4i-4j),
showed a higher activity as compared to those having an electropositive group (4f and 4g).
Apparently, the electronegative substituent on the phenyl ring of the ester moiety increases
the polarity of the molecule thus increasing the dipole-dipole association of the steroidal
molecule with the reactive site of the cell.

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Experimental section
Reagents and solvents were purchased from commercial sources and were used
without further purification. Melting points were determined on a Fisher Johns melting
1
point apparatus and are uncorrected. H NMR and 13C NMR were taken on a Varian
Gemini 200 and a VRX-300 respectively. Chemical shifts are given in ppm relative to that
of Me4Si (δ=0) in CDCl3 (the abbreviations of signal patterns are as follows: s, singlet; d,
doublet, t, triplet; m, multiplet). Mass spectra were obtained with a HP5985-B
spectrometer. IR spectra were recorded on a Perkin-Elmer 200 spectrometer.
Steroidal Synthesis
For the synthesis of the new esters derivatives described in this paper, we used the
commercially available dehydroepiandrosterone 1. The preparation of these compounds is
briefly described below.
General preparation of esters.
To a solution of steroid 1 (1g, 3 mmol), DCC (2.l47 g, 10.41 mmol), DMAP (0.848
g, 6.94 mmol) in chloroform (20 mL) was added the corresponding acid (14 mmol). The
resulting solution was stirred at room temperature for 2 hours. Hexane (15 mL) was added
and the precipitated dicyclohexyl urea was filtered. The organic phase was washed three
times with 10 % aqueous hydrochloric acid, 5 % aqueous sodium bicarbonate, water and
was dried over anhydrous sodium sulfate. The crude ester was recrystallized from
methanol.
17-oxaandrost-5-ene-3β-yl-cyclopropanoate (2a) Yield 92%.mp. 199-201°C, IR
1
(KBr) cm-1: 2947, 1736, 1712. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19),

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4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6). 13C-NMR (CDCl3) δ: 19.3 (C-
18), 20.3 (C-19), 73.6 (C-3), 121.7 (C-6), 139.9 (C-5), 174.2 (ester carbonyl), 220.9 (C-17).
HRMS cal. For C23H32O3 356.2351, found 356.2299.
17-oxaandrost-5-ene-3β-yl-cyclobutanoate (2b) Yield 98%.mp. 179-178°C, IR
1
(KBr) cm-1: 2942, 1735, 1720. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19),
4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6). 13C-NMR (CDCl3) δ: 19.3 (C-
18), 20.3 (C-19), 73.3 (C-3), 121.7 (C-6), 139.7 (C-5), 174.8 (ester carbonyl), 220.9 (C-17).
HRMS cal. For C24H34O3 370.2508, found 370.2256.
17-oxaandrost-5-ene-3β-yl-cyclopentanoate (2c) Yield 96%.mp. 176-178°C, IR
1
(KBr) cm-1: 2945, 1735, 1723. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19),
4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6). 13C-NMR (CDCl3) δ: 19.3 (C-
18), 20.3 (C-19), 73.2 (C-3), 121.7 (C-6), 140.3 (C-5), 176.1 (ester carbonyl), 221.0 (C-17).
HRMS cal. for C25H36O3 384.2664 found 384.2695.
17-oxaandrost-5-ene-3β-yl-cyclohexanoate (2d) Yield 95%.mp. 156-158°C, IR
1
(KBr) cm-1: 2928, 1736, 1725. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19),
4.6 (1H, m, H-3), 5.3 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6). 13C-NMR (CDCl3) δ: 19.3 (C-
18), 20.3 (C-19), 73.4 (C-3), 121.7 (C-6), 139.9 (C-5), 175.8 (ester carbonyl), 220.8 (C-17).
HRMS cal. for C26H38O3 398.2821 found 398.2885.
17-oxaandrost-5-ene-3β-yl-cycloheptanoate (2e) Yield 99%.mp. 161-163°C, IR
1
(KBr) cm-1: 2923, 1737, 1725. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19),
4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6). 13C-NMR (CDCl3) δ: 19.3 (C-
18), 20.3 (C-19), 73.2 (C-3), 121.7 (C-6), 139.9 (C-5), 176.6 (ester carbonyl), 221.4 (C-17).
HRMS cal. for C27H40O3 412.2977 found 412.2945.

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17-oxaandrost-5-ene-3β-yl-p-methoxybenzoate (2f) Yield 95%.mp. 209-211°C,
1
IR (KBr) cm-1:3034, 2936, 2890, 2853, 1736, 1702, 1606, 1512. H-NMR (CDCl3) δ: 0.9
(3H, s, H-18), 1.1 (3H, s, H-19), 3.86 (s, 3H, Ar-OCH3),4.8 (1H, m, H-3), 5.5 (1H, dd. J1=
4 Hz and J2= 2Hz, H-6), 6.9 (2H, d, J= 8 Hz, benzoic ring), 8.0 (2H, d, J = 8 Hz, benzoic
ring). 13C-NMR (CDCl3) δ: 13.7 (C-18), 19.5 (C-19), 74.1 (C-3), 122.0 (C-6), 140.2 (C-5),
113.6, 123.3, 131.7 and 163.2 (benzoic ring), 166.9 (ester carbonyl), 221.1 (C-17). HRMS
cal. for C27H34O4 422.5564 found 422.5612
17-oxaandrost-5-ene-3β-yl-p-methylbenzoate (2g) Yield 88%.mp. 220-222°C, IR
1
(KBr) cm-1: 3028, 2967, 2947, 2924, 2851, 2822, 1736, 1701, 1608, 1575. H-NMR
(CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 2.40 (s, 3H, Ar-CH3),4.8 (1H, m, H-3), 5.4
(1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 7.2 (2H, d, J= 8 Hz, benzoic ring), 7.9 (2H, d, J = 8
Hz, benzoic ring). 13C-NMR (CDCl3) δ: 13.7 (C-18), 19.5 (C-19), 74.2 (C-3), 122.1 (C-6),
140.1 (C-5), 128.1, 129.1, 129.7, 143.5 (benzoic ring), 166.2 (ester carbonyl), 221.3 (C-17).
HRMS cal. for C27H34O3 406.5570 found 406.5512
17-oxaandrost-5-ene-3β-yl-benzoate (2h) Yield 86%. mp. 242-246°C, IR (KBr)
1
cm-1: 3081, 2960, 2948, 2916, 2893, 2839, 2822, 1735, 1711, 1602, 1585. H-NMR
(CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19),4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
J2= 2Hz, H-6), 7.4 (2H, d, J= 8 Hz, benzoic ring), 7.6 (1H, t, J= 8 Hz, benzoic ring), 8.0
(2H, d, J = 8 Hz, benzoic ring). 13C-NMR (CDCl3) δ: 13.7 (C-18), 19.6 (C-19), 74.5 (C-3),
122.2 (C-6), 140.1 (C-5), 128.4, 129.7, 130.9, 132.9 (benzoic ring), 166.1 (ester carbonyl),
221.2 (C-17). HRMS cal. for C26H32O3 392.5304 found 392.5292
17-oxaandrost-5-ene-3β-yl-p-trifluoromethylbenzoate (2i) Yield 83%.mp. 225-
227°C, IR (KBr) cm-1:3068, 2934, 2886, 2857, 1793, 1721, 1650, 1585. 1H-NMR (CDCl3)

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δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2=
2Hz, H-6), 7.7 (2H, d, J= 8 Hz, benzoic ring), 8.1 (2H, d, J= 8 Hz, benzoic ring). 13C-NMR
(CDCl3) δ: 13.7 (C-18), 19.5 (C-19), 75.2 (C-3), 122.4 (C-6), 139.8 (C-5), 119.7, 122.4,
125.1, 127.9 (Ar-CF3), 125.4, 125.5, 133.9, 134.2, 134.6, 134.9, 134.1 (benzoic ring), 164.8
(ester carbonyl), 221.0 (C-17). HRMS cal. for C27H31F3O3 460.5284 found 460.5212
17-oxaandrost-5-ene-3β-yl-p-cyanobenzoate (2j) Yield 70%.mp. 236-238°C, IR
1
(KBr) cm-1: 3064, 2933, 2909, 2823, 2879, 3054, 2232, 1736, 1710, 1627, 1570. H-NMR
(CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.5 (1H, dd. J1= 4 Hz and
J2= 2Hz, H-6), 7.7 (2H, d, J= 8 Hz, benzoic ring), 8.1 (2H, d, J= 8 Hz, benzoic ring). 13C-
NMR (CDCl3) δ: 13.5 (C-18), 19.5 (C-19), 75.5 (C-3), 118.2 (Ar-CN), 122.5 (C-6), 139.7
(C-5), 116.4, 130.2, 132.3, 134.7(benzoic ring), 164.4 (ester carbonyl), 221.0 (C-17).
HRMS cal. for C27H31NO3 417.5399 found 417.5313
17-oxaandrost-5-ene-3β-yl-p-nitrobenzoate (2k) Yield 79%.mp. 204-206°C, IR
(KBr) cm-1:3113, 2943, 2890, 2853, 1736, 1716, 1525, 1605. 1H-NMR (CDCl3) δ: 0.9 (3H,
s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.5 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 8.2
13
(2H, d, J= 8 Hz, benzoic ring), 8.3 (2H, d, J= 8 Hz, benzoic ring). C-NMR (CDCl3) δ:
13.5 (C-18), 19.5 (C-19), 75.7 (C-3), 122.5 (C-6), 139.7 (C-5), 130.8, 123.6, 136.2,
150.6(benzoic ring), 164.2 (ester carbonyl), 221.1 (C-17). HRMS cal. for C26H31NO5
437.5285 found 437.5198
17-oxaandrost-5-ene-3β-yl-p-fluorobenzoate (2l) Yield 88%.mp. 208-209°C, IR
(KBr) cm-1:3111, 2948, 285, 2825, 1741, 1709. 1H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.1
(3H, s, H-19),4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 7.1 (2H, dd, J1= 8
13
Hz and J2= 2 Hz, benzoic ring), 8.1 (2H, dd, J1= 7 Hz, J2= 5 Hz, benzoic ring). C-NMR

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(CDCl3) δ: 13.7 (C-18), 19.6 (C-19), 74.6 (C-3), 122.2 (C-6), 139.9 (C-5), 115.4, 115.6,
127.1, 127.1, 132.1, 132.2, 164.5, 167.1(benzoic ring), 165.1 (ester carbonyl), 221.1 (C-
17). HRMS cal. for C26H31FO3 410.5209 found 410.4985
17-oxaandrost-5-ene-3β-yl-p-chlorobenzoate (2m) Yield 87%.mp. 231-234°C, IR
(KBr) cm-1:3041, 2947, 2888, 2844, 1739, 1719, 1626, 1592. 1H-NMR (CDCl3) δ: 0.9 (3H,
s, H-18), 1.1 (3H, s, H-19),4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 7.4
13
(2H, d, J= 8 Hz, benzoic ring), 8.0 (2H, d, J= 8 Hz, benzoic ring). C-NMR (CDCl3) δ:
13.7 (C-18), 19.6 (C-19), 74.8 (C-3), 122.3 (C-6), 139.9 (C-5), 128.8, 129.3, 131.1,
139.3(benzoic ring), 165.3 (ester carbonyl), 221.2 (C-17). HRMS cal. for C26H31ClO3
426.9755 found 426. 9712
17-oxaandrost-5-ene-3β-yl-p-bromobenzoate (2n) Yield 91%.mp. 223-225°C, IR
(KBr) cm-1:3035, 2927, 2849, 1736, 1703, 1622, 1570. 1H-NMR (CDCl3) δ: 0.9 (3H, s, H-
18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 7.6 (2H,
d, J= 8 Hz, benzoic ring), 7.9 (2H, d, J= 8 Hz, benzoic ring). 13C-NMR (CDCl3) δ: 13.7 (C-
18), 19.6 (C-19), 74.8 (C-3), 122.3 (C-6), 139.9 (C-5), 128.0, 129.8, 131.2, 131.8(benzoic
ring), 165.4 (ester carbonyl), 221.3 (C-17). HRMS cal. for C26H31BrO3 471.4265 found
471.4295
17-oxaandrost-5-ene-3β-yl-p-iodobenzoate (2o) Yield 89%.mp. 235-239°C, IR
(KBr) cm-1:3035, 2947, 2908, 2893, 2839, 1731, 1713, 1585. 1H-NMR (CDCl3) δ: 0.9 (3H,
s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 7.7
13
(2H, d, J= 8 Hz, benzoic ring), 7.8 (2H, d, J= 8 Hz, benzoic ring). C-NMR (CDCl3) δ:
13.7 (C-18), 19.6 (C-19), 74.8 (C-3), 122.3 (C-6), 139.9 (C-5), 100.7, 130.3, 131.2,

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137.7(benzoic ring), 165.6 (ester carbonyl), 221.2 (C-17). HRMS cal. for C26H31IO3
518.4270 found 518.4298
Synthesis of 17-oxoandrost-5-en-3-yl-3β-formate (2p)
In a round-bottomed flask, fitted with a magnetic stirrer, was added 500 mg of
dehydroepiandrosterone (1.7 mmol) and 40 mL of formic acid, the reaction was refluxed
for 5 hours. The solution was quenched a saturated solution of sodium bicarbonate, the
white precipitate was filtered under vacuum to obtain 490 mg of 3β-formyl-oxy-5-
androsten-17-one.Yield 89.3%. mp. 143-145°C, IR (KBr) cm-1: 2964.45, 2941.61, 2890.43,
1
2824.65, 1736.86, 1702.53. H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19), 4.6
(1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 8.03 (1H, s, formate).13C-NMR
(CDCl3) δ: 13.7 (C-18), 19.5 (C-19), 73.8 (C-3), 122.4 (C-6), 139.7 (C-5), 160.7 (formate),
221.1 (C-17). HRMS cal. for C20H28O3 316.4352 found 316.4312
Synthesis of 16-formyl-17-chloroandrosta-5,16-diene derivatives
Into a round-bottomed flask, fitted with a magnetic stirrer and cold
dymethylformamide (43 mmol) was added dropwise phosphorus oxychloride (36 mmol)
followed by a solution of the corresponding ester in chloroform (l mmol) (5 mL). The
mixture was refluxed for 5 h under N2. The reaction was quenched with a saturated solution
of sodium bicarbonate (10 mL). The compound was extracted with chloroform, and the
organic phase was washed with water and dried over anhydrous sodium sulfate. The
compound was purified on silica gel chromatography.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-cyclopropanoate (3a) Yield 72%.
1
mp. 124-126 °C; IR (KBr) cm-1: 2928, 1717, 1674, 1589. H-NMR (CDCl3) δ: 0.9 (3H, s,
H-18), 1.0 (3H, s, H-19), 4.6 (1H, m, H-3), 5.3 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 9.9

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13
(1H, s, aldehyde). C-NMR (CDCl3) δ: 19.2 (C-18), 20.3 (C-19), 73.5 (C-3), 121.7 (C-6),
136.4 (C-16), 140.1 (C-5), 162.2(C-17), 174.2 (ester carbonyl), 188.0 (aldehyde). HRMS
cal. for C24H31ClO3 402.1962 found 402.1922.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-cyclobutanoate (3b) Yield 69%.
1
mp. 152-154 °C; IR (KBr) cm-1: 2941, 1719, 1671, 1586. H-NMR (CDCl3) δ: 0.9 (3H, s,
H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 9.9
13
(1H, s, aldehyde). C-NMR (CDCl3) δ: 19.1 (C-18), 20.2 (C-19), 73.2 (C-3), 121.6 (C-6),
136.3 (C-16), 139.9 (C-5), 162.2 (C-17), 174.8 (ester carbonyl), 187.9 (aldehyde). HRMS
cal. for C25H33ClO3 416.2118 found 416.2189.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-cyclopentanoate (3c) Yield 70%.
1
mp. 180-182 °C; IR (KBr) cm-1: 2927, 1728, 1708, 1667, 1590. H-NMR (CDCl3) δ: 0.9
(3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-
6), 9.9 (1H, s, aldehyde). 13C-NMR (CDCl3) δ: 19.2 (C-18), 20.2 (C-19), 73.2 (C-3), 121.6
(C-6), 136.4 (C-16), 140.1 (C-5), 162.1 (C-17), 176.2 (ester carbonyl), 186.0 (aldehyde).
HRMS cal. for C26H35ClO3 430.2275 found 430.2210.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-cyclohexanoate (3d) Yield 71%.
1
mp. 166-168 °C; IR (KBr) cm-1: 2929, 1727, 1668, 1592. H-NMR (CDCl3) δ: 0.9 (3H, s,
H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2 Hz, H-6), 9.9
13
(1H, s, aldehyde). C-NMR (CDCl3) δ: 19.2 (C-18), 20.3 (C-19), 72.9 (C-3), 121.6 (C-6),
136.4 (C-16), 140.1 (C-5), 162.2 (C-17), 175.5 (ester carbonyl), 188.0 (aldehyde). HRMS
cal. for C27H37ClO3 444.2431 found 444.2487.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-cycloheptanoate (3e) Yield 70%.
1
mp. 215-217 °C; IR (KBr) cm-1: 2946, 1719, 1671, 1667, 1585. H-NMR (CDCl3) δ: 0.9

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(3H, s, H-18), 1.0 (3H, s, H-19), 4.5 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-
6), 9.9 (1H, s, aldehyde). 13C-NMR (CDCl3) δ: 19.2 (C-18), 20.3 (C-19), 73.1 (C-3), 121.6
(C-6), 136.4 (C-16), 140.1 (C-5), 162.2 (C-17), 176.5 (ester carbonyl), 188.0 (aldehyde).
HRMS cal. for C28H39ClO3 458.2588 found 458.2514.
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-methoxybenzoate (3f) Yield
62%. mp. 204-206 °C; IR (KBr) cm-1:3031, 2960, 2930, 2960, 2891, 2844, 2861, 1700,
1669, 1603, 1511. 1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 3.9 (3H, s, Ar-
CH3), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 6.6 (2H, d, J= 8 Hz,
benzoic ring), 8.0 (2H, d, J= 8 Hz, benzoic ring), 10.0 (1H, s, aldehyde). 13C-NMR
(CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 55.6 (Ar-OCH3), 74.1 (C-3), 113.6, 123.3, 131.7,
163.4(benzoic ring), 122.0 (C-6), 136.6 (C-16), 140.2 (C-5), 162.5 (C-17), 165.9 (ester
carbonyl), 188.3 (aldehyde). HRMS cal. for C28H33ClO4 469.0122 found 469.0195
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-methylbenzoate (3g) Yield 60%.
mp. 219-221 °C; IR (KBr) cm-1:3033, 2928, 2893, 2857, 2727, 1706, 1672, 1608, 1589.1H-
NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 3.9 (3H, s, Ar-CH3), 4.8 (1H, m, H-
3), 5.4 (1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 6.6 (2H, d, J= 8 Hz, benzoic ring), 8.0 (2H, d,
J= 8 Hz, benzoic ring), 10.0 (1H, s, aldehyde). 13C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-
19), 74.1 (C-3), 113.6, 123.3, 131.7, 163.4 (benzoic ring), 122.0 (C-6), 136.6 (C-16), 140.2
(C-5), 162.5 (C-17), 165.9 (ester carbonyl), 188.3 (aldehyde). HRMS cal. for C28H33ClO3
453.0128 found 453.0198
16-formyl-17-chloroandrost-5,16-diene-3β-yl-benzoate (3h) Yield 47%. mp. 177-
1
179 °C; IR (KBr) cm-1: 3032, 2940, 2926, 2892, 2833, 2860, 1708, 1664, 1590. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and

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J2= 2Hz, H-6), 7.4 (2H, t, J= 8 Hz, benzoic ring), 7.5 (1H, ddd, J1= 7,J2= 4, J3= 1 Hz,
benzoic ring), 8.0 (2H, t, J= 8 Hz, benzoic ring), 10.0 (1H, s, aldehyde). 13C-NMR (CDCl3)
δ: 15.1 (C-18), 19.4 (C-19), 74.4 (C-3), 128., 129.7, 130.8, 132.9(benzoic ring), 122.1 (C-
6), 136.6 (C-16), 140.1 (C-5), 162.5 (C-17), 166.1 (ester carbonyl), 188.2 (aldehyde).
HRMS cal. for C27H31ClO3 438.9862 found 438.9812
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-trifluorobenzoate (3i) Yield
83%. mp. 198-200 °C; IR (KBr) cm-1: 2948, 2912, 2869, 2825, 2723, 1716, 1674, 1588.
1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.5 (1H, dd. J1=
4 Hz and J2= 2Hz, H-6), 7.7 (2H, d, J= 8 Hz, benzoic ring), 8.1 (2H, d, J= 8 Hz, benzoic
ring), 10.0 (1H, s, aldehyde). 13C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 75.1 (C-
3),119.72, 122.43, 125.14, 127.86 (Ar-CF3), 125.4, 125.5, 130.1, 134.0, 134.1, 134.3,
134.6, 134.9 (benzoic ring), 122.2 (C-6), 136.6 (C-16), 139.9 (C-5), 162.3 (C-17), 164.9
(ester carbonyl), 188.2 (aldehyde). HRMS cal. for C28H30F3ClO3 506.9842 found 506.9812
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-cyanobenzoate (3j) Yield 36%.
mp. 212-215 °C; IR (KBr) cm-1: 3052, 2936, 2905, 2852, 2228, 1715, 1669, 1588. 1H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
J2= 2Hz, H-6), 7.7 (2H, d, J= 8 Hz, benzoic ring), 8.1 (2H, d, J= 8 Hz, benzoic ring), 10.0
13
(1H, s, aldehyde). C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 75.4 (C-3), 118.2 (Ar-
CN), 132.3, 134.7, 130.2, 116.4(benzoic ring), 122.5 (C-6), 136.6 (C-16), 139.8 (C-5),
162.4 (C-17), 164.4 (ester carbonyl), 188.2 (aldehyde). HRMS cal. for C28H30ClNO3
463.9957 found 463.9913
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-nitrobenzoate (3k) Yield 65%.
mp. 238-241 °C; IR (KBr) cm-1: 3057, 2950, 2936, 2912, 2958, 2824, 2724, 1711, 1668,

ACCEPTED MANUSCRIPT
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1586, 1526. 1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4
(1H, dd. J1= 4 Hz and J2= 2Hz, H-6), 8.2 (2H, d, J= 8 Hz, benzoic ring), 8.3 (2H, d, J= 8
13
Hz, benzoic ring), 10.0 (1H, s, aldehyde). C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19),
75.6 (C-3), 123.6, 130.8, 136.3, 150.6 (benzoic ring), 122.5 (C-6), 136.6 (C-16), 139.7 (C-
5), 162.3 (C-17), 164.2 (ester carbonyl), 188.2 (aldehyde). HRMS cal. for C27H30ClNO5
483.9843 found 483.9814
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-fluorobenzoate (3l) Yield 79%.
mp. 166-168 °C; IR (KBr) cm-1: 3052, 2977, 2957, 2932, 2853, 1714, 1663, 1601, 1585.
1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1=
4 Hz and J2= 2Hz, H-6), 7.1 (2H, dd, J1= 8 Hz, J2= 2 Hz benzoic ring), 8.0 (2H, ddd, J1=
5,J2= 2, J3= 1 Hz, benzoic ring), 10.0 (1H, s, aldehyde). 13C-NMR (CDCl3) δ: 15.1 (C-18),
19.4 (C-19), 74.6 (C-3), 115.4, 115.6, 127.1, 132.1, 132.2, 164.5, 167.1 (benzoic ring),
122.2 (C-6), 136.6 (C-16), 140.0 (C-5), 162.3 (C-17), 165.1 (ester carbonyl), 188.1
(aldehyde). HRMS cal. for C27H30ClFO3 456.9767 found 456.9716
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-chlorobenzoate (3m) Yield 46%.
mp. 186-187 °C; IR (KBr) cm-1: 3038, 2975, 2960, 2929, 2905, 2852, 2735, 1718, 1669,
1585. 1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H,
dd. J1= 4 Hz and J2= 2Hz, H-6), 7.4 (2H, d, J= 8 Hz, benzoic ring), 8.0 (2H, d, J1= 8 Hz,
13
benzoic ring), 10.0 (1H, s, aldehyde). C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 74.7
(C-3), 128.76, 129.32, 131.08, 139.35 (benzoic ring), 122.3 (C-6), 136.6 (C-16), 140.0 (C-
5), 162.4 (C-17), 165.2 (ester carbonyl), 188.2 (aldehyde). HRMS cal. for C27H30Cl2O3
473.4313 found 473.4378

ACCEPTED MANUSCRIPT
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16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-bromobenzoate (3n) Yield 65%.
1
mp. 178-180 °C; IR (KBr) cm-1: 3036, 2928, 2888, 2855, 1711, 1668, 1587. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
J2= 2Hz, H-6), 7.6 (2H, d, J= 8 Hz, benzoic ring), 7.9 (2H, d, J1= 8 Hz, benzoic ring), 10.0
(1H, s, aldehyde). 13C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 74.7 (C-3), 128.0, 129.7,
131.2, 131.7 (benzoic ring), 122.2 (C-6), 136.6 (C-16), 140.0 (C-5), 162.4 (C-17), 165.4
(ester carbonyl), 188.2 (aldehyde). HRMS cal. for C27H30BrClO3 517.8823 found 517.8869
16-formyl-17-chloroandrost-5,16-diene-3β-yl-p-iodobenzoate (3o) Yield 53%.
1
mp. 170-173 °C; IR (KBr) cm-1:3035, 2939, 2858, 1707, 1664, 1586.4. H-NMR (CDCl3)
δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.8 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and J2=
2Hz, H-6), 7.7 (2H, d, J= 8 Hz, benzoic ring), 7.8 (2H, d, J= 8 Hz, benzoic ring), 10.0 (1H,
s, aldehyde). 13C-NMR (CDCl3) δ: 15.1 (C-18), 19.4 (C-19), 74.8 (C-3), 100.7, 130.3,
131.1, 137.8 (benzoic ring), 122.2 (C-6), 136.6 (C-16), 140.0 (C-5), 162.4 (C-17), 165.6
(ester carbonyl), 188.2 (aldehyde). HRMS cal. for C27H30ClIO3 564.8827 found 564.8893
16-formyl-17-chloroandrost-5,16-diene-3β-yl-formate (3p) Yield 64%. mp. 159-
161 °C; IR (KBr) cm-1: 2952, 2922, 2905, 2893, 2866, 2854, 2830, 2730, 1712, 1665, 1584.
1H-NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.1 (3H, s, H-19), 4.7 (1H, m, H-3), 5.4 (1H, dd. J1=
4 Hz and J2= 2Hz, H-6), 8.0 (1H, s, formate), 10.0 (1H, s, aldehyde). 13C-NMR (CDCl3) δ:
15.1 (C-18), 19.3 (C-19), 73.7 (C-3), 122.3 (C-6), 136.6 (C-16), 139.8 (C-5), 162.4 (C-17),
162.4 (ester carbonyl), 188.2 (aldehyde). HRMS cal. for C21H27ClO3 362.8902 found
362.8975
Synthesis of 16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl
derivatives.

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A mixture of 1,2,4-triazole (3 mmol). K2CO3 (6 mmol) and the corresponding l6-
formyl-17-chloroandrosta-5,16-diene-3β-yl derivate (1 mmol) in dry DMF (5 mL) was
heated at 50 °C under N2 atmosphere for 3 h. Cold water was added and the solid was
filtered. The compound was purified by column chromatography on florisil.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-
cyclopropanoate (4a). Yield 93%.mp. 190-192 °C, IR (KBr) cm-1: 2933, 1721, 1662,
1613. 1H-NMR (CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H,
dd. J1= 4 Hz and J2= 2Hz, H-6), 8.1 (1H, s, aromatic), 8.4 (1H, s, aromatic), 10.1 (1H, s,
13
aldehyde). C-NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 73.5 (C-3), 121.7 (C-6), 132.3
(C-16), 140.5 (C-5), 143.7 (C-17), 153.1 (aromatic), 156.7 (aromatic), 174.2 (ester
carbonyl), 189.0 (aldehyde). HRMS cal. for C26H33N3O3 435.5585 found 435.5518.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-cyclobutanoate
1
(4b). Yield 89%.mp. 199-201 °C, IR (KBr) cm-1: 2940, 1724, 1664, 1613. H-NMR
(CDCl3) δ: 1.1 (3H, s, H-18), 1.2 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
13
J2= 2Hz, H-6), 8.1 (1H, s, aromatic), 8.4 (1H, s, aromatic), 10.1 (1H, s, aldehyde). C-
NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 73.2 (C-3), 121.6 (C-6), 132.3 (C-16), 140.0 (C-
5), 143.6 (C-17), 153.0 (aromatic), 156.7 (aromatic), 174.9 (ester carbonyl), 189.0
(aldehyde). HRMS cal. for C27H35N3O3 449.5851 found 449.5812.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-cyclopentanoate
1
(4c). Yield 91%.mp. 249-251 °C, IR (KBr) cm-1: 2937, 1728, 1667, 1618. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.5 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
13
J2= 2Hz, H-6), 8.1 (1H, s, aromatic), 8.4 (1H, s, aromatic), 10.1 (1H, s, aldehyde). C-
NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 73.2 (C-3), 121.6 (C-6), 132.3 (C-16), 140.1 (C-

ACCEPTED MANUSCRIPT
26
5), 143.6 (C-17), 152.9 (aromatic), 156.4 (aromatic), 175.9 (ester carbonyl), 189.1
(aldehyde). HRMS cal. for C28H37N3O3 463.6117 found 463.6167.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-cyclohexanoate
1
(4d). Yield 93%.mp. 232-234 °C, IR (KBr) cm-1: 2938, 1728, 1667, 1617. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.5 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
13
J2= 2Hz, H-6), 8.1 (1H, s, aromatic), 8.4 (1H, s, aromatic), 10.1 (1H, s, aldehyde). C-
NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 73.0 (C-3), 121.6 (C-6), 132.3 (C-16), 140.1 (C-
5), 143.8 (C-17), 153.0 (aromatic), 156.7 (aromatic), 175.5 (ester carbonyl), 189.9
(aldehyde). HRMS cal. for C29H39N3O3 477.6383 found 477.6319.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-cycloheptanoate
1
(4e). Yield 91%.mp. 195-197 °C, IR (KBr) cm-1: 2934, 1728, 1668, 1618. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.6 (1H, m, H-3), 5.4 (1H, dd. J1= 4 Hz and
13
J2= 2Hz, H-6), 8.1 (1H, s, aromatic), 8.4 (1H, s, aromatic), 10.1 (1H, s, aldehyde). C-
NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 73.0 (C-3), 121.1 (C-6), 132.3 (C-16), 140.1 (C-
5), 143.6 (C-17), 153.0 (aromatic), 156.6 (aromatic), 175.5 (ester carbonyl), 188.8
(aldehyde). HRMS cal. for C30H41N3O3 491.6648 found 491.6699.
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-4-methoxybenzoate
(4f). Yield 70.4% mp. 209-211 °C, IR (KBr) cm-1:3116, 2939, 1702, 1663, 1579, 1275. 1H-
NMR (CDCl3) δ: 1.1 (3H, s, H-18), 1.2 (3H, s, H-19), 3.85 (s, 3H, Ar-OCH3), 4.8 (1H, m,
H-3), 5.4 (1H, dd. J1= 4 Hz and J2=2 Hz, H-6), 6.9 (2H, d, J= 8 Hz, benzoic ring), 7.9 (2H,
t, J = 8 Hz, benzoic ring), 8.1 (1H, s, triazole ring), 8.4 (1H, s, triazole ring), 10.1 (1H, s,
aldehyde). 13C-NMR (CDCl3) δ: 19.2 (C-18), 20.4 (C-19), 55.4 (OCH3), 74.1 (C-3);
113.50, 123.1, 131.5 and 163.2 (benzoic ring); 121.7 (C-6), 132.3 (C-16), 156.7 (C-5),

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140.1 (C-17), 143.6 and 153.0 (triazole ring), 165.7 (ester carbonyl), 189.0 (aldehyde).
HRMS cal. for C30H35N3O4 501.6166, found 501.6113
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-p-
methylbenzoate (4g). Yield 68.6% mp. 239-241°C, IR (KBr) cm-1:3110, 2926, 1714, 1661,
1605, 1522. 1H-NMR (CDCl3) δ: 1.1 (3H, s, H-18), 1.2 (3H, s, H-19), 2.4 (3H, s, Ar-CH3);
4.8 (1H, m, H-3), 5.5 (1H, dd. J1= 4 Hz and J2= 2 Hz, H-6), 6.5 (1H, dd, J1 = 3 Hz, J2 =2
Hz, pyrazole ring); 7.2 (2H, d, J= 8 Hz, benzoic ring), 7.9 (2H, d, J = 8 Hz, benzoic ring),
8.1 (1H, s, triazole ring), 8.4 (1H, s, triazole ring), 10.1 (1H, s, aldehyde). 13C-NMR
(CDCl3) δ: 16.2 (C-18), 19.4 (C-19), 21.8 (Ar-CH3), 74.2 (C-3); 143.7 and 153.0 (triazole
ring); 128.2, 129.1, 129.7 and 143.5 (benzoic ring); 122.2 (C-6), 140.2 (C-5), 161.2 (C-17),
166.2 (ester carbonyl), 190.6 (aldehyde). HRMS cal. for C30H35N3O3 485.6172 found
485.6115
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-benzoate (4h).
1
Yield 74.3% mp. 239-241°C, IR (KBr) cm-1:3065, 2938, 1709, 1667, 1599. H-NMR
(CDCl3) δ: 1.1 (3H, s, H-18), 1.2 (3H, s, H-19), 4.8 (1H, m, H-3), 5.5 (1H, dd. J1= 4 Hz and
J2= 2 Hz, H-6), 7.4 (2H, t, J = 8 Hz benzoic ring); 7.5 (1H, t, J = 7.3 Hz, benzoic ring); 8.0
(2H, d, J = 8.0 Hz, benzoic ring); 8.1 (1H, s, triazole ring), 8.4 (1H, s, triazole ring), 10.1
13
(1H, s, aldehyde). C-NMR (CDCl3) δ: 16.2 (C-18), 19.4 (C-19), 74.4 (C-3),143.7 and
153.0 (triazole ring); 129.7, 128.4, 130.9, 132.9 (benzoic ring); 122.3 (C-6), 140.2 (C-5),
161.2 (C-17), 166.1 (ester carbonyl), 190.6 (aldehyde). HRMS cal. for C29H33N3O3
471.5906 found 471.5978
16-formyl-17-(1H-1,2,4-triazole-1-yl)androsta-5,16-diene-3β-yl-p-
trifluoromethylbenzoate (4i). Yield 73.7% mp.228-230 °C, IR (KBr) cm-1: 3065, 2938,